**The Shade Avoidance Syndrome Under the Sugarcane Crop**

Jocelyne Ascencio and Jose Vicente Lazo *Universidad Central de Venezuela, Facultad de Agronomía, Maracay Venezuela* 

#### **1. Introduction**

166 Crop Plant

Zhu, Q., Zheng, X., Luo, J., Gaut, B.S. and Ge, S*.* (2007). Multilocus analysis of nucleotide

domestication of rice*. Mol. Biol. Evol.,* 24*:* 875*-*888.

variation of *Oryza sativa* and its wild relatives: severe bottleneck during

Sugarcane is grown mainly for sugar and ethanol production, belongs to the Poaceae family, genus *Saccharum* native to Southest Asia and India and cultivated intensively in tropical and subtropical areas throughout the world, and it plays a significant role in the world economy and the area cultivated yields observed to have progressively increased to remarkable levels in the last 10 years (Azevedo et al., 2011). Commercial sugarcane, mainly the interspecific hybrids of *S*. *officinarum* and *S. spontaneum* would greatly benefit from biotechnological improvements due to the long duration (10-15 years) required to breed elite cultivars, more importantly there is an ongoing need to provide durable and disease and pest resistance in combination with superior agronomic performance (Lakshman et al., 2005).

There is an increasing pressure worldwide to enhance the productivity of sugarcane cultivation in order to sustain profitable sugar industries (Hanlon et al., 2000), for example, improvement of industrial processes along with strong sugarcane breeding programs in Brazil, brought technologies that currently support a cropland of 7 million hectares of sugarcane with an average yield of 75 tons/ha (Matsuoka et al., 2009). Besides, biomass has gained prominence in the last years as new technologies for energy production from crushed sugarcane stalks developed a sugarcane industry that currently is one the most efficient systems for the conversion of photosynthate into different forms of energy, for example, the production of ethanol as a liquid fuel.

The crop is vegetatively propagated by stalk cuttings, having one to three buds, known as seed pieces or setts, is a perennial crop regrowing from these vegetative buds after the crop has been harvested giving subsequent regrowth or crop cycles known as rattoning. The germinating bud develops its own root system, and several shoots arise by heavy tillering which produces a canopy of leaves during closing-in stages of crop growth; the term "closed crop" defines a community of plants, of uniform height, which extends indefinitely in the horizontal plane. Within a "closed crop" canopy, we might expect the leaves in any particular horizon to experience a uniform environment, and we might further expect the only significant source of environmental variation to be found in the vertical plane (Charles-Edwards, 1981), thus for the sugarcane crop the production of stalks, to quickly achieving a closed canopy, is important as a means of increasing competition with the weeds growing underneath and for crop protection against adverse conditions.

The Shade Avoidance Syndrome Under the Sugarcane Crop 169

conditions, slight differences in height, degree of tillering, earlier flowering and increase in the shoot/root ratio among different species, might imply a greater potential to survive escaping shade; thus recognition of plant species before and after canopy closure as well as the changes in the light profiles under the canopy, are important for weed detection in

Shade avoidance responses are mediated by multiple forms of phytochromes; despite of initial attempts to adscribe the SAS to the action of a single member of the phytochrome family (Franklin & Whitelam, 2005). In this context and according to Schmitt (1997), the shade avoidance response has undergone adaptive evolution as quantitative genetic variation in R:FR ratio sensitivity has been detected in wild populations. The "Shade Avoidance Syndrome" has been described by Morgan et al (2002); Smith (1982); Smith & Whitelam (1997) as an accelerated extension growth (as seen by an increase in shoot and petiole elongation), reduced branching (increase in apical dominance), earlier flowering (i.e., rate of flowering markedly accelerated) and increase in the shoot to root ratios, changes in plant architecture and leaf shape, among other responses not easily seen under field conditions, however, we have used the term "Agronomic Shade Avoidance Syndrome" to include species that persist and compete successfully with the crop after canopy closure, but not by means of growth responses normally associated to the SAS under field conditions, such as morphological changes in leaf shape, stem elongation or plant architecture. The persistence of such species after canopy closure, may be associated to the seed bank, sunflecks in gaps within the canopy and the production of underground organs (that become well established and almost impossible to control by shade) as well as climbing

From another point of view, the shade avoidance syndrome is not restricted to terrestrial ecosystems but has also been studied in connection to other stress responses, such as submergence (Pierik et al., 2005). In complete submergence, well-adapted plants may overcome the effects by adopting an avoidance strategy to induce growth responses, phenotypically similar to those described when plants are shaded by proximate neighbors. In this chapter, we will analyze light profiles within sugarcane canopies and how changing light conditions in a closed-in canopy, may affect the development and diversity of plant species, as seen in the field at the individual level. Then, based on the results of experimental trials and under controlled conditions, some of the strategies to escape or avoid shade and to capture light more efficiently, will be discussed for different species

Field experiments were conducted in two different sites where sugarcane is a mayor crop in Venezuela. The first experiment was established with sugarcane plants (*Saccharum* spp hybrid var. PR-692176 (first ratoon crop) in a 625 m2 plot inside a commercial regrowth of a sugarcane field in Chivacoa, Yaracuy State and the second experiment, in 2500 m2 experimental area in the Agricultural Experimental Station at the College of Agriculture in the Central University of Venezuela in Maracay, Aragua State, using droopy and erect leaf

commercial varieties planted from cane (initial or plant cane crop).

cultivated crops as a means to ascertain which species avoid shade.

strategies such as in tie-vines.

**2. Research methods 2.1 Field experiments** 

known to be noxious to the sugarcane crop.

Sugarcane uses the C4 pathway of photosynthesis where CO2 is efficiently captured, in the mesophyll cells giving a four-carbon organic acid, oxaloacetate which is the first product of CO2 fixation, and recycled inside the leaves because of the compartmentalized arrangement of leaf tissues into bundle sheath and mesophyll cells (Hatch & Slack, 1966). This photosynthetic specialization of cell types allows leaves to fix CO2 at higher rates and at lower stomatal conductance; however other C4 species dominate the list of the world´s worst weeds which in many cases, like for the sugarcane crop, are among the most noxious plants to the crop. Failure to control weeds during early stages of crop growth can reduce yields appreciably. As a C4 plant sugarcane grows better at high irradiances and temperatures and is also resistant to some environmental conditions very common in the field, especially in the tropics, such as water deficits. Because of these attributes improved cultivars with increased resistance to stressful conditions, adequate management of water and other resources have been developed.

Light interception is an important component of the environment within a crop canopy; in sugarcane solar radiation is intercepted by the extended leaves but canopy architecture can modify photosynthetic performance. Thus commercial sugarcane varieties may have erect or planophille leaves but in a closed canopy most of the light is intercepted by the top fully expanded leaves. Erect leaved varieties, appear to be more efficient capturing light than those with more planophille or droopy leaves, specially at high plant densities. In this context strategy for weed control in order to improve farm management must include the knowledge of the dynamic and biology of plants growing underneath the canopy.

Canopy shade is an important part of weed-crop interference, thus the effect of radiation quality (wavelength) and quantity (irradiance, photon flux) on the diversity of plant species is a serious constraint to production and crop management. The sugarcane crop canopy closes at about three months after planting/sprouting, and the population of plants under the canopy changes in number and diversity depending on the ability of some species to escape or avoid shade by a series of developmental changes at the individual and population levels. Under field conditions, for each sugarcane ratoon cycle (regrowth), recognition of the species diversity as well as their strategies for shade avoidance, before and after canopy closure, is relevant to agricultural applications and plant biology and as research on the shade avoidance syndrome has been mostly restricted to individual plants and under controlled conditions, the objective of the present study is to provide information about what happens under a sugarcane canopy under field conditions, as related to spectral shifts within the canopy and the changes in species diversity.

The shade avoidance syndrome (SAS) in plant neighborhoods such as those underneath a crop canopy, is associated with both the quality (wavelength) and quantity (energy) of light and the decrease in the red/far red ratio (R/FR), as the light environment becomes enriched in far red radiation that is reflected by the leaves of all plants, including the crop itself. A reduced R/FR is the proximity signal that is perceived by the plants alerting that they are being shaded, and in fact it is perceived in early developmental stages, as shown by Ballare & Casal, 2000; Ballare et al., 1991; Smith et al., 1990. Small changes in amounts of red relative to far red light have been shown to alter the equilibrium of different phytochrome forms appreciably, which plays a major role in plant development. Perception of light quality and quantity at the stem level may elicit morphological adaptations in the plants growing beneath the canopy, that may result in shade tolerance or avoidance, and under field

Sugarcane uses the C4 pathway of photosynthesis where CO2 is efficiently captured, in the mesophyll cells giving a four-carbon organic acid, oxaloacetate which is the first product of CO2 fixation, and recycled inside the leaves because of the compartmentalized arrangement of leaf tissues into bundle sheath and mesophyll cells (Hatch & Slack, 1966). This photosynthetic specialization of cell types allows leaves to fix CO2 at higher rates and at lower stomatal conductance; however other C4 species dominate the list of the world´s worst weeds which in many cases, like for the sugarcane crop, are among the most noxious plants to the crop. Failure to control weeds during early stages of crop growth can reduce yields appreciably. As a C4 plant sugarcane grows better at high irradiances and temperatures and is also resistant to some environmental conditions very common in the field, especially in the tropics, such as water deficits. Because of these attributes improved cultivars with increased resistance to stressful conditions, adequate management of water

Light interception is an important component of the environment within a crop canopy; in sugarcane solar radiation is intercepted by the extended leaves but canopy architecture can modify photosynthetic performance. Thus commercial sugarcane varieties may have erect or planophille leaves but in a closed canopy most of the light is intercepted by the top fully expanded leaves. Erect leaved varieties, appear to be more efficient capturing light than those with more planophille or droopy leaves, specially at high plant densities. In this context strategy for weed control in order to improve farm management must include the

Canopy shade is an important part of weed-crop interference, thus the effect of radiation quality (wavelength) and quantity (irradiance, photon flux) on the diversity of plant species is a serious constraint to production and crop management. The sugarcane crop canopy closes at about three months after planting/sprouting, and the population of plants under the canopy changes in number and diversity depending on the ability of some species to escape or avoid shade by a series of developmental changes at the individual and population levels. Under field conditions, for each sugarcane ratoon cycle (regrowth), recognition of the species diversity as well as their strategies for shade avoidance, before and after canopy closure, is relevant to agricultural applications and plant biology and as research on the shade avoidance syndrome has been mostly restricted to individual plants and under controlled conditions, the objective of the present study is to provide information about what happens under a sugarcane canopy under field conditions, as related to spectral

The shade avoidance syndrome (SAS) in plant neighborhoods such as those underneath a crop canopy, is associated with both the quality (wavelength) and quantity (energy) of light and the decrease in the red/far red ratio (R/FR), as the light environment becomes enriched in far red radiation that is reflected by the leaves of all plants, including the crop itself. A reduced R/FR is the proximity signal that is perceived by the plants alerting that they are being shaded, and in fact it is perceived in early developmental stages, as shown by Ballare & Casal, 2000; Ballare et al., 1991; Smith et al., 1990. Small changes in amounts of red relative to far red light have been shown to alter the equilibrium of different phytochrome forms appreciably, which plays a major role in plant development. Perception of light quality and quantity at the stem level may elicit morphological adaptations in the plants growing beneath the canopy, that may result in shade tolerance or avoidance, and under field

knowledge of the dynamic and biology of plants growing underneath the canopy.

shifts within the canopy and the changes in species diversity.

and other resources have been developed.

conditions, slight differences in height, degree of tillering, earlier flowering and increase in the shoot/root ratio among different species, might imply a greater potential to survive escaping shade; thus recognition of plant species before and after canopy closure as well as the changes in the light profiles under the canopy, are important for weed detection in cultivated crops as a means to ascertain which species avoid shade.

Shade avoidance responses are mediated by multiple forms of phytochromes; despite of initial attempts to adscribe the SAS to the action of a single member of the phytochrome family (Franklin & Whitelam, 2005). In this context and according to Schmitt (1997), the shade avoidance response has undergone adaptive evolution as quantitative genetic variation in R:FR ratio sensitivity has been detected in wild populations. The "Shade Avoidance Syndrome" has been described by Morgan et al (2002); Smith (1982); Smith & Whitelam (1997) as an accelerated extension growth (as seen by an increase in shoot and petiole elongation), reduced branching (increase in apical dominance), earlier flowering (i.e., rate of flowering markedly accelerated) and increase in the shoot to root ratios, changes in plant architecture and leaf shape, among other responses not easily seen under field conditions, however, we have used the term "Agronomic Shade Avoidance Syndrome" to include species that persist and compete successfully with the crop after canopy closure, but not by means of growth responses normally associated to the SAS under field conditions, such as morphological changes in leaf shape, stem elongation or plant architecture. The persistence of such species after canopy closure, may be associated to the seed bank, sunflecks in gaps within the canopy and the production of underground organs (that become well established and almost impossible to control by shade) as well as climbing strategies such as in tie-vines.

From another point of view, the shade avoidance syndrome is not restricted to terrestrial ecosystems but has also been studied in connection to other stress responses, such as submergence (Pierik et al., 2005). In complete submergence, well-adapted plants may overcome the effects by adopting an avoidance strategy to induce growth responses, phenotypically similar to those described when plants are shaded by proximate neighbors.

In this chapter, we will analyze light profiles within sugarcane canopies and how changing light conditions in a closed-in canopy, may affect the development and diversity of plant species, as seen in the field at the individual level. Then, based on the results of experimental trials and under controlled conditions, some of the strategies to escape or avoid shade and to capture light more efficiently, will be discussed for different species known to be noxious to the sugarcane crop.

### **2. Research methods**

### **2.1 Field experiments**

Field experiments were conducted in two different sites where sugarcane is a mayor crop in Venezuela. The first experiment was established with sugarcane plants (*Saccharum* spp hybrid var. PR-692176 (first ratoon crop) in a 625 m2 plot inside a commercial regrowth of a sugarcane field in Chivacoa, Yaracuy State and the second experiment, in 2500 m2 experimental area in the Agricultural Experimental Station at the College of Agriculture in the Central University of Venezuela in Maracay, Aragua State, using droopy and erect leaf commercial varieties planted from cane (initial or plant cane crop).

The Shade Avoidance Syndrome Under the Sugarcane Crop 171

For the second field experiment, with the objective of recognizing weed species before and after canopy closure for two commercial sugarcane varieties with contrasting growth habits, and to register radiation profiles within the canopy, a plant crop (first cycle) was established in 2500 m2 experimental area in the Agricultural Experimental Station at the College of Agriculture in the Central University of Venezuela in Maracay, Aragua State. The experimental area was divided into four 625 m2 plots where droopy and erect leaf commercial varieties, C 266-70, which is a fairly typical variety, with planophile or droopy leaves and RB 85-5035 with more erect leaves, were planted from initial plant canes. Crop management practices were as described for the first experiment except for the fact that 10 randomly located fixed wooden 1m2 squares, were used to collect the plants growing under the crop canopy and that weed control, in some previously selected drive areas between

Spectral profiles within sugarcane canopy: energy distribution of visible and near infrared radiation above, within, and on the soil below a canopy a sugarcane plants with droopy or erect leaf commercial varieties were registered and light spectra (Spectral Irradiance, W m-2 nm-1 and Quantum Intensity, µmol m-2 s-1 nm-1) were measured in the field plot during the growing season, at the beginning of canopy closure (three months from planting date), with a spectroradiometer ASD FieldSpec*pro* VNIR 350-1050 nm, using hyperspectral analysis, approximately at 20 cm above the crop, at 30 cm above and below the weed canopy and at

Three of the most abundant species known to be noxious weeds to the sugarcane crop and present in the experimental fields (*Rottboellia cochinchinensis, Leptochloa filiformis* and *Cyperus rotundus*), were selected for the study of growth responses associated to shade quality (wavelength) and quantity (Photon flux density, PFD 400 - 700 nm). Seeds of *Rottboellia* and *Leptochloa* and corms from *Cyperus* were sowed in pots, containing soil, shaded by cabinets covered with red, blue and green cellophane paper and under low PFD neutral shade while another group was left uncovered. Cabinets were directly exposed to daylight. Effects were compared separately for each species when plants in any of the groups showed visual

Under the sugarcane canopy in the **first field trial**, at 60 days after sprouting when canopy closure was not complete, a stratified pattern in plant height for the different species was observed. Plants species identified under the sugarcane canopy before canopy closure were (Lara & Ascencio, unpublished): *Ruellia tuberosa* L., *Trianthema portulacastrum* L., *Amaranthus dubius* Mart, *Eclipta alba* (L.) Hassk, *Tridax procumbens* L., *Lagascea mollis* Cav*., Heliotropium ternatum* Valhl and *H. indicum* L., *Cyperus rotundus* L*., Commelina difusa* Burm, *Ipomoea indica* (Burm.) Merr and *I. batatas* (L.) Lam, *Momordica charantia* L., *Cucumis dipsaceus* Ehremb. ex Spash, *Ceratosanthes palmata* (L.) Urb, *Euphorbia hirta* L. and *E. hypericifolia* L., *Croton lobatus* L. *Phylathus niruri* L., *Desmanthus virgatus* (L.) Willd, *Spigelia anthelmia* L., *Leptochloa filiformis*

**2.1.2 Second field experiment** 

rows, was manually performed.

**2.2 Greenhouse experiments** 

symptoms of deterioration.

**3. Research results 3.1 First field experiment** 

30 cm above the soil, in shaded and non shaded sites.

#### **2.1.1 First field experiment**

The objectives of the first trial were: 1) to acknowledge the species that were present before and after the sugarcane canopy closure; 2) to determine the effect of canopy shade on the developmental responses that could be associated to the SAS under field conditions and 3) to measure the amount of light in terms of photon flux density of photosynthetically active radiation (PAR), at different points within the crop and weed canopies.

 Standard management practices included hand planting of 50 cm long stalks or seed pieces placed at rates of 24000 stalks/ha in the bottom of furrows spaced 1,5 m and covering the sugarcane seed pieces with soil. Soil was regularly irrigated every two weeks, or as needed, and fertilized before planting with ammonium phosphate and with a second application of Urea+KCl at 45 days after planting. Chemical weed control in the drive areas of the experimental plot was not performed for the experiments discussed in this chapter. For species recognition, ten 0,5 m2 fixed wooden squares were randomly distributed in the field and all plant species (except for those of the crop) inside the squares were collected at 60 and 90 days after germination (sprouting of the buds from the seed pieces or stalks), to acknowledge for the presence and number of different species, and for plant height and leaf size determinations. Flowering and any other visual symptoms associated to the shade avoidance response were as well registered.

In order to compare the plants growing under a "canopy in a non-shaded condition", in some previously selected rows in the same plot the leaf arrangement of sugarcane plants, was artificially changed by loosely bounding the leaves in an upright position along the stem, simulating an plant biotype with erect leaves.

Light quantity was measured as Photosynthetically Active Radiation ( PAR as µmol m-2 s-1) using a quantum-radiometer-photometer LiCor 185B by positioning the quantum sensor at different heights above and below the sugarcane canopy and above the population of weeds growing in shaded and non-shaded sites beneath the crop, as shown in Fig. 1. Instant measurements, at five different points in each position (sites) within the canopy, were performed between 12M and 1PM at 60 days after germination of buds from the seed pieces or sugarcane stalks, for the two leaf arrangements described in the preceding paragraph.

Fig. 1. Sugarcane plants and weed underneath the canopy showing quantum sensor positions (lo, li, la, lm) to register PAR values shown in Table 1.

#### **2.1.2 Second field experiment**

170 Crop Plant

The objectives of the first trial were: 1) to acknowledge the species that were present before and after the sugarcane canopy closure; 2) to determine the effect of canopy shade on the developmental responses that could be associated to the SAS under field conditions and 3) to measure the amount of light in terms of photon flux density of photosynthetically active

 Standard management practices included hand planting of 50 cm long stalks or seed pieces placed at rates of 24000 stalks/ha in the bottom of furrows spaced 1,5 m and covering the sugarcane seed pieces with soil. Soil was regularly irrigated every two weeks, or as needed, and fertilized before planting with ammonium phosphate and with a second application of Urea+KCl at 45 days after planting. Chemical weed control in the drive areas of the experimental plot was not performed for the experiments discussed in this chapter. For species recognition, ten 0,5 m2 fixed wooden squares were randomly distributed in the field and all plant species (except for those of the crop) inside the squares were collected at 60 and 90 days after germination (sprouting of the buds from the seed pieces or stalks), to acknowledge for the presence and number of different species, and for plant height and leaf size determinations. Flowering and any other visual symptoms associated to the shade

In order to compare the plants growing under a "canopy in a non-shaded condition", in some previously selected rows in the same plot the leaf arrangement of sugarcane plants, was artificially changed by loosely bounding the leaves in an upright position along the

Light quantity was measured as Photosynthetically Active Radiation ( PAR as µmol m-2 s-1) using a quantum-radiometer-photometer LiCor 185B by positioning the quantum sensor at different heights above and below the sugarcane canopy and above the population of weeds growing in shaded and non-shaded sites beneath the crop, as shown in Fig. 1. Instant measurements, at five different points in each position (sites) within the canopy, were performed between 12M and 1PM at 60 days after germination of buds from the seed pieces or sugarcane stalks, for the two leaf arrangements described in the preceding paragraph.

Fig. 1. Sugarcane plants and weed underneath the canopy showing quantum sensor

positions (lo, li, la, lm) to register PAR values shown in Table 1.

radiation (PAR), at different points within the crop and weed canopies.

**2.1.1 First field experiment** 

avoidance response were as well registered.

stem, simulating an plant biotype with erect leaves.

For the second field experiment, with the objective of recognizing weed species before and after canopy closure for two commercial sugarcane varieties with contrasting growth habits, and to register radiation profiles within the canopy, a plant crop (first cycle) was established in 2500 m2 experimental area in the Agricultural Experimental Station at the College of Agriculture in the Central University of Venezuela in Maracay, Aragua State. The experimental area was divided into four 625 m2 plots where droopy and erect leaf commercial varieties, C 266-70, which is a fairly typical variety, with planophile or droopy leaves and RB 85-5035 with more erect leaves, were planted from initial plant canes. Crop management practices were as described for the first experiment except for the fact that 10 randomly located fixed wooden 1m2 squares, were used to collect the plants growing under the crop canopy and that weed control, in some previously selected drive areas between rows, was manually performed.

Spectral profiles within sugarcane canopy: energy distribution of visible and near infrared radiation above, within, and on the soil below a canopy a sugarcane plants with droopy or erect leaf commercial varieties were registered and light spectra (Spectral Irradiance, W m-2 nm-1 and Quantum Intensity, µmol m-2 s-1 nm-1) were measured in the field plot during the growing season, at the beginning of canopy closure (three months from planting date), with a spectroradiometer ASD FieldSpec*pro* VNIR 350-1050 nm, using hyperspectral analysis, approximately at 20 cm above the crop, at 30 cm above and below the weed canopy and at 30 cm above the soil, in shaded and non shaded sites.

#### **2.2 Greenhouse experiments**

Three of the most abundant species known to be noxious weeds to the sugarcane crop and present in the experimental fields (*Rottboellia cochinchinensis, Leptochloa filiformis* and *Cyperus rotundus*), were selected for the study of growth responses associated to shade quality (wavelength) and quantity (Photon flux density, PFD 400 - 700 nm). Seeds of *Rottboellia* and *Leptochloa* and corms from *Cyperus* were sowed in pots, containing soil, shaded by cabinets covered with red, blue and green cellophane paper and under low PFD neutral shade while another group was left uncovered. Cabinets were directly exposed to daylight. Effects were compared separately for each species when plants in any of the groups showed visual symptoms of deterioration.

#### **3. Research results**

#### **3.1 First field experiment**

Under the sugarcane canopy in the **first field trial**, at 60 days after sprouting when canopy closure was not complete, a stratified pattern in plant height for the different species was observed. Plants species identified under the sugarcane canopy before canopy closure were (Lara & Ascencio, unpublished): *Ruellia tuberosa* L., *Trianthema portulacastrum* L., *Amaranthus dubius* Mart, *Eclipta alba* (L.) Hassk, *Tridax procumbens* L., *Lagascea mollis* Cav*., Heliotropium ternatum* Valhl and *H. indicum* L., *Cyperus rotundus* L*., Commelina difusa* Burm, *Ipomoea indica* (Burm.) Merr and *I. batatas* (L.) Lam, *Momordica charantia* L., *Cucumis dipsaceus* Ehremb. ex Spash, *Ceratosanthes palmata* (L.) Urb, *Euphorbia hirta* L. and *E. hypericifolia* L., *Croton lobatus* L. *Phylathus niruri* L., *Desmanthus virgatus* (L.) Willd, *Spigelia anthelmia* L., *Leptochloa filiformis*

The Shade Avoidance Syndrome Under the Sugarcane Crop 173

Two growth strategies associated to the SAS as seen in the field were observed: increased internodes length and decreased leaf size. The species that showed higher sensitivity towards canopy shade were *Cyperus rotundus* and *Trianthema portulacastrum*, as plants eventually died in this condition apart from *Leptochloa filiformis* in which changes in leaf morphology, such as broader but shorter leaves were observed, as well as an early flowering of the individuals in order to produce seeds, which is also a means of escaping shade for population survival; however, the plants eventually died under the shade. Changes in leaf shape were also observed for plants escaping canopy shade through stem elongation, as in the case of *Rottboellia cocinchinensis* and *Panicum fasciculatum* were shorter leaves were

The effect of weed canopy shade on the development of plants in the neighborhood was observed when sugarcane leaves, in some selected rows, where loosely bound around the stem simulating an extreme erect biotype. As seen from Figure 3, at beginning of the crop cycle (60 days after sprouting) plant height for the different species were: *Rottboellia cochinchinensis* > *Leptochloa filiformis > Panicum fasciculatum* > *Heliotropium ternatum > Panicum maximum* > *Trianthema portulacastrum > Cynodon dactylon* > *Cyperus rotundus*, and after 90 days at full canopy closure (Fig.4), a steeper gradient for plant heights was observed as follows: *Rottboellia cochinchinensis* > *Panicum maximum* > *Panicum fasciculatum*  > *Heliotropium ternatum > Leptochloa filiformis* > *Trianthema portulacastrum* > *Cyperus rotundus*. Plants from *Cynodon dactylon* were absent from the stand, unable to tolerate

Species: 1. *Cyperus rotundus* L., 2. *Leptochloa filiformis* (Lam.) Beauv., 3. *Rottboellia cochinchinensis* (Lour.) W. Clayton, 4. Panicum fasciculatum Sw., 5. Heliotropium ternatum Vahl., 6. *Trianthema portulacastrum*

L., 7. *Panicum maximum* Jacq. y, 8. *Cynodon dactylon* (L.) Pers. (Lara & Ascencio, unpublished) Fig. 3. Plant height for species growing under a sugarcan (Saccharum spp hybrid var PR692176) canopy for 60 days after emergence with their leaves loosely bound to the stem

simulating and erect leaf arrangement.

observed as compared to those growing before canopy closure.

shade.

(Lam.) Beauv, *Rottboellia cochinchinensis* (Lour) W. Clayton, *Panicum fasciculatum* Sw., *Echinochloa colona* (L.) Link*, Eleusine indica* (L.) Gaerth, *Cynodon dactylon* (L.) Pers, *Dactyloctenium aegyptium* (L.) Wild, *Portulaca oleracea* L., *Capraria biflora* L., *Physalis angulata* L., *Corchorus orinocensis* Kunth, *Priva lappulaceae* (L.) Pers, *Kallstroemia maxima* (L.) Hook & Arn.

It is important to note that 23% of the species listed above belong to the Poaceae (as sugarcane) and that the rest are distributed in 18 families, thus 8 species known to be noxious for the sugarcane crop were selected for this study, where, except for *Trianthema postulacastrum*, *Heliotropium ternatum* and *Cyperus rotundus* the other five (*Leptochloa filiformis*, *Rottboellia cochinchinensis*, *Panicum fasciculatum, Panicum maximum* and *Cynodon dactylon*) belong to the Poaceae.

When the first evaluation was made in the field 30 days after sprouting a reduction in the number of plants for the species *Cyperus rotundus* and *Leptochloa filiformis* was observed at first sight under the shade of other plants (either the crop or other plants in the neighbourhood), but not for other species in the site, a first indication that shade was affecting plant performance, plant loss or even causing plant death. Differences in plant height (stem or internode elongation) for the species growing below the sugarcane canopy were observed after 60 days in the following order: *Rottboellia cochinchinensis* > *Panicum maximum* > *Panicum fasciculatum > Heliotropium ternatum* > *Trianthema portulacastrum* > *Cyperus rotundus*. Thus, these species except for *Cyperus rotundus*, *Trianthema portulacastrum* and *Heliotropium ternatum*, escaped canopy shade by an increase in plant height. Maximal plant height approaching that of the crop after full canopy closure (90 days) was observed for *Rottboellia exaltata* and *Panicum maximum*, which effectively escaped shade, competing successfully with the crop for light (Fig 2).

Species: 1. *Cyperus rotundus* L., 2. *Leptochloa filiformis* (Lam.) Beauv., 3. *Rottboellia cochinchinensis* (Lour.) W. Clayton, 4. *Panicum fasciculatum* Sw., 5. *Heliotropium ternatum* Vahl., 6. *Trianthema portulacastrum* L., 7. *Panicum maximum* Jacq., 8. *Cynodon dactylon* (L.) Pers. (Lara & Ascencio, unpublished).

Fig. 2. Plant height for species growing under a sugarcane (*Saccharum spp* hybrid) canopy with droopy leaves (*Saccharum spp* hybrid var PR692176) after 90 days of crope emergence.

(Lam.) Beauv, *Rottboellia cochinchinensis* (Lour) W. Clayton, *Panicum fasciculatum* Sw., *Echinochloa colona* (L.) Link*, Eleusine indica* (L.) Gaerth, *Cynodon dactylon* (L.) Pers, *Dactyloctenium aegyptium* (L.) Wild, *Portulaca oleracea* L., *Capraria biflora* L., *Physalis angulata* L., *Corchorus orinocensis* Kunth, *Priva lappulaceae* (L.) Pers, *Kallstroemia maxima* (L.) Hook & Arn.

It is important to note that 23% of the species listed above belong to the Poaceae (as sugarcane) and that the rest are distributed in 18 families, thus 8 species known to be noxious for the sugarcane crop were selected for this study, where, except for *Trianthema postulacastrum*, *Heliotropium ternatum* and *Cyperus rotundus* the other five (*Leptochloa filiformis*, *Rottboellia cochinchinensis*, *Panicum fasciculatum, Panicum maximum* and *Cynodon dactylon*)

When the first evaluation was made in the field 30 days after sprouting a reduction in the number of plants for the species *Cyperus rotundus* and *Leptochloa filiformis* was observed at first sight under the shade of other plants (either the crop or other plants in the neighbourhood), but not for other species in the site, a first indication that shade was affecting plant performance, plant loss or even causing plant death. Differences in plant height (stem or internode elongation) for the species growing below the sugarcane canopy were observed after 60 days in the following order: *Rottboellia cochinchinensis* > *Panicum maximum* > *Panicum fasciculatum > Heliotropium ternatum* > *Trianthema portulacastrum* > *Cyperus rotundus*. Thus, these species except for *Cyperus rotundus*, *Trianthema portulacastrum* and *Heliotropium ternatum*, escaped canopy shade by an increase in plant height. Maximal plant height approaching that of the crop after full canopy closure (90 days) was observed for *Rottboellia exaltata* and *Panicum maximum*, which effectively escaped shade, competing

Species: 1. *Cyperus rotundus* L., 2. *Leptochloa filiformis* (Lam.) Beauv., 3. *Rottboellia cochinchinensis* (Lour.) W. Clayton, 4. *Panicum fasciculatum* Sw., 5. *Heliotropium ternatum* Vahl., 6. *Trianthema portulacastrum* L., 7.

Fig. 2. Plant height for species growing under a sugarcane (*Saccharum spp* hybrid) canopy with

*Panicum maximum* Jacq., 8. *Cynodon dactylon* (L.) Pers. (Lara & Ascencio, unpublished).

droopy leaves (*Saccharum spp* hybrid var PR692176) after 90 days of crope emergence.

belong to the Poaceae.

successfully with the crop for light (Fig 2).

Two growth strategies associated to the SAS as seen in the field were observed: increased internodes length and decreased leaf size. The species that showed higher sensitivity towards canopy shade were *Cyperus rotundus* and *Trianthema portulacastrum*, as plants eventually died in this condition apart from *Leptochloa filiformis* in which changes in leaf morphology, such as broader but shorter leaves were observed, as well as an early flowering of the individuals in order to produce seeds, which is also a means of escaping shade for population survival; however, the plants eventually died under the shade. Changes in leaf shape were also observed for plants escaping canopy shade through stem elongation, as in the case of *Rottboellia cocinchinensis* and *Panicum fasciculatum* were shorter leaves were observed as compared to those growing before canopy closure.

The effect of weed canopy shade on the development of plants in the neighborhood was observed when sugarcane leaves, in some selected rows, where loosely bound around the stem simulating an extreme erect biotype. As seen from Figure 3, at beginning of the crop cycle (60 days after sprouting) plant height for the different species were: *Rottboellia cochinchinensis* > *Leptochloa filiformis > Panicum fasciculatum* > *Heliotropium ternatum > Panicum maximum* > *Trianthema portulacastrum > Cynodon dactylon* > *Cyperus rotundus*, and after 90 days at full canopy closure (Fig.4), a steeper gradient for plant heights was observed as follows: *Rottboellia cochinchinensis* > *Panicum maximum* > *Panicum fasciculatum*  > *Heliotropium ternatum > Leptochloa filiformis* > *Trianthema portulacastrum* > *Cyperus rotundus*. Plants from *Cynodon dactylon* were absent from the stand, unable to tolerate shade.

Species: 1. *Cyperus rotundus* L., 2. *Leptochloa filiformis* (Lam.) Beauv., 3. *Rottboellia cochinchinensis* (Lour.) W. Clayton, 4. Panicum fasciculatum Sw., 5. Heliotropium ternatum Vahl., 6. *Trianthema portulacastrum* L., 7. *Panicum maximum* Jacq. y, 8. *Cynodon dactylon* (L.) Pers. (Lara & Ascencio, unpublished)

Fig. 3. Plant height for species growing under a sugarcan (Saccharum spp hybrid var PR692176) canopy for 60 days after emergence with their leaves loosely bound to the stem simulating and erect leaf arrangement.

The Shade Avoidance Syndrome Under the Sugarcane Crop 175

sugarcane leaves were bound to the stalk allowing more light for the weeds to grow, so attenuation of radiation might have occurred by the scattering of radiation by the leaves. PAR values at soil level were dramatically lower under canopy shade (Im) as compared to that in between rows (Ir) with extinction values of -70 and -23% respectively; however it is worth noting that when these measurements were taken, crop canopy closure was about

20% of the maximum at full closure when values between rows may be much lower.

Crop (Io) (cm) Exposed to

642\*\* 66.4 <sup>582</sup>

1310 60.2 <sup>1150</sup>

Leaf

Droopy

Erect

\*\* Heavy cloud

is received.

arrangement

Above

 \* Weed canopy average height, 30 cm above soil

parenthesis are expressed as percentages of Io.

Crop canopy Weeds Underneath Crop

PAR Height PAR PAR

Light (Ii)

(-9.3%)

(-12.2%)

Table 1. Incident photosynthetically active radiation (PAR, µmol m-2 s-1) within a sugarcane

Incident radiation transmitted by the leaves is a function of the extinction coefficient, which quantifies the influence of the arrangement of the leaves in the canopy. According to Tolenaar & Dwyer 1999, in the three-dimensional space above the soil, the leaf area arrangement is determined by (1) plant height, (2) plant spacing (i.e., row width vs. distance between plants in the row), (3) leaf length and width, (4) leaf angle with respect to the horizontal plane, and (5) leaf orientation with respect to north and south (i.e., azimuth angle). The extinction coefficient is relatively constant during the middle of the daytime when close to 90% of the total daily incident photosynthetic photon flux density

Population dynamics (amount and diversity of species) below the canopy is highly influenced by the light environment (shade) and also by sunflecks which play an important role in the germination of new species not seen at the beginning of the crop cycle, as a great number of weed species have small, photoblastic seeds. This is discussed in the second field experiment as part of an strategy of escaping shade at a population level, as new species grown under shade conditions, are more likely to tolerate shade competing successfully with the crop for light. The importance of measurements of light (quantity and quality) in canopies is highlighted by Holt (1995) as a means to improve weed management as many plants possess the ability to adapt quickly to changes in light during the life cycle, species such as *Amaranthus palmeri*, *Crotalaria spectabilis*, *Cyperus rotundus*, *Cyperus esculentus*, *Imperata cylindrica* and *Abutilon theophrasti*, are mentioned as examples of weeds that respond to shade, thus, by understanding physiological and morphological mechanisms of

canopy (see Figure 1, for Io, Ii, Ia, Ir, and Im, quantum sensor positions). Values in

Canopy\* Soil Level

Exposed (Ir)

494 (-23.1%)

1032 (-21.2%) Shaded (Im)

194 (-70%)

236 (-82%)

Shaded (Ia)

396 (-38.3%)

460 (-65%)

Species: 1. Cyperus rotundus L., 2. Leptochloa filiformis (Lam.) Beauv., 3. Rottboellia cochinchinensis (Lour.) W. Clayton, 4. Panicum fascilatum Sw., 5. Heliotropium ternatum Vahl., 6. Trianthema portulacastrum L., 7. Panicum maximum Jacq. y 8. Cynodon dactylon (L.) Pers. (Lara & Ascencio, unplublished)

Fig. 4. Plant height for species growing under sugarcane (Saccharum spp hybrid var PR692176) canopy for 90 days after emergence with their leaves loosely bound to the stem simulating and erect leaf arrangement.

Light quantity, an important component of shade, was measured in shaded and non-shaded positions within the sugarcane canopy (see Fig. 1), and for two leaf arrangements along the stem: droopy (planophile leaves) and erect (leaves bound to the stem to an erect position). PAR was measured in full sunlight above the crop at different crop heights (Io), above the canopy of weeds at 30 cm above soil level (which is in average the height of the population of weeds underneath the crop), under shaded (Ia) and non shaded (Ii) positions, at soil level at sites not shaded by plants (Ir) and also under the canopy of weeds growing beneath the canopy of sugarcane leaves (Im). Values for (Ii) are more likely to be sunflecks reaching gaps inside the canopy, while Ia are PAR values above the canopy of weeds but underneath the sugarcane leaves.

As seen from the results shown in Table 1, maximum PAR values were observed above the crop (Io = 1310) and above the canopy of weeds exposed to light in the erect leaf arrangement (1150) growing below the crop canopy at 30 cm above soil level (Ii) . PAR was 50% higher for (Ii) than under the shade of leaves (Ia), with low extinction values from Io for droopy and erect leaf arrangement, equal to -9.3 and -12.2% respectively. Another characteristic of the light environment underneath a canopy of leaves, is that direct sunlight may arrive as high intensity light beams known as sunflecks. In Table 1, values for (Ii) are more likely to be sunflecks in drive areas between rows. For droopy leaf arrangement and underneath the crop canopy (Ia), extinction values from Io were -38.3% above the weed canopy, and -70% at soil level, while for the erect leaf arrangement, higher values equal to - 65.0 and -82.0% were observed for the extinction from Io. There is no clear explanation for this difference except for the fact that for the erect or vertically inclined leaf arrangement, sugarcane leaves were bound to the stalk allowing more light for the weeds to grow, so attenuation of radiation might have occurred by the scattering of radiation by the leaves. PAR values at soil level were dramatically lower under canopy shade (Im) as compared to that in between rows (Ir) with extinction values of -70 and -23% respectively; however it is worth noting that when these measurements were taken, crop canopy closure was about 20% of the maximum at full closure when values between rows may be much lower.


\* Weed canopy average height, 30 cm above soil

\*\* Heavy cloud

174 Crop Plant

Species: 1. Cyperus rotundus L., 2. Leptochloa filiformis (Lam.) Beauv., 3. Rottboellia cochinchinensis (Lour.) W. Clayton, 4. Panicum fascilatum Sw., 5. Heliotropium ternatum Vahl., 6. Trianthema portulacastrum L., 7. Panicum maximum Jacq. y 8. Cynodon dactylon (L.) Pers. (Lara & Ascencio,

Light quantity, an important component of shade, was measured in shaded and non-shaded positions within the sugarcane canopy (see Fig. 1), and for two leaf arrangements along the stem: droopy (planophile leaves) and erect (leaves bound to the stem to an erect position). PAR was measured in full sunlight above the crop at different crop heights (Io), above the canopy of weeds at 30 cm above soil level (which is in average the height of the population of weeds underneath the crop), under shaded (Ia) and non shaded (Ii) positions, at soil level at sites not shaded by plants (Ir) and also under the canopy of weeds growing beneath the canopy of sugarcane leaves (Im). Values for (Ii) are more likely to be sunflecks reaching gaps inside the canopy, while Ia are PAR values above the canopy of weeds but underneath the

As seen from the results shown in Table 1, maximum PAR values were observed above the crop (Io = 1310) and above the canopy of weeds exposed to light in the erect leaf arrangement (1150) growing below the crop canopy at 30 cm above soil level (Ii) . PAR was 50% higher for (Ii) than under the shade of leaves (Ia), with low extinction values from Io for droopy and erect leaf arrangement, equal to -9.3 and -12.2% respectively. Another characteristic of the light environment underneath a canopy of leaves, is that direct sunlight may arrive as high intensity light beams known as sunflecks. In Table 1, values for (Ii) are more likely to be sunflecks in drive areas between rows. For droopy leaf arrangement and underneath the crop canopy (Ia), extinction values from Io were -38.3% above the weed canopy, and -70% at soil level, while for the erect leaf arrangement, higher values equal to - 65.0 and -82.0% were observed for the extinction from Io. There is no clear explanation for this difference except for the fact that for the erect or vertically inclined leaf arrangement,

Fig. 4. Plant height for species growing under sugarcane (Saccharum spp hybrid var PR692176) canopy for 90 days after emergence with their leaves loosely bound to the stem

unplublished)

sugarcane leaves.

simulating and erect leaf arrangement.

Table 1. Incident photosynthetically active radiation (PAR, µmol m-2 s-1) within a sugarcane canopy (see Figure 1, for Io, Ii, Ia, Ir, and Im, quantum sensor positions). Values in parenthesis are expressed as percentages of Io.

Incident radiation transmitted by the leaves is a function of the extinction coefficient, which quantifies the influence of the arrangement of the leaves in the canopy. According to Tolenaar & Dwyer 1999, in the three-dimensional space above the soil, the leaf area arrangement is determined by (1) plant height, (2) plant spacing (i.e., row width vs. distance between plants in the row), (3) leaf length and width, (4) leaf angle with respect to the horizontal plane, and (5) leaf orientation with respect to north and south (i.e., azimuth angle). The extinction coefficient is relatively constant during the middle of the daytime when close to 90% of the total daily incident photosynthetic photon flux density is received.

Population dynamics (amount and diversity of species) below the canopy is highly influenced by the light environment (shade) and also by sunflecks which play an important role in the germination of new species not seen at the beginning of the crop cycle, as a great number of weed species have small, photoblastic seeds. This is discussed in the second field experiment as part of an strategy of escaping shade at a population level, as new species grown under shade conditions, are more likely to tolerate shade competing successfully with the crop for light. The importance of measurements of light (quantity and quality) in canopies is highlighted by Holt (1995) as a means to improve weed management as many plants possess the ability to adapt quickly to changes in light during the life cycle, species such as *Amaranthus palmeri*, *Crotalaria spectabilis*, *Cyperus rotundus*, *Cyperus esculentus*, *Imperata cylindrica* and *Abutilon theophrasti*, are mentioned as examples of weeds that respond to shade, thus, by understanding physiological and morphological mechanisms of

The Shade Avoidance Syndrome Under the Sugarcane Crop 177

Fig. 5. Quantum energy distribution of full sunlight above a sugarcane canopy in Maracay (10°11' N, 440 msl). Values are given as Quantum Intensity (QI micromol m-2 s-1 nm-1)

 The spectroradiometric measurements clearly show that the decreased QI of radiant energy within a sugarcane canopy is not uniform at all wavelengths and that spectral composition of shade light differs from that above the canopy (Fig. 1), because of the selective absorption of PAR (400-700 nm) by the leaves. Therefore, plant responses that may be attributed only to a reduced number of photons, or light intensity of radiant energy, could be confused with responses to a shift in the spectral composition of light received by the shaded leaves. In fact, a decreased R/FR is the most important radiation component within canopies as transmittance of far red radiation (730 nm) is substantial. This is shown in Figs 6 to 11 for the sugarcane canopies of this study. The population of weeds below the crop canopy and also crop architecture, influences the QI distribution of wavelengths reaching the soil at 30 cm from the ground, i.e. above the canopy of weeds. As compared to spectral distribution of sunlight (Fig 1) ,QI increases at wavelengths in the far red (radiation mostly reflected and transmitted by the leaves of the whole plant population crop+weeds) but differences are also found for QI values in canopies with planophile (Fig. 6 and 7 ) or erect leaves (Figs. 8 and 9) with low or high weed populations growing underneath. In deep shade QI values are

lower.

competition for light between weeds and crops will it be possible to manipulate crop canopies to suppress weeds.

#### **3.2 Second field experiment**

According to the results of the second field trial, a higher number of species survived after the closure of the canopy of sugarcane plants with erect leaves, as compared to those for droopy leaves, and new species appeared: *Amaranthus* sp, *Bidens pilosa*, *Cyperus rotundus*, *Euphorbia heterophylla*, *Sida* sp, *Aldana dentata*, *Desmodium* sp, *Phyllanthus niruri* and *Eclipta alba*; and the new species (*Aldana sp.*, *Phyllanthus sp.* and *Eclipta sp*) appeared. Under the canopy of sugarcane plants with droopy leaves, *Amaranthus* sp, *Commelina difusa* (new), *Bidens pilosa*, *Mimosa* sp, *Euphorbia heterophylla* and *Desmodium* sp, were observed. In the experimental field of the second experiment, *Rottboellia exaltata* (which is capable of avoiding shade) was not found, and plants of *Leptochloa filiformis*, *Cynodon dactylon*, *Echinochloa colona*, *Ipomoea sp*, *Cucumis melo*, *Ruellia tuberosa* and *Cyperus rotundus*, progressively died under the shade of the canopy with droopy leaves.

It is important to note that some of the species that persisted after canopy closure were located at points within the canopy where light penetration was higher, while others were part of the seed bank (new species that appeared after canopy closure) which, in our opinion, may be an strategy for "agronomic shade avoidance" at a population level. Plants are actually seen growing under the canopy, thus escaping or avoiding shade in some way. In this category, the morning glory group of species (*Ipomoea spp*) referred to as tie-vines, may also be included as they are capable of climbing and forming a dense mat that grows over the crop canopy, escaping shade. Perennial grasses, found under the sugarcane canopy are another example of "agronomic shade avoidance", as sugarcane itself is a grass and conditions able to its development are also conductive to grass weed growth. As seen from the results of this study, perennial grasses such as *Cynodon sp* and *Panicum spp,* persist after canopy closure. In short, during each crop cycle different species are found as part of the biodiversity of the seed bank and the changing environment associated to the quantity (PAR) and quality (wavelength) of light in a closed-in canopy.

Radiation measurements: Energy distribution of visible and near infrared radiation (irradiance and quantum intensity) was measured underneath the sugarcane canopy; as leaf angle with respect to the horizontal plane and leaf length and width, influence the extinction of light within the canopy, and that these variables are associated to variety types, in this second field trial radiation profiles were compared within sugarcane canopies with either planophile (droopy or horizontal leaf arrangement) or erect (vertically inclined) leaves and in selected rows with high and low weed populations.

The energy distribution of radiation above the sugarcane canopy (light profile), is shown in Fig. 5 in terms of Quantum Intensity (QI, in µmol m-2s-1nm-1) of full sunlight on a clear day in Maracay, Aragua (100 11'N, 440 msl). Figures 6 and 7, show energy distribution of visible and near infrared radiation within a canopy of sugarcane leaves with planophile leaves and with low and high populations of weeds growing underneath and Fig. 8 and 9 for a canopy with erect leaves with low or a high populations of weeds respectively.

competition for light between weeds and crops will it be possible to manipulate crop

According to the results of the second field trial, a higher number of species survived after the closure of the canopy of sugarcane plants with erect leaves, as compared to those for droopy leaves, and new species appeared: *Amaranthus* sp, *Bidens pilosa*, *Cyperus rotundus*, *Euphorbia heterophylla*, *Sida* sp, *Aldana dentata*, *Desmodium* sp, *Phyllanthus niruri* and *Eclipta alba*; and the new species (*Aldana sp.*, *Phyllanthus sp.* and *Eclipta sp*) appeared. Under the canopy of sugarcane plants with droopy leaves, *Amaranthus* sp, *Commelina difusa* (new), *Bidens pilosa*, *Mimosa* sp, *Euphorbia heterophylla* and *Desmodium* sp, were observed. In the experimental field of the second experiment, *Rottboellia exaltata* (which is capable of avoiding shade) was not found, and plants of *Leptochloa filiformis*, *Cynodon dactylon*, *Echinochloa colona*, *Ipomoea sp*, *Cucumis melo*, *Ruellia tuberosa* and *Cyperus rotundus*, progressively died under the

It is important to note that some of the species that persisted after canopy closure were located at points within the canopy where light penetration was higher, while others were part of the seed bank (new species that appeared after canopy closure) which, in our opinion, may be an strategy for "agronomic shade avoidance" at a population level. Plants are actually seen growing under the canopy, thus escaping or avoiding shade in some way. In this category, the morning glory group of species (*Ipomoea spp*) referred to as tie-vines, may also be included as they are capable of climbing and forming a dense mat that grows over the crop canopy, escaping shade. Perennial grasses, found under the sugarcane canopy are another example of "agronomic shade avoidance", as sugarcane itself is a grass and conditions able to its development are also conductive to grass weed growth. As seen from the results of this study, perennial grasses such as *Cynodon sp* and *Panicum spp,* persist after canopy closure. In short, during each crop cycle different species are found as part of the biodiversity of the seed bank and the changing environment associated to the quantity (PAR) and quality (wavelength) of light in a

Radiation measurements: Energy distribution of visible and near infrared radiation (irradiance and quantum intensity) was measured underneath the sugarcane canopy; as leaf angle with respect to the horizontal plane and leaf length and width, influence the extinction of light within the canopy, and that these variables are associated to variety types, in this second field trial radiation profiles were compared within sugarcane canopies with either planophile (droopy or horizontal leaf arrangement) or erect (vertically inclined) leaves and

The energy distribution of radiation above the sugarcane canopy (light profile), is shown in Fig. 5 in terms of Quantum Intensity (QI, in µmol m-2s-1nm-1) of full sunlight on a clear day in Maracay, Aragua (100 11'N, 440 msl). Figures 6 and 7, show energy distribution of visible and near infrared radiation within a canopy of sugarcane leaves with planophile leaves and with low and high populations of weeds growing underneath and Fig. 8 and 9 for a canopy

canopies to suppress weeds.

**3.2 Second field experiment** 

shade of the canopy with droopy leaves.

in selected rows with high and low weed populations.

with erect leaves with low or a high populations of weeds respectively.

closed-in canopy.

Fig. 5. Quantum energy distribution of full sunlight above a sugarcane canopy in Maracay (10°11' N, 440 msl). Values are given as Quantum Intensity (QI micromol m-2 s-1 nm-1)

 The spectroradiometric measurements clearly show that the decreased QI of radiant energy within a sugarcane canopy is not uniform at all wavelengths and that spectral composition of shade light differs from that above the canopy (Fig. 1), because of the selective absorption of PAR (400-700 nm) by the leaves. Therefore, plant responses that may be attributed only to a reduced number of photons, or light intensity of radiant energy, could be confused with responses to a shift in the spectral composition of light received by the shaded leaves. In fact, a decreased R/FR is the most important radiation component within canopies as transmittance of far red radiation (730 nm) is substantial. This is shown in Figs 6 to 11 for the sugarcane canopies of this study. The population of weeds below the crop canopy and also crop architecture, influences the QI distribution of wavelengths reaching the soil at 30 cm from the ground, i.e. above the canopy of weeds. As compared to spectral distribution of sunlight (Fig 1) ,QI increases at wavelengths in the far red (radiation mostly reflected and transmitted by the leaves of the whole plant population crop+weeds) but differences are also found for QI values in canopies with planophile (Fig. 6 and 7 ) or erect leaves (Figs. 8 and 9) with low or high weed populations growing underneath. In deep shade QI values are lower.

The Shade Avoidance Syndrome Under the Sugarcane Crop 179

Fig. 7. Quantum energy distribution within and below a closed canopy of field grown sugarcane with planophile leaves and a high population of weeds growing underneath the canopy. Measurements were taken between adjacent rows at 30 cm from the ground. Values

Fig. 8. Quantum energy distribution within and below a closed canopy of field grown sugarcane with erect leaves and a low population of weeds growing underneath the canopy. Measurements were taken between adjacent rows at 30 cm from the ground. Values are

are given as Quantum Intensity (QI micromol m-2 s-1 nm-1)

given as Quantum Intensity (QI micromol m-2 s-1 nm-1)

Fig. 6. Quantum energy distribution within and below a closed canopy of field grown sugarcan with planophile (droopy) leaves and a low population of weed growing underneath the canopy. Measurements were taken between adjacent rows at 30 cm from the ground. Values are given as Quantum Intensity (QI micromol m-2 s-1 nm-1)

Shifts in the amount of radiation beneath the canopy with planophile leaves and a low population of weeds, indicates that QI in the visible (400-700 nm) was lower than for a high population of weeds and, as could be expected, a higher QI was observed for wavelengths in the far red when the population of weeds was high (Fig.7). When comparing these results with the radiation profiles, as QI and irradiance, within a canopy of sugarcane leaves with erect leaves (Fig. 8 and 9), radiation reaching the soil in the visible was higher than for a canopy with planophile leaves, but still a higher QI for wavelengths in the near infrared was observed when the population of weeds was high. Except for the near infrared water sensitive portion (940-1040 nm), this is the shade that plants actually "see" and it is perceived at very early stages of development, alters phytochrome photoequilibrium and initiates growth responses to avoid the shade (shade avoidance syndrome).

Fig. 6. Quantum energy distribution within and below a closed canopy of field grown sugarcan with planophile (droopy) leaves and a low population of weed growing

ground. Values are given as Quantum Intensity (QI micromol m-2 s-1 nm-1)

growth responses to avoid the shade (shade avoidance syndrome).

underneath the canopy. Measurements were taken between adjacent rows at 30 cm from the

Shifts in the amount of radiation beneath the canopy with planophile leaves and a low population of weeds, indicates that QI in the visible (400-700 nm) was lower than for a high population of weeds and, as could be expected, a higher QI was observed for wavelengths in the far red when the population of weeds was high (Fig.7). When comparing these results with the radiation profiles, as QI and irradiance, within a canopy of sugarcane leaves with erect leaves (Fig. 8 and 9), radiation reaching the soil in the visible was higher than for a canopy with planophile leaves, but still a higher QI for wavelengths in the near infrared was observed when the population of weeds was high. Except for the near infrared water sensitive portion (940-1040 nm), this is the shade that plants actually "see" and it is perceived at very early stages of development, alters phytochrome photoequilibrium and initiates

Fig. 7. Quantum energy distribution within and below a closed canopy of field grown sugarcane with planophile leaves and a high population of weeds growing underneath the canopy. Measurements were taken between adjacent rows at 30 cm from the ground. Values are given as Quantum Intensity (QI micromol m-2 s-1 nm-1)

Fig. 8. Quantum energy distribution within and below a closed canopy of field grown sugarcane with erect leaves and a low population of weeds growing underneath the canopy. Measurements were taken between adjacent rows at 30 cm from the ground. Values are given as Quantum Intensity (QI micromol m-2 s-1 nm-1)

The Shade Avoidance Syndrome Under the Sugarcane Crop 181

Fig. 10. Spectral irradiance beneath the canopy of the populations of weeds growing under a

Fig. 11. Spectral irradiance above the canopy of the weed population growing under the

sugarcane crop canopy with planophile leaves. (Values are given in W m-2 nm-1

sugarcane canopy with planophile leaves. (Values are given in W m-2 nm-1)

Fig. 9. Spectral irradiance within and below a close canopy of field grown sugarcane with erect leaves and a high population of weeds growing underneath the canopy. Measurements were taken between adjacent rows at 30 cm from the ground. Values are given as irradiance (W m-2 nm-1)

In Fig 10 an 11 radiation profiles, are shown as spectral irradiance (energy units in Watt m-2nm-1), below and above the **canopy of the weeds** growing underneath the sugarcane canopy with planophille leaves. Under this circumstances, shade conditions are more accentuated and the amount of energy in the visible is almost at limits to sustain growth; in these figures extreme shade conditions are observed within a canopy architecture that favors shade conditions (as for planophile or more horizontal leaf arrangement).

Fig. 9. Spectral irradiance within and below a close canopy of field grown sugarcane with erect leaves and a high population of weeds growing underneath the canopy. Measurements were taken between adjacent rows at 30 cm from the ground. Values are given as irradiance

In Fig 10 an 11 radiation profiles, are shown as spectral irradiance (energy units in Watt m-2nm-1), below and above the **canopy of the weeds** growing underneath the sugarcane canopy with planophille leaves. Under this circumstances, shade conditions are more accentuated and the amount of energy in the visible is almost at limits to sustain growth; in these figures extreme shade conditions are observed within a canopy architecture that

favors shade conditions (as for planophile or more horizontal leaf arrangement).

(W m-2 nm-1)

Fig. 10. Spectral irradiance beneath the canopy of the populations of weeds growing under a sugarcane canopy with planophile leaves. (Values are given in W m-2 nm-1)

Fig. 11. Spectral irradiance above the canopy of the weed population growing under the sugarcane crop canopy with planophile leaves. (Values are given in W m-2 nm-1

The Shade Avoidance Syndrome Under the Sugarcane Crop 183

dry root and stolon and leaf biomass, which resulted in a lower total dry biomass and leaf area (Lazo & Ascencio 2010). Differences in growth were found between this environment and full exposure to daylight and under the red and blue filters. It is important to emphasize the larger leaf area ratio found under low PFD neutral shade, as resulted from a lower number and corm dry biomass as compared to the aerial part. This species, a C4 plant, is thus highly sensitive to shade as shown by its lower total dry biomass and leaf area under low PFD; this could possibly explain the wide distribution of *Cyperus rotundus* in high light intensity environments, which generally occurs in tropical areas (Bielinski, Morales-paya & Shilling, 1997). However, it is also seen under canopy shade, due to the corms germinating potential and that plants under the canopy flower ("emergency flowering"), to produce seeds that are promptly shed, thus enriching the seed bank. These features may explain its persistence from planting to canopy closure, where sunflecks may play an important role in

Different shade avoidance strategies in biomass production, tillering, leaf area, plant height and flowering, revealing different capacity of acclimation to shade are shown when comparing *Rottboellia exaltata* and *Leptochloa filiformis (*both of the Poaceae family as well as sugarcane). The effect on leaf dry biomass density in *Rottboellia* was similar to full exposure to daylight, under a shade with low PAR and artificial color filters, but a red stimulator effect was observed. In *Leptochloa,* on the other hand, it was observed under blue and red filters, but the differences were not significant when comparing groups among them. The effects on the accumulation of dry biomass of roots, showed higher values for *Rottboellia* in full exposure to daylight and red; in contrast, a remarkable increase in root biomass was observed in *Leptochloa* in full exposure to daylight, which was significantly higher than under low photon flux density shade and blue, red and green filters (Ascencio & Lazo 2009). These results show a higher sensitivity of *Leptochloa* to shade, as a consequence of a lower development of the root system, which did not permit a sustained growth of the plants. This hypothesis is supported by the fact that this species showed "emergency flowering" under conditions of low PAR neutral shade and blue filter, but not total solar exposition and red filters, which are non shade conditions (high R/FR). In contrast, *Rottboellia exaltata* showed shade avoidance responses such as increased petiole length and stem, rendering it capable of competing for a longer time with the sugarcane crop under cultivation. Early flowering was not observed. The effects of shading on *Rottboellia exaltata* were determined under controlled environment conditions by Patterson (1979) under 100, 60, 25 and 2% sunlight; according to the results, this species maintains the capacity for high photosynthetic rates and high growth rates when subsequently exposed to high irradiance, after being shaded, which may explain its

Besides accelerated shoot growth, decreased tillering and early flowering, and increase in the shoot to root ratio have been part of the responses that have been associated to the SAS; however, this is not always the case as increases or decreases in S/R have been observed. The apparent contradiction is probably due to the complex nature of plant weight as a character. The ability of *Rottboellia* to reduce the effects of shade appears related to increased dry matter partitioning to the shoots. In Table 2, shoot to root ratios on a dry basis are shown for the three species grown exposed to full sunlight, neutral shed of low photon flux density (PAR) and shed plant cabinets in open space under full sunlight and covered with

the maintenance of the plant population seen under cultivation.

competitiveness with crop species.

From the perspectives of this chapter, what is more important is to ascertain which species that were present before crop canopy closure persist. From the establishment of the crop to full canopy closure, different shade intensities are found, and also different species and plant types, then the question arises as to which are capable of developing a syndrome for shade avoidance? the first answer to this question is: those species capable of perceiving, early in their development, that they are being shaded and start building mechanisms or strategies to defend themselves from dying from the shade and to escape the shade; first thing, as seen under field conditions: acceleration of extension growth of stem and petioles. Adaptation takes a little longer.

In the next part of this chapter, experiments under controlled conditions are shown in order to investigate some of the growth strategies most commonly found for three important sugarcane weeds, under an artificial shade.

#### **3.3 Greenhouse experiments**

Experiments under controlled conditions are used to simulate and find the causes of plant behavior as seen in the field. The Shade Avoidance Syndrome has been mostly investigated in connection with the ratio of red to far red wavelengths as an indication of neighbor proximity and adaptive plasticity. According to the results obtained by Weing (2000), elongation responses to R:FR are more variable than previously realized and that the observed variability suggests competitive interactions in the natural environment. Other researchers have also stressed on the importance of plant development as influenced by light spectral quality and quantity (Rajcan et al. 2002; Wherley, Gardner & Metzger 2005); tillering dynamics in grasses in relation to R/FR (Evers et al. 2006; Monaco & Briske 2000) and also on the effects of canopy shade on morphological and phenological traits (Brainard, Bellinder & DiTommaso 2005). The effects of reduced irradiance and R/FR on the leaf development of papaya (*Carica papaya*) leaves to simulate canopy shade were studied by Buisson & Lee (1993) using experimental shadehouses; results indicate that although many morphological and anatomical characteristics were affected by reduction in irradiance, some were affected primarily by low R/FR. It is important to note that when vegetation shade is simulated by means of artificial filters in growth cabinets in which R/FR is low but PAR is sufficient to allow for sustained growth, phenological changes are exaggerated (Smith & Whiteham 1997).

In connection with these ideas, the results presented in this chapter were performed with simulated shades of different colors using cellophane paper to grow three of the more severe weeds for the sugarcane crop: *Cyperus rotundus* (purple nutsedge), *Rottboellia cochinchinensis* (ichgrass) and *Leptochloa filiformis*, in order to characterize their growth responses to different light qualities as an expression of the shade avoidance syndrome in these species.

The first species *(Cyperus rotundus*) have been studied mostly due to its susceptibility to canopy and artificial shading, which have been a basis, according to Neeser, Aguero & Swanton (1997) for integrated management under crop cultivation. For the experiment, the plants were grown for 48 days inside plant cabinets with red, blue and green artificial shade, low PFD neutral shade and full exposure to daylight. Results showed that *Cyperus rotundus* plants under low PFD neutral shade had lower values for the number of tillers and corms,

From the perspectives of this chapter, what is more important is to ascertain which species that were present before crop canopy closure persist. From the establishment of the crop to full canopy closure, different shade intensities are found, and also different species and plant types, then the question arises as to which are capable of developing a syndrome for shade avoidance? the first answer to this question is: those species capable of perceiving, early in their development, that they are being shaded and start building mechanisms or strategies to defend themselves from dying from the shade and to escape the shade; first thing, as seen under field conditions: acceleration of extension growth of stem and petioles.

In the next part of this chapter, experiments under controlled conditions are shown in order to investigate some of the growth strategies most commonly found for three important

Experiments under controlled conditions are used to simulate and find the causes of plant behavior as seen in the field. The Shade Avoidance Syndrome has been mostly investigated in connection with the ratio of red to far red wavelengths as an indication of neighbor proximity and adaptive plasticity. According to the results obtained by Weing (2000), elongation responses to R:FR are more variable than previously realized and that the observed variability suggests competitive interactions in the natural environment. Other researchers have also stressed on the importance of plant development as influenced by light spectral quality and quantity (Rajcan et al. 2002; Wherley, Gardner & Metzger 2005); tillering dynamics in grasses in relation to R/FR (Evers et al. 2006; Monaco & Briske 2000) and also on the effects of canopy shade on morphological and phenological traits (Brainard, Bellinder & DiTommaso 2005). The effects of reduced irradiance and R/FR on the leaf development of papaya (*Carica papaya*) leaves to simulate canopy shade were studied by Buisson & Lee (1993) using experimental shadehouses; results indicate that although many morphological and anatomical characteristics were affected by reduction in irradiance, some were affected primarily by low R/FR. It is important to note that when vegetation shade is simulated by means of artificial filters in growth cabinets in which R/FR is low but PAR is sufficient to allow for sustained growth, phenological changes are exaggerated (Smith &

In connection with these ideas, the results presented in this chapter were performed with simulated shades of different colors using cellophane paper to grow three of the more severe weeds for the sugarcane crop: *Cyperus rotundus* (purple nutsedge), *Rottboellia cochinchinensis* (ichgrass) and *Leptochloa filiformis*, in order to characterize their growth responses to different light qualities as an expression of the shade avoidance syndrome in

The first species *(Cyperus rotundus*) have been studied mostly due to its susceptibility to canopy and artificial shading, which have been a basis, according to Neeser, Aguero & Swanton (1997) for integrated management under crop cultivation. For the experiment, the plants were grown for 48 days inside plant cabinets with red, blue and green artificial shade, low PFD neutral shade and full exposure to daylight. Results showed that *Cyperus rotundus* plants under low PFD neutral shade had lower values for the number of tillers and corms,

Adaptation takes a little longer.

**3.3 Greenhouse experiments** 

Whiteham 1997).

these species.

sugarcane weeds, under an artificial shade.

dry root and stolon and leaf biomass, which resulted in a lower total dry biomass and leaf area (Lazo & Ascencio 2010). Differences in growth were found between this environment and full exposure to daylight and under the red and blue filters. It is important to emphasize the larger leaf area ratio found under low PFD neutral shade, as resulted from a lower number and corm dry biomass as compared to the aerial part. This species, a C4 plant, is thus highly sensitive to shade as shown by its lower total dry biomass and leaf area under low PFD; this could possibly explain the wide distribution of *Cyperus rotundus* in high light intensity environments, which generally occurs in tropical areas (Bielinski, Morales-paya & Shilling, 1997). However, it is also seen under canopy shade, due to the corms germinating potential and that plants under the canopy flower ("emergency flowering"), to produce seeds that are promptly shed, thus enriching the seed bank. These features may explain its persistence from planting to canopy closure, where sunflecks may play an important role in the maintenance of the plant population seen under cultivation.

Different shade avoidance strategies in biomass production, tillering, leaf area, plant height and flowering, revealing different capacity of acclimation to shade are shown when comparing *Rottboellia exaltata* and *Leptochloa filiformis (*both of the Poaceae family as well as sugarcane). The effect on leaf dry biomass density in *Rottboellia* was similar to full exposure to daylight, under a shade with low PAR and artificial color filters, but a red stimulator effect was observed. In *Leptochloa,* on the other hand, it was observed under blue and red filters, but the differences were not significant when comparing groups among them. The effects on the accumulation of dry biomass of roots, showed higher values for *Rottboellia* in full exposure to daylight and red; in contrast, a remarkable increase in root biomass was observed in *Leptochloa* in full exposure to daylight, which was significantly higher than under low photon flux density shade and blue, red and green filters (Ascencio & Lazo 2009). These results show a higher sensitivity of *Leptochloa* to shade, as a consequence of a lower development of the root system, which did not permit a sustained growth of the plants. This hypothesis is supported by the fact that this species showed "emergency flowering" under conditions of low PAR neutral shade and blue filter, but not total solar exposition and red filters, which are non shade conditions (high R/FR). In contrast, *Rottboellia exaltata* showed shade avoidance responses such as increased petiole length and stem, rendering it capable of competing for a longer time with the sugarcane crop under cultivation. Early flowering was not observed. The effects of shading on *Rottboellia exaltata* were determined under controlled environment conditions by Patterson (1979) under 100, 60, 25 and 2% sunlight; according to the results, this species maintains the capacity for high photosynthetic rates and high growth rates when subsequently exposed to high irradiance, after being shaded, which may explain its competitiveness with crop species.

Besides accelerated shoot growth, decreased tillering and early flowering, and increase in the shoot to root ratio have been part of the responses that have been associated to the SAS; however, this is not always the case as increases or decreases in S/R have been observed. The apparent contradiction is probably due to the complex nature of plant weight as a character. The ability of *Rottboellia* to reduce the effects of shade appears related to increased dry matter partitioning to the shoots. In Table 2, shoot to root ratios on a dry basis are shown for the three species grown exposed to full sunlight, neutral shed of low photon flux density (PAR) and shed plant cabinets in open space under full sunlight and covered with

The Shade Avoidance Syndrome Under the Sugarcane Crop 185

management strategies based on the knowledge of the dynamics and biology of plants that

When sucrose is the desired sugarcane product, sucrose yield is the ultimate concern, but when the production of ethanol is the main purpose, the accumulation of biomass is the goal to achieve. However, in both cases, the output is determined by the number of stalks, which in turn depends on adequate and timely management of weeds and the good use of agronomic practices. It is important to emphasize that one of the ways to control weeds in this perennial crop, is precisely to take advantage of the intense shading under the sugarcane canopy, which limits the density and biodiversity of plant population grown underneath the crop. At canopy closure, the light environment underneath can exclude a considerable number of plant species, since not all species can tolerate shade, without ruling out the possibility that there are some species that are able to tolerate the shade, and others remain in the seed bank, and become a delayed problem when it is activated by some environmental factor, such as sunflecks. In this connection, it is important to recognize which of the species can tolerate shade and which cannot; therefore, research and studies of the dynamics of weed populations in the sugar cane crop, are required from planting to canopy closure. Light profiles are rarely recorded in field conditions and may be the key to understanding some of the growth responses of different plant species under a crop canopy. Light quality (wavelength) and quantity (number of photons, irradiance) interact to control growth responses under vegetation canopies and some species underneath sugarcane canopies under field conditions can escape shade competing successfully with the crop for light. Two strategies associated to the Shade Avoidance Syndrome, as seen in the field, were observed: increased internodes length and decreased leaf size, while others species showed a higher sensitivity towards canopy shade and eventually died in this condition. Energy distributions of visible and near infrared radiation above, within, near the soil and above a canopy of weeds underneath a sugarcane canopy, showed that the decreased intensity of radiant energy was not uniform at all wavelengths. Some species are also seen under canopy shade, due to the corms or other subterranean organs and the germinating potential of the seed bank. These features may explain their persistence from time of planting to canopy closure, where sunflecks may play an important role in the maintenance of the plant population seen under crop cultivation. Different shade avoidance strategies in biomass production, tillering, leaf area, plant height and flowering, revealing different capacity of acclimation to shade were shown using experimental shadehouses; experiments under controlled conditions were useful to simulate and find the causes of plant behavior as seen in the field. Even though sugarcane is a crop economically important, research is limited in this area and in our opinion more has to be done on the biology and performance of the population of plants growing underneath the canopy from the beginning of the crop cycle, to improve weed control

We thank Fonacit government grant No. S1-2002000512 for financial support to this research and to Fernando Gil, M.Sc. (Fundacaña) and Jorge Ugarte, M.Sc. (UCV) for

grow under the canopy of the crop.

practices under cultivation.

**5. Acknowledgements** 

technical assistance in field experiments.

artificial filters made of red, blue and green cellophane paper. The values for S/R or dry matter partitioning are mainly related to biomass allocation to shoots in species capable of escaping shade as *Rottboellia*, however the highest value was found in full sunlight (2.27) but no significant differences were found for S/R with the rest of the light environments. In contrast, a remarkable increase in root biomass as compared to shoots, lowered the S/R in *Leptochloa* in full sunlight, while an increase was observed under shade conditions. The increase in S/A observed for this species and for *Cyperus* are more likely to be related to a lower root biomass allocation under shade conditions.


Values with the same letter, within the same column, do not differ according to Tukey HSD test; Homogenous Groups, alpha = .05000

Table 2. Shoot to root ratio (S/R) for *Rotboellia exaltata*, *Leptochloa filiformis* and *Cyperus rotundus* plants, grown: (1) exposed to full sunlight, (2) neutral shed of low PFD, and (3) shed, plant cabinets in open space under full sunlight and covered with red, blue and green cellophane paper.

The partitioning dry biomass to the roots and shoots for *Leptochloa* reflects a higher sensibility of this species to shade impairing the aerial parts to develop strategies to escape or avoid shade. This is shown in lower S/R for *Leptochloa* as compared to the other two species. The sensibility of *Cyperus rotundus* to shade is shown in the lower values of dry biomass for the subterranean parts (roots, corms and stolons) which produces higher values for S/R under low intensity neutral shade (2.63). This plant is totally intolerant to shade and under low intensity light, lower values for the number of tillers and corms, dry root, stolon and leaf biomass were observed (Lazo & Ascencio 2010). Because of the high plasticity of the S/R to environmental conditions, values are not easy to interpret and show less predictable responses to environmental variables such as light.

#### **4. Conclusion**

Sugarcane is an important crop in many tropical countries. Under these conditions, the major biotic limiting factor to productivity is the direct interference caused by weeds, especially during sprouting and the first three months of growth (initial stand establishment), when canopy closure has not been completed because crop growth is slow and foliage do not completely cover the area under cultivation. Weeds also serve as host plants of pests and diseases and interfere with crop management practices such as side dressing, sanitary inspection, sampling, maturation, etc., as well as with mechanical harvesting. Weed control is usually performed either by mechanical or the application of chemical herbicides, but the emergence and rapid evolution of weed species resistant to many herbicides, has prompted the search for new alternatives to control, and to consider

artificial filters made of red, blue and green cellophane paper. The values for S/R or dry matter partitioning are mainly related to biomass allocation to shoots in species capable of escaping shade as *Rottboellia*, however the highest value was found in full sunlight (2.27) but no significant differences were found for S/R with the rest of the light environments. In contrast, a remarkable increase in root biomass as compared to shoots, lowered the S/R in *Leptochloa* in full sunlight, while an increase was observed under shade conditions. The increase in S/A observed for this species and for *Cyperus* are more likely to be related to a

Exposed to Full Sunlight 2.27a 0.15b 0.74c Neutral Shade (low PAR) 0.93a 0.54b 2.63a Red cover 0.83a 0.83a,b 1.03c Blue cover 1.01a 0.48b 0.98c Green cover 1.58a 1.46a 1.82b Values with the same letter, within the same column, do not differ according to Tukey HSD test;

Table 2. Shoot to root ratio (S/R) for *Rotboellia exaltata*, *Leptochloa filiformis* and *Cyperus rotundus* plants, grown: (1) exposed to full sunlight, (2) neutral shed of low PFD, and (3) shed, plant cabinets in open space under full sunlight and covered with red, blue and green

The partitioning dry biomass to the roots and shoots for *Leptochloa* reflects a higher sensibility of this species to shade impairing the aerial parts to develop strategies to escape or avoid shade. This is shown in lower S/R for *Leptochloa* as compared to the other two species. The sensibility of *Cyperus rotundus* to shade is shown in the lower values of dry biomass for the subterranean parts (roots, corms and stolons) which produces higher values for S/R under low intensity neutral shade (2.63). This plant is totally intolerant to shade and under low intensity light, lower values for the number of tillers and corms, dry root, stolon and leaf biomass were observed (Lazo & Ascencio 2010). Because of the high plasticity of the S/R to environmental conditions, values are not easy to interpret and show less predictable

Sugarcane is an important crop in many tropical countries. Under these conditions, the major biotic limiting factor to productivity is the direct interference caused by weeds, especially during sprouting and the first three months of growth (initial stand establishment), when canopy closure has not been completed because crop growth is slow and foliage do not completely cover the area under cultivation. Weeds also serve as host plants of pests and diseases and interfere with crop management practices such as side dressing, sanitary inspection, sampling, maturation, etc., as well as with mechanical harvesting. Weed control is usually performed either by mechanical or the application of chemical herbicides, but the emergence and rapid evolution of weed species resistant to many herbicides, has prompted the search for new alternatives to control, and to consider

*Rottboellia exaltata Leptochloa filiformis Cyperus rotundus*  SA/SR SA/SR SA/SR

lower root biomass allocation under shade conditions.

responses to environmental variables such as light.

Homogenous Groups, alpha = .05000

cellophane paper.

**4. Conclusion** 

management strategies based on the knowledge of the dynamics and biology of plants that grow under the canopy of the crop.

When sucrose is the desired sugarcane product, sucrose yield is the ultimate concern, but when the production of ethanol is the main purpose, the accumulation of biomass is the goal to achieve. However, in both cases, the output is determined by the number of stalks, which in turn depends on adequate and timely management of weeds and the good use of agronomic practices. It is important to emphasize that one of the ways to control weeds in this perennial crop, is precisely to take advantage of the intense shading under the sugarcane canopy, which limits the density and biodiversity of plant population grown underneath the crop. At canopy closure, the light environment underneath can exclude a considerable number of plant species, since not all species can tolerate shade, without ruling out the possibility that there are some species that are able to tolerate the shade, and others remain in the seed bank, and become a delayed problem when it is activated by some environmental factor, such as sunflecks. In this connection, it is important to recognize which of the species can tolerate shade and which cannot; therefore, research and studies of the dynamics of weed populations in the sugar cane crop, are required from planting to canopy closure. Light profiles are rarely recorded in field conditions and may be the key to understanding some of the growth responses of different plant species under a crop canopy.

Light quality (wavelength) and quantity (number of photons, irradiance) interact to control growth responses under vegetation canopies and some species underneath sugarcane canopies under field conditions can escape shade competing successfully with the crop for light. Two strategies associated to the Shade Avoidance Syndrome, as seen in the field, were observed: increased internodes length and decreased leaf size, while others species showed a higher sensitivity towards canopy shade and eventually died in this condition. Energy distributions of visible and near infrared radiation above, within, near the soil and above a canopy of weeds underneath a sugarcane canopy, showed that the decreased intensity of radiant energy was not uniform at all wavelengths. Some species are also seen under canopy shade, due to the corms or other subterranean organs and the germinating potential of the seed bank. These features may explain their persistence from time of planting to canopy closure, where sunflecks may play an important role in the maintenance of the plant population seen under crop cultivation. Different shade avoidance strategies in biomass production, tillering, leaf area, plant height and flowering, revealing different capacity of acclimation to shade were shown using experimental shadehouses; experiments under controlled conditions were useful to simulate and find the causes of plant behavior as seen in the field. Even though sugarcane is a crop economically important, research is limited in this area and in our opinion more has to be done on the biology and performance of the population of plants growing underneath the canopy from the beginning of the crop cycle, to improve weed control practices under cultivation.

#### **5. Acknowledgements**

We thank Fonacit government grant No. S1-2002000512 for financial support to this research and to Fernando Gil, M.Sc. (Fundacaña) and Jorge Ugarte, M.Sc. (UCV) for technical assistance in field experiments.

The Shade Avoidance Syndrome Under the Sugarcane Crop 187

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sugar preices on farms-short and long term strategies. *Proc. Aust. Soc .Sugarcane* 

**6. References** 

0162.

0002-9122.

0264-6021.


**9** 

*1,3Canada 2Pakistan* 

**Molecular Genetics of Glucosinolate** 

**Manipulation and Application Aspects** 

**Biosynthesis in** *Brassicas***: Genetic** 

*1Department of Plant Science, University of Manitoba, Winnipeg,* 

*2Ex-Research Scientist, National Agriculture Research Centre, Islamabad, 3Lethbridge Research Centre, Agriculture and Agri-Food Canada, Lethbridge,* 

Glucosinolates are sulphur containing secondary metabolites biosynthesized by many plant species in the order *Brassicales*. Physical tissue or cell injury leads to the breakdown of glucosinolates through the hydrolytic action of the enzyme myrosinase, resulting in the production of compounds including isothiocynates, thiocyanates and nitriles. Derivative compounds of glucosinolates have a wide range of biological functions including anticarcinogenic properties in humans, anti-nutritional effects of seed meal in animals, insect pest repellent and fungal disease suppression (Mithen et al., 2000; Brader et al., 2006). Glucosinolates play important role in the nutritional qualities of *Brassica* products. *Brassica* products are consumed as oil, meal and as vegetables. Rapeseed (*B. napus*, *B. juncea* and *B. rapa*) is a source of oil and has a protein-rich seed meal. High glucosinolates in the seed meal pose health risks to livestock (Fenwick et al., 1983; Griffiths et al., 1998). Consequently, plant breeders have nearly eliminated erucic acid from the seed oil and have dramatically reduced the level of seed glucosinolates (>100 µmole/g seed to <30 µmole/g seed) via conventional breeding, allowing the nutritious seed meal to be used as an animal feed supplement. There is, however, a significant residual content of glucosinolates in rapeseed/canola seed meal (over 10 µmole/g seed) and further reduction of the total glucosinolate content would be nutritionally beneficial (McVetty et al., 2009). Therefore, to produce healthy seed meal from rapeseed, it is important to genetically manipulate glucosinolate content. *Brassica* vegetables (*B. rapa* and *B. oleracea*) are highly regarded for their nutritional qualities, they are a good source of vitamin A and C, dietary soluble fibres, folic acid, essential micro nutrients and low in calories, fat and health beneficial glucosinolates such as glucoraphanin and sulforaphane. Breeding objectives for these Brassica crops include the enhancement of beneficial glucosinolates and reduction of others. It is, therefore, important to understand the genetic, biosynthetic, transportation and

accumulation mechanisms for glucosinolates in *Brassica* species.

**1. Introduction** 

 \*

Corresponding author

Arvind H. Hirani1, Genyi Li1,\*, Carla D. Zelmer1, Peter B.E. McVetty1, M. Asif2 and Aakash Goyal3

Wherley,B.G., Gardner,D.S. & Metzger, J.D. (2005). Tall fescue photomorphogenesis as influenced by changes in the spectral composition and light intensity. *Crop Science* 45,2,(January 2005),pp.(562-568). ISSN 0011-183X

## **Molecular Genetics of Glucosinolate Biosynthesis in** *Brassicas***: Genetic Manipulation and Application Aspects**

Arvind H. Hirani1, Genyi Li1,\*, Carla D. Zelmer1,

Peter B.E. McVetty1, M. Asif2 and Aakash Goyal3 *1Department of Plant Science, University of Manitoba, Winnipeg,* 

*2Ex-Research Scientist, National Agriculture Research Centre, Islamabad, 3Lethbridge Research Centre, Agriculture and Agri-Food Canada, Lethbridge, 1,3Canada 2Pakistan* 

#### **1. Introduction**

188 Crop Plant

Wherley,B.G., Gardner,D.S. & Metzger, J.D. (2005). Tall fescue photomorphogenesis as

45,2,(January 2005),pp.(562-568). ISSN 0011-183X

influenced by changes in the spectral composition and light intensity. *Crop Science*

Glucosinolates are sulphur containing secondary metabolites biosynthesized by many plant species in the order *Brassicales*. Physical tissue or cell injury leads to the breakdown of glucosinolates through the hydrolytic action of the enzyme myrosinase, resulting in the production of compounds including isothiocynates, thiocyanates and nitriles. Derivative compounds of glucosinolates have a wide range of biological functions including anticarcinogenic properties in humans, anti-nutritional effects of seed meal in animals, insect pest repellent and fungal disease suppression (Mithen et al., 2000; Brader et al., 2006). Glucosinolates play important role in the nutritional qualities of *Brassica* products. *Brassica* products are consumed as oil, meal and as vegetables. Rapeseed (*B. napus*, *B. juncea* and *B. rapa*) is a source of oil and has a protein-rich seed meal. High glucosinolates in the seed meal pose health risks to livestock (Fenwick et al., 1983; Griffiths et al., 1998). Consequently, plant breeders have nearly eliminated erucic acid from the seed oil and have dramatically reduced the level of seed glucosinolates (>100 µmole/g seed to <30 µmole/g seed) via conventional breeding, allowing the nutritious seed meal to be used as an animal feed supplement. There is, however, a significant residual content of glucosinolates in rapeseed/canola seed meal (over 10 µmole/g seed) and further reduction of the total glucosinolate content would be nutritionally beneficial (McVetty et al., 2009). Therefore, to produce healthy seed meal from rapeseed, it is important to genetically manipulate glucosinolate content. *Brassica* vegetables (*B. rapa* and *B. oleracea*) are highly regarded for their nutritional qualities, they are a good source of vitamin A and C, dietary soluble fibres, folic acid, essential micro nutrients and low in calories, fat and health beneficial glucosinolates such as glucoraphanin and sulforaphane. Breeding objectives for these Brassica crops include the enhancement of beneficial glucosinolates and reduction of others. It is, therefore, important to understand the genetic, biosynthetic, transportation and accumulation mechanisms for glucosinolates in *Brassica* species.

<sup>\*</sup> Corresponding author

Molecular Genetics of Glucosinolate

rich animal feed supplement.

broccoli.

*oleracea* (CC, 2n=18).

**4. Genomic relationships in** *Brassica* **species** 

Biosynthesis in *Brassicas*: Genetic Manipulation and Application Aspects 191

Globally, rapeseed and canola oil is being utilized for human consumption, industrial applications and as a feedstock for biodiesel production. Canola oil is considered a healthy edible oil due to its high level of monounsaturated fatty acid (61%), lower level of saturated fatty acid (7%) and moderate amount of polyunsaturated fatty acid (22%) in its overall fatty acid profile (McVetty & Scarth, 2002). Rapeseed that has erucic acid levels greater than 45% also has many industrial applications such as plasticizers, slip agents for fibreglass and oil for the lubrication industry. Additionally, the seed meal is a marketable source of protein

Rapeseed is the world's third leading oil producing crop after palm and soybean, and it contributes about 15% to the global total vegetable oil production. Canada was the top rapeseed producing country in the world with 12.6 million MT productions in 2008 (FAO 2008). Canola/rapeseed contributes about \$14 billion annually to the Canadian economy along with the generation of about 200,000 jobs throughout Canada in the areas of production, transportation, exporting, crushing and refining (Canola Council of Canada, 2010b http://www.canolacouncil.org/canadian\_canola\_industry.aspx). Canola/rapeseed meal is the second most popular protein feed ingredient in the world after soybean meal. Protein content of canola/rapeseed meal ranges from 36 to 39%, with a good amino acid profile for animal feeding (Newkirk et al., 2003). The major producers and consumers of canola/rapeseed meal are Australia, Canada, China, European Union and India. Along with oil production, *Brassica* species also produce different forms of vegetables and are the most widely cultivated vegetable crops in the world. Most of the production is consumed locally with a small amount of international trade. *B. napus* and *B. juncea* are used as vegetables in Asian countries like China, Japan and India. *B. rapa* is differentiated into seven groups viz., var. *compestris*, *pekinensis*, *chinensis*, *parachinesis*, *narinosa*, *japonica* and *rapa*. *Brassica rapa* is cultivated for leafy and root vegetables in the form of Chinese cabbage, pak choi and turnip; *B. oleracea* is cultivated for leafy and floret vegetables in various morph types such as cabbage, cauliflower, kale, collard, kohlrabi, brussels and

Genomic relationships between the three diploid and three amphidiploid *Brassica* species were initially established in the 1930s based on various taxonomical and cytogenetic studies (Fig. 1) (Morinaga 1934; U 1935). Three allotetraploid *Brassica* species namely, *B. napus* (AACC, 2n=38), *B. juncea* (AABB, 2n=36) and *B. carinata* (BBCC, 2n=34) have been derived from three diploid elementary species, *B. rapa* (AA, 2n=20), *B. nigra* (BB, 2n=16) and *B.* 

The genomic relationships of *B. napus* with *B. rapa* and *B. oleracea* have been confirmed by the resynthesis of *B. napus* from *B. rapa* x *B. oleracea* crosses (U 1935; Downey et al., 1975; Olsson & Ellerstrom, 1980). The close relationship between the six *Brassica* species made it feasible to incorporate a trait from one species into others to make the crops more suitable to agricultural systems. Thus, complex traits like glucosinolates can also be manipulated as required through interspecific hybridization. It has been relatively easier to make interspecific crosses among some of these six species (e.g. *B. napus* x *B. rapa*) compared to others (e.g. *B. rapa* x *B. oleracea*). Wide hybridizations are normally performed by the application of embryo rescue techniques. The most recent advances in genome sequencing technology, bioinformatics and

#### **2. Historical background of** *Brassica* **species**

The crops belonging to the genus *Brassica* have been of great importance to humanity. Since ancient times, *Brassica* crops have been used for many purposes, including vegetables, oilseeds, feed, condiments, fodder, green manure and even medical treatments. Early history suggests that rapeseed has been cultivated for several thousand years with its origin in the Mediterranean region although exact time of domestication and the place of origin are still unknown. Sanskrit writings in 2000-1500 BC characterized species identified as *B. rapa* and *B. napus* as oleiferous forms and mustards, respectively. *Brassica juncea* and *B. rapa* are believed to have been crop plants in India long before the Christian era. The Greek, Roman and Chinese literature of 500-200 BC referred *B. rapa* as rapiferous forms and were also described for various medicinal properties (Downey & Röbellen, 1989). In early times, rapeseed oil was used as a lamp oil, which in later centuries led gradually to its use as a valuable cooking oil.

*Brassica* species are diverse in terms of morphology, agronomy and quality traits. Domestication of rapeseed in Europe seems to have begun in the early Middle Ages. In 1620, *B. rapa* was first recorded in Europe by the Swiss botanist Casper Banhin (Gupta & Pratap, 2007). As a result, *Brassica* crops were adapted and cultivated in many parts of the world (Mehra, 1966). Rapeseed was introduced in Canada before the Second World War (McVetty et al., 2009). Commercial cultivation in Canada began during the Second World War to supply lubricating oil for steamships. Canada's first *B. rapa* rapeseed cultivar, Arlo, with high erucic acid (40 to 45%) and high glucosinolate content (>150 µmole/g seed) was developed in 1958 using selection from open pollinated populations (McVetty et al., 2009). Initially, *B. rapa* was the dominant cultivated species of *Brassica* in western Canada. In late 1980s, a large acreage of *B. rapa* and *B. napus* was grown in the Prairie Provinces. Subsequently, the production area of *B. rapa* declined to about 15 – 20% of its former area in 1990s. The reduction in acreage of *B. rapa* resulted from the introduction of herbicide tolerance canola, which provided the early planting and high yield advantages of *B. napus* cultivars. Currently, *B. rapa* is still grown in small areas in Canada because of its early maturity. Research efforts are underway to develop disease resistant hybrid varieties to increase yield potential of *B. rapa*. *Brassica rapa* are grown as a winter sarson crop in Asian countries such as India, Pakistan, China and Bangladesh. Vegetable forms of *B. rapa* (Chinese cabbage, turnip, pak choi, komatsuna, mizuna green and rapini) are widely cultivated in many parts of the world (Prakash & Hinata, 1980; Takuno et al., 2007).

#### **3. Economic importance of** *Brassica* **species**

The family *Brassicaceae* (syn. *Cruciferae*) is one of the crucial plant families for humans and animals and supplies several products from various plant parts. The little cruciferous weed *A. thaliana* has become an important model organism for the study of plant molecular biology, including the related crop species. The mustard family (*Brassicaceae*) is the fifth largest monophyletic angiosperm family, comprising 338 genera and about 3700 species in 25 tribes (Beilstein et al., 2006). The genus *Brassica* is one of the 51 genera of the tribe *Brassiceae* and includes the economically valuable crop species. *B. napus*, *B. rapa*, *B. juncea, B. carinata* and *B. nigra* are grown for edible and industrial oil as well as nutritionally valued seed meal.

The crops belonging to the genus *Brassica* have been of great importance to humanity. Since ancient times, *Brassica* crops have been used for many purposes, including vegetables, oilseeds, feed, condiments, fodder, green manure and even medical treatments. Early history suggests that rapeseed has been cultivated for several thousand years with its origin in the Mediterranean region although exact time of domestication and the place of origin are still unknown. Sanskrit writings in 2000-1500 BC characterized species identified as *B. rapa* and *B. napus* as oleiferous forms and mustards, respectively. *Brassica juncea* and *B. rapa* are believed to have been crop plants in India long before the Christian era. The Greek, Roman and Chinese literature of 500-200 BC referred *B. rapa* as rapiferous forms and were also described for various medicinal properties (Downey & Röbellen, 1989). In early times, rapeseed oil was used as a lamp oil, which in later centuries led gradually to its use as a

*Brassica* species are diverse in terms of morphology, agronomy and quality traits. Domestication of rapeseed in Europe seems to have begun in the early Middle Ages. In 1620, *B. rapa* was first recorded in Europe by the Swiss botanist Casper Banhin (Gupta & Pratap, 2007). As a result, *Brassica* crops were adapted and cultivated in many parts of the world (Mehra, 1966). Rapeseed was introduced in Canada before the Second World War (McVetty et al., 2009). Commercial cultivation in Canada began during the Second World War to supply lubricating oil for steamships. Canada's first *B. rapa* rapeseed cultivar, Arlo, with high erucic acid (40 to 45%) and high glucosinolate content (>150 µmole/g seed) was developed in 1958 using selection from open pollinated populations (McVetty et al., 2009). Initially, *B. rapa* was the dominant cultivated species of *Brassica* in western Canada. In late 1980s, a large acreage of *B. rapa* and *B. napus* was grown in the Prairie Provinces. Subsequently, the production area of *B. rapa* declined to about 15 – 20% of its former area in 1990s. The reduction in acreage of *B. rapa* resulted from the introduction of herbicide tolerance canola, which provided the early planting and high yield advantages of *B. napus* cultivars. Currently, *B. rapa* is still grown in small areas in Canada because of its early maturity. Research efforts are underway to develop disease resistant hybrid varieties to increase yield potential of *B. rapa*. *Brassica rapa* are grown as a winter sarson crop in Asian countries such as India, Pakistan, China and Bangladesh. Vegetable forms of *B. rapa* (Chinese cabbage, turnip, pak choi, komatsuna, mizuna green and rapini) are widely

cultivated in many parts of the world (Prakash & Hinata, 1980; Takuno et al., 2007).

The family *Brassicaceae* (syn. *Cruciferae*) is one of the crucial plant families for humans and animals and supplies several products from various plant parts. The little cruciferous weed *A. thaliana* has become an important model organism for the study of plant molecular biology, including the related crop species. The mustard family (*Brassicaceae*) is the fifth largest monophyletic angiosperm family, comprising 338 genera and about 3700 species in 25 tribes (Beilstein et al., 2006). The genus *Brassica* is one of the 51 genera of the tribe *Brassiceae* and includes the economically valuable crop species. *B. napus*, *B. rapa*, *B. juncea, B. carinata* and *B. nigra* are grown for edible and industrial oil as well as nutritionally valued

**3. Economic importance of** *Brassica* **species** 

**2. Historical background of** *Brassica* **species** 

valuable cooking oil.

seed meal.

Globally, rapeseed and canola oil is being utilized for human consumption, industrial applications and as a feedstock for biodiesel production. Canola oil is considered a healthy edible oil due to its high level of monounsaturated fatty acid (61%), lower level of saturated fatty acid (7%) and moderate amount of polyunsaturated fatty acid (22%) in its overall fatty acid profile (McVetty & Scarth, 2002). Rapeseed that has erucic acid levels greater than 45% also has many industrial applications such as plasticizers, slip agents for fibreglass and oil for the lubrication industry. Additionally, the seed meal is a marketable source of protein rich animal feed supplement.

Rapeseed is the world's third leading oil producing crop after palm and soybean, and it contributes about 15% to the global total vegetable oil production. Canada was the top rapeseed producing country in the world with 12.6 million MT productions in 2008 (FAO 2008). Canola/rapeseed contributes about \$14 billion annually to the Canadian economy along with the generation of about 200,000 jobs throughout Canada in the areas of production, transportation, exporting, crushing and refining (Canola Council of Canada, 2010b http://www.canolacouncil.org/canadian\_canola\_industry.aspx). Canola/rapeseed meal is the second most popular protein feed ingredient in the world after soybean meal. Protein content of canola/rapeseed meal ranges from 36 to 39%, with a good amino acid profile for animal feeding (Newkirk et al., 2003). The major producers and consumers of canola/rapeseed meal are Australia, Canada, China, European Union and India. Along with oil production, *Brassica* species also produce different forms of vegetables and are the most widely cultivated vegetable crops in the world. Most of the production is consumed locally with a small amount of international trade. *B. napus* and *B. juncea* are used as vegetables in Asian countries like China, Japan and India. *B. rapa* is differentiated into seven groups viz., var. *compestris*, *pekinensis*, *chinensis*, *parachinesis*, *narinosa*, *japonica* and *rapa*. *Brassica rapa* is cultivated for leafy and root vegetables in the form of Chinese cabbage, pak choi and turnip; *B. oleracea* is cultivated for leafy and floret vegetables in various morph types such as cabbage, cauliflower, kale, collard, kohlrabi, brussels and broccoli.

#### **4. Genomic relationships in** *Brassica* **species**

Genomic relationships between the three diploid and three amphidiploid *Brassica* species were initially established in the 1930s based on various taxonomical and cytogenetic studies (Fig. 1) (Morinaga 1934; U 1935). Three allotetraploid *Brassica* species namely, *B. napus* (AACC, 2n=38), *B. juncea* (AABB, 2n=36) and *B. carinata* (BBCC, 2n=34) have been derived from three diploid elementary species, *B. rapa* (AA, 2n=20), *B. nigra* (BB, 2n=16) and *B. oleracea* (CC, 2n=18).

The genomic relationships of *B. napus* with *B. rapa* and *B. oleracea* have been confirmed by the resynthesis of *B. napus* from *B. rapa* x *B. oleracea* crosses (U 1935; Downey et al., 1975; Olsson & Ellerstrom, 1980). The close relationship between the six *Brassica* species made it feasible to incorporate a trait from one species into others to make the crops more suitable to agricultural systems. Thus, complex traits like glucosinolates can also be manipulated as required through interspecific hybridization. It has been relatively easier to make interspecific crosses among some of these six species (e.g. *B. napus* x *B. rapa*) compared to others (e.g. *B. rapa* x *B. oleracea*). Wide hybridizations are normally performed by the application of embryo rescue techniques. The most recent advances in genome sequencing technology, bioinformatics and

Molecular Genetics of Glucosinolate

to enhance genetic variability.

based on their biosynthetic origin

ii. Terpenoid compounds

**5. Plant secondary metabolites and their functions** 

i. Flavonoids and allied phenolic and polyphenolic compounds

status for research on secondary metabolites (Sønderby et al., 2010).

iii. Nitrogen and sulphur containing alkaloid compounds

**5.1 Glucosinolates as secondary metabolites** 

Biosynthesis in *Brassicas*: Genetic Manipulation and Application Aspects 193

synteny, there have been several reports of homoeologous recombination between the Aand C-genome of *Brassica* species (Udall et al., 2004; Leflon et al., 2006). Cytogenetic and molecular data revealed that small and large collinear genomic regions between the A- and C-genomes of *Brassica* species allow homoeologous recombination-based trait introgression

The sessile nature of plants requires them to produce a large numbers of defence compounds including primary and secondary metabolites. It is believed that the currently discovered plant metabolic compounds account for only about 10% of the actual compounds present naturally within the plant kingdom (Schwab, 2003; Wink, 2003). Plant secondary metabolites are organic biochemical compounds produced in plants during normal growth and development. While they are not directly involved in plant growth, development or reproduction, these secondary metabolites play vital roles in plant defence mechanisms, acting for example, phytoalexins and phytoanticipins. Phytoalexins are antimicrobial defence metabolites synthesized *de novo* in response to biotic and abiotic stresses. Phytoalexins are involved in induced plant defence mechanisms including lytic enzymes, oxidizing agents, cell wall lignifications and pathogenesis-related proteins and transcript stimulation (Pedras et al., 2008). Phytoanticipins are low molecular weight antimicrobial compounds which are constitutively active for defence. Their production may be increased under high biotic or abiotic stresses (Pedras et al., 2007). Certain classes of phytoanticipins require enzymatic modification and derivation in order to become active within the defence systems of the plant. Plant secondary metabolites are broadly categorized into three groups

Glucosinolates are sulphur rich, nitrogen containing anionic natural products, derived from specific amino acids and their precursors (Fenwick et al., 1983). Glucosinolates are reported almost exclusively from the order *Brassicales,* which possesses about 15 families such as *Brassica*ceae, *Capparaceae* and *Caricaceae*. Glucosinolates are also reported in a few members of the family *Euphorbiaceae*, a very distinct family to other glucosinolate containing families (Rodman et al., 1996). Glucosinolates coexist with endogenous thioglucosidases called myrosinases in cruciferous plant species and activate plant defence mechanism against biotic and abiotic stresses. Tissue disruption causes systemic interactions between glucosinolates and myrosinases in the presence of moisture. The interaction produces numerous compounds with diverse biological activities (Bones & Rossiter, 1996; Halkier, 1999). Glucosinolates are some of the most extensively studied plant secondary metabolites; various enzymes and transcription factors involved in biosynthesis have been studied in the model plant *Arabidopsis* and to some extent in *Brassica* crops species. The broad functionality, physiochemical and genetic studies of glucosinolates have led to a model

data mining have opened an avenue for comparative analysis of ESTs, BACs, genes (families), whole chromosomes and even entire genomes to determine evolutionary relationship between these species and their ancestors (Gao et al., 2004; Gao et al., 2006; Punjabi et al., 2008; Mun et al., 2009; Qiu et al., 2009; Nagoaka et al., 2010).

Fig. 1. U-triangle of genomic relationship between diploid and amphidiploid *Brassica* species (U 1935).

#### **4.1 Homoeology between the A, B and C genomes of** *Brassica* **species**

Genome homoeology has been characterized in *Brassica* species by comparative analyses of the genetic and physical maps of *Arabidopsis* with genetic maps of *Brassica* species (Osborn et al., 1997; Lan et al., 2000; Parkin et al., 2002; Lukens et al., 2003; Parkin et al., 2005). These studies indicate that each genomic region has had multiple events of polyploidization and chromosome rearrangements in the *Brassicaceae* lineage after the evolutionary divergence from *Arabidopsis* approximately 14.5 to 20.4 million years ago (MYA) (Yang et al., 1999; Parkin et al., 2002). In the *Brassicaceae*, the *B. nigra* (B) genome separated from the *B. rapa*/*B. oleracea* (AC) genome lineages about 7.9 MYA (Yang et al., 1999; Lysak et al., 2005).

There are high levels of homoeology among the A- and C-genomes of *B. rapa*, *B. oleracea* and *B. napus* (Parkin et al., 2005; Punjabi et al., 2008). Parkin et al., (2003) reported stretches of collinearity on the linkage groups N1 with N11, N2 with N12 and N3 with N13 of the Aand C-genomes, respectively. Similarly, Osborn et al., (2003) reported reciprocal interstitial translocations of homoeologous regions of linkage groups N7 and N16, and their effects on genome rearrangements and seed yield in *B. napus*. This suggests that inter-genomic translocations and rearrangements have taken place during the evolutionary divergence of *B. oleracea* and *B. rapa* from a polyploid ancestor (Sharpe et al., 1995). As a result of genomic

synteny, there have been several reports of homoeologous recombination between the Aand C-genome of *Brassica* species (Udall et al., 2004; Leflon et al., 2006). Cytogenetic and molecular data revealed that small and large collinear genomic regions between the A- and C-genomes of *Brassica* species allow homoeologous recombination-based trait introgression to enhance genetic variability.

#### **5. Plant secondary metabolites and their functions**

The sessile nature of plants requires them to produce a large numbers of defence compounds including primary and secondary metabolites. It is believed that the currently discovered plant metabolic compounds account for only about 10% of the actual compounds present naturally within the plant kingdom (Schwab, 2003; Wink, 2003). Plant secondary metabolites are organic biochemical compounds produced in plants during normal growth and development. While they are not directly involved in plant growth, development or reproduction, these secondary metabolites play vital roles in plant defence mechanisms, acting for example, phytoalexins and phytoanticipins. Phytoalexins are antimicrobial defence metabolites synthesized *de novo* in response to biotic and abiotic stresses. Phytoalexins are involved in induced plant defence mechanisms including lytic enzymes, oxidizing agents, cell wall lignifications and pathogenesis-related proteins and transcript stimulation (Pedras et al., 2008). Phytoanticipins are low molecular weight antimicrobial compounds which are constitutively active for defence. Their production may be increased under high biotic or abiotic stresses (Pedras et al., 2007). Certain classes of phytoanticipins require enzymatic modification and derivation in order to become active within the defence systems of the plant. Plant secondary metabolites are broadly categorized into three groups based on their biosynthetic origin


192 Crop Plant

data mining have opened an avenue for comparative analysis of ESTs, BACs, genes (families), whole chromosomes and even entire genomes to determine evolutionary relationship between these species and their ancestors (Gao et al., 2004; Gao et al., 2006; Punjabi et al., 2008; Mun et

Fig. 1. U-triangle of genomic relationship between diploid and amphidiploid *Brassica* species

Genome homoeology has been characterized in *Brassica* species by comparative analyses of the genetic and physical maps of *Arabidopsis* with genetic maps of *Brassica* species (Osborn et al., 1997; Lan et al., 2000; Parkin et al., 2002; Lukens et al., 2003; Parkin et al., 2005). These studies indicate that each genomic region has had multiple events of polyploidization and chromosome rearrangements in the *Brassicaceae* lineage after the evolutionary divergence from *Arabidopsis* approximately 14.5 to 20.4 million years ago (MYA) (Yang et al., 1999; Parkin et al., 2002). In the *Brassicaceae*, the *B. nigra* (B) genome separated from the *B. rapa*/*B.* 

There are high levels of homoeology among the A- and C-genomes of *B. rapa*, *B. oleracea* and *B. napus* (Parkin et al., 2005; Punjabi et al., 2008). Parkin et al., (2003) reported stretches of collinearity on the linkage groups N1 with N11, N2 with N12 and N3 with N13 of the Aand C-genomes, respectively. Similarly, Osborn et al., (2003) reported reciprocal interstitial translocations of homoeologous regions of linkage groups N7 and N16, and their effects on genome rearrangements and seed yield in *B. napus*. This suggests that inter-genomic translocations and rearrangements have taken place during the evolutionary divergence of *B. oleracea* and *B. rapa* from a polyploid ancestor (Sharpe et al., 1995). As a result of genomic

**4.1 Homoeology between the A, B and C genomes of** *Brassica* **species** 

*oleracea* (AC) genome lineages about 7.9 MYA (Yang et al., 1999; Lysak et al., 2005).

al., 2009; Qiu et al., 2009; Nagoaka et al., 2010).

(U 1935).

iii. Nitrogen and sulphur containing alkaloid compounds

#### **5.1 Glucosinolates as secondary metabolites**

Glucosinolates are sulphur rich, nitrogen containing anionic natural products, derived from specific amino acids and their precursors (Fenwick et al., 1983). Glucosinolates are reported almost exclusively from the order *Brassicales,* which possesses about 15 families such as *Brassica*ceae, *Capparaceae* and *Caricaceae*. Glucosinolates are also reported in a few members of the family *Euphorbiaceae*, a very distinct family to other glucosinolate containing families (Rodman et al., 1996). Glucosinolates coexist with endogenous thioglucosidases called myrosinases in cruciferous plant species and activate plant defence mechanism against biotic and abiotic stresses. Tissue disruption causes systemic interactions between glucosinolates and myrosinases in the presence of moisture. The interaction produces numerous compounds with diverse biological activities (Bones & Rossiter, 1996; Halkier, 1999). Glucosinolates are some of the most extensively studied plant secondary metabolites; various enzymes and transcription factors involved in biosynthesis have been studied in the model plant *Arabidopsis* and to some extent in *Brassica* crops species. The broad functionality, physiochemical and genetic studies of glucosinolates have led to a model status for research on secondary metabolites (Sønderby et al., 2010).

Molecular Genetics of Glucosinolate

**5.1.1.1 Aliphatic glucosinolates** 

oxidative stresses (Gao et al., 2001).

al., 1998).

amino acids. Each class is briefly discussed below.

Biosynthesis in *Brassicas*: Genetic Manipulation and Application Aspects 195

Glucosinolates are a uniform group of thioglucosides with an identical core structure called β-D-glucopyranose bound to a (Z)-N-hydroximinosulfate ester by a sulphur atom with a variable R group. Approximately 120 glucosinolates differing in their R group side chains have been identified (Halkier & Gershenzon, 2006). These glucosinolates are categorized into three classes based on their precursor amino acids and side chain modifications (Table 1). Kliebenstein et al., (2001a) suggested that these three classes of glucosinolates are independently biosynthesized and regulated by different sets of gene families from separate

Aliphatic glucosinolates are the major group of glucosinolates in *Brassica* species, contributing about 90% of the total glucosinolate content of the plant. Glucosinolates are constitutively biosynthesized *de novo* in cruciferous plants, although their degradation is highly regulated by spatial and temporal separation of glucosinolates and myrosinases within the plant based on environmental and biotic stresses (Drozdowska et al., 1992). Hydrolysis of glucosinolates produces a large number of biologically active compounds that have a variety of functions. The most common hydrolysis products of aliphatic glucosinolates in many cruciferous species are isothiocyanates that are formed by the rearrangement of aglycone with carbon oxime adjacent to the nitrogen at neutral pH while at acidic pH, nitriles are the predominant products (Fahey et al., 2001). These unstable compounds are cyclised to a class of substances responsible for goiter in animals (Griffiths et

By contrast, sulforaphane is one of the derivatives of glucoraphanin, an aliphatic glucosinolate that has several beneficial properties for humans and animals. It is known as an inducer of phase II enzymes such as glutathione transferases and quinone reductases of the xenobiotic pathway in human prostate cells (Zhang et al., 1992; Faulkner et al., 1998). The phase II enzymes are involved in the detoxification of electrophilic carcinogens that can lead to mutations in DNA and cause different types of cancers (Mithen et al., 2000). Enhanced consumption of cruciferous vegetables appears to reduce the risk of cancers (Nestle, 1997; Talalay 2000; Brooks et al., 2001). The sulforaphane content of these vegetables could be a leading factor in the reduction. Another less documented health benefit of sulforaphane is the inhibition of *Helicobacter pylori*, a pathogen of peptic ulcers and gastric cancer (Fahey et al., 2002). Sulforaphane also protects human retinal cells against severe

Isothiocyanates and other breakdown products of glucosinolates play important roles as repellents of certain insects and pests (Rask et al., 2000; Agrawal & Kurashige, 2003; Barth & Jander, 2006; Benderoth et al., 2006). Leaves of the mutant *myb28myb29* in *Arabidopsis* with low aliphatic glucosinolate content, when fed to the lepidopteran insect *Mamestra brassicae,*  enhanced larval weight by 2.6 fold (Beekwilder et al., 2008). Glucosinolates may have specific repellent or anti-nutritional effects on specific classes of insects and microorganisms. Some *in vitro* studies demonstrated that glucosinolate degradation products, isothiocyanates and nitriles, inhibited fungal and bacterial pathogen growth (Brader et al., 2001; Tierens et al., 2001). In *Arabidopsis,* over expression of *CYP79D2* from cassava increased accumulation of isopropyl and methylpropyl aliphatic glucosinolates and transformed plants showed

**5.1.1 Glucosinolates and their biological functions in agriculture and nature** 


Trivial name and chemical formula of R side-chains of glucosinolates identified in *Brassica* species, Mol. Wt.#: molecular weight of desulfoglucosinolates, RF: response factor (Haughn et al., 1991; Griffiths et al., 2000; Brown et al., 2003).

Table 1. Chemical structures of glucosinolates in Brassica species.

#### **5.1.1 Glucosinolates and their biological functions in agriculture and nature**

Glucosinolates are a uniform group of thioglucosides with an identical core structure called β-D-glucopyranose bound to a (Z)-N-hydroximinosulfate ester by a sulphur atom with a variable R group. Approximately 120 glucosinolates differing in their R group side chains have been identified (Halkier & Gershenzon, 2006). These glucosinolates are categorized into three classes based on their precursor amino acids and side chain modifications (Table 1). Kliebenstein et al., (2001a) suggested that these three classes of glucosinolates are independently biosynthesized and regulated by different sets of gene families from separate amino acids. Each class is briefly discussed below.

#### **5.1.1.1 Aliphatic glucosinolates**

194 Crop Plant

Trivial name and chemical formula of R side-chains of glucosinolates identified in *Brassica* species, Mol. Wt.#: molecular weight of desulfoglucosinolates, RF: response factor (Haughn et al., 1991; Griffiths et

Table 1. Chemical structures of glucosinolates in Brassica species.

al., 2000; Brown et al., 2003).

Aliphatic glucosinolates are the major group of glucosinolates in *Brassica* species, contributing about 90% of the total glucosinolate content of the plant. Glucosinolates are constitutively biosynthesized *de novo* in cruciferous plants, although their degradation is highly regulated by spatial and temporal separation of glucosinolates and myrosinases within the plant based on environmental and biotic stresses (Drozdowska et al., 1992). Hydrolysis of glucosinolates produces a large number of biologically active compounds that have a variety of functions. The most common hydrolysis products of aliphatic glucosinolates in many cruciferous species are isothiocyanates that are formed by the rearrangement of aglycone with carbon oxime adjacent to the nitrogen at neutral pH while at acidic pH, nitriles are the predominant products (Fahey et al., 2001). These unstable compounds are cyclised to a class of substances responsible for goiter in animals (Griffiths et al., 1998).

By contrast, sulforaphane is one of the derivatives of glucoraphanin, an aliphatic glucosinolate that has several beneficial properties for humans and animals. It is known as an inducer of phase II enzymes such as glutathione transferases and quinone reductases of the xenobiotic pathway in human prostate cells (Zhang et al., 1992; Faulkner et al., 1998). The phase II enzymes are involved in the detoxification of electrophilic carcinogens that can lead to mutations in DNA and cause different types of cancers (Mithen et al., 2000). Enhanced consumption of cruciferous vegetables appears to reduce the risk of cancers (Nestle, 1997; Talalay 2000; Brooks et al., 2001). The sulforaphane content of these vegetables could be a leading factor in the reduction. Another less documented health benefit of sulforaphane is the inhibition of *Helicobacter pylori*, a pathogen of peptic ulcers and gastric cancer (Fahey et al., 2002). Sulforaphane also protects human retinal cells against severe oxidative stresses (Gao et al., 2001).

Isothiocyanates and other breakdown products of glucosinolates play important roles as repellents of certain insects and pests (Rask et al., 2000; Agrawal & Kurashige, 2003; Barth & Jander, 2006; Benderoth et al., 2006). Leaves of the mutant *myb28myb29* in *Arabidopsis* with low aliphatic glucosinolate content, when fed to the lepidopteran insect *Mamestra brassicae,*  enhanced larval weight by 2.6 fold (Beekwilder et al., 2008). Glucosinolates may have specific repellent or anti-nutritional effects on specific classes of insects and microorganisms. Some *in vitro* studies demonstrated that glucosinolate degradation products, isothiocyanates and nitriles, inhibited fungal and bacterial pathogen growth (Brader et al., 2001; Tierens et al., 2001). In *Arabidopsis,* over expression of *CYP79D2* from cassava increased accumulation of isopropyl and methylpropyl aliphatic glucosinolates and transformed plants showed

Molecular Genetics of Glucosinolate

(Bodnaryk 1992; Brader et al., 2001).

**5.1.2 Biosynthesis of aliphatic glucosinolates** 

**5.1.1.3 Aromatic glucosinolates** 

Biosynthesis in *Brassicas*: Genetic Manipulation and Application Aspects 197

glucosinolate derived compound 4-methoxyglucobrassicin has strong insect deterrent activity (Kim & Jander, 2007; De Vos et al., 2008). Osbourn, (1996) reported antimicrobial activities of indole glucosinolates and their breakdown products in *Brassica* species. Several studies suggest that there is a metabolic association between indole glucosinolates and the plant hormone indole-3-acetic acid (IAA). In the consecutive reactions, indole glucosinolates are degraded into indole acetonitrile (IAN), which is then hydrolyzed by nitrilases into IAA (Fig. 2). In clubroot infected *Brassica* roots, indole glucosinolate-based induction of IAA was observed to be responsible for gall formation. The IAA production from indole glucosinolates during gall formation is associated with a signalling cascade of IAA and cytokinin complex (Ugajin et al., 2003). Structural similarity data indicates that the indole alkaloid, brassinin, and possibly other cruciferous phytoalexins are derived from glucobrassicin. Studies in rapeseed, mustard and *Arabidopsis* have suggested that methyl jasmonate and wounding induce the biosynthesis of particular indole glucosinolates

The third class of glucosinolates in cruciferous species is aromatic or benzylic glucosinolates, derived from the aromatic parental amino acids phenylalanine and tyrosine. Very limited information is available regarding aromatic glucosinolates at qualitative or quantitative levels. Aromatic glucosinolates are biosynthesized independently from other glucosinolates, which is apparently due to involvement of different amino acid precursors in the biosynthesis of the different classes of glucosinolates (Kliebenstein et al., 2001a). Cloning and functional characterization of the *CYP79A* gene of *Arabidopsis* suggests that cytochrome P450-dependent monooxygenase catalyzes the reaction from phenylalanine to phenylacetaldoxime in aromatic glucosinolate biosynthesis (Wittstock & Halkier, 2000). Five aromatic glucosinolates have been identified in *Brassicaceae*: glucotropaeolin, glucosinalbin, gluconasturtiin, glucobarbarin and glucomalcomiin. The distinctive aroma and spiciness of condiment *Brassica* plant parts, such as the leaves and seeds of white (*Sinapis alba*) and black (*B. nigra*) mustards, is due to the presence of these aromatic glucosinolates (Fenwich et al., 1983).

Aliphatic glucosinolates are the most abundant class in *Brassica* species, therefore, the genetic of biosynthesis is described in more detail. Aliphatic glucosinolates are biosynthesized from five amino acids (methionine, alanine, leucine, isoleucine and valine) (Halkier & Gershenzon, 2006). Biosynthesis of aliphatic glucosinolates occurs in three stages at two different locations. The first chain elongation step is catalyzed by *BCAT4* in the cytosol (Schuster et al., 2006), whereas development of core structures and secondary side chain modification reactions take place in the chloroplasts (Textor et al., 2007; Sawada et al., 2009). Chain elongation steps produce propyls (3C), butyls (4C), pentyls (5C), hexyls (6C), heptyls (7C) and octyls (8C) aliphatic glucosinolates in cruciferous species including *Arabidopsis*. Glucosinolate side chain modification reactions involve oxygenation, hydroxylation, alkenylation and benzoylation, which are controlled by several gene families. The pattern of glucosinolate biosynthesis varies from organ to organ within the plant; young leaves, buds, flowers and silique walls all have higher rates of glucosinolate biosynthesis than roots, old leaves and presumably seeds (Brown et al., 2003). Various studies also suggest that transportation of glucosinolates and their breakdown products from organ to

enhanced resistance against a bacterial soft-rot disease (Brader et al., 2006). Birch et al., (1992) reported that biotic stresses such as pest damage in *Brassica* species alters glucosinolate profiles in roots, stems, leaves and flowers. This suggests that a phytoanticipin property of glucosinolates is involved in the plant defence mechanisms of *Brassica*. Glucosinolates and their breakdown products have many biological functions, with a few compounds acting as biopesticides, biofungicides and soil fumigants, while others play roles in attraction of pollinators and provide oviposition cues to certain insects. The attraction of specialized insects could be due to the glucosinolate-sequestering phenomenon of some insects including harlequin bugs, sawflies, and some homoptera including aphids (Bridges et al., 2002; Mewis et al., 2002).

#### **5.1.1.2 Indole glucosinolates**

Indole (heterocyclic) glucosinolates in cruciferous plants (including *Arabidopsis*) are derived from tryptophan and possess variable R group side chains. The relatively high content of indole glucosinolates in the model plant *Arabidopsis* has enhanced our knowledge of the biosynthesis, transportation and functional properties of this class of glucosinolates (Petersen et al., 2002; Brown et al., 2003). Side chain modification in indole glucosinolates occurs through hydroxylations and methoxylations catalysed by several enzymes. Indole glucosinolate types and contents in different organs of the plant are strongly affected by environmental conditions. Four main indole glucosinolates have been identified in most cultivated *Brassica* species: glucobrassicin, neoglucobrassicin, 4-methoxyglucobrassicin and 4-hydroxyglucobrassicin. Similar to aliphatic glucosinolates, breakdown products of indole glucosinolates have multiple biological functions. Indole-3-carbinol derived from glucobrassicin has potent anticarcinogenic activity (Hrncirik et al., 2001). The indole

In this pathway, IAA produces from precursors and derivatives of 3-indolylmethyl glucosinolate by various nitrilases.

Fig. 2. Biosynthetic pathway and breakdown products of indole glucosinolates (De Vos et al., 2008).

glucosinolate derived compound 4-methoxyglucobrassicin has strong insect deterrent activity (Kim & Jander, 2007; De Vos et al., 2008). Osbourn, (1996) reported antimicrobial activities of indole glucosinolates and their breakdown products in *Brassica* species. Several studies suggest that there is a metabolic association between indole glucosinolates and the plant hormone indole-3-acetic acid (IAA). In the consecutive reactions, indole glucosinolates are degraded into indole acetonitrile (IAN), which is then hydrolyzed by nitrilases into IAA (Fig. 2). In clubroot infected *Brassica* roots, indole glucosinolate-based induction of IAA was observed to be responsible for gall formation. The IAA production from indole glucosinolates during gall formation is associated with a signalling cascade of IAA and cytokinin complex (Ugajin et al., 2003). Structural similarity data indicates that the indole alkaloid, brassinin, and possibly other cruciferous phytoalexins are derived from glucobrassicin. Studies in rapeseed, mustard and *Arabidopsis* have suggested that methyl jasmonate and wounding induce the biosynthesis of particular indole glucosinolates (Bodnaryk 1992; Brader et al., 2001).

#### **5.1.1.3 Aromatic glucosinolates**

196 Crop Plant

enhanced resistance against a bacterial soft-rot disease (Brader et al., 2006). Birch et al., (1992) reported that biotic stresses such as pest damage in *Brassica* species alters glucosinolate profiles in roots, stems, leaves and flowers. This suggests that a phytoanticipin property of glucosinolates is involved in the plant defence mechanisms of *Brassica*. Glucosinolates and their breakdown products have many biological functions, with a few compounds acting as biopesticides, biofungicides and soil fumigants, while others play roles in attraction of pollinators and provide oviposition cues to certain insects. The attraction of specialized insects could be due to the glucosinolate-sequestering phenomenon of some insects including harlequin bugs, sawflies, and some homoptera including aphids (Bridges

Indole (heterocyclic) glucosinolates in cruciferous plants (including *Arabidopsis*) are derived from tryptophan and possess variable R group side chains. The relatively high content of indole glucosinolates in the model plant *Arabidopsis* has enhanced our knowledge of the biosynthesis, transportation and functional properties of this class of glucosinolates (Petersen et al., 2002; Brown et al., 2003). Side chain modification in indole glucosinolates occurs through hydroxylations and methoxylations catalysed by several enzymes. Indole glucosinolate types and contents in different organs of the plant are strongly affected by environmental conditions. Four main indole glucosinolates have been identified in most cultivated *Brassica* species: glucobrassicin, neoglucobrassicin, 4-methoxyglucobrassicin and 4-hydroxyglucobrassicin. Similar to aliphatic glucosinolates, breakdown products of indole glucosinolates have multiple biological functions. Indole-3-carbinol derived from glucobrassicin has potent anticarcinogenic activity (Hrncirik et al., 2001). The indole

In this pathway, IAA produces from precursors and derivatives of 3-indolylmethyl glucosinolate by

Fig. 2. Biosynthetic pathway and breakdown products of indole glucosinolates (De Vos et

et al., 2002; Mewis et al., 2002). **5.1.1.2 Indole glucosinolates** 

various nitrilases.

al., 2008).

The third class of glucosinolates in cruciferous species is aromatic or benzylic glucosinolates, derived from the aromatic parental amino acids phenylalanine and tyrosine. Very limited information is available regarding aromatic glucosinolates at qualitative or quantitative levels. Aromatic glucosinolates are biosynthesized independently from other glucosinolates, which is apparently due to involvement of different amino acid precursors in the biosynthesis of the different classes of glucosinolates (Kliebenstein et al., 2001a). Cloning and functional characterization of the *CYP79A* gene of *Arabidopsis* suggests that cytochrome P450-dependent monooxygenase catalyzes the reaction from phenylalanine to phenylacetaldoxime in aromatic glucosinolate biosynthesis (Wittstock & Halkier, 2000). Five aromatic glucosinolates have been identified in *Brassicaceae*: glucotropaeolin, glucosinalbin, gluconasturtiin, glucobarbarin and glucomalcomiin. The distinctive aroma and spiciness of condiment *Brassica* plant parts, such as the leaves and seeds of white (*Sinapis alba*) and black (*B. nigra*) mustards, is due to the presence of these aromatic glucosinolates (Fenwich et al., 1983).

#### **5.1.2 Biosynthesis of aliphatic glucosinolates**

Aliphatic glucosinolates are the most abundant class in *Brassica* species, therefore, the genetic of biosynthesis is described in more detail. Aliphatic glucosinolates are biosynthesized from five amino acids (methionine, alanine, leucine, isoleucine and valine) (Halkier & Gershenzon, 2006). Biosynthesis of aliphatic glucosinolates occurs in three stages at two different locations. The first chain elongation step is catalyzed by *BCAT4* in the cytosol (Schuster et al., 2006), whereas development of core structures and secondary side chain modification reactions take place in the chloroplasts (Textor et al., 2007; Sawada et al., 2009). Chain elongation steps produce propyls (3C), butyls (4C), pentyls (5C), hexyls (6C), heptyls (7C) and octyls (8C) aliphatic glucosinolates in cruciferous species including *Arabidopsis*. Glucosinolate side chain modification reactions involve oxygenation, hydroxylation, alkenylation and benzoylation, which are controlled by several gene families. The pattern of glucosinolate biosynthesis varies from organ to organ within the plant; young leaves, buds, flowers and silique walls all have higher rates of glucosinolate biosynthesis than roots, old leaves and presumably seeds (Brown et al., 2003). Various studies also suggest that transportation of glucosinolates and their breakdown products from organ to

Molecular Genetics of Glucosinolate

Biosynthesis in *Brassicas*: Genetic Manipulation and Application Aspects 199

to its chain-elongated homolog leucine (Fig. 3). In *Arabidopsis*, a *bcat4* mutant showed about a 50% reduction in total aliphatic glucosinolates and at the same time increased the level of free methionine and S-methyl-methionine (Schuster et al., 2006). This suggests that the *BCAT4* gene produces an enzyme which is involved in the first deamination reaction. Subsequently, three consecutive reactions of transformations occur. The first is a transamination and condensation reaction with acetyl-CoA catalyzed by *GSL-ELONG* in *Brassica* species (Li & Quiros, 2002). This is homologous to *MAM1* in *Arabidopsis* (Campos de Quirose et al., 2000; Benderoth et al., 2006; Textor et al., 2007). The same reaction occurs for 3C aliphatic glucosinolates which is controlled by isopropylmalate synthase (*IPMS1*, *IPMS2*). Isopropylmalate synthase is homologous to *MAM1* in *Arabidopsis* (Kliebenstein et al., 2001b; Field et al., 2004) and to *GSL-PRO* in *Brassica* species (Li et al., 2003; Gao et al., 2006). The second isomerisation reaction is controlled by isopropylmalate isomerises (*IPMI*) and third reaction is oxidative decarboxylation controlled by isopropylmalate

These three consecutive reactions produce elongated 2-oxo acids with one or more methylene groups. These compounds are either transaminated by the BCAT enzyme to yield homo-methionine, which can enter into the core glucosinolate skeleton structure formation, or proceed through another round of chain elongation (Fig. 3). Overall, the methionine amino acid condensation pathway produces a range of methionine derivatives such as homo-methionine, dihomo-methionine, and trihomo-methionine, which proceed to

Glucosinolate core skeleton structure formation has been well characterized in *Arabidopsis*, with at least 13 enzymes and five different biochemical reactions, i.e., oxidation, oxidation with conjugation, C-S cleavage, glucosylation and sulfation (Grubb & Abel, 2006; Halkier & Gershenzon, 2006) involved in the formation. The precursors are catalyzed into aldoxime by cytochromes belonging to the *CYP79* gene family (Fig. 4). At least seven *CYP79s* were identified and functionally characterized in *Arabidopsis*. The *CYP79F1* gene converts all short chain methionine derivatives, whereas *CYP79F2* gene is involved in conversions of the long chain methionine derivatives. Similarly, *CYP79B2* and *CYP79B3* catalyze tryptophan derivatives, and *CYP79A2* catalyzes phenylalanine substrates (Fig. 4) (Zang et al., 2008). Subsequently, aldoximes are oxidized into either nitrile oxides or aci-nitro compounds by *CYP83A1* for methionine derivates and *CYP83B1* for tryptophan as well as phenylalanine derivates. This proceeds to a non-enzymatic conjugation to produce S-alklythiohydroximates. In this sulphur rich chemical pathway, the next step is C-S cleavage by C-S lyase from S-alkly-thiohydroximate to thiohydroximic acid; C-S lyase forms an enzymatic complex with an S-donating enzyme. The *c-s lyase* mutant of *Arabidopsis* showed complete lack of aliphatic and aromatic glucosinolates in *Arabidopsis*, suggesting that this single gene

In the glucosylation step, desulfoglucosinolate is formed by a member of the *UGT74* family. The final reaction of core skeleton formation is accomplished with sulfation of desulfoglucosinolates to produce intact glucosinolates by sulfotransferases *AtST5a*, *AtST5b* and *AtST5C* in *Arabidopsis*. Biochemical characterization of sulfotransferases in *Arabidopsis* revealed that *AtST5a* favour to sulfate phenylalanine and tryptophan derived

dehydrogenases (*IPM-DH*) (Fig. 3) (Wentzell et al., 2007; Sawada et al., 2009).

the next biosynthesis step called glucosinolate core skeleton formation (Fig. 3).

family has a crucial role in skeleton processing (Mikkelsen et al., 2004).

**5.1.2.2 Glucosinolate core skeleton formation** 

organ via phloem occurs upon requirement to protect the plant. Seeds, however, are the most important store of total glucosinolates produced by the plants (Brudnell et al., 1999). Seeds contain much higher glucosinolates concentrations than other plant parts and it is thought that leaf glucosinolates are the basis for accumulations of total glucosinolates in seeds (Klienbestein et al., 2001a). This suggests that long distance transportation of glucosinolates from source to sink occurs. A few reports discuss an independent pathway of glucosinolate biosynthesis in seeds, resulting in the high concentration of glucosinolate in seeds (Du & Halkier, 1998; Osbourn, 1996). Experimental evidence, however, is not strong enough to support a separate pathway at this time.

#### **5.1.2.1 Parental amino acid biosynthesis and condensation**

Methionine is the main precursor of aliphatic glucosinolates in *Brassica* species. The enzyme BCAT4 catalyzes the initial chain elongation reaction to produce 2-oxo acid from methionine, an analogous process to the formation of the branched chain amino acid valine

All the reactions are catalyzed by *BCATs*, *ELONGs*, *IPMIs* and *IPM-DHs* gene families for 3C, 4C and 5C glucosinolates. Genes shown in gray boxes and derivative products shown in blue boxes. BCATbranched chain amino transferase, MTOB- 4-methylthio-2-oxobutanoate, MTOP- 6 methylthio-2 oxopentanoate, MTOHX- 4-methylthio-2-oxohexanoate, IPMI- isopropylmalate isomerases, IPMDHisopropylmalate dehydrogenases, MOB- methyl-2-oxobutanoate, MOP- methyl-2-oxopentanoate, AHAS- acetohydroxyacid synthase, KARI- ketolacid reductoisomerase, DHAD- dihydroxyacid dehydratase, 2AL- 2-acetolactate, 2A2HB- 2-aceto-2-hydroxybutyrate, 2OB- 2-oxobutyrate, 2,3DH3MB-2,3-dihydroxy-3-methylbutyrate, 2,3DH3MP- 2,3-dihydroxy-3-methylpentanoate.

Fig. 3. Methionine amino acid condensation pathway regulated by several gene families (Kroymann et al., 2001; Sawada et al., 2009).

organ via phloem occurs upon requirement to protect the plant. Seeds, however, are the most important store of total glucosinolates produced by the plants (Brudnell et al., 1999). Seeds contain much higher glucosinolates concentrations than other plant parts and it is thought that leaf glucosinolates are the basis for accumulations of total glucosinolates in seeds (Klienbestein et al., 2001a). This suggests that long distance transportation of glucosinolates from source to sink occurs. A few reports discuss an independent pathway of glucosinolate biosynthesis in seeds, resulting in the high concentration of glucosinolate in seeds (Du & Halkier, 1998; Osbourn, 1996). Experimental evidence, however, is not strong

Methionine is the main precursor of aliphatic glucosinolates in *Brassica* species. The enzyme BCAT4 catalyzes the initial chain elongation reaction to produce 2-oxo acid from methionine, an analogous process to the formation of the branched chain amino acid valine

All the reactions are catalyzed by *BCATs*, *ELONGs*, *IPMIs* and *IPM-DHs* gene families for 3C, 4C and 5C glucosinolates. Genes shown in gray boxes and derivative products shown in blue boxes. BCATbranched chain amino transferase, MTOB- 4-methylthio-2-oxobutanoate, MTOP- 6 methylthio-2 oxopentanoate, MTOHX- 4-methylthio-2-oxohexanoate, IPMI- isopropylmalate isomerases, IPMDHisopropylmalate dehydrogenases, MOB- methyl-2-oxobutanoate, MOP- methyl-2-oxopentanoate, AHAS- acetohydroxyacid synthase, KARI- ketolacid reductoisomerase, DHAD- dihydroxyacid dehydratase, 2AL- 2-acetolactate, 2A2HB- 2-aceto-2-hydroxybutyrate, 2OB- 2-oxobutyrate, 2,3DH3MB-

Fig. 3. Methionine amino acid condensation pathway regulated by several gene families

2,3-dihydroxy-3-methylbutyrate, 2,3DH3MP- 2,3-dihydroxy-3-methylpentanoate.

(Kroymann et al., 2001; Sawada et al., 2009).

enough to support a separate pathway at this time.

**5.1.2.1 Parental amino acid biosynthesis and condensation** 

to its chain-elongated homolog leucine (Fig. 3). In *Arabidopsis*, a *bcat4* mutant showed about a 50% reduction in total aliphatic glucosinolates and at the same time increased the level of free methionine and S-methyl-methionine (Schuster et al., 2006). This suggests that the *BCAT4* gene produces an enzyme which is involved in the first deamination reaction. Subsequently, three consecutive reactions of transformations occur. The first is a transamination and condensation reaction with acetyl-CoA catalyzed by *GSL-ELONG* in *Brassica* species (Li & Quiros, 2002). This is homologous to *MAM1* in *Arabidopsis* (Campos de Quirose et al., 2000; Benderoth et al., 2006; Textor et al., 2007). The same reaction occurs for 3C aliphatic glucosinolates which is controlled by isopropylmalate synthase (*IPMS1*, *IPMS2*). Isopropylmalate synthase is homologous to *MAM1* in *Arabidopsis* (Kliebenstein et al., 2001b; Field et al., 2004) and to *GSL-PRO* in *Brassica* species (Li et al., 2003; Gao et al., 2006). The second isomerisation reaction is controlled by isopropylmalate isomerises (*IPMI*) and third reaction is oxidative decarboxylation controlled by isopropylmalate dehydrogenases (*IPM-DH*) (Fig. 3) (Wentzell et al., 2007; Sawada et al., 2009).

These three consecutive reactions produce elongated 2-oxo acids with one or more methylene groups. These compounds are either transaminated by the BCAT enzyme to yield homo-methionine, which can enter into the core glucosinolate skeleton structure formation, or proceed through another round of chain elongation (Fig. 3). Overall, the methionine amino acid condensation pathway produces a range of methionine derivatives such as homo-methionine, dihomo-methionine, and trihomo-methionine, which proceed to the next biosynthesis step called glucosinolate core skeleton formation (Fig. 3).

#### **5.1.2.2 Glucosinolate core skeleton formation**

Glucosinolate core skeleton structure formation has been well characterized in *Arabidopsis*, with at least 13 enzymes and five different biochemical reactions, i.e., oxidation, oxidation with conjugation, C-S cleavage, glucosylation and sulfation (Grubb & Abel, 2006; Halkier & Gershenzon, 2006) involved in the formation. The precursors are catalyzed into aldoxime by cytochromes belonging to the *CYP79* gene family (Fig. 4). At least seven *CYP79s* were identified and functionally characterized in *Arabidopsis*. The *CYP79F1* gene converts all short chain methionine derivatives, whereas *CYP79F2* gene is involved in conversions of the long chain methionine derivatives. Similarly, *CYP79B2* and *CYP79B3* catalyze tryptophan derivatives, and *CYP79A2* catalyzes phenylalanine substrates (Fig. 4) (Zang et al., 2008). Subsequently, aldoximes are oxidized into either nitrile oxides or aci-nitro compounds by *CYP83A1* for methionine derivates and *CYP83B1* for tryptophan as well as phenylalanine derivates. This proceeds to a non-enzymatic conjugation to produce S-alklythiohydroximates. In this sulphur rich chemical pathway, the next step is C-S cleavage by C-S lyase from S-alkly-thiohydroximate to thiohydroximic acid; C-S lyase forms an enzymatic complex with an S-donating enzyme. The *c-s lyase* mutant of *Arabidopsis* showed complete lack of aliphatic and aromatic glucosinolates in *Arabidopsis*, suggesting that this single gene family has a crucial role in skeleton processing (Mikkelsen et al., 2004).

In the glucosylation step, desulfoglucosinolate is formed by a member of the *UGT74* family. The final reaction of core skeleton formation is accomplished with sulfation of desulfoglucosinolates to produce intact glucosinolates by sulfotransferases *AtST5a*, *AtST5b* and *AtST5C* in *Arabidopsis*. Biochemical characterization of sulfotransferases in *Arabidopsis* revealed that *AtST5a* favour to sulfate phenylalanine and tryptophan derived

Molecular Genetics of Glucosinolate

Biosynthesis in *Brassicas*: Genetic Manipulation and Application Aspects 201

the 4-methylthiobutyl to 4-methylsulfinyl reaction and *GSL-FMOOX5* is involved in the Soxygenation of long chain glucosinolates in *Arabidopsis* (Li et al., 2008). In *Brassica* vegetables, products of *GSL-FMOs* catalyses are the sources of anticancer compounds from aliphatic glucosinolates. It will be beneficial to identify these genes/loci in *Brassica* species so that they might be further used to manipulate aliphatic glucosinolates towards favourable forms.

A second round of binary side chain modification changes methylsulfinyl to alkenyl- and to hydroxyl- aliphatic glucosinolates (Fig. 5). In *Arabidopsis* these reactions are controlled by a *GSL-ALK*/*GSL-OHP* locus that has three tandem repeats (*GSL-AOP1*, *GSL-AOP2* and *GSL-AOP3*), which encode 2-oxoglutarate-dependent dioxygenases located on chromosome IV. Functional characterization indicates that *GSL-AOP2* catalyzes the reaction to alkenyl, whereas *GSL-AOP3* controls the reaction toward hydroxyalkenyl. The function of *GSL-AOP1*, however, is not clear in *Arabidopsis*, it might be involved in both reactions (Fig. 5) (Hall et al., 2001; Kliebenstein et al., 2001c; Mithen et al., 1995). The *GSL-ALK* and *GSL-OHP* are either closely linked on the same genomic region or allelic variants of a single genetic locus though they may show variable functions. In *Arabidopsis*, *GSL-OHP* catalyzes the reaction only for 3C aliphatic glucosinolate branches, whereas *GSL-ALK* is involved in 3C, 4C and 5C aliphatic glucosinolate branches. There is no clear functional information available for long chain (6C and so on) aliphatic glucosinolate branches and presumably

Fig. 5. Glucosinolate core structure and side chain modification pathway for 3C, 4C and 5C aliphatic glucosinolates. In the biosynthesis steps, gene symbols ending with A indicate A genome, C for C genome and At for *A. thaliana* (Magrath et al., 1994; Mithen et al., 1995; Li &

Quiros, 2003; Mahmood et al., 2003).

desulfoglucosinolates, whereas *AtST5b* and *AtST5c* favour to sulfate long chain aliphatic glucosinolates (Piotrowski et al., 2004). In a comparative analysis study between *Arabidopsis* and *B. rapa*, at least 12 paralogs of sulfotransferases were known to be responsible for this reaction (Zang et al., 2008). In glucosinolate skeleton formation reactions, the first four biosynthesis reactions take place in the chloroplast and the last reaction of sulfation occurs in the cytosol. This suggests that shuttle transporters play important roles in the entire biosynthesis process (Klein et al., 2006).

ST- sulfotransferase, UGT- glucuronosyltransferases, GST- glutathione S-transferase.

Fig. 4. Glucosinolate core skeleton structure formation by cytochromes. Methionine amino acid precursors produce aliphatic, tryptophan produces indole and phenylalanine produces aromatic glucosinolate core structures (Grubb & Abel, 2006; Halkier & Gershenzon, 2006).

#### **5.1.2.3 Side chain modification in aliphatic glucosinolates**

After glucosinolate core skeleton structure formation, the core skeletons are subjected to a set of reactions known as side chain modification or secondary transformation. Side chain modifications of glucosinolates are the last crucial enzymatic reactions on intact glucosinolates before their transport to sinks or biological degradation by myrosinases occur. Hydrolysis products of individual glucosinolates are recognized based on side chain variation in R groups. A hydrolysis product of glucoraphanin has anticancer properties. The R group modifications of glucoraphanin change their chemical properties, therefore, hydrolysis products have anticarcinogenic functions. Hydrolysis products of progoitrin, however, have anti-nutritional effect in animals, which reduce the palatability of rapeseed meal.

Side chain modification begins with the oxidation of sulphur in the methylthio precursor to produce methylsulfinyl and then methylsulfonyl moieties (Fig. 5). In *Arabidopsis*, this reaction is catalyzed by the flavin monooxygenases, *GSL-FMOOX1-5* located within the *GSL-OX1* locus on chromosome I. Phylogenetic analysis revealed a main group of *GSL-FMOs* for cruciferous species, which is further categorized according to subspecies, indicating that functional diversity of S-oxygenation of glucosinolates exists (Hansen et al., 2007; Li et al., 2008). Knockout mutant and over expression studies suggested that *GSL-FMOOX1-4* catalyzes

desulfoglucosinolates, whereas *AtST5b* and *AtST5c* favour to sulfate long chain aliphatic glucosinolates (Piotrowski et al., 2004). In a comparative analysis study between *Arabidopsis* and *B. rapa*, at least 12 paralogs of sulfotransferases were known to be responsible for this reaction (Zang et al., 2008). In glucosinolate skeleton formation reactions, the first four biosynthesis reactions take place in the chloroplast and the last reaction of sulfation occurs in the cytosol. This suggests that shuttle transporters play important roles in the entire

ST- sulfotransferase, UGT- glucuronosyltransferases, GST- glutathione S-transferase.

**5.1.2.3 Side chain modification in aliphatic glucosinolates** 

meal.

Fig. 4. Glucosinolate core skeleton structure formation by cytochromes. Methionine amino acid precursors produce aliphatic, tryptophan produces indole and phenylalanine produces aromatic glucosinolate core structures (Grubb & Abel, 2006; Halkier & Gershenzon, 2006).

After glucosinolate core skeleton structure formation, the core skeletons are subjected to a set of reactions known as side chain modification or secondary transformation. Side chain modifications of glucosinolates are the last crucial enzymatic reactions on intact glucosinolates before their transport to sinks or biological degradation by myrosinases occur. Hydrolysis products of individual glucosinolates are recognized based on side chain variation in R groups. A hydrolysis product of glucoraphanin has anticancer properties. The R group modifications of glucoraphanin change their chemical properties, therefore, hydrolysis products have anticarcinogenic functions. Hydrolysis products of progoitrin, however, have anti-nutritional effect in animals, which reduce the palatability of rapeseed

Side chain modification begins with the oxidation of sulphur in the methylthio precursor to produce methylsulfinyl and then methylsulfonyl moieties (Fig. 5). In *Arabidopsis*, this reaction is catalyzed by the flavin monooxygenases, *GSL-FMOOX1-5* located within the *GSL-OX1* locus on chromosome I. Phylogenetic analysis revealed a main group of *GSL-FMOs* for cruciferous species, which is further categorized according to subspecies, indicating that functional diversity of S-oxygenation of glucosinolates exists (Hansen et al., 2007; Li et al., 2008). Knockout mutant and over expression studies suggested that *GSL-FMOOX1-4* catalyzes

biosynthesis process (Klein et al., 2006).

the 4-methylthiobutyl to 4-methylsulfinyl reaction and *GSL-FMOOX5* is involved in the Soxygenation of long chain glucosinolates in *Arabidopsis* (Li et al., 2008). In *Brassica* vegetables, products of *GSL-FMOs* catalyses are the sources of anticancer compounds from aliphatic glucosinolates. It will be beneficial to identify these genes/loci in *Brassica* species so that they might be further used to manipulate aliphatic glucosinolates towards favourable forms.

A second round of binary side chain modification changes methylsulfinyl to alkenyl- and to hydroxyl- aliphatic glucosinolates (Fig. 5). In *Arabidopsis* these reactions are controlled by a *GSL-ALK*/*GSL-OHP* locus that has three tandem repeats (*GSL-AOP1*, *GSL-AOP2* and *GSL-AOP3*), which encode 2-oxoglutarate-dependent dioxygenases located on chromosome IV. Functional characterization indicates that *GSL-AOP2* catalyzes the reaction to alkenyl, whereas *GSL-AOP3* controls the reaction toward hydroxyalkenyl. The function of *GSL-AOP1*, however, is not clear in *Arabidopsis*, it might be involved in both reactions (Fig. 5) (Hall et al., 2001; Kliebenstein et al., 2001c; Mithen et al., 1995). The *GSL-ALK* and *GSL-OHP* are either closely linked on the same genomic region or allelic variants of a single genetic locus though they may show variable functions. In *Arabidopsis*, *GSL-OHP* catalyzes the reaction only for 3C aliphatic glucosinolate branches, whereas *GSL-ALK* is involved in 3C, 4C and 5C aliphatic glucosinolate branches. There is no clear functional information available for long chain (6C and so on) aliphatic glucosinolate branches and presumably

Fig. 5. Glucosinolate core structure and side chain modification pathway for 3C, 4C and 5C aliphatic glucosinolates. In the biosynthesis steps, gene symbols ending with A indicate A genome, C for C genome and At for *A. thaliana* (Magrath et al., 1994; Mithen et al., 1995; Li & Quiros, 2003; Mahmood et al., 2003).

Molecular Genetics of Glucosinolate

of their oils and seed meals.

Biosynthesis in *Brassicas*: Genetic Manipulation and Application Aspects 203

spring rape cultivar, Bronowski, with low glucosinolate content was discovered by The Agriculture Canada Research Station in Saskatoon (Kondra & Stefansson, 1970). This sole genetic source of the low glucosinolate trait has been used to develop all the low glucosinolate cultivars in *B. napus* and *B. rapa* worldwide through conventional plant breeding. *B. napus* and *B. rapa* cultivars with low content of erucic acid and glucosinolate were developed, which ushered in a new era for *Brassica* crop production and its consumption. The world's first double low (low erucic acid and low glucosinolate content) *B. napus* and *B. rapa* cultivars, Tower and Candle, respectively, were developed by pedigree selection in the progenies of interspecific crosses in 1970s (Stefansson & Downey, 1995; McVetty et al., 2009). In Canada, this new type of oilseed rape was designated "Canola". The term "Canola" applies to any rapeseed cultivars with erucic acid content of <2% and glucosinolates content of <30 µmol/g in oil-free seed meal. The Canola term is a registered trademark of the Canadian Canola Association. The name is derived from **Can**adian **O**il **L**ow **A**cid (Canola Council of Canada, 2010a, http://www.canola-council.org/ canola\_the\_official\_definition.aspx). Currently, most rapeseed (high erucic acid) and canola cultivars have glucosinolate levels <15 µmole/g in oil-free seed meal. The development of low erucic acid and low glucosinolate cultivars has also been undertaken for other *Brassica* rapeseed species (e.g. *B. juncea*) and in other parts of the world for the quality improvement

**7. Quantitative trait loci for glucosinolates in major** *Brassica* **species** 

Glucosinolate biosynthesis in *Brassica* crops has quantitative inheritance, which is regulated by complex genetic factors and affected by environmental factors. Glucosinolates are functionally diverse and well recognized plant secondary metabolites; so they have been extensively studied in terms of QTL mapping, biosynthesis gene cloning and functional characterization in *Arabidopsis* (Kliebenstein et al., 2001a; Kliebenstein et al., 2001c; Compos de Quiros et al., 2000; Brown et al., 2003; Benderoth et al., 2006; Textor et al., 2007; Li et al., 2008). However, very limited genetic, biochemical and metabolomic information is available on glucosinolate biosynthesis, transport and final product utilization in *Brassica* crops including *B. rapa*. There has been a few QTL mapping studies reported for major *Brassica* crop seed glucosinolates. Uzunova et al., (1995) mapped four QTL for total seed glucosinolate content in a *B. napus* DH population, which accounted 61% total phenotypic variance. Similarly, Toroser et al., (1995), based on a RFLP linkage map, identified two larger and three small effect QTL for total aliphatic glucosinolate content using a DH population in *B. napus*. These QTL explained 70% of the total phenotypic variance. This suggests that several loci with additive or epistatic effect are involved in total seed glucosinolate biosynthesis in different genetic backgrounds. Howell et al., (2003) reported QTL mapping for total seed glucosinolates analyzed by X-ray fluorescence (XRF) and near-infrared reflection spectroscopy (NIRS) in two inter-varietal *B. napus* backcross populations. They identified four QTLs accounting for 76% of the phenotypic variance in the Victor x Tapidor population. These three QTL accounted for 86% of phenotypic variance in this second population. These studies, however, were limited to either total seed glucosinolates or 3C, 4C and 5C aliphatic glucosinolates, and did not infer the genetic loci for individual aliphatic, indole or aromatic glucosinolates. Furthermore, there were no reports of publicly available

*GSL-ALK* accomplishes these reactions in *Arabidopsis* (Kliebenstein et al., 2001c; Parkin et al., 1994). In *B. oleracea*, *GSL-ALK* was inferred by positional cloning and biochemical analysis. The functional allele in collard and the non-functional allele (with 2 bp deletion creating a frame-shift mutation) in broccoli were confirmed. A locus or loci of *GSL-ALK* is also believed to have a role in the catalysis of methylsulfinyl to alkenyl glucosinolates (Li & Quiros, 2003). Hydroxylation changes alkenyl to hydroxy aliphatic glucosinolate (in butyls, pentyls, hexyls and so on) biosynthesis branches in *Arabidopsis* and *Brassica* species; these sets of reactions are controlled by *GSL-OH* dependant on the presence of both *GSL-AOP2*  and *GSL-ELONG*. In *Brassica*, the final product of this reaction in 4C glucosinolate biosynthesis is progoitrin and its hydrolytic derivative, oxazolidine-2-thione which causes goiter in animals. These compounds are major obstacles to the use of *Brassica* crops as animal feed (Fenwick et al., 1983).

#### **5.1.3 Diversity of glucosinolates in** *Brassicaceae*

Glucosinolates are united by their unique basic skeleton (β-D-glucopyranose) but glucosinolates are diverse in their origins, side chain modifications, degradations and final biological functions. In addition to structural diversity, a diversity of glucosinolates is seen between families, genera, species, subspecies and different accessions of subspecies. This diversity provides insight into glucosinolate biosynthesis at the genomic, physiological, biochemical and host-pathogen interaction levels. The natural variation of glucosinolate profiles between species or different cultivars of same species permits the investigation of the effects of QTL or genes and gene interactions. This can be utilized for advanced breeding applications like MAS, trait introgression and gene pyramiding for beneficial glucosinolates. In *Arabidopsis*, naturally occurring variations in glucosinolates were identified and quantified for 34 types of glucosinolates in the leaves of 39 ecotypes (Hogge et al., 1988; Reichelt et al., 2002). Similarly, different morphotypes of *B. rapa* possess eight different glucosinolates with gluconapin and glucobrassicanapin as predominant aliphatic glucosinolates (He et al., 2000). Padilla et al., (2007) reported 16 different glucosinolates among 116 accessions of turnip greens.

The wide range of variation in glucosinolate profiles provides the opportunity to study individual glucosinolates for their potent biological activities *in planta*. Within different forms of *B. oleracea*, 12 different glucosinolates have been detected. The beneficial glucosinolate glucoraphanin showed significant variation ranging from 44 to 274 µmole/g seed in different genotypes of broccoli (Mithen et al., 2000; Rangkadilok et al., 2002). Furthermore, variation in concentration of individual glucosinolates also exists in cultivars of the same species.

#### **6. Low glucosinolate rapeseed and canola**

Early forms of domesticated rapeseed and their cultivars possessed a high concentration of glucosinolates (100 to 180 µmole/g) in their oil-free seed meal. The presence of glucosinolates in rapeseed had hindered the use of rapeseed meal in livestock industries due to anti-nutritional effects of its hydrolysis products in animals. As a result, in the 1970s, plant breeders searched germplasm collections for low glucosinolate contents. A Polish

*GSL-ALK* accomplishes these reactions in *Arabidopsis* (Kliebenstein et al., 2001c; Parkin et al., 1994). In *B. oleracea*, *GSL-ALK* was inferred by positional cloning and biochemical analysis. The functional allele in collard and the non-functional allele (with 2 bp deletion creating a frame-shift mutation) in broccoli were confirmed. A locus or loci of *GSL-ALK* is also believed to have a role in the catalysis of methylsulfinyl to alkenyl glucosinolates (Li & Quiros, 2003). Hydroxylation changes alkenyl to hydroxy aliphatic glucosinolate (in butyls, pentyls, hexyls and so on) biosynthesis branches in *Arabidopsis* and *Brassica* species; these sets of reactions are controlled by *GSL-OH* dependant on the presence of both *GSL-AOP2*  and *GSL-ELONG*. In *Brassica*, the final product of this reaction in 4C glucosinolate biosynthesis is progoitrin and its hydrolytic derivative, oxazolidine-2-thione which causes goiter in animals. These compounds are major obstacles to the use of *Brassica* crops as

Glucosinolates are united by their unique basic skeleton (β-D-glucopyranose) but glucosinolates are diverse in their origins, side chain modifications, degradations and final biological functions. In addition to structural diversity, a diversity of glucosinolates is seen between families, genera, species, subspecies and different accessions of subspecies. This diversity provides insight into glucosinolate biosynthesis at the genomic, physiological, biochemical and host-pathogen interaction levels. The natural variation of glucosinolate profiles between species or different cultivars of same species permits the investigation of the effects of QTL or genes and gene interactions. This can be utilized for advanced breeding applications like MAS, trait introgression and gene pyramiding for beneficial glucosinolates. In *Arabidopsis*, naturally occurring variations in glucosinolates were identified and quantified for 34 types of glucosinolates in the leaves of 39 ecotypes (Hogge et al., 1988; Reichelt et al., 2002). Similarly, different morphotypes of *B. rapa* possess eight different glucosinolates with gluconapin and glucobrassicanapin as predominant aliphatic glucosinolates (He et al., 2000). Padilla et al., (2007) reported 16 different glucosinolates

The wide range of variation in glucosinolate profiles provides the opportunity to study individual glucosinolates for their potent biological activities *in planta*. Within different forms of *B. oleracea*, 12 different glucosinolates have been detected. The beneficial glucosinolate glucoraphanin showed significant variation ranging from 44 to 274 µmole/g seed in different genotypes of broccoli (Mithen et al., 2000; Rangkadilok et al., 2002). Furthermore, variation in concentration of individual glucosinolates also exists in cultivars

Early forms of domesticated rapeseed and their cultivars possessed a high concentration of glucosinolates (100 to 180 µmole/g) in their oil-free seed meal. The presence of glucosinolates in rapeseed had hindered the use of rapeseed meal in livestock industries due to anti-nutritional effects of its hydrolysis products in animals. As a result, in the 1970s, plant breeders searched germplasm collections for low glucosinolate contents. A Polish

animal feed (Fenwick et al., 1983).

among 116 accessions of turnip greens.

**6. Low glucosinolate rapeseed and canola** 

of the same species.

**5.1.3 Diversity of glucosinolates in** *Brassicaceae*

Agriculture Canada Research Station in Saskatoon (Kondra & Stefansson, 1970). This sole genetic source of the low glucosinolate trait has been used to develop all the low glucosinolate cultivars in *B. napus* and *B. rapa* worldwide through conventional plant breeding. *B. napus* and *B. rapa* cultivars with low content of erucic acid and glucosinolate were developed, which ushered in a new era for *Brassica* crop production and its consumption. The world's first double low (low erucic acid and low glucosinolate content) *B. napus* and *B. rapa* cultivars, Tower and Candle, respectively, were developed by pedigree selection in the progenies of interspecific crosses in 1970s (Stefansson & Downey, 1995; McVetty et al., 2009). In Canada, this new type of oilseed rape was designated "Canola". The term "Canola" applies to any rapeseed cultivars with erucic acid content of <2% and glucosinolates content of <30 µmol/g in oil-free seed meal. The Canola term is a registered trademark of the Canadian Canola Association. The name is derived from **Can**adian **O**il **L**ow **A**cid (Canola Council of Canada, 2010a, http://www.canola-council.org/ canola\_the\_official\_definition.aspx). Currently, most rapeseed (high erucic acid) and canola cultivars have glucosinolate levels <15 µmole/g in oil-free seed meal. The development of low erucic acid and low glucosinolate cultivars has also been undertaken for other *Brassica* rapeseed species (e.g. *B. juncea*) and in other parts of the world for the quality improvement of their oils and seed meals.

### **7. Quantitative trait loci for glucosinolates in major** *Brassica* **species**

Glucosinolate biosynthesis in *Brassica* crops has quantitative inheritance, which is regulated by complex genetic factors and affected by environmental factors. Glucosinolates are functionally diverse and well recognized plant secondary metabolites; so they have been extensively studied in terms of QTL mapping, biosynthesis gene cloning and functional characterization in *Arabidopsis* (Kliebenstein et al., 2001a; Kliebenstein et al., 2001c; Compos de Quiros et al., 2000; Brown et al., 2003; Benderoth et al., 2006; Textor et al., 2007; Li et al., 2008). However, very limited genetic, biochemical and metabolomic information is available on glucosinolate biosynthesis, transport and final product utilization in *Brassica* crops including *B. rapa*. There has been a few QTL mapping studies reported for major *Brassica* crop seed glucosinolates. Uzunova et al., (1995) mapped four QTL for total seed glucosinolate content in a *B. napus* DH population, which accounted 61% total phenotypic variance. Similarly, Toroser et al., (1995), based on a RFLP linkage map, identified two larger and three small effect QTL for total aliphatic glucosinolate content using a DH population in *B. napus*. These QTL explained 70% of the total phenotypic variance. This suggests that several loci with additive or epistatic effect are involved in total seed glucosinolate biosynthesis in different genetic backgrounds. Howell et al., (2003) reported QTL mapping for total seed glucosinolates analyzed by X-ray fluorescence (XRF) and near-infrared reflection spectroscopy (NIRS) in two inter-varietal *B. napus* backcross populations. They identified four QTLs accounting for 76% of the phenotypic variance in the Victor x Tapidor population. These three QTL accounted for 86% of phenotypic variance in this second population. These studies, however, were limited to either total seed glucosinolates or 3C, 4C and 5C aliphatic glucosinolates, and did not infer the genetic loci for individual aliphatic, indole or aromatic glucosinolates. Furthermore, there were no reports of publicly available

Molecular Genetics of Glucosinolate

labelling (Kiddle et al., 2001).

2001b).

Biosynthesis in *Brassicas*: Genetic Manipulation and Application Aspects 205

indole glucosinolate conten and three QTL for leaf aromatic glucosinolates in two DH populations of *B. rapa* using an AFLP and SSR based linkage map. There was no information regarding QTL for seed glucosinolates. Glucosinolate content varies greatly between leaves and seeds (Brown et al., 2003). As well, there is variation in the expression patterns of the genetic loci underpinning glucosinolate production in leaves and seeds (Kliebenstein et al.,

Early analysis of glucosinolates began with detection of glucosinolates and possible hydrolysis products by paper and thin-layer chromatography. The paper chromatography was applied in combination with high voltage electrophoresis, but it had many complications and low yield (Greer, 1962). Danielak & Borkowski, (1969) analyzed glucosinolates from seeds of 150 different cruciferous species using thin-layer chromatography. Since then, numerous techniques have been employed for quantification of total glucosinolate content with various modifications including steam distillation and titration of isothiocyanates, ELISA, sulfate-release assay, UV spectroscopy and gas chromatography of isothiocyanates. Near infrared reflectance spectroscopy (NIRS) is one of the widely used techniques for seed total glucosinolates quantification, which detects N ̶ H, C ̶ H and O ̶ H groups of total glucosinolates. NIRS is a preferred technique because it can simultaneously quantify oil and protein along with total glucosinolates in canola/rapeseed (Velasco & Becker, 1998). Individual intact glucosinolates can be determined using techniques such as reverse phase HPLC-MS, thermospray LC with tandem MS in the two

Desulfoglucosinolates usually are analyzed by reverse phase HPLC or by X-ray fluorescence spectroscopy (XRF). The reverse-phase HPLC analytical approach has been widely used for quantification of individual intact or desulfo- glucosinolates. The technique was developed in 1984 with UV based detection of either intact glucosinolates or an on-column enzymatic desulfation from plant extracts (Spinks et al., 1984). The photodiode array (PDA) with UV detector can distinguish spectra of aliphatic from indole and aromatic glucosinolates; the indole and aromatic glucosinolates spectra end with a shoulder. This widely applicable method for glucosinolate separation is yet subject to difficulties in interpretation of results because of differences in the time and enzymatic activity for the desulfation reaction, pH effects and mobile phase solvents with an appropriate gradient. Desulfoglucosinolates also have been analyzed by the determination of the sulfur content of the seeds using X-ray fluorescence spectroscopy (XRF) (Schnug & Haneklaus, 1990). The hydrolysis products of glucosinolates, isothiocyanates, nitriles, thiocyanates and benzenedithiol, have been analyzed using techniques including GC or GC-MS and HPLC with or without fluorescent

**8. Glucosinolate identification and quantification approaches** 

most common interfaces (ESI or APCI), capillary GC-MS and GC-MS-MS.

**9. Molecular markers and their applications for glucosinolates** 

Molecular markers are efficient, reliable, time saving and cost effective tools that may enhance the capacity of conventional breeding for improvement in agronomy, quality and yield related traits of crop species without adverse effects. Morphological traits such as petal

molecular markers for marker assisted selection of glucosinolates. Such markers, if developed, could be used in breeding to manipulate glucosinolate profiles and contents in *Brassica* crop species.

In another amphidiploid species, *B. juncea,* several studies were conducted for QTL mapping of seed glucosinolates. Cheung et al., (1998) detected two QTL for 2-propenyl and three QTL for 3-butenyl glucosinolates which explained between 89% and 81% of total phenotypic variance. This QTL mapping study was carried out in a DH population derived from the F1 of two *B. juncea* parental lines, J90-4317 (low glucosinolates) and J90-2733 (high glucosinolates). Mahmood et al., (2003) reported three QTL for 2-propenyl glucosinolate content which explained 78% of the phenotypic variance, while five QTL for total seed aliphatic glucosinolates explained phenotypic variance between 30% and 45%. In this study a DH population and an RFLP linkage map was used. Similarly, Ramchiary et al., (2007) reported six QTL for seed glucosinolate content in the F1DH and advanced backcross DH (BC4DH) of *B. juncea*. Some of the large effect QTL in advanced backcross (BC4DH) of *B. juncea* were fine mapped using a candidate gene approach and comparative sequence analyses of *Arabidopsis* and *B. oleracea* (Bisht et al., 2009). The results suggested that epistasis and additive effects of glucosinolate genes in different genetic backgrounds in *B. juncea* exist. This study, however, could not explain the homoeologous effects of genes/loci from the A- and B-genomes on the individual or total seed glucosinolate content.

In *B. oleracea*, *BoGSL-ELONG* a side chain elongation gene was cloned based on the *Arabidopsis* sequence information, and functionally characterized using an RNA interference (RNAi) approach. The results suggested that *BoGSL-ELONG* is involved in 4C and 5C aliphatic glucosinolate biosynthesis in *Brassica* species. The RNAi lines displayed an increased level of propyl glucosinolates suggesting that the precursor homo-methionine concentration enhances the activity of 3C aliphatic glucosinolate biosynthesis in *B. napus* (Li & Quiros, 2002, Liu et al., 2010). A natural mutation in *BoGSL-ELONG* resulting in the failure of excision of the third intron and thus producing a long cDNA fragment has been identified in a white cauliflower genotype (*B. oleracea*) lacking 4C and 5C aliphatic glucosinolates (Li & Quiros, 2002). A molecular marker for this mutation would be useful in *Brassica* breeding programs for modification of glucosinolate profiles. Additionally, a gene *BoGSL-PRO* which control propyl glucosinolate biosynthesis in *B. oleracea* was sequenced using comparative analysis of the *MAM* (*methylthioalkylmalate synthase*) gene family in *Arabidopsis* (Li et al., 2003; Gao et al., 2006).

A glucosinolate side chain modification gene, *BoGSL-ALK,* was cloned using a positional cloning approach based on a closely linked SRAP marker in *B. oleracea* (Li & Quiros, 2003). Functional characterization of *BoGSL-ALK* by overexpression in *Arabidopsis* and RNA interference (RNAi) in *B. napus* suggests that *BoGSL-ALK* is involved in catalyzation of either sulfinylbutyl to butenyl or hydoxybutenyl with high functional redundancy (Li & Quiros, 2003, Liu et al., 2012). Interestingly, a natural frame shift mutation of 2 bp deletions was identified in broccoli, which accumulates sulfinylbutyl glucosinolate by ceasing downstream biosynthesis of other 4C aliphatic glucosinolates.

In *B. rapa*, a single QTL mapping study for leaf glucosinolates has been reported, although it is one of the widely distributed *Brassica* species for oil and vegetable production. Lou et al., (2008) identified six QTL for leaf total aliphatic glucosinolate content, three QTL for total leaf

molecular markers for marker assisted selection of glucosinolates. Such markers, if developed, could be used in breeding to manipulate glucosinolate profiles and contents in

In another amphidiploid species, *B. juncea,* several studies were conducted for QTL mapping of seed glucosinolates. Cheung et al., (1998) detected two QTL for 2-propenyl and three QTL for 3-butenyl glucosinolates which explained between 89% and 81% of total phenotypic variance. This QTL mapping study was carried out in a DH population derived from the F1 of two *B. juncea* parental lines, J90-4317 (low glucosinolates) and J90-2733 (high glucosinolates). Mahmood et al., (2003) reported three QTL for 2-propenyl glucosinolate content which explained 78% of the phenotypic variance, while five QTL for total seed aliphatic glucosinolates explained phenotypic variance between 30% and 45%. In this study a DH population and an RFLP linkage map was used. Similarly, Ramchiary et al., (2007) reported six QTL for seed glucosinolate content in the F1DH and advanced backcross DH (BC4DH) of *B. juncea*. Some of the large effect QTL in advanced backcross (BC4DH) of *B. juncea* were fine mapped using a candidate gene approach and comparative sequence analyses of *Arabidopsis* and *B. oleracea* (Bisht et al., 2009). The results suggested that epistasis and additive effects of glucosinolate genes in different genetic backgrounds in *B. juncea* exist. This study, however, could not explain the homoeologous effects of genes/loci from

the A- and B-genomes on the individual or total seed glucosinolate content.

downstream biosynthesis of other 4C aliphatic glucosinolates.

In *B. oleracea*, *BoGSL-ELONG* a side chain elongation gene was cloned based on the *Arabidopsis* sequence information, and functionally characterized using an RNA interference (RNAi) approach. The results suggested that *BoGSL-ELONG* is involved in 4C and 5C aliphatic glucosinolate biosynthesis in *Brassica* species. The RNAi lines displayed an increased level of propyl glucosinolates suggesting that the precursor homo-methionine concentration enhances the activity of 3C aliphatic glucosinolate biosynthesis in *B. napus* (Li & Quiros, 2002, Liu et al., 2010). A natural mutation in *BoGSL-ELONG* resulting in the failure of excision of the third intron and thus producing a long cDNA fragment has been identified in a white cauliflower genotype (*B. oleracea*) lacking 4C and 5C aliphatic glucosinolates (Li & Quiros, 2002). A molecular marker for this mutation would be useful in *Brassica* breeding programs for modification of glucosinolate profiles. Additionally, a gene *BoGSL-PRO* which control propyl glucosinolate biosynthesis in *B. oleracea* was sequenced using comparative analysis of the *MAM* (*methylthioalkylmalate synthase*) gene family in *Arabidopsis* (Li et al., 2003; Gao et al., 2006). A glucosinolate side chain modification gene, *BoGSL-ALK,* was cloned using a positional cloning approach based on a closely linked SRAP marker in *B. oleracea* (Li & Quiros, 2003). Functional characterization of *BoGSL-ALK* by overexpression in *Arabidopsis* and RNA interference (RNAi) in *B. napus* suggests that *BoGSL-ALK* is involved in catalyzation of either sulfinylbutyl to butenyl or hydoxybutenyl with high functional redundancy (Li & Quiros, 2003, Liu et al., 2012). Interestingly, a natural frame shift mutation of 2 bp deletions was identified in broccoli, which accumulates sulfinylbutyl glucosinolate by ceasing

In *B. rapa*, a single QTL mapping study for leaf glucosinolates has been reported, although it is one of the widely distributed *Brassica* species for oil and vegetable production. Lou et al., (2008) identified six QTL for leaf total aliphatic glucosinolate content, three QTL for total leaf

*Brassica* crop species.

indole glucosinolate conten and three QTL for leaf aromatic glucosinolates in two DH populations of *B. rapa* using an AFLP and SSR based linkage map. There was no information regarding QTL for seed glucosinolates. Glucosinolate content varies greatly between leaves and seeds (Brown et al., 2003). As well, there is variation in the expression patterns of the genetic loci underpinning glucosinolate production in leaves and seeds (Kliebenstein et al., 2001b).

### **8. Glucosinolate identification and quantification approaches**

Early analysis of glucosinolates began with detection of glucosinolates and possible hydrolysis products by paper and thin-layer chromatography. The paper chromatography was applied in combination with high voltage electrophoresis, but it had many complications and low yield (Greer, 1962). Danielak & Borkowski, (1969) analyzed glucosinolates from seeds of 150 different cruciferous species using thin-layer chromatography. Since then, numerous techniques have been employed for quantification of total glucosinolate content with various modifications including steam distillation and titration of isothiocyanates, ELISA, sulfate-release assay, UV spectroscopy and gas chromatography of isothiocyanates. Near infrared reflectance spectroscopy (NIRS) is one of the widely used techniques for seed total glucosinolates quantification, which detects N ̶ H, C ̶ H and O ̶ H groups of total glucosinolates. NIRS is a preferred technique because it can simultaneously quantify oil and protein along with total glucosinolates in canola/rapeseed (Velasco & Becker, 1998). Individual intact glucosinolates can be determined using techniques such as reverse phase HPLC-MS, thermospray LC with tandem MS in the two most common interfaces (ESI or APCI), capillary GC-MS and GC-MS-MS.

Desulfoglucosinolates usually are analyzed by reverse phase HPLC or by X-ray fluorescence spectroscopy (XRF). The reverse-phase HPLC analytical approach has been widely used for quantification of individual intact or desulfo- glucosinolates. The technique was developed in 1984 with UV based detection of either intact glucosinolates or an on-column enzymatic desulfation from plant extracts (Spinks et al., 1984). The photodiode array (PDA) with UV detector can distinguish spectra of aliphatic from indole and aromatic glucosinolates; the indole and aromatic glucosinolates spectra end with a shoulder. This widely applicable method for glucosinolate separation is yet subject to difficulties in interpretation of results because of differences in the time and enzymatic activity for the desulfation reaction, pH effects and mobile phase solvents with an appropriate gradient. Desulfoglucosinolates also have been analyzed by the determination of the sulfur content of the seeds using X-ray fluorescence spectroscopy (XRF) (Schnug & Haneklaus, 1990). The hydrolysis products of glucosinolates, isothiocyanates, nitriles, thiocyanates and benzenedithiol, have been analyzed using techniques including GC or GC-MS and HPLC with or without fluorescent labelling (Kiddle et al., 2001).

#### **9. Molecular markers and their applications for glucosinolates**

Molecular markers are efficient, reliable, time saving and cost effective tools that may enhance the capacity of conventional breeding for improvement in agronomy, quality and yield related traits of crop species without adverse effects. Morphological traits such as petal

Molecular Genetics of Glucosinolate

chromosomes

rapeseed meal.

**11. References** 

**10. Acknowledgments** 

of distinct chromosome behaviour

Manitoba, Canada for reviewing this chapter.

trichome evolution. *Am J Bot*. 93: 607-619.

*Natl Acad Sci USA* 103(24): 9118-9123.

Biosynthesis in *Brassicas*: Genetic Manipulation and Application Aspects 207

i. duplicated or triplicated genomic regions may mask the effect of the single locus being

ii. lack of similarity of gene and spacer sequences between alien and host chromosomes in

iv. directional exchange of genetic materials in trivalent formations during meiosis because

v. host genome chromosome numbers and amount of homology between host and alien

Several traits in *Brassica* species have been improved through introgression of functional genes from allied species through interspecific or inter-generic crosses such as *B. rapa* x *B. oleracea* and *B. rapa* x *B. oxyrrhina* (Srinivasan et al., 1998). In near future, development of molecular markers using sequenced genome information of *B. rapa* and *Arabidopsis* will hasten marker assisted selection of glucosinolates to increase beneficial glucosinolates such as glucoraphanin and glucoerucin in *Brassica* vegetables and to reduce total glucosinolates in

The authors are grateful to the Genome Canada/Genome Alberta/Genome Prairie and Manitoba Provincial Government for financial support. The authors also extend their thanks to Dr. Habibur Rahman, Department of Agricultural, Food & Nutritional Science, University of Alberta, Canada and Dr. Anne Worley, Department of Biological Sciences, University of

Agrawal, A. A. & Kurashige, N. S. (2003) A role for isothiocyanates in plant resistance against the specialist herbivore *Pieris rapae*. *J Chem Ecol*. 29: 1403-1415. Barth, C. & Jander, G. (2006) *Arabidopsis* myrosinases TGG1 and TGG2 have redundant function in glucosinolate breakdown and insect defense. *Plant J*. 46: 549-562. Beekwilder, J., Leeuwen, W., Dam, N. M., Bertossi, M., Grandi,V., Mizzi, L., Soloviev, M.,

glucosinolates on insect herbivory in *Arabidopsis*. *PLoS One*. 3(4): e2068. Beilstein, M. A., Al-Shehbaz, I. A. & Kellogg, E. A. (2006) *Brassicaceae* phylogeny and

Benderoth, M., Textor, S., Windsor, A. J., Mitchell-Olds, T., Gershenzon, J. & Kroymann, J.

Birch, A. N. E., Griffiths, D. W., Hopkins, R. J., Smith, W. H. M. & McKinlay, R. G. (1992)

turnip root fly (*Delia floralis*) larvae. *J Sci Food Agric*. 60: 1-9.

Szabados, L., Molthoff, J. W., Schipper, B., Verbocht, H., Vos, R. C. H., Morandini, P., Aarts, M. G. M. & Bovy, A. (2008) The impact of the absence of aliphatic

(2006) Positive selection driving diversification in plant secondary metabolism. *Proc* 

Glucosinolate responses of swede, kale, forage and oilseed rape to root damage by

replaced for a quantitative traits like glucosinolate profile and concentration

iii. presence of active homoeologous recombination regulator genes during meiosis

monosomic or disomic alien chromosome addition lines

color, leaf shape *etc* were used as markers in classical breeding, where significant time and effort was required to refine crosses. There have been many practical difficulties with the use of morphological markers, including:


Advancements in molecular biology tools and techniques have overcome some of the difficulties of classical breeding. Different types of DNA molecular markers (hybridization based e.g. RFLP; PCR based e.g. SSR, RAPD, SCAR, and SRAP) have been used for gene/QTL mapping, cloning, genetic map construction and marker assisted selection in plant breeding. Most recently, the conversion of various molecular markers (RFLP, RAPD, SRAP, AFLP, SSR, SNP etc.) to simple PCR based SCAR markers for marker assisted selection has overcome the difficulties of other markers. It is feasible and cost effective to use SCAR markers for marker assisted selection of populations.

Marker assisted selection in plant breeding is well supported by the availability of molecular maps developed using various marker systems in different mapping populations. The use of molecular markers has facilitated introgression of important traits through intra or interspecific as well as inter-generic crosses. Similar to agronomic, disease resistance and yield related traits, seed quality traits such as glucosinolates can be genetically manipulated using interspecific hybridization followed by marker assisted selection for introgression or replacement of a native gene with the allied gene. Natural mutations for glucosinolate biosynthesis genes have been identified in accessions of *B. oleracea* (Li & Quiros, 2002; 2003) and molecular markers have been developed. These molecular markers have been employed for the manipulation of glucosinolate profiles in *Brassica* through interspecific hybridization and marker assisted selection. In our QTL mapping study in *B. rap*a RIL mapping population, we identified single major QTL for 5C aliphatic glucosinolates (glucobrassicanapin, glucoalyssin and gluconapoleiferin) on chromosome A3 and gene specific SCAR molecular markers were developed and utilized that markers for marker assisted selection in other *Brassica* interspecific crosses (unpublished). Hasan et al., (2008) reported linkage of SSR markers to candidate genetic loci of glucosinolate biosynthesis genes in *Brassica napus* through structure-based allele-trait association studies, and found potential application of these markers in marker assisted selection for glucosinolates.

On the other hands, Niu (2008) attempted to replace the functional *GSL-ALK* gene of *B. rapa* by the null allele from *B. oleracea* (broccoli) using a gene specific SCAR marker. However, introgression of the *GSL-ALK* null allele or replacement of a single locus with small effect did not change the glucosinolate profile of the *B. rapa* in this study. This suggests that multiple loci with functional redundancy play important roles in glucosinolate biosynthesis in *Brassica* species. This approach has met with very little or no success. This might be due to many reasons, such as:


Several traits in *Brassica* species have been improved through introgression of functional genes from allied species through interspecific or inter-generic crosses such as *B. rapa* x *B. oleracea* and *B. rapa* x *B. oxyrrhina* (Srinivasan et al., 1998). In near future, development of molecular markers using sequenced genome information of *B. rapa* and *Arabidopsis* will hasten marker assisted selection of glucosinolates to increase beneficial glucosinolates such as glucoraphanin and glucoerucin in *Brassica* vegetables and to reduce total glucosinolates in rapeseed meal.

#### **10. Acknowledgments**

206 Crop Plant

color, leaf shape *etc* were used as markers in classical breeding, where significant time and effort was required to refine crosses. There have been many practical difficulties with the

i. a paucity of suitable markers and associations with agriculturally important traits

ii. undesirable pleiotropic effects of many morphological markers on plant phenotypes

iv. trait of interest easily can be lost in a breeding cycle if there is no strong linkage

Advancements in molecular biology tools and techniques have overcome some of the difficulties of classical breeding. Different types of DNA molecular markers (hybridization based e.g. RFLP; PCR based e.g. SSR, RAPD, SCAR, and SRAP) have been used for gene/QTL mapping, cloning, genetic map construction and marker assisted selection in plant breeding. Most recently, the conversion of various molecular markers (RFLP, RAPD, SRAP, AFLP, SSR, SNP etc.) to simple PCR based SCAR markers for marker assisted selection has overcome the difficulties of other markers. It is feasible and cost effective to use

Marker assisted selection in plant breeding is well supported by the availability of molecular maps developed using various marker systems in different mapping populations. The use of molecular markers has facilitated introgression of important traits through intra or interspecific as well as inter-generic crosses. Similar to agronomic, disease resistance and yield related traits, seed quality traits such as glucosinolates can be genetically manipulated using interspecific hybridization followed by marker assisted selection for introgression or replacement of a native gene with the allied gene. Natural mutations for glucosinolate biosynthesis genes have been identified in accessions of *B. oleracea* (Li & Quiros, 2002; 2003) and molecular markers have been developed. These molecular markers have been employed for the manipulation of glucosinolate profiles in *Brassica* through interspecific hybridization and marker assisted selection. In our QTL mapping study in *B. rap*a RIL mapping population, we identified single major QTL for 5C aliphatic glucosinolates (glucobrassicanapin, glucoalyssin and gluconapoleiferin) on chromosome A3 and gene specific SCAR molecular markers were developed and utilized that markers for marker assisted selection in other *Brassica* interspecific crosses (unpublished). Hasan et al., (2008) reported linkage of SSR markers to candidate genetic loci of glucosinolate biosynthesis genes in *Brassica napus* through structure-based allele-trait association studies, and found

potential application of these markers in marker assisted selection for glucosinolates.

On the other hands, Niu (2008) attempted to replace the functional *GSL-ALK* gene of *B. rapa* by the null allele from *B. oleracea* (broccoli) using a gene specific SCAR marker. However, introgression of the *GSL-ALK* null allele or replacement of a single locus with small effect did not change the glucosinolate profile of the *B. rapa* in this study. This suggests that multiple loci with functional redundancy play important roles in glucosinolate biosynthesis in *Brassica* species. This approach has met with very little or no success. This might be due to

use of morphological markers, including:

iii. high linkage drag (Ranade et al., 2001), and

between marker and traits (Ranade et al., 2001).

SCAR markers for marker assisted selection of populations.

(Ranade et al., 2001),

(Ranade et al., 2001),

many reasons, such as:

The authors are grateful to the Genome Canada/Genome Alberta/Genome Prairie and Manitoba Provincial Government for financial support. The authors also extend their thanks to Dr. Habibur Rahman, Department of Agricultural, Food & Nutritional Science, University of Alberta, Canada and Dr. Anne Worley, Department of Biological Sciences, University of Manitoba, Canada for reviewing this chapter.

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**10** 

*Uruguay* 

**Legume Crops, Importance and Use of Bacterial** 

Legumes are flowering plants that produce seedpods. They have colonized several ecosystems (from rain forests and arctic/alpine regions to deserts; Schrire et al., 2005), and have been found in most of the archaeological record of plants. Early in 37 B.C. Varro said "Legumes should be planted in light soils, not so much for their own crop as for the good they do to subsequent crops" (Graham & Vance, 2003), recognizing the importance of

Leguminosae or Fabaceae is the third most populous family of flowering plants (behind Asteraceae and Orchidaceae) with 670 to 750 genera and 18,000 to 19,000 species. Legumes include important grain, pasture and agro-forestry species. They are harvested as crops for human and animal consumption as well as used as pulp for paper production, fuel-woods, timber, oil production, sources of chemicals and medicines, and are also cultivated as

The legumes provide many benefits to the soil so they are usually utilized as cover crop, intercropped with cereals and other staple foods. They do produce substantial amounts of organic nitrogen (see below, Improving legume yield by inoculation with rhizobia), increase soil organic matter, improve soil porosity and structure, recycle nutrients, decrease soil pH, reduce soil compaction, diversify microorganisms and mitigate disease problems (U.S Department of Agriculture [USDA], 1998). In rotation with cereals, legumes provide a source of slow-release nitrogen that contributes to sustainable cropping systems. The improvement in the production of these crops will therefore contribute substantially to

Based on total harvested area and production, cereals are the most important crops, and they are followed by legumes (Fig.1). Close up to 180 million Ha (12-15% of the Earth's arable surface) are worldwide used to produce grain and forage legumes. These numbers point the central importance of world legumes production. In addition, the promise of lowcost production of legume biomass, mainly soybean, for bioenergy purpose focus attention of investors in the improvement of legume production, and deserves an entirely section for

ornamental, used as living fences and firebreaks among others (Lewis et al., 2005).

**1. Introduction** 

discussion.

 \*

multiple cropping and intercropping production.

better human nutrition and soil health (Popelka et al., 2004).

M. Morel and V. Braña contributed equally to this chapter

**Inoculation to Increase Production** 

María A. Morel1\*, Victoria Braña1\*\*and Susana Castro-Sowinski1, 2 *1Molecular Microbiology, Biological Sciences Institute Clemente Estable 2Biochemistry and Molecular Biology, Faculty of Science, Montevideo,* 

Zhang, Y., Talalay, P., Cho, C. G. & Posner, G. H. (1992) A major inducer of anticarcinogenic protective enzymes from broccoli: isolation and elucidation of structure. *Proc Natl Acad Sci USA*. 89: 2399-2403.

## **Legume Crops, Importance and Use of Bacterial Inoculation to Increase Production**

María A. Morel1\*, Victoria Braña1\*\*and Susana Castro-Sowinski1, 2 *1Molecular Microbiology, Biological Sciences Institute Clemente Estable 2Biochemistry and Molecular Biology, Faculty of Science, Montevideo, Uruguay* 

#### **1. Introduction**

216 Crop Plant

Zhang, Y., Talalay, P., Cho, C. G. & Posner, G. H. (1992) A major inducer of anticarcinogenic

*Acad Sci USA*. 89: 2399-2403.

protective enzymes from broccoli: isolation and elucidation of structure. *Proc Natl* 

Legumes are flowering plants that produce seedpods. They have colonized several ecosystems (from rain forests and arctic/alpine regions to deserts; Schrire et al., 2005), and have been found in most of the archaeological record of plants. Early in 37 B.C. Varro said "Legumes should be planted in light soils, not so much for their own crop as for the good they do to subsequent crops" (Graham & Vance, 2003), recognizing the importance of multiple cropping and intercropping production.

Leguminosae or Fabaceae is the third most populous family of flowering plants (behind Asteraceae and Orchidaceae) with 670 to 750 genera and 18,000 to 19,000 species. Legumes include important grain, pasture and agro-forestry species. They are harvested as crops for human and animal consumption as well as used as pulp for paper production, fuel-woods, timber, oil production, sources of chemicals and medicines, and are also cultivated as ornamental, used as living fences and firebreaks among others (Lewis et al., 2005).

The legumes provide many benefits to the soil so they are usually utilized as cover crop, intercropped with cereals and other staple foods. They do produce substantial amounts of organic nitrogen (see below, Improving legume yield by inoculation with rhizobia), increase soil organic matter, improve soil porosity and structure, recycle nutrients, decrease soil pH, reduce soil compaction, diversify microorganisms and mitigate disease problems (U.S Department of Agriculture [USDA], 1998). In rotation with cereals, legumes provide a source of slow-release nitrogen that contributes to sustainable cropping systems. The improvement in the production of these crops will therefore contribute substantially to better human nutrition and soil health (Popelka et al., 2004).

Based on total harvested area and production, cereals are the most important crops, and they are followed by legumes (Fig.1). Close up to 180 million Ha (12-15% of the Earth's arable surface) are worldwide used to produce grain and forage legumes. These numbers point the central importance of world legumes production. In addition, the promise of lowcost production of legume biomass, mainly soybean, for bioenergy purpose focus attention of investors in the improvement of legume production, and deserves an entirely section for discussion.

<sup>\*</sup> M. Morel and V. Braña contributed equally to this chapter

Legume Crops, Importance and Use of Bacterial Inoculation to Increase Production 219

The soybean (U.S.) (*Glycine max*), also called soya bean (U.K.), is an annual summer legume native of South-eastern Asia, which is used as human food (Liu, 1999) and livestock feed as well as for several industrial purposes (Ali, 2010). According to the newest available information, this legume is one of the main crops cultivated for oil extraction (35.9 million tons oil and 57% global oilseed production), preceded only by the oil of palm (FAO, 2011). Interestingly, over half of the world´s 2007 soybean crop (58.6%) was genetically modified (GM), achieving 77% in the year 2009. These GM-soybeans possess a gene that confers herbicide resistance. The nations that produce almost exclusively GM-soybean are U.S. (85%) and Argentina (98%), tending to 100%. The global production and utilization of soybean have increased by ten during the last century (Qiu & Chang, 2010). In 2009, world´s soybean cultivated area and production were 99.5 million Ha and 223.2 million tons (FAO, 2011), respectively. U.S. is the world´s leader soybean producer and exporter, responsible of 41% global production, followed by Brazil (26%), Argentina (14%), China (7%) and India

In U.S. the soybean farm gate value raised more than double, ranging from 12.6 billion USD (in 2001) to 29.6 billion USD (in 2009). The price of soybean has increased more than 80% because of soybean-oil's use in soy-biodiesel and as feed for fish farming. Biodiesel is in demand and soybean represents about 25% total worldwide global biodiesel raw material (Pahl, 2008). The net energy balance when the soybean-oil is used for fuel has improved since soybean is a legume, it fixes nitrogen and does not require nitrogen fertilizer (see

Leguminous plants are relevant economic and cultural important crops because their exceptional diversity, manifested in variety of vegetable forms that adapted to a wide range of ecological conditions, the high protein content of some grains, their use as pastures, increased world production and commodities. In this scenario, many farm investors, industries and researchers have focussed attention in the development of biological and ecofriendly technologies for legume growth improvement and establishment. The ability of many legumes to form associations with bacteria that fix atmospheric nitrogen (the symbiotic association that improve growth) is thus a big matter of ecological and economic

Microorganisms are essential to the Earth's nitrogen cycle and to the Biological Nitrogen Fixation (BNF) process in leguminous plants, playing a very important role in terms of plant production in agriculture. Nitrogen fixing microorganisms could be used in live formulations (biofertilizer) that when applied to seed, root or soil colonize the rhizosphere, or the interior of the plant, and promote growth by increasing the nitrogen supply to the host plant and building up soil health. The evaluation, in terms of economic and ecological costs, between chemical- and biological-nitrogen fertilizers support that BNF represents an economic, sustainable and environmentally friendly resource to guarantee the nitrogen

**1.2 Soybean – The new legume-star** 

(4%) (FAO, 2011).

below) (Kinney & Clemente, 2010).

interest (Zahran, 2009).

requirement of an agro-ecosystem.

**2. Improving legume yield by inoculation with rhizobia** 

**2.1 Biological vs chemical nitrogen fertilization** 

Fig. 1. Net worldwide harvested area for several crops, in the year 2009. Data obtained from Food and Agriculture Organization database [FAO]

(http://faostat.fao.org/site/567/default.aspx#ancor) and Graham & Vance (2003).

#### **1.1 Forage and grain legumes**

Forage legumes play an important role in dairy and meat production being sources of protein, fibre and energy. They are usually richer in protein, calcium, and phosphorus than other nonlegume forages, such as grass. They include alfalfa (*Medicago sativa*), clover (*Trifolium* spp.), birdsfoot trefoil (*Lotus corniculatus*) and vetch (*Vicia* spp.) among others. Alfalfa is one of the most important forage crops. In 2006, the worldwide production was around 436 million tons. U.S. is the largest alfalfa producer, with 15 million Ha planted in 2010. Canada, Argentina (primarily grazed), Southern Europe, Australia, South Africa, and the Middle East have also considerable production (FAO, 2011).

Grain legumes also called pulses, which according to FAO are crops harvested exclusively for the dry seeds, play an important role in the nutrition of many people due to their high protein content in seeds. They represent a major source of protein in many developing countries, especially among the poorest population, and are rich in essential amino acids such as lysine, supplementing thus the nutritional value of cereal and tuber diets (Graham & Vance, 2003). The world pulse production has almost increased by half during the period of 1980 – 2004, overtaking the 60 million tons in 2005 (FAO, 2005). According to FAO Statistical Yearbook 2010, in the year 2008, Canada, China and United States were the main exporters of pulses (28%, 12% and 11% of total exports, respectively). Interestingly, India, the world's 12th largest economy and the third largest in Asia behind Japan and China, is the main importer, responsible of 21% of global trade in of pulses (2.5-3.5 million tons). India produces (15-18 million tons; the world's largest producer), imports and consumes (18.5-20.5 million tons) a wide range of pulses. Thus, considering pulse relevance in the world´s largest economies such as U.S., China and India, incomes and a raising world population, it is obvious the interest of farmers and investors for improving pulse production.

#### **1.2 Soybean – The new legume-star**

218 Crop Plant

19,36%

 Legumes Wheat Rice Maize Barley Potatos Cassava

Food and Agriculture Organization database [FAO]

**1.1 Forage and grain legumes** 

considerable production (FAO, 2011).

19,61%

27,7% 22,06%

Fig. 1. Net worldwide harvested area for several crops, in the year 2009. Data obtained from

Forage legumes play an important role in dairy and meat production being sources of protein, fibre and energy. They are usually richer in protein, calcium, and phosphorus than other nonlegume forages, such as grass. They include alfalfa (*Medicago sativa*), clover (*Trifolium* spp.), birdsfoot trefoil (*Lotus corniculatus*) and vetch (*Vicia* spp.) among others. Alfalfa is one of the most important forage crops. In 2006, the worldwide production was around 436 million tons. U.S. is the largest alfalfa producer, with 15 million Ha planted in 2010. Canada, Argentina (primarily grazed), Southern Europe, Australia, South Africa, and the Middle East have also

Grain legumes also called pulses, which according to FAO are crops harvested exclusively for the dry seeds, play an important role in the nutrition of many people due to their high protein content in seeds. They represent a major source of protein in many developing countries, especially among the poorest population, and are rich in essential amino acids such as lysine, supplementing thus the nutritional value of cereal and tuber diets (Graham & Vance, 2003). The world pulse production has almost increased by half during the period of 1980 – 2004, overtaking the 60 million tons in 2005 (FAO, 2005). According to FAO Statistical Yearbook 2010, in the year 2008, Canada, China and United States were the main exporters of pulses (28%, 12% and 11% of total exports, respectively). Interestingly, India, the world's 12th largest economy and the third largest in Asia behind Japan and China, is the main importer, responsible of 21% of global trade in of pulses (2.5-3.5 million tons). India produces (15-18 million tons; the world's largest producer), imports and consumes (18.5-20.5 million tons) a wide range of pulses. Thus, considering pulse relevance in the world´s largest economies such as U.S., China and India, incomes and a raising world population, it

is obvious the interest of farmers and investors for improving pulse production.

(http://faostat.fao.org/site/567/default.aspx#ancor) and Graham & Vance (2003).

2,33% 2,33%

6,62%

The soybean (U.S.) (*Glycine max*), also called soya bean (U.K.), is an annual summer legume native of South-eastern Asia, which is used as human food (Liu, 1999) and livestock feed as well as for several industrial purposes (Ali, 2010). According to the newest available information, this legume is one of the main crops cultivated for oil extraction (35.9 million tons oil and 57% global oilseed production), preceded only by the oil of palm (FAO, 2011). Interestingly, over half of the world´s 2007 soybean crop (58.6%) was genetically modified (GM), achieving 77% in the year 2009. These GM-soybeans possess a gene that confers herbicide resistance. The nations that produce almost exclusively GM-soybean are U.S. (85%) and Argentina (98%), tending to 100%. The global production and utilization of soybean have increased by ten during the last century (Qiu & Chang, 2010). In 2009, world´s soybean cultivated area and production were 99.5 million Ha and 223.2 million tons (FAO, 2011), respectively. U.S. is the world´s leader soybean producer and exporter, responsible of 41% global production, followed by Brazil (26%), Argentina (14%), China (7%) and India (4%) (FAO, 2011).

In U.S. the soybean farm gate value raised more than double, ranging from 12.6 billion USD (in 2001) to 29.6 billion USD (in 2009). The price of soybean has increased more than 80% because of soybean-oil's use in soy-biodiesel and as feed for fish farming. Biodiesel is in demand and soybean represents about 25% total worldwide global biodiesel raw material (Pahl, 2008). The net energy balance when the soybean-oil is used for fuel has improved since soybean is a legume, it fixes nitrogen and does not require nitrogen fertilizer (see below) (Kinney & Clemente, 2010).

#### **2. Improving legume yield by inoculation with rhizobia**

Leguminous plants are relevant economic and cultural important crops because their exceptional diversity, manifested in variety of vegetable forms that adapted to a wide range of ecological conditions, the high protein content of some grains, their use as pastures, increased world production and commodities. In this scenario, many farm investors, industries and researchers have focussed attention in the development of biological and ecofriendly technologies for legume growth improvement and establishment. The ability of many legumes to form associations with bacteria that fix atmospheric nitrogen (the symbiotic association that improve growth) is thus a big matter of ecological and economic interest (Zahran, 2009).

#### **2.1 Biological vs chemical nitrogen fertilization**

Microorganisms are essential to the Earth's nitrogen cycle and to the Biological Nitrogen Fixation (BNF) process in leguminous plants, playing a very important role in terms of plant production in agriculture. Nitrogen fixing microorganisms could be used in live formulations (biofertilizer) that when applied to seed, root or soil colonize the rhizosphere, or the interior of the plant, and promote growth by increasing the nitrogen supply to the host plant and building up soil health. The evaluation, in terms of economic and ecological costs, between chemical- and biological-nitrogen fertilizers support that BNF represents an economic, sustainable and environmentally friendly resource to guarantee the nitrogen requirement of an agro-ecosystem.

Legume Crops, Importance and Use of Bacterial Inoculation to Increase Production 221

The current taxonomy of rhizobia consists of several genera in the subclass Alpha- and Beta-Proteobacteria. *Rhizobium*, *Mesorhizobium*, *Ensifer* (formerly *Sinorhizobium*), *Azorhizobium*, *Methylobacterium*, *Bradyrhizobium*, *Phyllobacterium*, *Devosia* and *Ochrobactrum* are genera that belong to rhizobial Alpha-Proteobacteria. In rhizobial Beta-Proteobacteria the following genera have been described: *Burkholderia*, *Herbaspirillum* and *Cupriavidus* (NZ Rhizobia, 2011). It is important to clarify that this classification is based on taxonomically important strains that may not necessarily be important reference strains for legume growth improvement. Rhizobial strains commonly used in inoculants have good field performance and stability of symbiotic properties in culture, but are not necessarily well documented or used in taxonomy or molecular biology studies (Lindström et al., 2010). The legumerhizobia association is specific (each rhizobial strain establishes a symbiosis with only a limited set of host plants and *vice versa*). Thus, there is a restricted number of inoculants that fit with a leguminous plant, and farmers must know which inoculant must be applied according plants and characteristics of soil (Mabrouk & Belhadj, 2010). In other words "Be sure to buy the right inoculant for the legume the farmer intends to plant". Such information must be given by the manufacturer and should be clearly specified in the label. Plants mutually compatible with the same species of rhizobia were listed in earlier years in so-called "crossinoculation groups" (Table 1). This concept was used in rhizobial taxonomy, but is it unreliable as taxonomic marker because of aberrant cross-infection among plant groups.

**Rhizobia Legume Cross-inoculation group**

*Rhizobium leguminosarum* bv *trifolii* Clover Group (Clover I, II, III and IV): clovers

*Rhizobium leguminosarum* bv *viciae* Pea Group: peas (*Pisum* spp.), lentil (*Lens culinaris*),

*Rhizobium leguminosarum* bv *phaseoli* Bean Group: beans (*Phaseolus vulgaris*), scarita

*Mesorhizobium loti* Chickpea Group: chickpea (*Cicer* spp.),

*Rhizobium lupini* Group Lupines *Rhizobium* spp Crownvetch

Table 1. Cross-inoculation group and *Rhizobium*-legume association

*Bradyrhizobium japonicum* Soybean Group: soybean (*Glycine max*)

Alfalfa Group: alfalfa (*Medicago sativa*),

fenugreek (*Trigonella* spp.)

puero (*Pueraria phaseoloides*)

runner bean (*Phaseolus coccineus*)

Birdsfoot trefoil (*Lotus corniculatus* L.)

(*Trifolium* spp.)

sweet clover (*Melilotus* spp.) (yellow and white),

Cowpea Group: pigeon pea (*Cajanus cajan*); peanut (*Arachis hypogaea*); cowpea, mungbean, black gram, rice bean (*Vigna* spp.); lima bean (*Phaseolus lunatus*); *Acacia mearnsii; A. mangium; Albizia* spp.; *Enterlobium* spp., *Desmodium* spp., *Stylosanthes* spp., Kacang bogor (*Voandzeia subterranea*), *Centrosema* sp., winged bean (*Psophocarpus tetragonolobus*), hyacinth bean (*Lablab purpureus*), siratro (*Macroptilium atropurpureum*), guar bean (*Cyamopsis* 

*tetragonoloba*), calopo (*Calopogonium mucunoides*),

vetches (*Vicia* spp.), faba bean (*Vicia faba*)

**2.3 Rhizobia: The master microbe** 

*Ensifer meliloti* 

*Bradyrhizobium* spp.

Chemical-fertilizer demand has historically been influenced by changing and often interrelated factors such as increasing populations and economic growth, agricultural production, prices, and government policies. In 2007, the production of chemical nitrogen fertilizers was 130 million tons which is likely to increase further in the coming years (FAO, 2011). Their production requires a great consumption of fossil fuels (1-2 % global fossil fuel) and is subjected of constant variations in prices (Vieira et al., 2010). Although their direct contribution to energy consumption seems minimal, it is unnecessary and unsustainable. On average, U.S. farmers apply 30-40 % more chemical nitrogen than is needed for optimal crop yield, thus wasting most of the applied chemical nitrogen. Given the rising cost of chemical nitrogen fertilizers, nitrogen fixation cover crops offer significant economic benefits. In 2006, the price of nitrogen fertilizers in U.S. raised to 521 USD per ton (Huang, 2007), estimating an over cost of 7 to 10 billion USD annually compared with FBN. For instance, the modest use of alfalfa in rotation with corn by U.S. farmers saved 200 to 300 million USD (Graham & Vance, 2003).

In addition to the inconvenience of increasing prices, chemical nitrogen fertilization is associated with environmental problems because watershed contamination by nitrogen leaching, volatilization and denitrification. These problems could be avoided offering to farmers low-cost biofertilizer technologies. These are ecologically sound and their application could help to minimize the global warming as well as to reduce the fertilizer input in farming practices (Herridge et al., 2008a).

#### **2.2 The biological nitrogen fixation (BNF)**

BNF benefits not only the legumes themselves but also any intercropped or succeeding crop, reducing or removing the need for nitrogen fertilization. In soils with low mineral nitrogen content, nitrogen fixing microorganisms provide ammonium into the legume biomass, allowing faster growing than their plant competitors. In contrast, if nitrogen is abundant, nitrogen fixing microorganisms tend to be competitively excluded by non-fixing species because the nitrogen fixation process is bio-energetically costly (Houlton et al., 2008). It means that there is a range of physiological and ecological situations that tend to constrain BNF in legume systems, mainly by the nitrogen demand of the plant and by the C:N stoichiometry of the ecosystem. In fact, the hypothesis of a feedback control between legume demand and BNF in a particular ecosystem has been now supported by evidence from both experimental and theoretical models (Soussana & Tallec, 2010).

There is the potential to increase BNF by the use of well adapted and efficient nitrogen fixing microorganisms and/or genetic modified plant species to ensure legume crop at high levels of productivity. Farmers are familiar with the application of commercially available microorganisms (inoculants) that have been especially selected for their ability to effectively nodulate plants and to fix nitrogen from the atmosphere. These kind of microbial inoculants, also known as soil inoculants, are agricultural amendments that use microorganisms known as rhizobia to promote legume growth. These bacteria form symbiotic relationships with the target leguminous plant, and both parts benefit. The legume supplies energy and photosynthates to rhizobia, and rhizobia provide the legume with nitrogen, mainly in the form of ammonium (Howard & Rees, 1996). The symbiosis is initiated through the legume root infection by the rhizobia and formation of root nodules where BNF occurs through the action of a bacterial enzyme, called "Nitrogenase" (Masson-Boivin et al., 2009).

#### **2.3 Rhizobia: The master microbe**

220 Crop Plant

Chemical-fertilizer demand has historically been influenced by changing and often interrelated factors such as increasing populations and economic growth, agricultural production, prices, and government policies. In 2007, the production of chemical nitrogen fertilizers was 130 million tons which is likely to increase further in the coming years (FAO, 2011). Their production requires a great consumption of fossil fuels (1-2 % global fossil fuel) and is subjected of constant variations in prices (Vieira et al., 2010). Although their direct contribution to energy consumption seems minimal, it is unnecessary and unsustainable. On average, U.S. farmers apply 30-40 % more chemical nitrogen than is needed for optimal crop yield, thus wasting most of the applied chemical nitrogen. Given the rising cost of chemical nitrogen fertilizers, nitrogen fixation cover crops offer significant economic benefits. In 2006, the price of nitrogen fertilizers in U.S. raised to 521 USD per ton (Huang, 2007), estimating an over cost of 7 to 10 billion USD annually compared with FBN. For instance, the modest use of alfalfa in rotation with corn by U.S. farmers saved 200 to 300 million USD (Graham &

In addition to the inconvenience of increasing prices, chemical nitrogen fertilization is associated with environmental problems because watershed contamination by nitrogen leaching, volatilization and denitrification. These problems could be avoided offering to farmers low-cost biofertilizer technologies. These are ecologically sound and their application could help to minimize the global warming as well as to reduce the fertilizer

BNF benefits not only the legumes themselves but also any intercropped or succeeding crop, reducing or removing the need for nitrogen fertilization. In soils with low mineral nitrogen content, nitrogen fixing microorganisms provide ammonium into the legume biomass, allowing faster growing than their plant competitors. In contrast, if nitrogen is abundant, nitrogen fixing microorganisms tend to be competitively excluded by non-fixing species because the nitrogen fixation process is bio-energetically costly (Houlton et al., 2008). It means that there is a range of physiological and ecological situations that tend to constrain BNF in legume systems, mainly by the nitrogen demand of the plant and by the C:N stoichiometry of the ecosystem. In fact, the hypothesis of a feedback control between legume demand and BNF in a particular ecosystem has been now supported by evidence from both

There is the potential to increase BNF by the use of well adapted and efficient nitrogen fixing microorganisms and/or genetic modified plant species to ensure legume crop at high levels of productivity. Farmers are familiar with the application of commercially available microorganisms (inoculants) that have been especially selected for their ability to effectively nodulate plants and to fix nitrogen from the atmosphere. These kind of microbial inoculants, also known as soil inoculants, are agricultural amendments that use microorganisms known as rhizobia to promote legume growth. These bacteria form symbiotic relationships with the target leguminous plant, and both parts benefit. The legume supplies energy and photosynthates to rhizobia, and rhizobia provide the legume with nitrogen, mainly in the form of ammonium (Howard & Rees, 1996). The symbiosis is initiated through the legume root infection by the rhizobia and formation of root nodules where BNF occurs through the

action of a bacterial enzyme, called "Nitrogenase" (Masson-Boivin et al., 2009).

Vance, 2003).

input in farming practices (Herridge et al., 2008a).

experimental and theoretical models (Soussana & Tallec, 2010).

**2.2 The biological nitrogen fixation (BNF)** 

The current taxonomy of rhizobia consists of several genera in the subclass Alpha- and Beta-Proteobacteria. *Rhizobium*, *Mesorhizobium*, *Ensifer* (formerly *Sinorhizobium*), *Azorhizobium*, *Methylobacterium*, *Bradyrhizobium*, *Phyllobacterium*, *Devosia* and *Ochrobactrum* are genera that belong to rhizobial Alpha-Proteobacteria. In rhizobial Beta-Proteobacteria the following genera have been described: *Burkholderia*, *Herbaspirillum* and *Cupriavidus* (NZ Rhizobia, 2011). It is important to clarify that this classification is based on taxonomically important strains that may not necessarily be important reference strains for legume growth improvement. Rhizobial strains commonly used in inoculants have good field performance and stability of symbiotic properties in culture, but are not necessarily well documented or used in taxonomy or molecular biology studies (Lindström et al., 2010). The legumerhizobia association is specific (each rhizobial strain establishes a symbiosis with only a limited set of host plants and *vice versa*). Thus, there is a restricted number of inoculants that fit with a leguminous plant, and farmers must know which inoculant must be applied according plants and characteristics of soil (Mabrouk & Belhadj, 2010). In other words "Be sure to buy the right inoculant for the legume the farmer intends to plant". Such information must be given by the manufacturer and should be clearly specified in the label. Plants mutually compatible with the same species of rhizobia were listed in earlier years in so-called "crossinoculation groups" (Table 1). This concept was used in rhizobial taxonomy, but is it unreliable as taxonomic marker because of aberrant cross-infection among plant groups.


Table 1. Cross-inoculation group and *Rhizobium*-legume association

Legume Crops, Importance and Use of Bacterial Inoculation to Increase Production 223

range from 15.00 to 18.00 (US) per Ha (Xavier et al., 2004). But, inoculants need only a modest increase in yield to offset the cost. A good inoculant will usually provide at least a

The annual input of fixed nitrogen was calculated to be 2.95 Mton for the pulses and 18.5 Mton for the oilseed legumes, being the soybean the dominant crop legume (50% global crop legume area and 68% global production). In addition to the annual legume nitrogen fixation inputs of 12-15 Mton (pasture and fodder legumes), there is an input by nitrogen fixation in rice (5 Mton), sugar cane (0.5 Mton), non-legume crop lands (<4 Mton) and extensive savannas (<14 Mton). Thus, the total overall estimated in agricultural systems is of 50–70 Mton biologically fixed nitrogen (Herridge et al., 2008a). These numbers show that the process of BNF is an economically attractive and eco-friendly alternative to reduce the external nitrogen (chemical

A successful BNF is capable of improving agricultural productivity while minimizing soil loss and ameliorating adverse edaphic conditions. Conditions such as drought, salinity, unfavorable soil pH, nutrient deficiency, mineral toxicity, high temperature, insufficient or excessive soil moisture, inadequate photosynthesis, and plant diseases conspire against a successful symbiotic process. Many inoculant manufactures worldwide have developed formulations with high symbiotic efficiency under stress conditions. However, the actual view of plant growth promoting preparations focuses their investigations in the design and development of new-formulations supplemented with plant and/or microbe exudates. These exudates contain molecules involved in the microbe-plant interaction: flavonoids, sugars, acids, amino acids, amines and other low molecular weight compounds that promote plant growth (Skorupska et al., 2010; Garg & Geetanjali, 2009). Macchiavelli & Brelles-Mariño (2004) showed increased plant nodulation treating *Medicago truncatula* roots and seeds with Nod Factors prior to inoculation. Lipo-chito-oligosaccharides (LCOs), or Nod Factors (NFs), are biosignals produced by the rhizobia which act as bacteria-to-plant communication molecule that mediates recognition and nodule organogenesis (Masson-Boivin et al., 2009). The inclusion of NFs in formulations might have technological applications since presoaking seeds with submicromolar concentrations of this oligo-saccharide before sowing leaded to increased nodulation under field conditions. In fact, a soybean inoculant based on NFs technology was introduced on the market many years ago (Zhang & Smith, 2002). Currently, many companies like Rizobacter (www.rizobacter.com.ar) and Nitragin (www.nitragin.com.ar) are marketing formulations with bio-signals that improve the symbiotic relationship, activate mechanisms to

fertilizers) input, which improves the quality and quantity of crop resources.

resist abiotic stress conditions, and induce defensive response.

**3. The use of microbial consortium in legume agronomic production** 

soils (Gamalero et al., 2009) when the formulation contains different PGPB.

The new fashion in agriculture is the use of microbial consortiums of plant-growth promoting bacteria (PGPB, which includes rhizobia). PGPB are exogenous bacteria introduced into agricultural ecosystems that act positively upon plant development (Castro-Sowinski et al., 2007). It is possible to increase agricultural productivity and, eliminate or decrease the use of chemical fertilizers and pesticides (Adesemoye et al., 2009a; Vessey, 2003) even in marginal

70- to 140-Kg per Ha return on yield.

**2.5 The input of BNF in legume yield** 

The occurrence of a wide diversity of microorganisms in a particular soil increases the opportunity for a legume host to find compatible rhizobia. The principle of specific legumerhizobia association is commonly used for the isolation of well adapted and efficient rhizobial strains (Castro-Sowinski et al., 2002; Florentino et al., 2010). Usually trap-plants are used to catch the rhizobial strain with highest performance and the strain is used for the design of new inoculants. Details about inoculation technology for legumes can be read in Herridge (2008b).

#### **2.4 Formulation and low-cost are crucial aspect of producing inoculants**

Formulation is the industrial "art" of converting a promising laboratory-proven microorganism into a commercial field product. But, the development of successful inoculants involves more than the selection of the most efficient rhizobial strain, it involves the choice of a carrier (powder, granule, and liquid), packaging and marketing, avoiding of microbial contaminations. Inoculant preparations for agricultural use constitute a stressful environment because bacterial cells may have to be stored for long periods, and should survive desiccation and transportation conditions. Some aspects related to inoculant preparation, production and application are described by Hungria et al. (2005).

The formulation should maintain or enhance activity in field. In order to survive in nutrientpoor ecosystems, bacteria use different strategies, among them, the use of polyhydroxyalkanoates (PHA) as intracellular carbon storage compounds. Cells with higher PHA content can survive longer than those with lower amounts, and PHA degraded elements can be used rapidly for numerous metabolic needs. Accumulation of PHA can provide the cell with the ability to endure a variety of harmful physical and chemical stresses (Castro-Sowinski et al., 2010; Kadouri et al., 2005).

A good formulation contains microorganisms (active ingredient) in an active metabolic state, immersed in a suitable carrier together with additives that are responsible for the microbial cells stabilization and protection during storage and transportation. Most of the research done in the improvement of inoculant quality is based on improving carrier properties, by adding elements that can prolong survival, such as nutrients, or other synthetic products (López et al., 1998). Most commercial inoculants are in powder (finely ground peat mixed with the nitrogen-fixing bacteria), ready for mixing with the seed. Granular formulations are designed to be placed in the seed rut at planting. Liquid inoculants and other non-peat-based inoculants are also being used. Liquid inoculants simplify the production of the inoculant and the application to seeds or field. However, bacterial survival in the inoculant and on inoculated seeds is not as good as when using peat as a carrier, because bacteria lack carrier protection (Tittabutr et al., 2007). Peat provides bacterial protection and prevents drying and death, compared to the inoculants that do not contain peat. However, alternative substrates to peat can be used as carriers: compost cork, perlite, volcanic pumice, alginate beads and coal, among many others, also gave good results in terms of supporting bacterial growth and long survival, as well survival on seeds (Albareda et al., 2008; Ben Rebah et al., 2007).

Another important consideration in formulation is the cost-effectiveness that must be low enough to allow sufficient incoming compared to chemical fertilization. In U.S. and Canada, a seed inoculant is sell for 5.00 and 2.50 USD per Ha, respectively, while granular inoculants range from 15.00 to 18.00 (US) per Ha (Xavier et al., 2004). But, inoculants need only a modest increase in yield to offset the cost. A good inoculant will usually provide at least a 70- to 140-Kg per Ha return on yield.

#### **2.5 The input of BNF in legume yield**

222 Crop Plant

The occurrence of a wide diversity of microorganisms in a particular soil increases the opportunity for a legume host to find compatible rhizobia. The principle of specific legumerhizobia association is commonly used for the isolation of well adapted and efficient rhizobial strains (Castro-Sowinski et al., 2002; Florentino et al., 2010). Usually trap-plants are used to catch the rhizobial strain with highest performance and the strain is used for the design of new inoculants. Details about inoculation technology for legumes can be read in

Formulation is the industrial "art" of converting a promising laboratory-proven microorganism into a commercial field product. But, the development of successful inoculants involves more than the selection of the most efficient rhizobial strain, it involves the choice of a carrier (powder, granule, and liquid), packaging and marketing, avoiding of microbial contaminations. Inoculant preparations for agricultural use constitute a stressful environment because bacterial cells may have to be stored for long periods, and should survive desiccation and transportation conditions. Some aspects related to inoculant

The formulation should maintain or enhance activity in field. In order to survive in nutrientpoor ecosystems, bacteria use different strategies, among them, the use of polyhydroxyalkanoates (PHA) as intracellular carbon storage compounds. Cells with higher PHA content can survive longer than those with lower amounts, and PHA degraded elements can be used rapidly for numerous metabolic needs. Accumulation of PHA can provide the cell with the ability to endure a variety of harmful physical and chemical

A good formulation contains microorganisms (active ingredient) in an active metabolic state, immersed in a suitable carrier together with additives that are responsible for the microbial cells stabilization and protection during storage and transportation. Most of the research done in the improvement of inoculant quality is based on improving carrier properties, by adding elements that can prolong survival, such as nutrients, or other synthetic products (López et al., 1998). Most commercial inoculants are in powder (finely ground peat mixed with the nitrogen-fixing bacteria), ready for mixing with the seed. Granular formulations are designed to be placed in the seed rut at planting. Liquid inoculants and other non-peat-based inoculants are also being used. Liquid inoculants simplify the production of the inoculant and the application to seeds or field. However, bacterial survival in the inoculant and on inoculated seeds is not as good as when using peat as a carrier, because bacteria lack carrier protection (Tittabutr et al., 2007). Peat provides bacterial protection and prevents drying and death, compared to the inoculants that do not contain peat. However, alternative substrates to peat can be used as carriers: compost cork, perlite, volcanic pumice, alginate beads and coal, among many others, also gave good results in terms of supporting bacterial growth and long survival, as well survival on seeds

Another important consideration in formulation is the cost-effectiveness that must be low enough to allow sufficient incoming compared to chemical fertilization. In U.S. and Canada, a seed inoculant is sell for 5.00 and 2.50 USD per Ha, respectively, while granular inoculants

**2.4 Formulation and low-cost are crucial aspect of producing inoculants** 

preparation, production and application are described by Hungria et al. (2005).

stresses (Castro-Sowinski et al., 2010; Kadouri et al., 2005).

(Albareda et al., 2008; Ben Rebah et al., 2007).

Herridge (2008b).

The annual input of fixed nitrogen was calculated to be 2.95 Mton for the pulses and 18.5 Mton for the oilseed legumes, being the soybean the dominant crop legume (50% global crop legume area and 68% global production). In addition to the annual legume nitrogen fixation inputs of 12-15 Mton (pasture and fodder legumes), there is an input by nitrogen fixation in rice (5 Mton), sugar cane (0.5 Mton), non-legume crop lands (<4 Mton) and extensive savannas (<14 Mton). Thus, the total overall estimated in agricultural systems is of 50–70 Mton biologically fixed nitrogen (Herridge et al., 2008a). These numbers show that the process of BNF is an economically attractive and eco-friendly alternative to reduce the external nitrogen (chemical fertilizers) input, which improves the quality and quantity of crop resources.

A successful BNF is capable of improving agricultural productivity while minimizing soil loss and ameliorating adverse edaphic conditions. Conditions such as drought, salinity, unfavorable soil pH, nutrient deficiency, mineral toxicity, high temperature, insufficient or excessive soil moisture, inadequate photosynthesis, and plant diseases conspire against a successful symbiotic process. Many inoculant manufactures worldwide have developed formulations with high symbiotic efficiency under stress conditions. However, the actual view of plant growth promoting preparations focuses their investigations in the design and development of new-formulations supplemented with plant and/or microbe exudates. These exudates contain molecules involved in the microbe-plant interaction: flavonoids, sugars, acids, amino acids, amines and other low molecular weight compounds that promote plant growth (Skorupska et al., 2010; Garg & Geetanjali, 2009). Macchiavelli & Brelles-Mariño (2004) showed increased plant nodulation treating *Medicago truncatula* roots and seeds with Nod Factors prior to inoculation. Lipo-chito-oligosaccharides (LCOs), or Nod Factors (NFs), are biosignals produced by the rhizobia which act as bacteria-to-plant communication molecule that mediates recognition and nodule organogenesis (Masson-Boivin et al., 2009). The inclusion of NFs in formulations might have technological applications since presoaking seeds with submicromolar concentrations of this oligo-saccharide before sowing leaded to increased nodulation under field conditions. In fact, a soybean inoculant based on NFs technology was introduced on the market many years ago (Zhang & Smith, 2002). Currently, many companies like Rizobacter (www.rizobacter.com.ar) and Nitragin (www.nitragin.com.ar) are marketing formulations with bio-signals that improve the symbiotic relationship, activate mechanisms to resist abiotic stress conditions, and induce defensive response.

#### **3. The use of microbial consortium in legume agronomic production**

The new fashion in agriculture is the use of microbial consortiums of plant-growth promoting bacteria (PGPB, which includes rhizobia). PGPB are exogenous bacteria introduced into agricultural ecosystems that act positively upon plant development (Castro-Sowinski et al., 2007). It is possible to increase agricultural productivity and, eliminate or decrease the use of chemical fertilizers and pesticides (Adesemoye et al., 2009a; Vessey, 2003) even in marginal soils (Gamalero et al., 2009) when the formulation contains different PGPB.

2007).

Soybean (*Glycine max*)

**Legume Bacterial system**

*B. japonicum - Serratia* spp*.* 

*B. japonicum -* 

*B. japonicum - S. proteamaculans /B. subtilis*

*B. japonicum -* 

*B. japonicum - A. brasilense* 

*E. fredii - Chryseobacterium balustinum* 

*B. japonicum - P. putida* 

Legume Crops, Importance and Use of Bacterial Inoculation to Increase Production 225

involved in stress mitigation is still unknown (Figueiredo et al., 2008; Furina & Bonartseva,

**3.2 Enhancing the legume – Rhizobia symbiosis by co-inoculation: Modes of action**  Many evidences have been accumulated showing that co-inoculation with beneficial microorganisms, having different mechanisms of plant-growth promotion, have additive or synergistic effect on plant growth and crop yield (Table 2). Diverse mechanisms are implicates in the co-inoculation benefits and some of them have been discussed in Barea at al. (2005).

> **Experiments done in**

*B. cereus* 10 in SDW Field Unknown Bullied et al.,

30 in NN Greenhouse Production of

Laboratory

Laboratory

Greenhouse (saline stress)

**Proposed mechanism of action** 

LCO- analogue

Laboratory Unknown Zhang et al.,

Limited Nauptake

Production of IAA, GA3 and Zeatin

Laboratory Unknown Molla et al.,

plant hormones

(saline stress) Unknown Estevez et al.,

P-solubilization and production of siderophores

Greenhouse Production of

**Reference** 

Bai et al., 2002a Bai et al., 2002b

1996

2002

Han & Lee, 2005

Cassán et al., 2009

2001a

Molla et al., 2001b

2009

Rosas et al., 2006

**Increase (%) compared to single rhizobial inoculation** 

50 in NN; 30 in SDW; 32 in RDW

40 in NN under suboptimal temperature

12 in SDW; 10 in P-uptake

*A. brasilense* 47 in NN Laboratory

16-40 in RDW; 200-700 in total RL

56 and 44 in SDW; 100 and 200 in RDW; 155 and 286 in NN under non-saline and saline conditions respectively

40 in SDW; 80 in NN; 45 in RDW

#### **3.1 Getting more from legumes**

Current studies indicate that we are still detecting new bacteria and fungi with diverse growth-promoting characteristics, and that the combination of different PGPB into a singleformulation increases plant yield, compared with single-inoculation. On the other hand, efforts have been done manipulating PGPB to produce master inoculants by the introduction of foreign DNA that provides new abilities (GMM, Genetic Modified Microorganisms). Globally, it was expected a big explosion in this area of research, the use of recombinant DNA-technological tools for the production of inoculants (Barea et al., 2005; Valdenegro et al., 2001). However, the use of GMM is in discussion and needs clear regulatory policies, controls and suitable legislation (Fedoroff et al., 2010).

Some cooperative microbial activities can be exploited for developing new sustainable, environmentally-friendly, agro-technological practices (Barea et al., 2005). In this regard, the plant co-inoculation with rhizobia and other PGPB received considerable attention for legume growth promotion (Cassán et al., 2009; Bai et al., 2002a; 2002b; Zhang et al., 1996). Results from many studies concerning the effect of co-inoculation on legume growth are summarized in Table 2. Several genera of bacteria have been identified as "*helpers*" of the rhizobia-legume symbiotic process (Beattie, 2006). Examples are bacteria of the genus *Azospirillum* (Cassán et al., 2009; Itzigsohn et al., 1993), *Azotobacter* (Qureshi et al., 2009; Yasari et al., 2008), *Bacillus* (Bullied et al., 2002), *Pseudomonas* (Barea et al., 2005; Fox et al., 2011), *Serratia* (Bai et al., 2002b; Lucas-Garcia et al., 2004a; Zhang et al., 1996), *Thiobacillus* (Anandham et al., 2007), and *Delftia* (Morel et al., 2011), among many other. The stimulation of the legume–rhizobia symbiosis by nonrhizobial-PGPB implicates different processes such as production of phytohormones (usually indole-acetic acid; IAA) that stimulates root growth; qualitative change of flavonoids pattern secreted for the plant; solubilization of non-available nutrients (mainly re-fixation of exogenously applied phosphorus), among others (Medeot et al., 2010). In this section, we summarize the knowledge about bacteria that promote the symbiotic relationship between legumes and rhizobia (from now, the symbiotic enhancer), and the mechanisms involved in this phenomenon. The effect of other microorganisms, such as micorrhizal fungi is not discussed.

Probably the most studied bacterial consortium is the rhizobia-azospirilla one. Azospirilla species are being used as seed inoculants under field conditions for more than a decade (Dobbelaere et al., 2001; Puente et al., 2009). The positive effect of *Azospirillum* in the nodulation and nitrogen fixation by rhizobia on several forage legumes was early reported (Yahalom et al., 1987). Since then, many works have been done and mostly are summarized in Bashan et al. (2004). It proven that the combined inoculation with rhizobia and azospirilla increases the shoot length and weight, root hairs number, root diameter, the main- and totalroot nodule number and the percentage of infected root hairs, thus resulting in increased legume yields (Cassán et al., 2009). Worldwide, salinity is one of the most important abiotic stresses that limit crop growth and productivity. It was shown that the rhizobia-azospirilla co-inoculation significantly reduces the negative effects of abiotic stresses (such as caused by irrigation with saline water) on root development and nodulation (Dardanelli et al., 2008).

Under stress conditions, such as drought, salinity, S-deficient or heavy metal (HM) contaminated soils, several associations between plants and beneficial bacteria showed a defensive response and an increased yield (Anandham et al., 2007; Dary et al., 2010; Fuentes-Ramírez & Caballero-Mellado, 2005; Han & Lee, 2005). However, the physiological mechanism

Current studies indicate that we are still detecting new bacteria and fungi with diverse growth-promoting characteristics, and that the combination of different PGPB into a singleformulation increases plant yield, compared with single-inoculation. On the other hand, efforts have been done manipulating PGPB to produce master inoculants by the introduction of foreign DNA that provides new abilities (GMM, Genetic Modified Microorganisms). Globally, it was expected a big explosion in this area of research, the use of recombinant DNA-technological tools for the production of inoculants (Barea et al., 2005; Valdenegro et al., 2001). However, the use of GMM is in discussion and needs clear

Some cooperative microbial activities can be exploited for developing new sustainable, environmentally-friendly, agro-technological practices (Barea et al., 2005). In this regard, the plant co-inoculation with rhizobia and other PGPB received considerable attention for legume growth promotion (Cassán et al., 2009; Bai et al., 2002a; 2002b; Zhang et al., 1996). Results from many studies concerning the effect of co-inoculation on legume growth are summarized in Table 2. Several genera of bacteria have been identified as "*helpers*" of the rhizobia-legume symbiotic process (Beattie, 2006). Examples are bacteria of the genus *Azospirillum* (Cassán et al., 2009; Itzigsohn et al., 1993), *Azotobacter* (Qureshi et al., 2009; Yasari et al., 2008), *Bacillus* (Bullied et al., 2002), *Pseudomonas* (Barea et al., 2005; Fox et al., 2011), *Serratia* (Bai et al., 2002b; Lucas-Garcia et al., 2004a; Zhang et al., 1996), *Thiobacillus* (Anandham et al., 2007), and *Delftia* (Morel et al., 2011), among many other. The stimulation of the legume–rhizobia symbiosis by nonrhizobial-PGPB implicates different processes such as production of phytohormones (usually indole-acetic acid; IAA) that stimulates root growth; qualitative change of flavonoids pattern secreted for the plant; solubilization of non-available nutrients (mainly re-fixation of exogenously applied phosphorus), among others (Medeot et al., 2010). In this section, we summarize the knowledge about bacteria that promote the symbiotic relationship between legumes and rhizobia (from now, the symbiotic enhancer), and the mechanisms involved in this phenomenon. The effect of other microorganisms, such as micorrhizal fungi is not discussed.

Probably the most studied bacterial consortium is the rhizobia-azospirilla one. Azospirilla species are being used as seed inoculants under field conditions for more than a decade (Dobbelaere et al., 2001; Puente et al., 2009). The positive effect of *Azospirillum* in the nodulation and nitrogen fixation by rhizobia on several forage legumes was early reported (Yahalom et al., 1987). Since then, many works have been done and mostly are summarized in Bashan et al. (2004). It proven that the combined inoculation with rhizobia and azospirilla increases the shoot length and weight, root hairs number, root diameter, the main- and totalroot nodule number and the percentage of infected root hairs, thus resulting in increased legume yields (Cassán et al., 2009). Worldwide, salinity is one of the most important abiotic stresses that limit crop growth and productivity. It was shown that the rhizobia-azospirilla co-inoculation significantly reduces the negative effects of abiotic stresses (such as caused by irrigation with saline water) on root development and nodulation (Dardanelli et al., 2008). Under stress conditions, such as drought, salinity, S-deficient or heavy metal (HM) contaminated soils, several associations between plants and beneficial bacteria showed a defensive response and an increased yield (Anandham et al., 2007; Dary et al., 2010; Fuentes-Ramírez & Caballero-Mellado, 2005; Han & Lee, 2005). However, the physiological mechanism

regulatory policies, controls and suitable legislation (Fedoroff et al., 2010).

**3.1 Getting more from legumes** 

involved in stress mitigation is still unknown (Figueiredo et al., 2008; Furina & Bonartseva, 2007).

#### **3.2 Enhancing the legume – Rhizobia symbiosis by co-inoculation: Modes of action**

Many evidences have been accumulated showing that co-inoculation with beneficial microorganisms, having different mechanisms of plant-growth promotion, have additive or synergistic effect on plant growth and crop yield (Table 2). Diverse mechanisms are implicates in the co-inoculation benefits and some of them have been discussed in Barea at al. (2005).


Legume Crops, Importance and Use of Bacterial Inoculation to Increase Production 227

**Experiments done in** 

120 in RDW Greenhouse Production of

Laboratory and Field

Field (two levels of Nfertilization)

Greenhouse (S-deficiency) and Field

in NN Laboratory Production of B-

Field (Heavy metal contaminated soil)

nodulation rate Laboratory IAA production Morel et al.,

**Proposed mechanism of action** 

flavonoid-like

Laboratory Unknown Goel et al.,

Laboratory IAA production Malik &

N-fixation by *B. subtilis* or/and P-solubilization by *B. megaterium*

by PGPB

group vitamins

Laboratory IAA production Morel et al.,

Phytostabilization: Biosorption of heavy metals by bacterial biomass

Field P-solubilization

**Reference** 

Parmar & Dadarwal, 1999

2002

Sindhu., 2011

Elkoca et al., 2008

2009

Wani et al., 2007

al., 2007

Marek-Kozaczuk & Skorupska, 2001

2011

Dary et al., 2010

2011

Unknown Qureshi et al.,

S-oxidation Anandham et

**Increase (%) compared to single rhizobial inoculation** 

20 in SDW; 30-

70 in NN; 30 in SDW, 30 in Nuptake

1,2-1,86 in NN; 1,3-2,11 NFW; 1-2,93 in PDW

18 in SDW; 16- 30 in RDW; 14 in total biomass yield in field

15 in NN; 25 in P-soil availability

20 in PDW; 30 in NN; 100 in P-uptake

50 in PDW; 80 in NN

20 in SDW; 100

50 in SDW and 80 in nodulation rate

66 in SDW and 20-40, 25, and 30-50 decrease in Cd, Cu and Zn accumulation in roots, respectively

10 in SDW; 30 in

**Legume Bacterial system**

*Rhizobium spp - Pseudomonas/ Bacillus* spp*.* 

*Mesorhizobium*  sp*. Cicer - Pseudomonas* spp*.*

> *Rhizobium - B. subtilis/ megaterium*

*M. ciceri - Azotobacter chroococcum* 

*M. ciceri - Pseudomonas* sp*/ Bacillus* sp*.* 

*Thiobacillus* sp*. - Rhizobium* sp*.* 

*R. leguminosarum bv.trifolii - P. fluorescens* 

*R. leguminosarum bv. trifolii - Delftia* sp*.* 

*Bradyrhizobium*  sp*. - Pseudomonas* sp*./ Ochrobactrum cytisi* 

> *S. meliloti - Delftia* sp*.*

Chickpea (*Cicer arietinum*)

Peanut

(*Arachis* 

Clover (*Trifolium* 

Altramuz (*Lupinus* 

Alfalfa

(*Medicago* 

*sativa*)

*luteus*)

*repens*)

*hypogaea*)


**Experiments done in** 

Hydroponic (saline stress)

Laboratory

Greenhouse (drought stress)

Greenhouse (two levels of P-fertilization)

Field

**Proposed mechanism of action** 

Production of flavonoid-like compounds

(saline stress) Unknown Estevez et al.,

IAA production or 1 aminocycloprop ane-1 carboxylate (ACC) deaminase activity

P-solubilization; auxin and siderophores production

70 in NN Hydroponic IAA production Remans et al.,

30 total yield Field IAA production Remans et al.,

**Reference** 

Dardanelli et al., 2008

2009

al., 2008

Remans et al., 2007

2008a

2008b

Yadegari et al., 2010

Unknown Figuereido et

**Increase (%) compared to single rhizobial inoculation** 

18-35 and 20- 70 in RDW; 29 and 28 in SDW under non saline and saline conditions, respectively

35 in SDW; 35 in NN under non-saline conditions; and 39 in SDW; 63 in RDW under saline conditions

50 in NN; 40 in N uptake in non-drought stress

30 in NN; 20 in SDW; 30-45 in RDW

25 in NN; 13 in SDW; 74 in seed yield

**Legume Bacterial system**

*R. tropici/etli - A. brasilense* 

*R. etli - C. balustinum* 

*R. tropici - Paenibacillus polymyxa* 

*Rhizobium* spp. *- A. brasilense /B. subtilis/P. putida*

*Rhizobium* spp*. - A. brasilense* 

*Rhizobium* spp*. - P. fluorescens /A. lipoferum* 

Common bean (*Phaseolus vulgaris*)


Legume Crops, Importance and Use of Bacterial Inoculation to Increase Production 229

Probably, the most reported mechanism that explains the improved rhizobia-legume association by other PGPB is the production of plant-hormones (phytohormones), such as gibberellic acid (GA3) or auxin-type phytohormones (mainly indole-3-acetic acid; IAA; Beattie, 2006). That is the case for *Pseudomonas* (Egamberdieva et al. 2010; Malik & Sindhu, 2011) and *Azospirillum* (Cassán et al., 2009; Dobbelaere et al., 2001; Okon, 1994; Perrig et al., 2007). For information about IAA production and effects, we recommend Baca & Elmerich (2007) and Spaepen et al. (2007). However, the main mechanism involved in improved rhizobia-legume association is still under investigation (Dobbelaere & Okon, 2007). It might be possible that multiple mechanisms, rather than only one are acting. This is known as the

Many other signal molecules or analogues involved in plant-rhizobia communication, different than phytohormones but produced by the non-rhizobial co-inoculant strain, have been implicated in the rhizobia-plant association (Lucas-Garcia et al., 2004b; Mañero et al., 2003). Some direct evidence suggests that the presence of *Pseudomonas* spp. (Parmar & Dadarwal, 1999) and *Azospirillum* spp. cells (Burdman et al., 1996; Dardanelli et al., 2008, Volpin et al., 1996) induce the synthesis of flavonoids by roots of chickpea, common bean and alfalfa, in experiment of co-inoculation with rhizobia. Interestingly, it is not strictly necessary the presence of the bacteria, the application of bacteria-free exudates of symbiotic enhancers to the root exert similar effect that during bacterial-co-inoculation (Molla et al., 2001b). For example, the application of NFs analogues produced by *Serratia proteamaculans* 1-102 promotes soybeanbradyrhizobia nodulation and soybean growth (Bai et al., 2002b). The list of metabolites produced by symbiotic enhancers might become bigger: vitamins that may supplement the nutritional requirement of rhizobia (Marek-Kozaczuk & Skorupska, 2001); hydrolytic enzymes that assist during rhizobial penetration in the root hair, or attack phytopathogenic fungi (Egamberdieva et al., 2010; Sindhu & Dadarwal, 2001; Sindhu et al., 2002); or P-solubilizing acids that increase phosphorus availability (Elkoca et al., 2008). However, in most cases the mechanism underlying the plant growth promotion by co-inoculation is unknown (Bullied et

"Additive Hypothesis" (Bashan et al., 2004; Bashan & de-Bashan, 2010).

al., 2002; Goel et al., 2002; Lucas García et al., 2004a, 2004b; Vessey & Buss, 2002).

On average, an increase of 4-5% in crop yield has an important impact in agricultural production. The data obtained in different growth-systems (gnotobiotic laboratory conditions, hydroponics, greenhouse and field) shows that co-inoculation produces a major increase in legume yield compared with single inoculation (Table 2), overwhelming the

Inoculation and co-inoculation experiments must be done in field to provide a realistic assessment of the performance of a living-formulation in practical farming conditions. Table 2 shows examples of legume co-inoculation in field experiments. An increase of 74% in seed yield was detected when *Phaseolus vulgaris* was co-inoculated with *P. fluorescens* or *A. brasilense* compared with single-inoculation with *Rhizobium* spp. (Yadegari et al., 2010). As well, 14% total biomass chickpea yield was detected during co-inoculation with P-solubilizing *Bacillus* isolates compared with single-inoculation with *Rhizobium* sp (Elkoca et al., 2008). Vast areas of agricultural land are not appropriated for cropping because the soil has P-deficiency and the co-inoculation of legumes with rhizobia and P-solubilizing bacteria might supply nitrogen and phosphorus to these poor lands. The examples above provided show a huge increase in yield

**3.3 Increasing crop yield by co-inoculation** 

agronomic expectations.


Table 2. Ten years of studies on legume co-inoculation (2001-2011). Increase in legume symbiotic parameters and yield by co-inoculation compared to single-inoculation with rhizobia. Abbreviations are as follows: RDW: root dry weight; SDW: shoot dry weight; RL: root length; NN: nodule number; NFW: Nodule fresh weight; PDW: plant dry weight; PFW: plant fresh weight.

**Experiments done in** 

Greenhouse

30 in SDW Greenhouse IAA production

Hydroponic

Greenhouse (*Fusarium oxysporum* infected soils)

Laboratory and greenhouse

Laboratory and greenhouse

Greenhouse (sterile soil)

Greenhouse

**Proposed mechanism of action** 

Production of IAA and/or cellulase by *Pseudomonas* spp.

and increased root secretion of flavonoids

Antifungal activity by production of siderophores

Cross-utilization of siderophores produced by *Bacillu*s sp. and *Rhizobium*

> Reduced ethylene production

grain yield Greenhouse Unknown Tilak et al.,

**Reference** 

Egamberdieva et al., 2010

Star et al., 2011

Kumar et al., 2001

2009

2009

Rajendran et al., 2008

2006

Shaharoona et al., 2006

Unknown Mishra et al.,

Unknown Mishra et al.,

**Increase (%) compared to single rhizobial inoculation** 

70 in SDW; 60 in RDW; 30 in NN; 44 in Nuptake

nod gene induction and decreased in indoles content

1,3 in Pea DW; 0,5-0,69 in plants with disease

> 84 times in NN; 15 in SDW; 15 in RDW

73 in NN; 5 in SDW; 10-30 in RDW

50 in PFW; 300 in NN

73 in NN; 30 in

20 in total biomass; 48 in NN

Table 2. Ten years of studies on legume co-inoculation (2001-2011). Increase in legume symbiotic parameters and yield by co-inoculation compared to single-inoculation with rhizobia. Abbreviations are as follows: RDW: root dry weight; SDW: shoot dry weight; RL: root length; NN: nodule number; NFW: Nodule fresh weight; PDW: plant dry weight; PFW:

**Legume Bacterial system**

*R. galegae* bv. *orientalis - Pseudomonas* spp*.*

*R. leguminosarum*  bv*. viciae - A. brasilense* 

*R. leguminosarum bv viceae - P. fluorescens* 

*R. leguminosarum-B. thuringeinsis*

*R. leguminosarum-B. thuringeinsis*

*Rhizobium* sp.*- Bacillus* spp*.* 

*Rhizobium* sp. *- P.putida/ P. fluorescens/ B. cereus* 

*B. japonicum - P. putida* 

Galega

Vetch

Pea (*Pisum sativum* L.

Lentin

(*Lens* 

Pigeon pea

Mung bean

(*Vigna radiata* 

*L.)*

plant fresh weight.

(Cajanus cajan)

*culinaris*

L.)

cv. Capella)

(*Vicia sativa*)

(*Galega* 

*orientalis*)

Probably, the most reported mechanism that explains the improved rhizobia-legume association by other PGPB is the production of plant-hormones (phytohormones), such as gibberellic acid (GA3) or auxin-type phytohormones (mainly indole-3-acetic acid; IAA; Beattie, 2006). That is the case for *Pseudomonas* (Egamberdieva et al. 2010; Malik & Sindhu, 2011) and *Azospirillum* (Cassán et al., 2009; Dobbelaere et al., 2001; Okon, 1994; Perrig et al., 2007). For information about IAA production and effects, we recommend Baca & Elmerich (2007) and Spaepen et al. (2007). However, the main mechanism involved in improved rhizobia-legume association is still under investigation (Dobbelaere & Okon, 2007). It might be possible that multiple mechanisms, rather than only one are acting. This is known as the "Additive Hypothesis" (Bashan et al., 2004; Bashan & de-Bashan, 2010).

Many other signal molecules or analogues involved in plant-rhizobia communication, different than phytohormones but produced by the non-rhizobial co-inoculant strain, have been implicated in the rhizobia-plant association (Lucas-Garcia et al., 2004b; Mañero et al., 2003). Some direct evidence suggests that the presence of *Pseudomonas* spp. (Parmar & Dadarwal, 1999) and *Azospirillum* spp. cells (Burdman et al., 1996; Dardanelli et al., 2008, Volpin et al., 1996) induce the synthesis of flavonoids by roots of chickpea, common bean and alfalfa, in experiment of co-inoculation with rhizobia. Interestingly, it is not strictly necessary the presence of the bacteria, the application of bacteria-free exudates of symbiotic enhancers to the root exert similar effect that during bacterial-co-inoculation (Molla et al., 2001b). For example, the application of NFs analogues produced by *Serratia proteamaculans* 1-102 promotes soybeanbradyrhizobia nodulation and soybean growth (Bai et al., 2002b). The list of metabolites produced by symbiotic enhancers might become bigger: vitamins that may supplement the nutritional requirement of rhizobia (Marek-Kozaczuk & Skorupska, 2001); hydrolytic enzymes that assist during rhizobial penetration in the root hair, or attack phytopathogenic fungi (Egamberdieva et al., 2010; Sindhu & Dadarwal, 2001; Sindhu et al., 2002); or P-solubilizing acids that increase phosphorus availability (Elkoca et al., 2008). However, in most cases the mechanism underlying the plant growth promotion by co-inoculation is unknown (Bullied et al., 2002; Goel et al., 2002; Lucas García et al., 2004a, 2004b; Vessey & Buss, 2002).

#### **3.3 Increasing crop yield by co-inoculation**

On average, an increase of 4-5% in crop yield has an important impact in agricultural production. The data obtained in different growth-systems (gnotobiotic laboratory conditions, hydroponics, greenhouse and field) shows that co-inoculation produces a major increase in legume yield compared with single inoculation (Table 2), overwhelming the agronomic expectations.

Inoculation and co-inoculation experiments must be done in field to provide a realistic assessment of the performance of a living-formulation in practical farming conditions. Table 2 shows examples of legume co-inoculation in field experiments. An increase of 74% in seed yield was detected when *Phaseolus vulgaris* was co-inoculated with *P. fluorescens* or *A. brasilense* compared with single-inoculation with *Rhizobium* spp. (Yadegari et al., 2010). As well, 14% total biomass chickpea yield was detected during co-inoculation with P-solubilizing *Bacillus* isolates compared with single-inoculation with *Rhizobium* sp (Elkoca et al., 2008). Vast areas of agricultural land are not appropriated for cropping because the soil has P-deficiency and the co-inoculation of legumes with rhizobia and P-solubilizing bacteria might supply nitrogen and phosphorus to these poor lands. The examples above provided show a huge increase in yield

Legume Crops, Importance and Use of Bacterial Inoculation to Increase Production 231

Chickpea is the most largely produced pulse crop in India accounting for 40% of total pulse crops production, being the leading chickpea producing country in the world. India annually produces around 6 Million tons of chickpea and contributes of approximately 70% in the total world production. On the other hand, Brazil is the world leader in dry bean production (3.3 Million ton), followed by India (3.0 Millon ton) and China (1.9 Millon ton). All these countries belong to "the BRICs". In economics, BRIC is a grouping acronym that refers to Brazil, Russia, India and China, which are considered to be at a similar stage of newly advanced economic development. The BRIC thesis, by Goldman Sachs, recognizes that Brazil, Russia, India and China have changed their political systems to embrace global capitalism, and predicts that China and India, respectively, will become the dominant global suppliers of manufactured goods and services, while Brazil and Russia will become similarly dominant as suppliers of raw materials. In this scenario, of countries with growing world economies and important production and consumption of pulses, the development of new formulations based in bacterial consortiums are being encouraged. However, a major constraint for exploiting living-formulation technologies has been that most farmers are not

Some bacterial symbiotic enhancers are promising microorganisms that would be used for the design of new formulations. These formulations could contain different bacteria in one pack, ready for direct placing in the seed at planting. However, some manufacturers also produce formulations that do contain non-rhizobial PGPB, but that can be mixed with rhizobial-formulation at the moment of planting. Information on both kinds of formulations

Despite the great progress and the increasing interest in mixed formulations for legumes inoculation, there are few commercial products with different bacteria. Most of these products are based on *Bacillus* spp. *Azospirillum*-based inoculants are also abundant in the market, but most of them are available for non-legumes crops (Figueiredo et al., 2010). Most commercially available biofertilizers are biopesticides and biofunguicides, but they are not

The doubling time world's current growth is 54 years and we can expect the world's population to become 12 billion by 2054. This demographic growth has to be accompanied by an increase in food production. Thus, the humanity has to face a new challenge, by doing a good use of soils (Fedoroff et al., 2010; Godfray et al., 2010) and developing new technologies (Pretty, 2008), mainly based in eco-friendly microorganisms that control pest and improve plant growth. In such scenario, the use of biofertilizers, rhizobia or consortium of plant-beneficial microbes (rhizobia and symbiotic enhancers) in formulations provides a potential solution. The data showed in this chapter support that the design of new formulations with cooperative microbes might contribute to the growth improvement of legumes. The co-inoculation has a positive effect in growth stimulation of legume crops; however, we believe it is necessary

aware of the technology and its benefits.

is provided in Table 3.

described in this chapter.

**4. Concluding remarks** 

to continue studying this subject.

**3.4 New formulations: The use of bacterial consortium** 


during co-inoculation in field experiments, pointing the economically relevance of coinoculation practices in countries with high pulse crop production.

a – compared to single inoculation

b –the formulation contains both rhizobia and non-rhizobial PGPB in the same package c – recommended for all kind of legumes

d – recommended for many crops, including legumes

e – the formulation does not contain rhizobia, but it can be used with rhizobial-formulation

Table 3. Some available commercial formulations (containing two PGPB) for legume crops. Note: mycorrhiza and bio-control bacteria are not included in this list.

Chickpea is the most largely produced pulse crop in India accounting for 40% of total pulse crops production, being the leading chickpea producing country in the world. India annually produces around 6 Million tons of chickpea and contributes of approximately 70% in the total world production. On the other hand, Brazil is the world leader in dry bean production (3.3 Million ton), followed by India (3.0 Millon ton) and China (1.9 Millon ton). All these countries belong to "the BRICs". In economics, BRIC is a grouping acronym that refers to Brazil, Russia, India and China, which are considered to be at a similar stage of newly advanced economic development. The BRIC thesis, by Goldman Sachs, recognizes that Brazil, Russia, India and China have changed their political systems to embrace global capitalism, and predicts that China and India, respectively, will become the dominant global suppliers of manufactured goods and services, while Brazil and Russia will become similarly dominant as suppliers of raw materials. In this scenario, of countries with growing world economies and important production and consumption of pulses, the development of new formulations based in bacterial consortiums are being encouraged. However, a major constraint for exploiting living-formulation technologies has been that most farmers are not aware of the technology and its benefits.

#### **3.4 New formulations: The use of bacterial consortium**

Some bacterial symbiotic enhancers are promising microorganisms that would be used for the design of new formulations. These formulations could contain different bacteria in one pack, ready for direct placing in the seed at planting. However, some manufacturers also produce formulations that do contain non-rhizobial PGPB, but that can be mixed with rhizobial-formulation at the moment of planting. Information on both kinds of formulations is provided in Table 3.

Despite the great progress and the increasing interest in mixed formulations for legumes inoculation, there are few commercial products with different bacteria. Most of these products are based on *Bacillus* spp. *Azospirillum*-based inoculants are also abundant in the market, but most of them are available for non-legumes crops (Figueiredo et al., 2010). Most commercially available biofertilizers are biopesticides and biofunguicides, but they are not described in this chapter.

### **4. Concluding remarks**

230 Crop Plant

during co-inoculation in field experiments, pointing the economically relevance of co-

<sup>b</sup>*rasilense* soybean Co-inoculant 10 www.nitrosoil.com.ar

*B. megaterium* All cropsd Inoculante 10 www.rajshreesugars.com

*aurantia* All crops Inoculant 10-20 www.manidharmabiotech.com

b –the formulation contains both rhizobia and non-rhizobial PGPB in the same package c – recommended for all kind of legumes

Note: mycorrhiza and bio-control bacteria are not included in this list.

e – the formulation does not contain rhizobia, but it can be used with rhizobial-formulation

Table 3. Some available commercial formulations (containing two PGPB) for legume crops.

**Yield increase (%)a**

Co-inoculantb 4-6 www.histicknt.com

Co-inoculant 8-30 www.intxmicrobials.com

Undeclared www.manidharmabiotech.com

www.brettyoung.ca

soybean Co-inoculant 5-10 www.inoculantespalaversich.com

legumesc Co-inoculant Undeclared www.iabiotec.com

All crops Inoculant 10-15 www.gsfclimited.com

All crops Inoculant 20-30 www.varshabioscience.com

*(B.napus)* Inoculant Undeclared Banerjee & Yesmin, 2004

**Reference** 

inoculation practices in countries with high pulse crop production.

**Formulation**

**Bacteria Target** 

Rhizobia - *B. subtilis* 

Rhizobia - *A. brasilense* 

Rhizobia – *A*. *brasilense* - *P. fluorescens* 

Rhizobia – *A*.

Rhizobia - *B. megaterium* - *Saccharomyces cerevisiae* 

P-solubilizing bacteria (genus undeclared)

P-solubilizing bacteria (genus undeclared)

*Frateuria* 

*Delftia acidovorans*  **crop** 

Soybean; peanuts; dry beans

Soybean; peanut; pea; vetch

All

Canola

d – recommended for many crops, including legumes

a – compared to single inoculation

The doubling time world's current growth is 54 years and we can expect the world's population to become 12 billion by 2054. This demographic growth has to be accompanied by an increase in food production. Thus, the humanity has to face a new challenge, by doing a good use of soils (Fedoroff et al., 2010; Godfray et al., 2010) and developing new technologies (Pretty, 2008), mainly based in eco-friendly microorganisms that control pest and improve plant growth. In such scenario, the use of biofertilizers, rhizobia or consortium of plant-beneficial microbes (rhizobia and symbiotic enhancers) in formulations provides a potential solution. The data showed in this chapter support that the design of new formulations with cooperative microbes might contribute to the growth improvement of legumes. The co-inoculation has a positive effect in growth stimulation of legume crops; however, we believe it is necessary to continue studying this subject.

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### *Edited by Aakash Goyal*

This book provides us a thorough overview of Crop Plant with current advance in research. Divided into two section based on the chapters contents. Chapter 1 provides information about markers and next generation sequencing technology and its use. Chapter 2 is about how we can use Silicon for Drought tolerance. Chapter 3 is to deal with the major problem of rising CO2 and O3 causing environmental pollution. Chapter 4 covers the phenomena of RNAi and its use, application in crop science. Chapter 5 is a review for boron deficiency in soils and how to deal with it for better crops. Chapter 6-10 provide some information regarding recent works going on in crop science.

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Crop Plant

Crop Plant