Responses of explant type of wheat (Triticum aestivum L.) genotypes to different tissue culture media

Wheat recalcitrance to in vitro culture is one of the most important and crucial steps for its genetic transformation, regenerate, composition of the media and genotype. Callus induction and regeneration potential of various explants, viz, mature embryo, immature embryo, endosperm supported (ES) mature embryo and shoot apical meristem, taken from the same investigated using Murashige and Skoog (MS) basal callus 2,4-dichlorophenoxyacetic acid (2,4-D) and regeneration media comprising various combinations of auxins and cytokinins. In vitro culture response of the explants taken from the same and media composition. Immature embryo culture of genotype AS-2002 exhibited the highest callusing ability (84.75 %) (78.50 %). Similarly, immature embryos cultured on MS basal exhibited the highest callusing ability of 90.83 and 88.33 %, regeneration medium comprising 0.4 mg/L 2,4-D, 1.0 mg/L potentials of 59.67 and 59.0 %, respectively. Appraisal of the supported mature embryo is the most suitable explant for plant .


INTRODUCTION
billion by 2020 (Wagiran et al., 2010). Efforts are being biotic and abiotic stresses through genetic transformation, out through gene gun and agrobacterium-mediated et al., the genes but the establishment of long-term cultures and Wheat (Triticum aestivum L.) is amongst the most recalcitrant cereals to in vitro et al., 2011), since its embryogenic callus formation and regeneration of transformed plants, besides genotypic and media composition (Chen et al., 2006;Mahmood et al., et al., 2014), are explant dependent (Sarker et al et al., 2014). In cereal tissue culture, not all cells express totipotency (Shah et al., 2003) and the choice of a favourable explant and genotype is required for the success in tissue culture approaches. Therefore, the success of any callus-based transformation strategy depends largely upon the regeneration capability of the target explant efforts are being made to increase their callusing and of phytohormones (Mahmood et al., 2012c). source for tissue culture and genetic transformation Vendruscolo et al et al., 2011;Mahmood et al et al., 2014; Yang et al embryos throughout the year is a space, time and labour intensive task and appears to be not feasible. In addition, the lack of competence of immature embryos in certain elite lines is still a barrier to routine production of transgenic commercial genotypes. Therefore, alternative transformation of cereal crops to avoid or reduce the above limitations.
The selection of a promising explant, genotype and et al., 2012c). Explants such as the shoot apex (Aguadoet al., 2011), mature seed (Shah et al., 2003), isolated mature embryos (Vendruscolo et al., 2008;et al., 2014), endosperm supported (ES) mature et al., 2011) and coleoptiles (Benkirane et al., 2000) are deployed to study convenient and easily available throughout the year. regeneration potential of these explants taken from the same plants of various genotypes in response to various The present study is an attempt to test the hypothesis the same plant differ from each other and is dependent on the genotype and media composition. In addition, callusing and regeneration potential of various explants (mature embryo, immature embryo, ES mature embryo explored, and a reliable callus induction and regeneration aim of exploitation of somaclonal variation, and biolistic and agrobacterium-mediated genetic transformation for breeding biotic and abiotic stress tolerant plants of

Plant material
tissue culture responses among seven genotypes tested (Mahmood et al., 2012c). Therefore, genotypes study. Tissue culture responses of various explants, viz, immature embryo, mature embryo, ES mature embryo and shoot apical meristem, taken from the same plants standardised for each explant.

Immature embryo
caryopses at the milk ripe to soft dough stages (about scutellum facing up. The remaining spikes/caryopses of mature embryo, ES mature embryo and shoot epical meristem explants.

Mature embryo
facing the scutellum side up.

Endosperm supported mature embryo
induction medium.

Callus induction
to autoclaving at 121 o C for 20 min, pH of the medium darkness at 25 ± 1 o C for 21 ds, replacing the media after

Regeneration
The regeneration potential of the explants (immature embryo, mature embryo, ES mature embryo and shoot each explant using their respective best suited callus induction medium (MS basal medium + 30 g/L sucrose + 6 g/L agar and best suited concentration of 2,4-D for callus induction for the respective explants of each genotype), as determined in the previous step of this medium (half of the best suited concentration of 2, 4-D for callus induction of the respective genotype) for a regeneration media (MS basal medium + 30 g/L sucrose + 6 g/L agar + various combinations of phytohormones). o regardless of the number of shoots and roots. The various regeneration protocols tested are given in Table 1.

RESULTS AND DISCUSSION
The callusing and regeneration potential of various explants (mature embryo, immature embryo, ES mature genotypes, viz by using various callus induction and regeneration protocols. The callogenesis response of various explants       Table 4). In general, regeneration medium M 3 (0.4 mg/L of 2,4-D, 1.0 mg/L 2 respectively (Table 5).
Auxins play a vital role in gene activation involved in cell division and dedifferentiation (Dudits et al., 1991). Also, cell division, callus proliferation, regeneration and recovery of transformed plants are mainly determined by the type and concentration of auxins (Barro et al., 1999). Unlike callogenesis from immature embryos at calli from mature embryos, ES mature embryos and epical meristem (Table 3)   0.5 mg/L IAA and 40 mg/L tyrosine have been reported best for shoot regeneration of mature embryonic calli of In cereal tissue culture, embryogenic callus formation and regeneration potential besides genotype and media composition (Mahmood et al., 2012c), are also dependent on the explant source because not all cells are totipotenic (Shah et al., 2003). Various explants taken even from the same plant of a genotype differ in their regeneration of a favourable explant is inevitable for the success of tissue culture based transformation of cereals. Most embryos are the best explants in terms of regeneration (Yang et al., 2015). The results reported in the present study also indicated that explants of both genotypes have different callusing abilities (Table 2), and that the callogenesis response of explants is genotype and media dependent (Table 3). Yang et al. (2015) have also reported explant types, and revealed that different explants of higher differentiation and regeneration potentials than those from mature embryos (Yang et al., 2015). Similarly cultured in the absence of endosperm (non-endosperm supported culture), regardless of the 2,4-D concentration (2 or 8 mg/L 2,4-D) used in induction medium (Chen et al a higher regeneration potential than the non-endosperm supported ones mainly because of higher oxalate oxidase activity (Chen et al., 2006).
The results presented here depicted that immature differences in regeneration potential under the tested conditions (Figure 1), and ES mature embryo can be a

CONCLUSION
In vitro culture response of explants taken from the same Immature and mature embryos had the highest callusing ability (80.21 and 79.86 %, respectively)