**1. Introduction**

262 Gamma Radiation

[68] Seo, K. H., and J. F. Frank. 1999. Attachment of *Escherichia coli* 0157:H7 to lettuce leaf

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[70] Solomon, E.B., S. Yaron and K. R. Matthews. 2002. Transmission of *Escherichia coli*

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[74] Tauxe, R. V. 1997. Emerging food borne diseases: an evolving public health challenge.

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[77] USFDA. U. S. Food and Drug Administration.1999. Potential for infiltration, survival

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[79] Warner, G. 1997. Fresh packers await new food safety guidelines. Good fruit Grower

[80] WHO. 1981. Wholesomeness of irradiated foods. Technical Report Series 659. World

[82] WHO. 1999. High-dose irradiation: Wholesomeness of food irradiated with doses above

[83] Report of a joint FAO/IAEA/WHO study group. WHO technical report series 890.

[85] Williams, C., 1995. Healthy eating: clarifying advice about fruit and vegetables. Brit.

[86] Wimberly R.C., B. J. Vander, B. L Wells. 2003. The globalization of food and how

[87] Xuetong, Fan, A. Brendan A. Niemira, and A. Prakash. Irradiation of fresh fruit and

20of%20Fresh%20Fruits%20and%20Vegetables-IFT%20report. Food Tech.

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using confocal scanning laser microscopy. *J. Food Prot.* 6:3-9.

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and growth of human pathogens within fruits and vegetables.

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O157:H7 from contaminated manure and irrigation water to lettuce plant tissue and its subsequent internalization. *Applied and Environmental Microbiology* 68:397-

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M. Tatsuya, K. Yukio, and K. Shinichi. 2009. Effect of gamma-irradiation on the survival of Listeria monocytogenes and allergenicity of cherry tomatoes. Radiation

attached to the surface of cantaloupe and cell transfer to fresh-cut tissues during

O157:H7 with pre-harvest leaf lettuce upon exposure to contaminated irrigation

It is well known that ionizing radiation is currently a very important way to create genetic variability that is not exists in nature or that is not available to the breeder (Ahloowalia & Maluszynski, 2001; Lemus *et al.*, 2002). Therefore, there are many papers aimed to determine the best radiation dose to applied in plant breeding work. As a result it has been defined intervals gamma radiation useful for many cultivated species, though the determination of the radiosensitivity of tissues by exposure to different intensities of radiation (De la Fe *et al.*, 1996; Castillo *et al.*, 1997; Fuchs *et al.*, 2002; Lemus *et al.*, 2002; Fuentes *et al.*, 2004; Ramírez *et al.*, 2006). However, most studies have been conducted have been designed to evaluate the biological response to high doses of radiation, while in relatively few studies have used low doses to stimulate physiological processes (radiostimulation) although the ionizing radiation hormesis has been widely supported (Luckey, 1980). Hormesis is the excitation, or stimulation, by small doses of any agent in any sistem (Luckey, 2003). The beneficial effect of hormesis has been well documented in species of agricultural importance (Zaka *et al.*, 2004; Kim *et al.*, 2005). However, there is not enough information about its use in forestry. Although little is known about the basic nature of this phenomenon, Vaiserman (2010) had indicated the possible relationship between the hormesis and epigenetic effects. The application of low-dose ionizing radiation could produce in coniferous species hormetics radiostimulants effects through genetic and epigenetic changes that manifest as adaptive responses.

In Mexico and especially in many natural populations of conifers from Veracruz such as *Pinus hartwegii* Lindl., and *Abies religiosa* Kunth (Schltdl.) *et.* Cham., both located in Cofre de Perote, Ver., are seriously affected mainly by the high load of lethal alleles which are causing a serious reduction in reproductive rate and a significant decrease in the production and quality of its seed (Iglesias *et al.,* 2006).

Despite the usefulness of using ionizing radiation to increase the germination potential and generating useful mutations in forestry, there are not many references in the literature on the use of nuclear techniques in these species (Iglesias *et al.,* 2010). Therefore, in this

Current Importance and Potential Use of Low Doses of Gamma Radiation in Forest Species 265

Ionizing radiation can be quantified in terms of absorbed dose (D) which is the amount of ionizing radiation energy deposited per unit mass of irradiated material. Initially, the magnitude was measured in roentgens, called "radiation dose". A Roentgen is a unit of measure used to quantify the radiometric exposure, in other words, the total charge of ions released per unit mass of dry air at standard conditions of pressure and temperature, but now the most often unit used to quantify the biological effects of ionizing radiation is the gray (Gy), which is a unit derived from the international system of units to measure the absorbed dose of ionizing radiation for a certain material. One gray is equivalent to the absorption of one joule of radiation energy per kilogram of irradiated material. Radiation doses are divided into three broad categories:high (> 10 kGy), medium (1 to 10 kGy), and

Gamma rays are widely used for mutation induction in plants. Therefore many studies have been done on the dose-response effects of ionizing radiation, specifically gamma radiation on several growth and yield traits in plants with high-doses of ionizing radiation (Table 1).

*vitro*.

Some reports (Gunckel & Sparrow, 1961; Ikram *et al.*, 2010) have been shown higher exposures of gamma rays produce generally negative effects on plant growth and development although the effect of dose rate on mutation frequency might differs among plant species. These effects include cytological, anatomy, genetical, biochemical, physiological and morphogenetic changes in cells and tissues. Many changes in the plant cellular structure and metabolism e.g., dilation of thylakoid membranes, alteration in photosynthesis, modulation of the antioxidative system and accumulation of phenolic compounds had been documented in different plant species (Kim *et al.*, 2004; Wi *et al.*, 2005). Higher exposures of gamma rays usually produce inhibitor effects on Gymnosperm and Angiosperm seed germination (Kumari & Singh, 1996) whereas lower exposures produce

It is important define the threshold between high-doses, in several cases dangerous, and low-doses with stimulatory effects. A radiostimulant low-dose is defined as any doses from

metaphase.

**doses Effect Reference** 

Bassam & Simon (1996)

Bassam *et al.* (2000)

Chauhan *et al.* (2009)

Akgün & Tosun (2004)

Kim *et al.* (2005)

germination of carrot seeds.

Low doses stimulated the

Low doses increase the number of microtubes *in* 

structures by mutation.

Increase the frequency of cells in anaphase and

growth and stress resistance.

low (<1 kGy).

**Species Gamma rays** 

*Capsicum annuum* L. 2, 4, 8

*Solanum tuberosum* L. 2.5, 5, 10

*Secale montanum* 

Guss.

*Daucus carota* L. 0.5 and 1 kR Irradiation accelerated

and 16 Gy

and 15 Gy.

0,2,4,6,8,10,12,1 4,16,20,25 and 30 kR

*Beta vulgaris* L. 20 kR Induced abnormal floral

sometimes a stimulatory effect (Raghava & Raghava, 1989; Thapa, 1999).

Table 2. Examples of hormetic effects in plants by low doses of ionizing radiation.

chapter will be give a review on the use of low doses of ionizing radiation on forest species, and it will perform a particular consideration to the effect of low doses of gamma radiation on germination and growth of some variables forest species such as *P. hartwegii* and *A. religiosa*.

## **2. Uses of low doses of ionizing radiation on plant species**

Ionizing radiation is defined as the energy that propagates in the form of photons (X-rays and γ) or in the form of subatomic particles (α, β, neutrons and protons). Among them, gamma rays have been reported to be the most efficient ionizing radiation of creating mutants in plants. Gamma rays belonging to ionizing radiation group are the most energetic form of electromagnetic radiation (Ikram *et al.*, 2010). This kinds of rays possesses the energy level from 10 keV to several hundred kiloelectron volts, and they are considered as the most penetrating physical mutagenic agent in comparison to other radiation source such as alpha and beta rays (Kovács & Keresztes, 2002). Like other ionining radiation gamma rays interacts with atoms or molecules to produce free radicals in cells. These radicals can induce high mutation in plants because it could produce serious cell damage or afectations in important plant cells components (Kovács & Keresztes, 2002).


Table 1. Application of high-doses of ionizing radiation in plant breeding.

chapter will be give a review on the use of low doses of ionizing radiation on forest species, and it will perform a particular consideration to the effect of low doses of gamma radiation on germination and growth of some variables forest species such as *P. hartwegii*

Ionizing radiation is defined as the energy that propagates in the form of photons (X-rays and γ) or in the form of subatomic particles (α, β, neutrons and protons). Among them, gamma rays have been reported to be the most efficient ionizing radiation of creating mutants in plants. Gamma rays belonging to ionizing radiation group are the most energetic form of electromagnetic radiation (Ikram *et al.*, 2010). This kinds of rays possesses the energy level from 10 keV to several hundred kiloelectron volts, and they are considered as the most penetrating physical mutagenic agent in comparison to other radiation source such as alpha and beta rays (Kovács & Keresztes, 2002). Like other ionining radiation gamma rays interacts with atoms or molecules to produce free radicals in cells. These radicals can induce high mutation in plants because it could produce serious cell damage or afectations in

Wilczek. 40 - 80 kR Increase polygenic variability. Sangwan & Singh

**doses Effects Reference** 

Improve the germination

Percentage of abnormal seedlings increased with increase in radiation dose.

Irradiated seeds showd superiority over control population for several traits.

Mutagenic effects by intergenomic chromosomal

rearrangements.

above the non-irradiated rice.

The regeneration rate decrease with increase in the total dose of radiation.

rate of the seeds.

tillering and grain yield. Iqbal (1980)

seedling height and tillering. Iqbal (1980)

growth rate. Gehring (1985)

(1977)

Bhargava & Khalatkar (1987)

(2003)

(2003)

(2009)

(2010)

Amjad & Akbar

Ali & Manzor

Yamaguchi *et al.* (2008)

Singh & Balyan

Kumar & Singh

**2. Uses of low doses of ionizing radiation on plant species** 

important plant cells components (Kovács & Keresztes, 2002).

*Triticum aestivum* L. 0.5–7 kR Stimulatory effect on height,

*Sorghum vulgare* L. 1–10 kR Large reduction in mean

*Salix nigra* Marsh. 0.1 – 100 kR Low doses increasing the

and 50 kR

and 100 kR

15, 30 and 60 Gy

and 40 kR

and 800 Gy

Table 1. Application of high-doses of ionizing radiation in plant breeding.

Basmati rice varieties 150 – 300 Gy Increase of total spikelets

**Species Gamma rays** 

*Tectona grandis* L. f. 10, 20, 30, 40

*Allium cepa* L. 10, 20, 40, 80,

*Triticum aestivum* L. 10, 20, 30

*Sesamum indicum* L. 200, 400, 600

and *A. religiosa*.

*Vigna radiata* (L.)

*Chrysanthemum morifolium* cv.

Ionizing radiation can be quantified in terms of absorbed dose (D) which is the amount of ionizing radiation energy deposited per unit mass of irradiated material. Initially, the magnitude was measured in roentgens, called "radiation dose". A Roentgen is a unit of measure used to quantify the radiometric exposure, in other words, the total charge of ions released per unit mass of dry air at standard conditions of pressure and temperature, but now the most often unit used to quantify the biological effects of ionizing radiation is the gray (Gy), which is a unit derived from the international system of units to measure the absorbed dose of ionizing radiation for a certain material. One gray is equivalent to the absorption of one joule of radiation energy per kilogram of irradiated material. Radiation doses are divided into three broad categories:high (> 10 kGy), medium (1 to 10 kGy), and low (<1 kGy).

Gamma rays are widely used for mutation induction in plants. Therefore many studies have been done on the dose-response effects of ionizing radiation, specifically gamma radiation on several growth and yield traits in plants with high-doses of ionizing radiation (Table 1).


Table 2. Examples of hormetic effects in plants by low doses of ionizing radiation.

Some reports (Gunckel & Sparrow, 1961; Ikram *et al.*, 2010) have been shown higher exposures of gamma rays produce generally negative effects on plant growth and development although the effect of dose rate on mutation frequency might differs among plant species. These effects include cytological, anatomy, genetical, biochemical, physiological and morphogenetic changes in cells and tissues. Many changes in the plant cellular structure and metabolism e.g., dilation of thylakoid membranes, alteration in photosynthesis, modulation of the antioxidative system and accumulation of phenolic compounds had been documented in different plant species (Kim *et al.*, 2004; Wi *et al.*, 2005). Higher exposures of gamma rays usually produce inhibitor effects on Gymnosperm and Angiosperm seed germination (Kumari & Singh, 1996) whereas lower exposures produce sometimes a stimulatory effect (Raghava & Raghava, 1989; Thapa, 1999).

It is important define the threshold between high-doses, in several cases dangerous, and low-doses with stimulatory effects. A radiostimulant low-dose is defined as any doses from

Current Importance and Potential Use of Low Doses of Gamma Radiation in Forest Species 267

Recent developments in biotechnology—especially in understanding the structure and function of plant genomes—confirms *in vitro* mutation induction as one of the most efficient and cost-effective tools for functional genomics projects dealing with both forward and reverse genetics strategies (Jain, 2001; Shu & Lagoda, 2007). The high number of research reports suggests also that mutagenesis in combination with tissue culture has high potential in plant breeding programs. It has been indicated (Maluszynski *et al*., 1995), that the use of tissue culture techniques can overcome some of the limitations in the application of mutation techniques; these are the lack of effective mutant screening techniques and the unrealistically large but necessary size of the mutated population, calculated on the basis of an expected mutation frequency for a desired trait. The determination of radio-sensitivity tests, irradiation with optimal doses and multiplication of irradiated material through in vitro mutation techniques has assumed a new dimension (Ahloowalia & Maluszynski, 2001). An example of setting the boundary between and tissue damage from ionizing radiation was shown by Fuchs *et al.* (2002). These authors found in callus culture of *Saccharum sp*. (sugarcane), dose of greater than 4 kR of gamma radiation eliminated any possibility to induce an organogenetic process in this tissue. In this case, the hormetic threshold (2 to 4 kR) was much lower that applied to seeds of other species (4 to 20 kR)

(Lemus *et al.*, 2002; Ramírez-Calderón *et al.*, 2003; González *et al.*, 2004).

longevity, and reproduction.

**3. Hormesis and molecular mechanisms of the adaptive response** 

The hormesis term comes from greek meaning "to excite". Exposure of sublethal doses of ionizing radiation can induce protective mechanisms against a subsequent higher dose irradiation. So, the hormesis is the excitement and stimulation by small doses of any agent on any system. Luckey (1980), in his book entitled "Hormesis", documented thousand experiments where fungi and other lower life forms were seen to prosper with doses of radiation exceeding their normal background exposures with ionizing radiation. In a second book entitled "Radiation Hormesis" (Luckey, 1991) examined hundreds of studies on animals and humans, showing that low levels of radiation were beneficial to health,

Many studies have been also indicated that pre-expose to low dose radiation (or some other genotoxic agent) can change radiosensitivity, reducing score of chromosomal aberrations, micronuclei and mutations. This phenomenon is called adaptation and could be related with defense mechanisms some of them have evolved to minimize genotoxic damage. One of these is induced radioresistance or adaptive response (AR). The term "adaptive response" usually means that a relatively small "conditioning" radiation dose induces increased radioresistance when the cells are irradiated with higher doses several hours later (Hillova & Drasil, 1967). Thus, radioadaptive response induction expresses the ability of low dose radiation to induce cellular changes that alter the level of subsequent radiation-induced or spontaneous damage (Amundson *et al*., 1999). The exposure to minimal stress inducing a very low level of damage can trigger an AR resulting in increased resistance to higher levels of the same or of other types of stress (Patra *et al*., 2003; Asad *et al*., 2004; Girigoswami & Ghosh, 2005; Yan *et al*., 2006). The AR could be considered a nonspecific phenomenon and have been confirmed but not explained by many studies. Adaptation after preexposure to

chronic or prolonged exposure to low-level radiation doses was not often described.

environmental radiation levels and the threshold that marks the boundary between positive and negative biological effect (Luckey, 2003). This radiostimulatory effects which has been observed in different plant species (Table 2), through the use of low-doses of ionizing gamma radiation could be considered an interesting alternative somewhat unexplored in agriculture and forestry practice.

It has been recognized low-doses of radiation promote increased of cell respiration, enzyme activation, increase in the threshold of lethal doses of radiation, increasing the production of reproductive structures, higher growth, early maturation, accelerated development and disease resistance (Luckey, 1980; 1998). However, most of the works done in this way have been addressed to find the boundaries between hormesis and tissue damage (Castillo *et al.*, 1997; Fuchs *et al.*, 2002; Lemus *et al.*, 2002; Fuentes *et al.*, 2004; Ramírez *et al.*, 2006). There is few researches have been conducted to evaluate the effect of the radiation on the full cycle of the organisms (Cepero *et al.*, 2002; Ramírez *et al.*, 2006). The results obtained in the works of radiostimulation or radiohormesis revealed increases (10-40%) in agricultural yields, seed germination, contents of carotenes and vitamin C in some vegetables and protein and fat in cereals, finally resistance to diseases and abiotic factors (González *et al.*, 2002; Vasilevski, 2003). On other hand, chronic radiation is another kind of irradiation treatment used to increase variation in different plant species (Sparow & Woodwell, 1962). This type of irradiation could produce at the plant population level, most severe effects on sexual reproduction because during and after meiosis: (1) nuclear volume is high; (2) chromosome number is reduced after meiosis; (3) the rate of nuclear division may be low, some species requiring two years between meiosis and full maturation of seeds; (4) meiotic pairing and reduction tend to enhance the damage wrought by aberrations which may survive in diploid somatic cells. In forestry was evaluated too the effects of chronic ionizing radiation of low intensity (3–15 r/20 hr day) over a period of several years on the reproductive capacity of the trees, floral abnormalities, as well as growth of their progenies (Mergen & Stairs, 1962). These authors were found for *Pinus rigida* a decrease in cone length and in seed germination and seedling height for plants grown from irradiated cones that was associated with an increase in the chronic gamma radiation accumulated by the trees. For *Quercus alba* was observed visual aberrations in flower morphology in trees receiving from 6 to 12 r/day and a decrease on survival percentage and height growth of seedlings with radiation level (Mergen & Stairs, 1962). In this case like others mutagenic treatments the relative dosage levels necessary to produce specified responses in growth rate, reproductive capacity or in degree of mortality vary greatly within a species.

Irradiation treatments performed at *in vitro* culture has been also employed to increase genetic variability and mutants as a potential source of new commercial cultivars (Rasheed *et al.*, 2003; Orbovic´ *et al.*, 2008). So, tissue culture techniques offer a wide choice of explants (initial plant material) for gamma radiation treatment (cells, tissues, somatic embryos and organs). These explants will give origin to complete plants composed of a few or even of one cell with a higher probability for find mutated cells. *In vitro* culture also allows for the handling of unlimited vegetative material for radiation treatment, aseptic and controlled *in vitro* selection, and micropropagation of selected variants (so called somaclones). In addition, according to Predieri (2001), tissue culture increases the efficiency of mutagenic treatments for variation induction, handling of large populations, use of ready selection methods, and rapid cloning of selected variants.

environmental radiation levels and the threshold that marks the boundary between positive and negative biological effect (Luckey, 2003). This radiostimulatory effects which has been observed in different plant species (Table 2), through the use of low-doses of ionizing gamma radiation could be considered an interesting alternative somewhat unexplored in

It has been recognized low-doses of radiation promote increased of cell respiration, enzyme activation, increase in the threshold of lethal doses of radiation, increasing the production of reproductive structures, higher growth, early maturation, accelerated development and disease resistance (Luckey, 1980; 1998). However, most of the works done in this way have been addressed to find the boundaries between hormesis and tissue damage (Castillo *et al.*, 1997; Fuchs *et al.*, 2002; Lemus *et al.*, 2002; Fuentes *et al.*, 2004; Ramírez *et al.*, 2006). There is few researches have been conducted to evaluate the effect of the radiation on the full cycle of the organisms (Cepero *et al.*, 2002; Ramírez *et al.*, 2006). The results obtained in the works of radiostimulation or radiohormesis revealed increases (10-40%) in agricultural yields, seed germination, contents of carotenes and vitamin C in some vegetables and protein and fat in cereals, finally resistance to diseases and abiotic factors (González *et al.*, 2002; Vasilevski, 2003). On other hand, chronic radiation is another kind of irradiation treatment used to increase variation in different plant species (Sparow & Woodwell, 1962). This type of irradiation could produce at the plant population level, most severe effects on sexual reproduction because during and after meiosis: (1) nuclear volume is high; (2) chromosome number is reduced after meiosis; (3) the rate of nuclear division may be low, some species requiring two years between meiosis and full maturation of seeds; (4) meiotic pairing and reduction tend to enhance the damage wrought by aberrations which may survive in diploid somatic cells. In forestry was evaluated too the effects of chronic ionizing radiation of low intensity (3–15 r/20 hr day) over a period of several years on the reproductive capacity of the trees, floral abnormalities, as well as growth of their progenies (Mergen & Stairs, 1962). These authors were found for *Pinus rigida* a decrease in cone length and in seed germination and seedling height for plants grown from irradiated cones that was associated with an increase in the chronic gamma radiation accumulated by the trees. For *Quercus alba* was observed visual aberrations in flower morphology in trees receiving from 6 to 12 r/day and a decrease on survival percentage and height growth of seedlings with radiation level (Mergen & Stairs, 1962). In this case like others mutagenic treatments the relative dosage levels necessary to produce specified responses in growth rate, reproductive capacity or in

Irradiation treatments performed at *in vitro* culture has been also employed to increase genetic variability and mutants as a potential source of new commercial cultivars (Rasheed *et al.*, 2003; Orbovic´ *et al.*, 2008). So, tissue culture techniques offer a wide choice of explants (initial plant material) for gamma radiation treatment (cells, tissues, somatic embryos and organs). These explants will give origin to complete plants composed of a few or even of one cell with a higher probability for find mutated cells. *In vitro* culture also allows for the handling of unlimited vegetative material for radiation treatment, aseptic and controlled *in vitro* selection, and micropropagation of selected variants (so called somaclones). In addition, according to Predieri (2001), tissue culture increases the efficiency of mutagenic treatments for variation induction, handling of large populations, use of ready selection

agriculture and forestry practice.

degree of mortality vary greatly within a species.

methods, and rapid cloning of selected variants.

Recent developments in biotechnology—especially in understanding the structure and function of plant genomes—confirms *in vitro* mutation induction as one of the most efficient and cost-effective tools for functional genomics projects dealing with both forward and reverse genetics strategies (Jain, 2001; Shu & Lagoda, 2007). The high number of research reports suggests also that mutagenesis in combination with tissue culture has high potential

in plant breeding programs. It has been indicated (Maluszynski *et al*., 1995), that the use of tissue culture techniques can overcome some of the limitations in the application of mutation techniques; these are the lack of effective mutant screening techniques and the unrealistically large but necessary size of the mutated population, calculated on the basis of an expected mutation frequency for a desired trait. The determination of radio-sensitivity tests, irradiation with optimal doses and multiplication of irradiated material through in vitro mutation techniques has assumed a new dimension (Ahloowalia & Maluszynski, 2001). An example of setting the boundary between and tissue damage from ionizing radiation was shown by Fuchs *et al.* (2002). These authors found in callus culture of *Saccharum sp*. (sugarcane), dose of greater than 4 kR of gamma radiation eliminated any possibility to induce an organogenetic process in this tissue. In this case, the hormetic threshold (2 to 4 kR) was much lower that applied to seeds of other species (4 to 20 kR) (Lemus *et al.*, 2002; Ramírez-Calderón *et al.*, 2003; González *et al.*, 2004).
