Flowering of Sweet Cherries "*Prunus avium*" in Tunisia

*Thouraya Azizi-Gannouni and Youssef Ammari*

### **Abstract**

In Tunisia, the development of cherry growing is limited by two major constraints, namely, the chilling requirements and the self-incompatibility of some cultivars. In order to contribute to the development of this high added-value culture, which is capable to play an important socioeconomic role in rural and semi-forestry places, this study has set the main objective, characterization, and selection of best-suited cultivars to mild winter based on the blooming period. The plant materials used for this study are composed of the introduced cultivars, which are "Napoleon," "Van," "Moreau," "Sunburst," and "Stella," and unknown cultivars, which are "V1," "V2," "V3," "V4," and "V5," and a local one "Bouargoub." Differential behavior between cultivars was shown for phenological stages (budbreak, flowering, maturity, and leaf fall), and this behavior is dependent in some cases on the cold requirement [chilling requirements (CR)]. The local cultivar "Bouargoub" recorded the lowest "CR" with early flowering and maturity.

**Keywords:** behavior, chilling requirement, flowering, mild winter

#### **1. Introduction**

Sweet cherry tree is a hardy species capable of adapting to various soil and climatic conditions; but its development is surrounded by climatic and physiological constraints. The sweet cherry (Prunus avium L.) from Rosaceae family, with a number of chromosomes (2n = 2x = 16), is an allogamous species that is adopted to a self-incompatibility system to ensure cross-fertilization. Self-compatibility in sweet cherry occurs rarely in nature and, consequently, there are a reduced number of self-compatible cultivars [1]. Sweet cherry is the first fruit of summer season and is highly appreciated by consumers, cultivated for its edible fruits and its wood.

According to FAO [2], world production of sweet cherries has been estimated at 2,245,826 tones. The largest cherry-producing countries are Turkey, the United States, Iran, Italy, and Spain [2]. The cultivation of sweet cherries in Tunisia covers an area of about 961 ha [3]. This species is particularly cultivated in the region of northern Tunisia, where the winter is mild and spring frosts are rare. National production is estimated at 5187 tons [3]. Despite the favorable conditions, sweet cherry is poorly valued and only some regions practice this culture in small-scale along Tunisia.

The local cultivar is poorly valued, given the predominance of introduced cultivars which have high productivity and good adaptation to the mild North African climate. The characterization of this variety and the comparison with other introduced varieties is essential for its conservation. In this context, the present work is focused on the study of the phonological characteristics such as blooming stage of some cultivars of sweet cherry trees in relation to the conditions of the environment. Flowering is a determining factor in fruit production based on pollen self-incompatibility. The cultivation of cherry trees is limited in the north of Tunisia in the high altitudes to fill chill requirement. Therefore, this study is part of the evaluation, the development of genetic resources in fruit arboriculture in Tunisia, and the extension of the cultivation of cherry trees in regions at medium altitude.

For sweet cherry, like for other temperate-zone fruit species, when chilling requirements are not adequately satisfied, negative repercussions on productivity occur. Insufficient chilling can lead to erratic, delayed budbreak, and heterogeneous flowering. Chilling increased the flower size, pedicel length, and fruit set [4].

In many perennial species, it has been shown that increase in temperatures during the last dormant stage (autumn, winter) was responsible for advancing blooming dates, leading to an increased risk of damage caused by late frosts, phenological disorders, with a large spread of flowering dates and difficult synchronization of flowering with the activity of pollinators.

In a Mediterranean climate and specifically in Tunisia, development of sweet cherry growing (Prunus avium) shows several problems related to floral biology, chilling requirement, appearance of bud anomalies, and inconstant and extremely low yield**.**

The aim of our study was to investigate the blooming phenophase and the effect of temperature during flowering period on the fruit set and the production of 11 cultivars of sweet cherry in the climate condition of Tunisia, from which researchers and orchard managers will get reliable information for their study or planting.

#### **2. Experimental sites and plant materials**

The behavior of the different cultivars was monitored in three experimental sites located in three regions of northwestern Tunisia with different pedo-climatic characteristics:

The site of Ain-Draham is located at latitude 36°46′34″ North and longitude 8°41′05″ East, with an altitude of 800 m. The average annual rainfall was about 1040 mm. The lowest average temperature was about 6.08°C during February and the warmest was 26°C during July. The bioclimatic floor is humid superior with temperate winter. The site of Bousalem is at latitude 36°36′34″ North and longitude 8°58′17″ East, with an altitude of 127 m above sea level. The average annual rainfall was about 57.24 mm. The lowest average temperature was around 10.03°C during February and the highest temperature was 35.50°C during July. The bioclimatic floor is subhumid with temperate winter. The site of Tibar is at latitude 36°31′21″ North and longitude 9°06′22″ East, with an altitude of 328 m. The average annual rainfall was about 540 mm. The lowest average temperature was about 8.37°C during February and the hottest temperature was around 29°C during July–August. The bioclimatic floor is subhumid with mild winter.

The plant materials used in this study are composed of 11 cultivars of local and introduced sweet cherries (Prunus avium L.) of known and unknown origins. These cultivars are unequally distributed between the three experimental sites (**Table 1**).

The different studied flower traits are the length of the pistil (LPIST), the ovary area (SROV) and the number of stamens (NBET), length (Lopt) and width of petals (Larpt) and flower diameter (DFL), shape of petals (SHPE), and the arrangement of petals (ARPE).

**63**

*Flowering of Sweet Cherries "*Prunus avium*" in Tunisia DOI: http://dx.doi.org/10.5772/intechopen.93234*

**Sites**

**Cultivar Origin Ain-Draham Bousalem Tibar (In)** 

Bouargoub Tunisia + — — \*

Napoléon Germany + — + III S3S4 Van Canada + — + II S1S3 Moreau French + — + XVI S3S9 Sunburst Canada + — + \*\*SC S3S4' Stella Canada + — — \*\*SC S3s4'

V1 unknown — — + — XVI S3S9 V2 unknown — — + — \*\*SC S3S4' V3 unknown — — + — XVIII S1S9 V4 unknown — — + — \*\*SC S3S4' V5 unknown — — + — II S1S3

**compatibility groups\***

XLII S2S10

**S allele composition\***

with electronic scanning microscope (Leica).

*+, indicates that the variety is tested in the relevant site.*

*The S-genotype and incompatibility groups [5] according to Schuster [6].*

*Name, origin, distribution, and S-genotype of the studied cultivars per experimental site.*

*\**

**Table 1.**

*\*\*SC: Self Compatible.*

**3. Flowering of sweet cherry**

flowering and fruiting [8].

means were separated by the LSD test (α ≤ 0.05).

sion that flowering is dependent on warm temperatures.

The flowers were collected in full bloom by using five flowers per tree on five trees by cultivar and site. The different measurements were carried out using a vernier caliper for measuring the length and width of petals and flower diameter. However, the ovary area, pistil length, and number of stamens were carried out

• Statistical analyses were performed using SAS 9.1. ANOVA was carried out and

The transition from the vegetative state to a reproductive state is a crucial stage of development in fruit trees, and this transition is marked by floral induction. During the vegetative phase, the vegetative meristems produce leaves and stems necessary for the accumulation of sufficient reserves to eventually lead to the growth of the tree depending on its genotype and environmental conditions [7]. These meristems become inflorescential, producing flowers. The success of this sexual reproduction depends both on the sufficient accumulation of reserves and on a synchronous reproductive phase with optimal environmental conditions for

The response of flowering at room temperature is variable depending on the species and genotypes. Studies carried out on different accessions of *Arabidopsis* have shown that high temperature favors flowering [9], which leads to the conclu-

The trunk and branches carry spurs (**Figure 1**) called "bouquets of May" because their development is generally completed at the end of May. The flowers appear in all cases at the base of the annual shoots of the previous year, whether it is long shoots of the trunk and branches or bouquets of May. On a cherry tree a


*+, indicates that the variety is tested in the relevant site.*

*\* The S-genotype and incompatibility groups [5] according to Schuster [6].*

*\*\*SC: Self Compatible.*

#### **Table 1.**

Prunus

low yield**.**

characteristics:

introduced varieties is essential for its conservation. In this context, the present work is focused on the study of the phonological characteristics such as blooming stage of some cultivars of sweet cherry trees in relation to the conditions of the environment. Flowering is a determining factor in fruit production based on pollen self-incompatibility. The cultivation of cherry trees is limited in the north of Tunisia in the high altitudes to fill chill requirement. Therefore, this study is part of the evaluation, the development of genetic resources in fruit arboriculture in Tunisia, and the extension of the cultivation of cherry trees in regions at medium altitude. For sweet cherry, like for other temperate-zone fruit species, when chilling requirements are not adequately satisfied, negative repercussions on productivity occur. Insufficient chilling can lead to erratic, delayed budbreak, and heterogeneous flowering. Chilling increased the flower size, pedicel length, and fruit set [4].

In many perennial species, it has been shown that increase in temperatures during the last dormant stage (autumn, winter) was responsible for advancing blooming dates, leading to an increased risk of damage caused by late frosts, phenological disorders, with a large spread of flowering dates and difficult synchronization of

In a Mediterranean climate and specifically in Tunisia, development of sweet cherry growing (Prunus avium) shows several problems related to floral biology, chilling requirement, appearance of bud anomalies, and inconstant and extremely

The aim of our study was to investigate the blooming phenophase and the effect of temperature during flowering period on the fruit set and the production of 11 cultivars of sweet cherry in the climate condition of Tunisia, from which researchers and orchard managers will get reliable information for their study or planting.

The behavior of the different cultivars was monitored in three experimental sites located in three regions of northwestern Tunisia with different pedo-climatic

The site of Ain-Draham is located at latitude 36°46′34″ North and longitude 8°41′05″ East, with an altitude of 800 m. The average annual rainfall was about 1040 mm. The lowest average temperature was about 6.08°C during February and the warmest was 26°C during July. The bioclimatic floor is humid superior with temperate winter. The site of Bousalem is at latitude 36°36′34″ North and longitude 8°58′17″ East, with an altitude of 127 m above sea level. The average annual rainfall was about 57.24 mm. The lowest average temperature was around 10.03°C during February and the highest temperature was 35.50°C during July. The bioclimatic floor is subhumid with temperate winter. The site of Tibar is at latitude 36°31′21″ North and longitude 9°06′22″ East, with an altitude of 328 m. The average annual rainfall was about 540 mm. The lowest average temperature was about 8.37°C during February and the hottest temperature was around 29°C during July–August. The

The plant materials used in this study are composed of 11 cultivars of local and

The different studied flower traits are the length of the pistil (LPIST), the ovary area (SROV) and the number of stamens (NBET), length (Lopt) and width of petals (Larpt) and flower diameter (DFL), shape of petals (SHPE), and the arrange-

introduced sweet cherries (Prunus avium L.) of known and unknown origins. These cultivars are unequally distributed between the three experimental sites

flowering with the activity of pollinators.

**2. Experimental sites and plant materials**

bioclimatic floor is subhumid with mild winter.

**62**

(**Table 1**).

ment of petals (ARPE).

*Name, origin, distribution, and S-genotype of the studied cultivars per experimental site.*

The flowers were collected in full bloom by using five flowers per tree on five trees by cultivar and site. The different measurements were carried out using a vernier caliper for measuring the length and width of petals and flower diameter. However, the ovary area, pistil length, and number of stamens were carried out with electronic scanning microscope (Leica).

• Statistical analyses were performed using SAS 9.1. ANOVA was carried out and means were separated by the LSD test (α ≤ 0.05).

#### **3. Flowering of sweet cherry**

The transition from the vegetative state to a reproductive state is a crucial stage of development in fruit trees, and this transition is marked by floral induction. During the vegetative phase, the vegetative meristems produce leaves and stems necessary for the accumulation of sufficient reserves to eventually lead to the growth of the tree depending on its genotype and environmental conditions [7].

These meristems become inflorescential, producing flowers. The success of this sexual reproduction depends both on the sufficient accumulation of reserves and on a synchronous reproductive phase with optimal environmental conditions for flowering and fruiting [8].

The response of flowering at room temperature is variable depending on the species and genotypes. Studies carried out on different accessions of *Arabidopsis* have shown that high temperature favors flowering [9], which leads to the conclusion that flowering is dependent on warm temperatures.

The trunk and branches carry spurs (**Figure 1**) called "bouquets of May" because their development is generally completed at the end of May. The flowers appear in all cases at the base of the annual shoots of the previous year, whether it is long shoots of the trunk and branches or bouquets of May. On a cherry tree a

#### Prunus

**Figure 1.** *Spurs (bouquet of May) in Ain-Draham site.*

few years old, most of the flowering intended for fruit production is carried by the bouquets of May. The good development of these bouquets is very important to maintain a good quality of cherries production [10].

The flower of this genus is generally characterized by the following features: flower with five petals and five sepals, solitary carpel with a terminal style [11]. It is a hermaphrodite flower and the fruit is a drupe [12]. These drupes are most often edible and delicious but sometimes bitter or sour (cherries, sloes), more rarely toxic (fruits of the cherry laurel).

The development of flower buds is under biochemical control. This biochemical signal allows the tissue to change from the vegetative state to the reproductive state [13]. It occurs due to a balance between gibberellic acid, auxin, cytokinins, and ethylene-type hormones [14]. The floral initiation (sepals, petals, stamens, and pistil) of sweet cherry occurs after harvest [14].

### **3.1 Different characteristics of flowers traits in relation to the environment conditions**

The morphology of fruit species provides information on the adaptation and behavior of these species with regard to their environmental conditions. Indeed, the size of the flowers is generally considered to be the most important factor for pollinators.

For each experimental site, the results of the multiple comparisons of means for the different flower traits (**Figure 2**) are presented in **Table 2** and in the study of Azizi-Gannouni et al. [15].

Azizi-Gannouni et al. [15] showed significant variability in the number of stamens for the same variety between the two different sites (Ain-Draham and Tibar). The local cultivar "Bouargoub" has a longer pistil compared to other cultivars, while V4 (Bousalem site) recorded the shortest pistil (**Table 2**). Genotypic differences that control the dependence of these floral parameters on its genetic potential are to be excluded in view of the different behaviors of the same cultivar in pedoclimatically different experimental sites [15] (**Figure 3**).

Flowers with large diameters generally attract more pollinators [16]. This directly affects the pollination of flowers and therefore their setting and their production. Morphological monitoring carried out on all the cultivars in the three sites shows that in Bousalem site the flowers of small diameter have a high fruit production (**Figure 4**), which contradicts the results of the work of Johnson et al. [17] and Wetzstein et al. [18].

**65**

**Table 2.**

*Flowering of Sweet Cherries "*Prunus avium*" in Tunisia DOI: http://dx.doi.org/10.5772/intechopen.93234*

**Sites Cultivars DFL** 

Ain-Draham

**Figure 2.**

**(mm)**

*The different studied flower traits of cultivars in Ain-Draham site.*

**Lopt (mm)**

Moreau 25.80c 11.4c 10.2d Broad

Stella 41.60a 18.8a 14.94b Medium

Tibar Napoleon 44.12a 19.56a 18.9a Circular Intermediate

Bousalem V1 41a 18a 17a Circular Intermediate V2 33.6b 14.8b 14.7b Medium

V4 32.04b 15.02b 12.9c Medium

V5 43.8a 18.9a 17.26a Broad

*Different small letters in the same column indicate significantly different values within cultivars at* α *≤ 0.05.*

*Mean of the parameters measured on sweet cherry flowers (*Prunus avium *L.) at three sites.*

Moreau 39b 17.5b 16.78b Broad

Bouargoub 41.66a 19.33a 17.96a Broad

**Larpt (mm)**

Napoléon 34.60b 13.8b 12.2c Circular Intermediate Van 28.40c 11.2c 11.6c Circular Intermediate

Sunburst 40.40a 18.2a 11.8c Circular Disjoints free

Van 38.2b 16.6b 15.2c Circular Intermediate

Sunburst 29.16c 12.58c 9.78d Circular Disjoints free

V3 43.96a 19.48a 15.28b Circular Intermediate

**SHPE ARPE**

Disjoints free

Disjoints free

Overlapping

Disjoints free

Disjoints free

Disjoints free

Overlapping

obovate

obovate

obovate

obovate

obovate

obovate

obovate


#### **Figure 2.**

Prunus

**Figure 1.**

few years old, most of the flowering intended for fruit production is carried by the bouquets of May. The good development of these bouquets is very important to

The flower of this genus is generally characterized by the following features: flower with five petals and five sepals, solitary carpel with a terminal style [11]. It is a hermaphrodite flower and the fruit is a drupe [12]. These drupes are most often edible and delicious but sometimes bitter or sour (cherries, sloes), more rarely toxic

The development of flower buds is under biochemical control. This biochemical signal allows the tissue to change from the vegetative state to the reproductive state [13]. It occurs due to a balance between gibberellic acid, auxin, cytokinins, and ethylene-type hormones [14]. The floral initiation (sepals, petals, stamens, and

**3.1 Different characteristics of flowers traits in relation to the environment** 

The morphology of fruit species provides information on the adaptation and behavior of these species with regard to their environmental conditions. Indeed, the size of the flowers is generally considered to be the most important factor for

For each experimental site, the results of the multiple comparisons of means for the different flower traits (**Figure 2**) are presented in **Table 2** and in the study of

Azizi-Gannouni et al. [15] showed significant variability in the number of stamens for the same variety between the two different sites (Ain-Draham and Tibar). The local cultivar "Bouargoub" has a longer pistil compared to other cultivars, while V4 (Bousalem site) recorded the shortest pistil (**Table 2**). Genotypic differences that control the dependence of these floral parameters on its genetic potential are to be excluded in view of the different behaviors of the same cultivar in pedo-

Flowers with large diameters generally attract more pollinators [16]. This directly affects the pollination of flowers and therefore their setting and their production. Morphological monitoring carried out on all the cultivars in the three sites shows that in Bousalem site the flowers of small diameter have a high fruit production (**Figure 4**), which contradicts the results of the work of Johnson et al.

maintain a good quality of cherries production [10].

pistil) of sweet cherry occurs after harvest [14].

climatically different experimental sites [15] (**Figure 3**).

(fruits of the cherry laurel).

*Spurs (bouquet of May) in Ain-Draham site.*

**conditions**

Azizi-Gannouni et al. [15].

[17] and Wetzstein et al. [18].

pollinators.

**64**

*The different studied flower traits of cultivars in Ain-Draham site.*


#### **Table 2.**

*Mean of the parameters measured on sweet cherry flowers (*Prunus avium *L.) at three sites.*

**Figure 3.** *Shape of Petals of the four cultivars (*Prunus avium *L.) in Tibar site.*

**Figure 4.** *Yield (Kg/tree; Rdt) and Maximum temperature during blooming at Ain-Draham site.*

Furthermore, in the three study sites, the collected data show a significant effect of the site on the morphology and size of all the tested cultivars (identified and unidentified). This confirms the important effect of climatic conditions on this parameter, a result confirmed by the work of Niu et al. [19], who showed that the diameter of the flower is more influenced by daytime temperatures than by nighttime temperatures and this dimension has no relation to the difference of temperatures between day and night.

Whereas, in the Ain-Draham site, it is observed that the local cultivar "Bouargoub" has the largest flower diameter compared to "Van" and "Moreau" cultivars (**Table 2**). Likewise, it has the longest style but the smallest ovary surface with an intermediate number of stamens [15].

Lu [20] has shown that cultivars grown in a warm winter climate give flowers with longer styles than these grown in a cold winter climate, which contradicts the results of the present work (**Table 2**), where the cultivar "Napoleon" shows a longer style in Ain-Draham (climate with cold winters) than in Tibar (climate with mild winters).

**67**

the present [21].

*Flowering of Sweet Cherries "*Prunus avium*" in Tunisia DOI: http://dx.doi.org/10.5772/intechopen.93234*

as self-incompatible "V1," "V3," and "V5" [5].

of Tibar and Ain-Draham.

Site\*

**Table 3.**

*NS, not significant. \*Significant at 0.05. \*\*\*Significant at 0.001.*

the priority sites for in situ conservation.

**4. Climatic change and chilling requirement**

Referring to the hypothesis relating the floral diameter to pollen self-compatibility, our results show the invalidity of this assumption since the self-compatible cultivars "Sunburst," "Stella," and "Bouargoub" show large floral diameters as well

Site 303.92\*\*\* 363.73\*\*\* 854\*\*\* Cultivars 102.74\*\*\* 63.61\*\*\* 235\*\*\*

cultivars 334.71\*\*\* 348.13\*\*\* 258\*\*\*

**DFL Lopt Larpt**

The shape and arrangement of the petals do not change according to the pedo-

The variance analysis (**Table 3**) makes it possible to test for the different flower parameters, the effect of the cultivars, the effect of the sites, and the effect of the interaction "cultivar x sites." The inter-sites' analyses were done for the four commoncultivars (Napoleon, Van, Moreau, and Sunburst) at the two experimental sites

The effect of the interaction "cultivars x sites" was significant (*p* < 0.05 to *p* < 0.001) for the morphometric measured characteristics for the flower (**Table 3**). The "Genotype × Environment" interactions were observed for flowers' quantitative parameters [15] (**Table 2**). This indicates that the sources of variation are both genetic and environmental. In addition, the differences in altitude and soilclimatic conditions of the two study sites may explain the observed variations. In our study, all of the cultivars cultivated at the three sites showed the inter-varietal variability in flower traits and therefore these three geographical areas should be

Global warming, the phenomenon of sustainable rise in ocean and atmospheric temperatures, is the main form of climate change. Terrestrial temperature

Human activities are therefore the dominant cause of the warming observed over the last 50 years on Earth [22]. This climate change is already having consequences on the biodiversity and ecosystems [8]. Temperature is an influencing factor on the development and growth of plants. Climate change can therefore have a major impact on their phenology. Changes in the phenological stages such as the date of leaf coloring [23] and blooming [24] have already been observed. The

measurements made during the twentieth century show an increase in the average temperature. This warming would have taken place during the twentieth century in two phases, the first from 1910 to 1945, and the second from 1976 to

advance of the growing period has been linked to climate [25, 26].

climatic conditions of the experimental site, and it can be said that these two

**3.2 The effect of the environment and genotype on the floral parameters**

morphological traits depend on the genetic potential of the cultivar.

*Inter-site variance analysis: Values and significance of the F-test for flower traits.*


**Table 3.**

Prunus

**Figure 3.**

**66**

**Figure 4.**

tures between day and night.

with an intermediate number of stamens [15].

Furthermore, in the three study sites, the collected data show a significant effect

Lu [20] has shown that cultivars grown in a warm winter climate give flowers with longer styles than these grown in a cold winter climate, which contradicts the results of the present work (**Table 2**), where the cultivar "Napoleon" shows a longer style in Ain-Draham (climate with cold winters) than in Tibar (climate with mild winters).

of the site on the morphology and size of all the tested cultivars (identified and unidentified). This confirms the important effect of climatic conditions on this parameter, a result confirmed by the work of Niu et al. [19], who showed that the diameter of the flower is more influenced by daytime temperatures than by nighttime temperatures and this dimension has no relation to the difference of tempera-

*Yield (Kg/tree; Rdt) and Maximum temperature during blooming at Ain-Draham site.*

*Shape of Petals of the four cultivars (*Prunus avium *L.) in Tibar site.*

Whereas, in the Ain-Draham site, it is observed that the local cultivar "Bouargoub" has the largest flower diameter compared to "Van" and "Moreau" cultivars (**Table 2**). Likewise, it has the longest style but the smallest ovary surface *Inter-site variance analysis: Values and significance of the F-test for flower traits.*

Referring to the hypothesis relating the floral diameter to pollen self-compatibility, our results show the invalidity of this assumption since the self-compatible cultivars "Sunburst," "Stella," and "Bouargoub" show large floral diameters as well as self-incompatible "V1," "V3," and "V5" [5].

The shape and arrangement of the petals do not change according to the pedoclimatic conditions of the experimental site, and it can be said that these two morphological traits depend on the genetic potential of the cultivar.

#### **3.2 The effect of the environment and genotype on the floral parameters**

The variance analysis (**Table 3**) makes it possible to test for the different flower parameters, the effect of the cultivars, the effect of the sites, and the effect of the interaction "cultivar x sites." The inter-sites' analyses were done for the four commoncultivars (Napoleon, Van, Moreau, and Sunburst) at the two experimental sites of Tibar and Ain-Draham.

The effect of the interaction "cultivars x sites" was significant (*p* < 0.05 to *p* < 0.001) for the morphometric measured characteristics for the flower (**Table 3**).

The "Genotype × Environment" interactions were observed for flowers' quantitative parameters [15] (**Table 2**). This indicates that the sources of variation are both genetic and environmental. In addition, the differences in altitude and soilclimatic conditions of the two study sites may explain the observed variations. In our study, all of the cultivars cultivated at the three sites showed the inter-varietal variability in flower traits and therefore these three geographical areas should be the priority sites for in situ conservation.
