Androgenesis Induced in Nicotiana alata and the Effect of Gamma Irradiation

Nicotiana alata anthers cultured on different modified media based on MS, MT and N were used to obtain haploid plants through direct and indirect ways. The haploid plants resulting on MS medium ranged from 52-80%, on MT medium ranged from 32-52% and on N medium ranged from 28-44%. Accordingly, the best medium used for haploid induction was MS supplemented with 0.2 mgl NAA + 0.5 mgl KIN. On the other hand, MS medium supplemented with 0.4 mgl NAA + 0.5 mgl KIN or 1.0 mgl BAP + 0.5 mgl NAA were the best mediums for callus induction and plant regeneration, respectively. Morphologically, the leaf size, stem highest and diameter, flower size and diameter, anther length and number were about 67% of the diploid plants growth. Irradiated anthers with doses of 0, 2.5, 5, 7.5, 10, 15, 20 and 25 Gy caused reducing the number of haploid plants with increasing gamma radiation dose. For the haploid plants irradiated with same doses, the mortality percentage of bud survival was increasing with increasing gamma radiation dose. The irradiated callus with doses of 0, 5, 10, 15 and 20 Gy was affected negatively on growth rate and morphology. Proline content in irradiated plantlets increased with increasing gamma radiation dose. As well, total soluble protein content was increased with gamma irradiation up to 10 Gy. However, the higher doses caused a severe decrease of total soluble proteins. The production of proline and total soluble proteins in haploid plants were 48.6% and 69.5%, respectively comparing with diploid plants.


Introduction
Nicotiana alata is an ornamental plant, member from Solanaceae family and the diploid cells contain 18 chromosomes.Tobacco is an ideal plant for obtaining haploid cultures in direct way.Tobacco cultures produce an explosion of haploids, which are now used in hybridization processes.Some authors stimulated the production of female gametes (gynogenesis) or male gametes (androgenesis) in haploid individuals produced directly.In androgenesis, which is carried out only in vitro, vegetative or generative nuclei from pollen grains are stimulated to develop haploid plants without fertilization.The literature on in vitro androgenesis (Bhojwani and Razdan, 1986;Pierik, 1979) clearly shows that species from the Solanaceae family are capable for regeneration of haploids from isolated anthers.Haploid plants can be obtained by isolation of anthers in vitro in two ways: directly, with formation of embryo from the pollen grains (microspore), and indirectly, with callus development and formation of haploid embryoids or adventive buds (Pierik, 1979).Gamma rays are often used on plants in developing varieties that are agriculturally an economically important and have high productivity potential (Jain et al., 1998).They are useful for mutations in breeding programs and in vitro mutagenesis in order to develop required features of plants and increase the genetic variability.Many mutant varieties, which are resistant to biotic and abiotic stress and with high quality, have been developed (Jain et al., 1998).Several attempts of mutagenic treatment on cultured anthers have been reported in higher plants (Ling et al., 1991;MacDonald et al., 1988;Sangwan and Sangwan, 1986).However, the protocol of mutation induction at haploid level has not been established.
Gamma rays belong to ionizing radiation and interact to atoms or molecules to produce free radicals in cells.These radicals can damage or modify important components of plant cells and have been reported to affect differentially the morphology, anatomy, biochemistry and physiology of plants depending on the irradiation level.These effects include 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 (Kim et al., 2004;Kovacs and Keresztes, 2002;Wi et al., 2005).
The aim of this work was to investigate some composition of culture media to induce haploid plants through direct and indirect ways and the effect of gamma rays at different stages of microspore development in Nicotiana alata anther culture.

Materials and methods
The anthers of Nicotiana alata were excised from flower buds and surface sterilized by dipping in Clorox (30%) for ten minutes followed by three times rinses in sterile distilled water.Anthers were aseptically removed and placed on nutrient medium and cultured for 8 weeks at 25± 2 ° C.They were then exposed to 16/8 photoperiod (3,000 lux).

Determination of the ploidy level
Cytologically, immature floral buds and root meristems were collected from test plants, fixed in Carnoy's fluid and stored at 4 °C.Slides were prepared using the squash method and pollen mother cells were stained using acetocarmine according to Ramana and Parkken (1967).
Morphologically, plantlets derived from anther culture, cultivated in sterile jars containing peat moss and sand with ratio 1:1.Plastic caps were removed and covered with plastic boring sheets.After one week the plastic cover sheets were removed and the anthers were left in growth chamber one week before transfer the green house (Fig. 1).
Irradiation was carried out with 60 Co source at the dose rate 1 Krad/ min at National Centre for Radiation Research and Technology, Cairo, Egypt.
The proline content was estimated according to Batels et al. (1973) in irradiated and non-irradiated plantlets.
The total soluble protein in irradiated and non-irradiated plantlets was estimated according to Bradford (1976).

Results and discussions
Over 43 modified culture media based on those of Murashige and Skoog (1962)-MS, Murashige and Tuker (1969)-MT and (Nitsch, 1969)-N were used to obtain haploid plants through direct way with formation of embryoid by isolation of anthers in vitro (microspore) and through indirect way with callus development and formation of haploid embryoid or adventive buds.
Data in Table 1 illustrated that all the composition media used for haploid induction in Nicotiana alata were positive, but the percentage of haploid plants was variable, dependent on the type of medium and hormone combinations (direct way).Twenty five anthers were tested for each culture medium.The results showed that higher haploid plant numbers were on MS medium, whereas the haploid plants produced a range from 13 to 20 anther cultures (52% -80%), consequently MT medium with a range of 8 to 13 anther cultures (32% -52%) and the last performant was N medium with a of range 7-11 anther culture 68 Fig. 2. The effect of MS medium with different BAP and NAA concentrations on plant regeneration (28% -44%).Accordingly, the best results for haploid plants induction were observed with MS medium, especially with NAA and KIN addition.Therefore, MS medium supplemented with 0.2 mgl -l NAA + 0.5 mgl -l KIN were used for further experiments.
On the other hand, the induction of haploid plants through callus culture (indirect way) was also promising.MS medium was used for callus induction and plant regeneration  The MS medium supplemented with 0.4 mgl -l NAA + 0.5 mgl -l KIN gave the best results for callus induction.The obtained calli were transferred to regeneration medium, using MS medium containing different hormones combination (BAP and/or NAA).The regeneration of plants was observed after 40 days and ranged between 10 to 33 plantlets/callus (Fig. 2).The best combination of hormones used with MS medium for plant regeneration was 1.0 mgl -l BAP + 0.5 mgl -l NAA.The ability to form callus mainly depends on the physiological state of the donor plants and the season during which the anthers are explanted.It has been reported that flower buds which have developed after plant regeneration give rise to pollen grains of flower androgenetic ability (Maheshvari et al., 1980;Shtereva et al., 1998;Zhou, 1996).Microspore developmental stage at the time of anther excision is one factor determining the induction of androgenesis.Some researchers indicate early meiosis as the Fig. 5a.Effect of gamma radiation doses on anthers (to produce haploid plants) Fig. 5b.The effect of gamma irradiation on anther optimal stage, while according to others it is the uninucleate stage (Sasmita et al., 2001;Summers et al., 1992;Zagoraska et al., 1982).The growth regulator specific action depended both on genotype and physiological state of the donor plant.A similar tendency of phytohormone influence on organogenesis was also observed by Dewi et al. (2004), Miceska (2011), Shtereva et al. (1998).
The regenerated plants can be obtained by isolation of anther in vitro, whether by direct or indirect way, therefore it was investigated morphologically and cytologically to confirm the haploidy.
Morphologically, it has been proved by observation and measurements that the highest haploid plant was of 104 cm comparing with diploid plant (155 cm); measurements were also made for steam diameter, leaf size, flower size and diameter, anther length and number.The haploid plants presented about 67% growth of the diploid plants (Figs. 3 and 6b).
Cytologically, with used squash method for each pollen grains and root meristem examination, the pollen grains in haploid plants were invalid.However, the root meristem investigation confirmed the haploid level got half of 69 Fig. 6b.The difference between diploid (D), haploid (H) and irradiated haploid leaves  chromosomes number set (Fig. 4).These results are in agreement with Zagorska et al. (1998) and Miceska (2011).
The various techniques for anther culture and induction of androgenesis are summarized by Chlyah et al. (1990) and Summers (1997) for tomato and Vagera (1990) for pepper.

Anther radio-sensitivity in Nicotiana alata
The investigation of produced haploid plants from anther culture by direct way with sensitivity gamma radiation doses (0.0, 2.5, 5, 7.5, 10, 15, 20 and 25 Gy) proved that irradiated anthers decreased the number of haploid plants and also shoot length with increasing gamma radiation dose (Figs.5a  and b).As well as when haploid segments were irradiated with same doses it was observed the same results (Table 2, Figs.6a and b).On the other hand, the effect of gamma radiation doses (5, 10, 15 and 20 Gy) on callus morphology and growth rate was investigated.The callus morphology changed from friable to massive greenish and the callus growth rate decreased with increasing gamma radiation dose (Fig. 7).However, the irradiated callus failed to regenerate Fig. 8. Effect of gamma radiation doses on total soluble protein in haploid plants plants.The results showed by the radiation sensitivity test based on survival percentage of irradiated and non-irradiated plantlets that a significant reduction in survival percentage was observed with increasing gamma dosage.These results were in accordance with radiation sensitivity test done by Hasegawa et al. (1995) for tobacco, El-Fiki (1997) for potato, El-Fiki et al. (2005a and b) for alfalfa, Norfadzrin et al. (2007) for tomato and okra and Kiong et al. (2008) for Orthosiphon stamineus.

Proline content estimation
Proline content in irradiated plants with doses of 2.5, 5, 7.5, 10, 15, 20 and 25 Gy and non-irradiated plants was estimated.The results showed that the proline content was increased with increasing gamma radiation dose (Fig. 8).The haploid plants contained 0.18 mg/ 100 g fresh weight proline, while diploid plants had 0.37 mg/ 100 g fresh weight (48.6%).

Total soluble protein
Total soluble protein was estimated in irradiated plants with different doses (2.5,5,7.5,10,15,20 and 25 Gy) and nonirradiated plants.The results demonstrated that the total soluble protein was increased with increasing gamma radiation dose up to 10 Gy.However, the higher doses have had a negative impact on total soluble protein, as they began to decline until it reached the lowest for the dose of 25 Gy (Fig. 9).The amount of total soluble proteins in haploid plants was equivalent to 69.5% compared to the amount found in diploid plants.The total soluble proteins in diploid plants was 1.41 mg/ 100 g fresh weight, while the haploid plants were containing 0.98 mg/ 100 g fresh weight total soluble proteins.
The most crucial function of plant cell is to respond to gamma stress by developing defense mechanisms.This defense may be affected by alteration in the pattern of gene expression (Corthals et al., 2000), which may led to modulation of certain metabolic and defensive pathways (Zolla et al., 2003).Owing to gene expression altered under gamma stress, qualitative and quantitative changes in total soluble protein contents were obvious in the study of Corthals et al. (2000).These proteins play an important role in signal transduction, antioxidative defense, antifreezing, heat shock, metal binding, antipathogenesis or osmolyte synthesis, which are essential to a plant's function and growth (El-Fiki et al., 2003, 2004;Gygi et al., 1999).

Conclusions
Nicotiana alata was considered a model plant for production of haploid plants.Through direct and indirect ways, haploid plants were obtained.Gamma radiation doses have had a negative impact of haploid production, while the irradiated callus did not have positive effect on plant regeneration.The haploid plants irradiated with gamma rays and cultured on MS medium had a negative effect on vitality and growth rate.
Fig. 1.Adaptation stages of haploid plants

Fig. 4 .
Fig. 4. The pollen grains in diploid and haploid plants and cells from root meristem of a haploid regeneration

Fig. 6a .
Fig. 6a.Effect of gamma radiation doses on % of bud survival in haploid plants

Table 1 .
Composition of culture media

Table 2 .
Effect of gamma irradiation on in vivo Nicotiana alata haploid plants