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Optimization of regeneration using differential growth regulators in indica rice cultivars

Paddy is a staple crop and being grown in major parts of India, but similarly it is facing some major challenges like abiotic stresses (drought, salinity, etc.) by which its productivity is greatly affected. De-husked seeds of 21 cultivars of indica rice were cultured onto MS medium for screening of their micropropagation capabilities. All the selected cultivars are being cultivated by farmers of Andhra Pradesh and Rajasthan states of India, these cultivars bear good quality of grain and high yield. Similarly, both states are facing the problem of drought so; we selected these cultivars for the micropropagation and transformation studies. The induction of callus and regeneration in these selected cultivars were achieved on MS culture medium with supplementation of various growth regulators. Maximum callus induction and regeneration frequency were observed in Sambha mahsuri at the concentration of 3.0 mg L−1 of 2,4-D and BAP (5.0 mg L−1) while the moderate response achieved in other cultivars of rice. Sambha mahsuri showed promising results in terms of callus induction frequency, and regeneration potentialities of embryogenic callus so, there is need to develop a rice cultivars that could thrive under drought condition if the country is to sustain rice production. Finally, regenerated plantlets were successfully transferred to natural conditions (in pots) after acclimatization.


Introduction
Oryza sativa L. is the most economically important cultivated rice species of the Oryzeae tribe of Poaceae family. Rice is the world's single most important staple food crop, and it represents a primary food for more than one-third of its population (Ilahi et al. 2005). Tissue culture techniques have become necessary for the production of the transgenic rice plants (Peng et al. 1992), hybrids (Mariam et al. 1996), and for the recovery of germplasm when seed availability is limited. These methods are based on adventitious shoot culture (Shahsavari 2011) and somatic embryogenesis (Amarasinghe 2009) which resulted into genetic variations in rice cultivars (Mannan et al. 2013). Micropropagation protocols have been established for japonica and indica rice cultivars using as explants like as mature seeds (Ullah et al. 2007), shoot segments (Verma et al. 2011), embryos (Ali et al. 2004;Evangelista et al. 2009), anthers (Khatun et al. 2010) which produced regenerated plantlets. Fifteen rice cultivars of indica rice were screened for induction of callus as well as subsequent regeneration of plantlets through plant tissue culture (Hertke and Lörz 1989). Similarly, 500 cultivars of rice were comprehensively studied for the induction of callus as well as regeneration of plantlets by Kamia et al. (1988). It was also reported that an individual genotype of rice plays a significant role in the induction of callus and regeneration of plantlets (Hoque and Mansfield 2004;Islam et al. 2005). Sambha mahsuri, Cotton Dora sannalu, PR-115 and PR-116 are high yielding cultivars in Telangana, Andhra Pradesh and Rajasthan states of India so, we focused our study on these cultivars of indica rice.
The authors designed the general experimental conditions as follows: (a) conditions for callus induction-each concentration of growth regulators, authors kept two replicas and five culture tubes for each concentration, i.e., ten tubes for each growth regulator concentration studied and one control for each concentration without addition of growth regulators; (b) multiple shoot formation-for each concentration of growth regulator, kept two replicas and five culture tubes for each concentration, i.e., ten tubes for each growth regulator concentration studied and one control for each concentration without addition of growth regulators; (c) profuse rooting-for each concentration of growth regulator, kept two replicas and five culture tubes for each concentration, i.e., ten tubes for each growth regulator concentration studied and one control for each concentration without addition of growth regulators.

Culture conditions
De-husked seeds of the indica rice cultivars were washed with tap water to remove dust and other particles. The cleaned de-husked seeds were placed in 70 % alcohol for 2.0 min and surface sterilized with 1.0 % (w/v) mercuric chloride solution for 3.0 min. The treated seeds were then rinsed three times with sterilized distilled water to remove all the traces of mercuric chloride. The sterilized seeds were inoculated onto MS (Murashige and Skoog 1962) (Table 1). Besides Sambha mahsuri and PR-116 cultivar, embryogenic calli with mild growth were also observed in PR-115 and Erramallelu cultivars of indica rice. Non-embryogenic callus formed in Sarsu-52 cultivar of indica rice at the dose of 3.0 mg L -1 of 2,4-D (Table 1). There were some variations in calli also observed among all the 21 cultivars of indica rice qualitatively, i.e., size and color of calli, growth of callus. The observed variations in induction and proliferation of callus in different cultivars seemed to be mainly due to the physical factors like as position and location of explants onto the culture medium because of the same genetic make-up of explants. As evident from the observed results, meristemoids developed in these calli which exhibited sufficient reasonable regeneration capability. The 2,4-D supplementation in MS culture medium hastened the induction of callus in 11 cultivars of indica rice individually as well as in combination with cytokinins. Similarly, the effects of cytokinins (kinetin, BAP) in induction of calli were also observed in 11 cultivars of indica rice (Table 1). It was also observed that kinetin 2.5 mg L -1 was more Rammapa, Khushava. When the concentration of kinetin was further increased to the level of 1.0 mg L -1 , i.e., from 2.5 to 3.5 mg L -1 keeping the concentration of IAA as Those with best average values were considered in 21 varieties for example average response (%) below 50 % was not considered, below 8 multiple shoots were not considered and below 8 roots were not considered. Number of tubes per each concentration studied = 5; Number of replicas for each concentration studied = 2; Total number of tubes per each concentration studied = 5 9 2 = 10; Number of de-husked seeds used per tube = 1; Number of de-husked seeds used per concentration studied = 10 a Average response % stable (2.5 mg L -1 ), excellent (e.g., weight) callus induction and growth was observed within 3-4 weeks in Sambha mahsuri, Erramallelu, Pothana (Table 1).
Profuse rooting was achieved in four cultivars of indica rice (PR-116, PR-115, Sambha mahsuri and Erramallelu) when, cultured onto MS culture medium with supplementation of IBA (1.0 mg L -1 ), in 18-20 days, respectively ( Table 3). The maximum rooting (average number 13.63 ± 0.83 roots per shoot) was achieved in Sambha mahsuri cultivar of indica rice with supplementation of IBA (1.0 mg L -1 ) after 18 days (Table 3; Fig. 1). Similarly, moderate rooting was also observed in Erramallelu (average number 10.44 ± 0.73 roots per shoot) cultivar of indica rice at concentration of IBA (1.0 mg L -1 ), in 19 days, respectively (Table 3). The in vitro regenerated plantlets after attaining a size of 2-3 inches were then taken out from aseptic containers and transferred to the natural conditions after acclimatization.

Discussion
In the current study, 21 cultivars of indica rice were used for micropropagation through de-husked seeds. The effects of 2,4-D were studied and observed that 2,4-D enhanced the frequency of induction and growth of callus (Deo et al. 2009). Similarly, in Sambha mahsuri cultivar, maximum multiple shoots regeneration was achieved in supplementation of MS ? BAP (5.0 mg L -1 ). Simultaneously, stimulatory effects of BAP in combination with IAA and NAA have previously been reported to facilitate regeneration in indica rice cell cultures (Mandal et al. 2003;Karthekeyan et al. 2009). The observed results revealed that kinetin was found to be effective for the induction and proliferation of shoots and roots. This may be because cytokinin causes development of single pole and formation of meristematic dome from which the shoot primordium develops. The role of cytokinins on somatic embryogenesis has been explained by enhanced cell division of pre-embryogenically determined cells (Kintzois et al. 2002). In addition to these reports, kinetin increases proliferation and regeneration of callus by affecting mitosis, cytokinesis, total protein synthesis, lignin biosynthesis, vascular differentiation and BAP 6-Benzyl amino purine, KIN kinetin, 2,4-D 2,4-Dichlorophenoxy acetic acid, NAA 1-Naphthaleneacetic acid a Determination of shoots = Number of replicates for each concentration was two differentiation of mature chloroplast from protoplastids (Wan et al. 1988). An important aspect of the present protocol was that all the plantlets regenerated from mature embryo-derived calli developed good root system. Moreover, after transferring to pots, in vitro regenerated plantlets of four cultivars survived, which in turn indicated good adaptability of somaclones in ambient environmental conditions. High auxin concentration leads to development of root primordium leading to root development. However, one of our cultivar Sambha mahsuri decreases in root development at high auxin concentration. Thus, in turn, suggested that genotypes also possessed variability in their capability of regeneration as reported in previous studies (Kumaria et al. 2000). The present study establishes a successful protocol for the induction and growth of callus, somatic embryogenesis in four diverse cultivars (PR-115, PR-116, Sambha mahsuri, and Erramallelu) of Indian sub-continent, and the in vitro regenerated plants were showed yield potential at par to that of direct seeded plants in ambient conditions. The present protocol would be useful for genetic transformation in elite genotypes of rice using any selected gene transfer method in near future.

Conclusion
Maximum frequency of induction and growth of callus, shoot proliferation was achieved in Sambha mahsuri cultivar of indica rice cultured onto MS culture medium with supplementation of 2,4-D (3.0 mg L -1 ) and BAP (5.0 mg L -1 ) in 27 days, while slow induction in other studied cultivars of indica rice. Thus, there is a need to develop new rice cultivars that could cope up to drought conditions and flourish into good quality of grain and higher yield. In our next course of the study, drought tolerant gene DREB1A will be introduced into Sambha mahsuri cultivar by particle bombardment method to improve of its growth and high yield under drought conditions for incremental benefits of farmers.