EFFECT OF GENOTYPES AND THEIR INTERACTION WITH 2 , 4 DICHLOROPHENOXY ACETIC ACID ( 2 , 4-D ) LEVELS ON WHEAT ' S IMMATURE EMBRYO CULTURE RESPONSE REHAB

heat is one of the most important cereal crops in the world. Therefore, it has been extensively investigated with respect to plant regeneration from in vitro culture. Shoot regeneration is of crucial importance in the realization of the potential of cell and tissue culture techniques for plant improvement (Purnhauser et al., 1987). Tissue culture technique also offers creation of variation through somaclonal and gametoclonal variations. These variations could be exploited for crop improvement program. Therefore, plant regeneration from callus cultures could provide useful new germplasm for plant breeding program. In vitro regeneration of wheat is possible from different explants such as mature and immature embryos, seeds, endosperm, leaves, shoot bases and root tips (Sarker and Biswas, 2002). Among them the immature embryo was reported as the best organ for callus induction and shoot regeneration (Arzani and Mirodjagh, 1999; Hou et al., 1997; Wu et al., 2002; Pellegrineschi et al., 2004). Callus induction and regeneration potential are affected by cultivars, explants and carbohydrate sources, plant growth regulators, basal salts of culture medium and culture conditions. Cultivars and culture medium play an important role in callus induction and plant regeneration in wheat (Ozias-Akins and Vasil, 1982 and 1983; He et al., 1986; Armstrong et al., 1987; Ben Amer et al., 1992; Machii et al., 1998).


EFFECT OF GENOTYPES AND THEIR INTERACTION WITH 2,4 DICHLOROPHENOXY ACETIC ACID (2,4-D) LEVELS ON WHEAT ' S IMMATURE EMBRYO CULTURE RESPONSE
heat is one of the most important cereal crops in the world. Therefore, it has been extensively investigated with respect to plant regeneration from in vitro culture. Shoot regeneration is of crucial importance in the realization of the potential of cell and tissue culture techniques for plant improvement (Purnhauser et al., 1987). Tissue culture technique also offers creation of variation through somaclonal and gametoclonal variations. These variations could be exploited for crop improvement program. Therefore, plant regeneration from callus cultures could provide useful new germplasm for plant breeding program. In vitro regeneration of wheat is possible from different explants such as mature and immature embryos, seeds, endosperm, leaves, shoot bases and root tips (Sarker and Biswas, 2002). Among them the immature embryo was reported as the best organ for callus induction and shoot regeneration (Arzani and Mirodjagh, 1999;Hou et al., 1997;Wu et al., 2002;Pellegrineschi et al., 2004). Callus induction and regeneration potential are affected by cultivars, explants and carbohydrate sources, plant growth regulators, basal salts of culture medium and culture conditions. Cultivars and culture medium play an important role in callus induction and plant regeneration in wheat (Ozias-Akins andVasil, 1982 and1983;He et al., 1986;Armstrong et al., 1987;Ben Amer et al., 1992;Machii et al., 1998).
Several studies have been reported on the effect of 2, 4-D on callus induction and growth from explants of graminious species (Conger et al., 1978;Deambrogio and Dale, 1980;Lu et al., 1982;Thomas and Scott, 1985;Fladung and Hesselbach, 1986;Kamil, 2002;Sikandar et al. 2007;Rahman et al., 2008;Afzal et al., 2010). In most of these studies, an optimal concentration of 2,4-D for callus induction and growth was investigated which varied with the species.
Thus, this investigation aimed to study the effects of genotype, levels of 2,4-D and their interaction on immature embryo induction in wheat. In addition, to partition the phenotypic variances presented in this process to its components and subsequently, estimation of heritability percentages for in vitro traits.

Plant material
In this investigation six wheat varieties were used. Three varieties were Sakha 8, Sakha 93 and Sakha 94 belong to the species (Triticum aestivum, L.) and another three varieties were Sohag 3, Benysweif 4 and Benysweif 5 belongs to the species (Triticum durum L.). All these varieties were supplied by Field Crops Research Institute (FCRI), Agriculture Research Center (ARC), Giza, Egypt.

Embryo culture procedure
During winter season (2010/2011), seeds of these varieties were cultivated at the Experimental Station, Faculty of Agriculture, Mansoura University, Egypt. At the flowering stage, all varieties were bagged to keep them self-pollinated in order to increase seeds. The flowing season 2011/2012, selfed seeds from these varieties were sown. Young spikes were collected about 15 days post anthesis from each genotype. Immature caryopses were surface sterilized under sterile conditions by immersing them for one minute in 75% ethanol followed by immersion in 0.1% mercuric chloride solution with 2 drops of Tween 20 as a wetting agent for 20 minutes. Then, it was rinsed three times in sterile double distilled water. Immature embryos were excised from each grain aseptically and cultured with the scutellm side up on induction medium under sterile conditions. The induction medium used in this study was MS medium, which recommended by Murashige and Skoog (1962) containing 3% sucrose and sup-plemented with three different concentrations of 2,4-dichlorophenoxy acetic acid (2,4-D) as the following: A: MS medium with 2 mg/l 2,4-D B: MS medium with 4 mg/l 2,4-D C: MS medium with 6 mg/l 2,4-D The cultures were incubated in darkness at 25C ± 2C for four weeks. Then, the total number of responding embryo, the total number of calli and the fresh weight of calli were recorded. The produced calli were transferred to regeneration medium with 1 mg/l 1-Naphthylacetic acid (NAA) and 0.5 mg/l kinetin. The cultures were kept under 16hour's illumination (fluorescent light) at 22C ± 2C for four weeks. The regenerable calli, which have green shoot primordial were counted and transferred to the same regeneration medium under the same condition for shoot development for four weeks. When the regenerated plants were about 5-8 cm length and have normal roots and shoots were carefully pulled up from the containers and their roots were washed with tap water to remove the remained parts of medium. Then, the total number of plantlet, plant weight, plant length, root length and number of tiller were recorded. These plants were sown in 12 cm plastic pots and kept in a growth room at 20C, R.H 80-90% and 16 h photoperiod for four weeks.

Experimental design
The experiments were designed as a complete randomized for the wheat gen-otypes used under three different levels of 2,4-D. Each treatment was replicated three times. The specific MS medium for each treatment was distributed into culture Petri dishes (12 cm inner diameter); each one contained 20 ml of medium. The culture Petri dishes were autoclaved at 121C, 15 pis for 20 min. One Petri dish containing 10 sterilized immature embryos is considered as one of experimental unit.

RESULTS AND DISCUSSION
As can be seen in Table (1), test of significance on the mean squares of genotypes at the three levels of 2,4-D, which indicated the presence of significant differences between these genotypes for all in vitro traits with respect to the three 2,4-D levels except for responding embryos and number of regenerated plants at B level as well as number of tillers per plant at A and B levels. However, replications of mean squares were not significant in most of occasions, indicating that there was no difference between spikes, which were collected from each genotype of immature embryos. In addition, the data which were obtained from the three 2,4-D levels for varieties were set up in a combined analysis of variance and the obtained results are presented in Table (2). Significance testes on the mean squares of genotype were highly significant for all studied in vitro traits. These traits were: responding embryos (R.E.) (Fig. 1 Table (3). The results showed that no specific variety was superior or inferior for all studied traits at different levels of 2,4-D. However, the six varieties at level A, regarding durum wheat, the greatest means of responding embryo, plant weight and number of tiller were observed in Sohag 3 with means of 0.957, 0.858 and 2.020, respectively, but greatest mean of plant weight was observed in Benysweif 4, while the greatest means values for callus weight, number of calli, number of green point, number of plants, plant length and root length were observed in Benysweif 5. Regarding aestivum wheat, the greatest values for responding embryo, number of plants and number of tiller were observed in Sakha 93 with means of 0.833, 0.277 and 1.567, respectively and the greatest values for callus weight and number of calli were observed in Sakha 94 with means 0.083 and 6.633, respectively. The greatest values for number of green point, plant weight, plant length and root length were detected in Sakha 8 with means of 2.643, 0.784, 17.083 and 4.847, respectively. Although, the greatest values for responding embryo and number of tiller were recorded in Sohag 3 with means of 0.957 and 2.020, respectively, the greatest values for plant weight were observed in Sohag 3 and Benysweif 4 with the same mean value which equal to 0.858. In addition, the greatest values for callus weight, number of plants, plant length and root length were observed in Benysweif 5 with means of 0.111, 0.553, 22.760 and 7.230, respectively. Regarding the number of calli and number of green point Sakha 8 seemed to be the best variety with means of 6.633 and 2.643, respectively. On the other hand, the lowest mean values for responding embryo and number of calli were observed in Benysweif 4 with means of 0.677 and 3.177, respectively. The lowest means value for callus weight was recorded in Sakha 8 with mean 0.056 and the lowest means value for number of green point was observed in Sohag 3 with mean 0.567, whereas lowest means values for number of plants, plant length and number of tiller were observed in Sakha 94 with means of 0.087, 11.417 and 1.337, respectively. Sakha 93 gave the lowest means values for plant weight and root length with means of 0.408 and 1.893, respectively.
At level B, regarding durum wheat (Sohag 3, Benysweif 4 and Benysweif 5), although the greatest mean values for responding embryos, callus weight, number of green point, number of plants, plant weight, plant length and root length were recorded in Benysweif 5, while the greatest mean value for number of tiller was recorded in Benysweif 4 but Sohag 3 was the greatest value for number of calli. For aestivum wheat (Sakha 8, Sakha 93 and Sakha 94) the greatest values for responding embryo, number of calli, plant weight, plant length and root length were recorded in Sakha 94, while the greatest values for number of green points, number of plants and number of tiller were recorded in Sakha 8. Whereas, Sakha 93 gives the greatest value for callus weight. The greatest overall values for responding embryo and plant weight were recorded in Sakha 94, while the greatest values for callus weight, number of green points, number of plants, plant length and root length were recorded in Benysweif 5 and the greatest value for number of tiller was recorded in Benysweif 4. Whereas, the greatest value for number of calli was recorded in Sohag 3. On the other hand, the lowest overall for responding embryo and number of green points were observed in Sohag 3. While, Sakha 8 was recorded the lowest value for callus weight but Sakha 93 were the lowest values for number of calli, plant weight, plant length, root length and number of tillers per plant. Whereas, Sakha 94 was the lowest value for number of plants. In general these values ranged from 0.943 to 1.00 for Sohag 3 and Sakha 94, respectively with respect to responding embryos, while it ranged from 0.480 to 3.243 in the case of regeneration ability (green point percentages) for Sohag 3 and Benysweif 5, respectively. These findings indicated that the variety Sohag 3 was the inferior one among the studied varieties for immature embryos culture purpose with respect to level B.
Regarding level C, although the greatest mean values for responding embryo, number of green points, and root length were recorded in Benysweif 5 with percentages values of 1.00, 4.04 and 7.36, respectively, Benysweif 4 was the best for number of plants, plant weight, plant length and number of tillers with percentages values of 0.29, 0.94, 20.24 and 2.47, respectively. Whereas, Sohag 3 showed the same percentage value with Benyseweif 5 in the case of responding embryos and it was the best one for callus weight with percentage value of 0.16. On the other hand, Sakha 93 and Sakha 94 seemed to be the inferior varieties for regeneration ability which recorded the lowest percentages values for number of plants, plant weight, plant height, root length and number of tillers per plant with respect to MS basal medium supplemented with 6 mg/l 2,4-D (C level).
Since, these genotypes which included (Triticum aestivum L.) and (Triticum durum L.) varieties gave different performance with different 2,4-D levels as observed in Table (4) for most of studied in vitro traits. Therfore, the combined data over the three 2,4-D levels could be more precise to present information concerning the behavior of these genotypes. For that, the average means of six varieties were determined from the data combined over the three levels of 2,4-D and the obtained results are shown in Table ( In general, from the previous results it could be observed that durum varieties are more suitable for immature embryo culture compared to aestivum varieties with respect to the genotypes used in this study. These results could be in line with the results obtained by Ahmed and Allam (2003). They found that the hybrid pro-embryos of hexaploid wheat x rye were much more responsive than those of tetraploid x rye. Nasircilar et al. (2006) reported that among the T. aestivum cultivars, Yakar had the highest regeneration capacity in two induction media. In T. durum cultivars, Kiziltan gave the highest regeneration capacity on MS+2,4-D medium and Yilmaz gave the highest regeneration capacity on MS+NAA medium.
Owing to the 2,4-D levels effect as observed earlier, it could be more informative to average the performances of all studied genotypes over each level. Therefore, the average means of the three 2,4-D levels over all genotypes for all in vitro studied traits are presented in Table  (5). The results indicated that the greatest means values were observed in the level (C) for all studied traits except number of green points. These findings revealed that the addition of 6 mg/l 2,4-D to MS nutrient medium and 1 mg/l NAA and 0.5 mg/l Kinetin in regeneration medium could be the best concentrations for immature embryo culture purpose in wheat. In this respect, Hassanien et al. (2000) reported that the highest concentration of the auxin (6 mg/l) gave the best callus induction and plantlet regeneration in wheat. Sarkar and Biswas (2002) indicated that the MS medium supplemented with 6.0 mg/l of 2,4-D showed the best response for callus induction from mature wheat embryos. Rahman et al. (2008) found that maximum number of calli were produced on MS medium supplemented with 6.0 mg/l of 2,4-D. But comparatively larger calli were produced on MS medium supplemented with 4.0 mg/l of 2,4-D. Mahmood et al. (2012) indicated that cultivars AS-2002 and GA-2002 produced maximum number of calli on induction media comprising 4 mg/l 2,4-D; while, cv. Chakwal-50 performed best at 6 mg/l of 2,4-D while, Shah et al. (2003) found that good callus formation was obtained on MS medium containing 3.5 mg/l 2,4-D, Raziuddin et al. (2010) found that MS medium containing 2 mg l -1 2, 4-D produced the greatest number of calli. Whereas, El-Wafa and Ismail (1999) reported that the application of 2 mg 2,4-D/litre gave the greatest response for all studied characters.
The genetic variation and heritability in broad (H b %) sense were estimated within each 2,4-D levels for all in vitro studied traits and the obtained results are presented in Table (6). The results revealed that the magnitude of genetic variation was positive for all in vitro traits at the three levels of 2,4-D. The results showed that genetic variation was high for all traits with respect to the three levels of 2,4-D (2 mg, 4mg and 6 mg/l) except for responding embryos and number of plants at level B (4 mg/l) and number of tillers/plant at levels A and B. These results confirmed by the values of heritability, which ranged from 66.67 to 98.7% for callus weight at level A and plant weight at level C, respectively. Moreover, the results also showed that heritability in broad sense (H b %) was high (more than 85%) for all traits in the high concentration of 2,4-D (6 mg/l). The results showed that increasing concentrations of 2,4-D led to increase heritability for most in vitro traits. Thus, it could be more precise to estimate these parameter from the data combined over three levels of 2,4-D concentrations. Therefore, the relative magnitudes of these parameters were estimated for all studied in vitro traits from the combined data over the three levels and the obtained results are shown in Table (7). The results revealed that the genetic variations were high and positive for most of studied traits. This finding is emphasized by the heritability values, which were more than 60% for all studied traits except for responding embryos, plant weight and number of tillers/plant. In addition, the values of genetic by levels interaction variations were high and positive in all studied in vitro traits, especially in the cases of responding embryos, plant weight and number of tillers/plant. These findings explain the low values of heritability in these traits as well as the genes control these traits are highly affected by media composition. In this respect, El-Wafa (1999) found that the genetic variance was extremely large in magnitude relative to environmental variance for the studied traits. In addition, the traits showed high heritability, El-Wafa (1999) reported that the genetic variance relative to environmental variance was large in magnitude for studied traits. Heritability (broadsense) and phenotypic and genotypic coefficients of variability were high for fresh weight of callus at one month and increasing weight of callus at one month. Lange et al. (1998) found that estimated genetic variance and heritability in the broad sense had very distinct values among the crosses of the traits.

Genotypic and phenotypic correlation
Genotypic (r g ) and phenotypic (r ph ) correlation among different pairs of the traits were studied from the data combined over the three levels of 2,4-D concentrations and the obtained results are shown in

SUMMARY
The main objective of this investigation was to study the effects of genotype, levels of 2,4-D (2,4-dichlorophynoxyacetic acid) concentrations and their interaction on immature embryo induction in wheat. In addition, to partition the phenotypic variances presented in this process to its components and subsequently, estimating the heritability percentages for in vitro traits. The genetic materials used in this study were six varieties. Three varieties belong to the species (Triticum aestivum L.) and another three varieties belong to the species (Triticum durum L.). These genotypes were in vitro evaluated for immature embryos culture ability at three levels of 2,4-D. The results showed the presence of significant differences among genotypes at the three levels of 2,4-D for all in vitro traits except for responding embryos and number of regenerated plants at B level as well as number of tillers per plant at A and B levels. Furthermore, levels and genotype x levels interaction of mean squares were highly significant with respect to all the studied in vitro traits except in a few cases. This indicates that these genotypes gave different response at different 2,4-D levels. The results revealed that greatest percentages values for responding embryo, green points, number of plantlets, plant height and root length were recorded for Benysweif 5. In addition, Sohag 3 was the best variety for responding embryos, callus weight and number of calli. Whereas, the greatest values for plant weight and number of tiller were observed in Benysweif 4. Therefore, durum varieties are more suitable for immature embryo culture compared to aestivum varieties. Furthermore, the results revealed that the genetic variation were high and positive for most of studied traits. This finding is emphasized by the heritability values, which were more than 60% for all studied traits except for responding embryos, plant weight and number of tillers/plant. In addition, the values of genetic levels of interaction variations were high and positive in all studied in vitro traits. This finding explains the low values of heritability in these traits as well as the genes control these traits are highly affected by media compositions.
Plant regeneration from cultured immature embryos and inflorescences of Triticum aestivum L.