**4. Current and potential use of low ionizing radiation in forestry: A case study**

At present there are virtually no studies of hormesis by ionizing radiation in forest species. Most of the work focused radiation treatment of species of agronomic interest since they have shorter lifetime and germination time, the tissues of this type of seeds have a greater amount of water, which maximizes the effect of radiation; and the generation of seedlings is much easier in the herbaceous form.

One of the few jobs that exist in tree species was conducted with *Araucaria angustifollia* (Bert) O. Kuntze (Ferreira *et al*., 1980). The study showed a hormetic effect on seed germination and seedling growth at low doses of gamma radiation (0.1 to 0.4 kR). This first study showed the effectiveness of ionizing radiation to improve seed germination in species of trees, one of the main agronomic traits for forest management.

*Abies religiosa* (fir) and *Pinus hartwegii* are two conifer species that develop on the National Park Cofre de Perote, Veracruz, Mexico. Both have great ecological (*P. harwegii* is the conifer species taking place at higher altitudes) and economical importance (in particular, the fir is valuable for its timber, trementine production and as an ornamental Christmas trees). These forests have protective functions to other resources to cushion the effects of environmental pollution and contribute as a regulation of the hydrological cycle (Solís, 1994).

In Veracruz, these populations develop principally in the National Park Cofre de Perote and Pico de Orizaba between 2 800 y 3 500 m.a.s.l. in 17°30' to 20°00' N and 97°104' W (Manzanilla 1974; Sánchez-Velásquez & Pineda-López 1993). Both species have been seriously affected by fire and logging clandestine, has resulted in a reduction of the effective size of the same low viability and high percentage of abortive seeds, and a significant decrease in reproductive rate, apparently due to manifestation of the phenomenon of inbreeding depression, common in coniferous species (Williams & Savolaienen, 1996). But, in both species, is common a low reproductive rate (Franklin, 1974).

Current Importance and Potential Use of Low Doses of Gamma Radiation in Forest Species 271

Fig. 2. Effect of the gamma radiation on seed germination in *Abies religiosa* and *Pinus* 

thickened short roots and therefore were less vigorous and did not survive.

on the characters of the seedling height and number of needles (Figure 3 a,b).

was most effective to induce a similar effect on the fir.

seedlings (b), *in Pinus hartwegii* and *Abies religiosa.* 

According to these results the radiostimulatory effect was more pronounced in *Pinus hartwegii* since treatment of 2 Gy resulted in a high percentage of germination. 5 Gy dose

These results are consistent with the radiostimulatory effect observed by Rudolph (1979) and Sokolov *et al.* (1998) to evaluate the germination of seeds of *Pinus bankasiana* and *P. sylvestris*, respectively. On the other hand, showed that, like as detected by Nwachukwu *et al*. (1994) and Lemus *et al*. (2002); the frequency of mutation increased with the percentages of germination. The few seedlings that managed to germinate at the highest doses of gamma radiation showed

Low doses of gamma radiation used in both species showed similar radiostimulatory effects

**b)**

**a)**

Fig. 3. Effect of the low-doses of gamma radiation on height (a) and number of needles in

*hartwegii*.

Despite the potential utility of the low doses of ionizing radiation for the induction of hormetic effects in these species, to date have not implemented these techniques to increase the germination potential and generating useful mutation in forestry. Therefore, we study the mutagenic effect of gamma radiation and know whether low doses of radiation can have a stimulatory effect on germination and development of *P. hartwegii* and *A. religiosa*. Twenty-five to thirty cones of *A. religiosa* and *P. hartwegii* were collected in populations that develop on 3510 m.a.s.l., in the locality "El Conejo", both located in the National Park "Cofre de Perote" Veracruz, Mexico. These populations are fragmented and have been affected by significant changes in land use to agricultural crops (Sánchez-Velásquez *et al*., 1991).

To apply the mutagenic treatments, seeds were extracted from the cones collected, and kept under controlled conditions at a relative humidity of 8%. Two replicates of 100 seeds of each species at low doses (2, 5, 10, 15 and 20 Gy) of gamma radiation (Co60) were made. The seeds for irradiation were placed at a distance of 80 cm (for a field of 30 x 30 cm), with the help of a head of the Cobalt 60 Unit (Theraton 780e) in the "Centro de Cancerologia de Xalapa, Veracruz, Mexico; and plastic tray, which was secured in a cage at 50 cm from the radiation source (Figure 1). Subsequently, the irradiated seeds of each species and their corresponding controls were planted separately under greenhouse conditions in trays containing a mixture of forest soil and sand in a 1:1 ratio. In both cases we used a randomized complete block design with three replications. Seedlings were transferred to plastic bags for study.

Fig. 1. Cobalt 60 Unit (Theraton 780e), used for irradiation of seeds of *Abies religiosa* and *Pinus hartwegii*, located in Centro Estatal de Cancerología de Xalapa, Veracruz, Mex.

To evaluate the effect of the applied radiation dose was counted the number of seeds germinated at 90 days for each dose studied; the percentage of germination was calculated. Was considered germinated seeds showed a greater than 5 mm radical length. At 45 days, was evaluated the height and number of needles of each seedling. Height (cm) was measured with a millimeter rule from the base of the stem of the root to the terminal bud. From the measurements we calculated the percentages of germination, plant height and number of needles as a relative value with respect to control.

The results showed a significant radiostimulating effect on the germination of the seeds of *Abies religiosa* and *Pinus hartwegii* treated with low doses of gamma radiation (Figure 2).

Despite the potential utility of the low doses of ionizing radiation for the induction of hormetic effects in these species, to date have not implemented these techniques to increase the germination potential and generating useful mutation in forestry. Therefore, we study the mutagenic effect of gamma radiation and know whether low doses of radiation can have a stimulatory effect on germination and development of *P. hartwegii* and *A. religiosa*. Twenty-five to thirty cones of *A. religiosa* and *P. hartwegii* were collected in populations that develop on 3510 m.a.s.l., in the locality "El Conejo", both located in the National Park "Cofre de Perote" Veracruz, Mexico. These populations are fragmented and have been affected by

significant changes in land use to agricultural crops (Sánchez-Velásquez *et al*., 1991).

design with three replications. Seedlings were transferred to plastic bags for study.

Fig. 1. Cobalt 60 Unit (Theraton 780e), used for irradiation of seeds of *Abies religiosa* and *Pinus hartwegii*, located in Centro Estatal de Cancerología de Xalapa, Veracruz, Mex.

number of needles as a relative value with respect to control.

To evaluate the effect of the applied radiation dose was counted the number of seeds germinated at 90 days for each dose studied; the percentage of germination was calculated. Was considered germinated seeds showed a greater than 5 mm radical length. At 45 days, was evaluated the height and number of needles of each seedling. Height (cm) was measured with a millimeter rule from the base of the stem of the root to the terminal bud. From the measurements we calculated the percentages of germination, plant height and

The results showed a significant radiostimulating effect on the germination of the seeds of *Abies religiosa* and *Pinus hartwegii* treated with low doses of gamma radiation (Figure 2).

To apply the mutagenic treatments, seeds were extracted from the cones collected, and kept under controlled conditions at a relative humidity of 8%. Two replicates of 100 seeds of each species at low doses (2, 5, 10, 15 and 20 Gy) of gamma radiation (Co60) were made. The seeds for irradiation were placed at a distance of 80 cm (for a field of 30 x 30 cm), with the help of a head of the Cobalt 60 Unit (Theraton 780e) in the "Centro de Cancerologia de Xalapa, Veracruz, Mexico; and plastic tray, which was secured in a cage at 50 cm from the radiation source (Figure 1). Subsequently, the irradiated seeds of each species and their corresponding controls were planted separately under greenhouse conditions in trays containing a mixture of forest soil and sand in a 1:1 ratio. In both cases we used a randomized complete block

Fig. 2. Effect of the gamma radiation on seed germination in *Abies religiosa* and *Pinus hartwegii*.

According to these results the radiostimulatory effect was more pronounced in *Pinus hartwegii* since treatment of 2 Gy resulted in a high percentage of germination. 5 Gy dose was most effective to induce a similar effect on the fir.

These results are consistent with the radiostimulatory effect observed by Rudolph (1979) and Sokolov *et al.* (1998) to evaluate the germination of seeds of *Pinus bankasiana* and *P. sylvestris*, respectively. On the other hand, showed that, like as detected by Nwachukwu *et al*. (1994) and Lemus *et al*. (2002); the frequency of mutation increased with the percentages of germination. The few seedlings that managed to germinate at the highest doses of gamma radiation showed thickened short roots and therefore were less vigorous and did not survive.

Low doses of gamma radiation used in both species showed similar radiostimulatory effects on the characters of the seedling height and number of needles (Figure 3 a,b).

Fig. 3. Effect of the low-doses of gamma radiation on height (a) and number of needles in seedlings (b), *in Pinus hartwegii* and *Abies religiosa.* 

Current Importance and Potential Use of Low Doses of Gamma Radiation in Forest Species 273

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**6. References** 

As for the variable germination, was found at 2 Gy a radiostimulatory effect on height and number of needles of the seedlings of *P. hartwegii*. In fir, this effect is slightly higher.

In addition, there was a trend towards reduction in height and number of needles in both species as they increased the dose of radiation. This effect was more pronounced for the number of needles of *P. hartwegii* (Figure 3 b).

According to Olvera & West(1985); reducing the growth of seedlings generated from seeds treated with high doses of radiation is mainly due to the destruction of auxin and its precursors. It should be noted that the height variable is used in this type of study as the most sensitive indicator of radiation.

Based on our results consider the possibility of using the aforementioned dose to induce mutations that may be of interest in these species. However, all the applied doses produced a negative effect on the number of needles per plant, which are fundamental in the production of Christmas trees. Doses of 2, 5 and 10 Gy gave average values ranging from 3.3 to 6.8 needles per seedlings, well below the average control value, which was 27.7 in *P. hartwegii*. Contravention in the production because mutations are required to encourage a more fodder for a demanding market of this product.

It has been suggested in this regard that high doses of radiation cause damage that affects physiological character related to growth, especially with the number of needles. High doses of radiation can alter in a direct or indirect the DNA, causing damage of bases, single strand breaks and chromosomal alterations, serious and irreversible destruction of the membrane system of mitochondria and chloroplasts (Ladanova, 1993). However, it will take more repetitions to achieve seedlings with large needles and branches, or select for traditional breeding seedlings with large needles for future generations.

In summary, prolonged exposure to radiation by gamma rays produced a severe effect on almost all variables; this effect was greater in the seeds irradiated with 15 and 20 Gy. In the range of medium dose (5 Gy), radiation induced lesions can eventually lead to an important radiobiological response, which at the cellular level can alter the viability and even cause cell death (Ward, 1988).

This response was manifested by affectations in traits related to germination and seedling growth, as the germination percentage and number of needles that were most affected. On the other hand, not all variables were impacted in the same direction, since a dose of 5 Gy showed the presence of a radiopositive effect in the percentage of germination for fir, and at doses of 5 - 10 Gy there was a negative effect at the height of the plant and number of needles, with respect to control. However, when analyzing these results it is recommended to fully explore dose of 5 Gy for *A. religiosa*, and other below 2 Gy for *P. hartwegii*, in accordance with the sensitivity shown by this species to gamma radiation for improve the germination rate.
