The mature seeds of paddy were dehusked manually prior to surface sterilization. The seeds were washed 2 to 3 times with sterile distilled water to remove any impurity present. This was done under aseptic conditions. The seeds were then surface sterilized with 70 per cent ethanol for 120 sec followed by 0.1 per cent mercuric chloride solution for 6 - 8 min. The seeds were then rinsed 3 to 4 times thoroughly with sterile distilled water. Surface sterilized seeds were inoculated under aseptic conditions, in the callus induction medium and direct embryo contact with the medium was avoided. Similarly, the immature embryos were collected from the healthy and disease free plant when the seeds were in milky stage. The stage was identified by pressing the spikelet in between thumb and fore finger and the milky ooze coming out of the spikelet indicated the correct stage for collecting immature embryos. The spikelets were dissected and the immature embryos were taken out under aseptic condition. Generally in rice genotypes the immature embryos formed 12–14 days after flowering. The callus tubes were incubated under controlled environment conditions in the growth room. The cultures were subjected to light and dark cycle of 16/8 hrs per day. The light (2000 lux) was provided by a cool, white lamp (Philips 40w). The cultures were maintained at a temperature of 25 ± 20 °C and 70 per cent relative humidity. For callus initiation, the tubes were kept under dark conditions. Afterwards tubes were taken outside and exposed to light for callus development.

When sufficient calli were obtained, enough embryogenic calli were selected and transferred to regeneration medium with a change in type and concentration of hormones. BAP was tried at 0.5, 1.0, 1.5, 2.0 mg l^-1; NAA at 0.5, 1.0 mg l^-1 keeping 1.0 mg l^-1 of kinetin as constant. In the regeneration medium, growth hormones were combined at various levels to initiate shooting and rooting simultaneously and the treatment combinations were as follows R1 = 0.5 mg l^-1 BAP + 0.5 mg l^-1 NAA + 1.0 mg l^-1 kin, R2 = 0.5 mg l^-1 BAP + 1.0 mg l^-1 NAA + 1.0 mg l^-1 kin, R3 = 1.0 mg l^-1 BAP + 0.5 mg l^-1 NAA + 1.0 mg l^-1 kin, R4 = 1.0 mg l^-1 BAP + 1.0 mg l^-1 NAA + 1.0 mg l^-1 kin, R5 = 1.5 mg l^-1 BAP + 0.5 mg l^-1 NAA + 1.0 mg l^-1 kin, R6 = 1.5 mg l^-1 BAP + 1.0 mg l^-1 NAA + 1.0 mg l^-1 kin, R7 = 2.0 mg l^-1 BAP + 0.5 mg l^-1 NAA + 1.0 mg l^-1 kin, R8 = 2.0 mg l^-1 BAP + 1.0 mg l^-1 NAA + 1.0 mg l^-1 kin.

Young plantlets with good shoot and root growth were taken out of the tubes for hardening. Various hardening procedures tried were as follows.

The observations recorded were statistically analyzed by subjecting the data to Factorial Completely Randomized Design (Gomez and Gomez 1984). Level of significance was determined by using standard analysis of variance (ANOVA) (Panse and Sukhatme, 1964).Differences among mean values were assessed by LSD (Least Significant Difference).The data obtained with per cent values were subjected to arc sine transformation (where ever necessary) and analysed using the statistical software AGRES.

Callus induction and plant regeneration from callus need homogenous population of totipotent and embryogenic cells (Agrawal et al. 2006). The potential for callus formation and regeneration have been reported to be varietal characteristics and efficient regeneration in indica rice still poses a major problem for genetic manipulation through innovative approaches (Saharan et al. 2004). While it has been possible to obtain high plant regeneration frequencies in japonica rice varieties, the success for reproducible fertile regeneration has been limited in indica rice varieties. As a result progress towards the transfer of useful genes into indica rice has been slow. Wide differences existed between the tissue culturability of the two sub species viz., indica and japonica. It is important to establish parameters for the transformation of popular indicia rice varieties (Visarada et al. 2002 and Tyagi et al. 2007). Availability of an efficient regeneration system is a prerequisite for undertaking any transformations study. The optimum media composition and other culture condition are required for efficient plant regeneration (Khanna and Raina, 1997 and Saharan et al. 2004). Agrobacterium- based transformation of rice, needs efficient regeneration systems, including highly responsive indica genotypes as well as culture conditions are critical factors to be identified and standardized.

Therefore, the present investigation is performed to standardize the tissue culture technique with respect to media composition, genotype, explant, hormonal combination, regeneration and hardening.

The selection of explant plays an important role in successful callus induction and plant regeneration. In the present study seed and immature embryo were used as explants and the performance of immature embryo was better than seed explant in forming profuse and embryogenic callus and this observation is in accordance with previous findings reported by Aldemita and Hodges (1996), Sudha (2000), Vinothini (2004). The present study revealed that even explants from a single genotype do not respond identically in culture. For example seed explant of Pusa Basmati 1 gave 62.82 per cent callus induction and immature embryo explant of the same genotype gave 75.55 per cent callus induction. Most likely it was due to the varying gradients of endogenous hormones. Similar observations were reported by (Lakshmi Narayanan 1997 and Sudha 2000). Immature embryo explants have proven to be the best source for the establishment of embryogenic callus and plant regeneration. The genetic basis of variability in tissue culture response and morphogenesis is most likely due to differences in hormone metabolism within the explant (Norstog 1970). Immature embryo explant used in this study was taken from 12 and 14 days after pollination to get maximum response. Vasil (1988) also indicated that only explants cultured at a specific developmental stage gave regenerable cultures.

Rice callus induction is known to depend on the type of medium used (Zhao 1999 and Lee et al. 2002). The implication of such result is that there may still be room for further modification of the subculture to improve both the growth rate and quality of the calli. In this study, the maximum callus induction per cent was noticed on MS media and it was lower on N6 media .Similar results were already reported by Sudha (2000) and Khaleda and Al-Forkan (2006). In contrast Lee et al. (2002) reported N6 media showed higher callus induction and embryogenic callus formation. Induction of embryogenic calli in rice is considered as the most critical step. These results were in complete agreement with those of (Saharan et al. 2004; Khaleda and Al-Forkan 2006).

Thus the results of the present study supplement the earlier reports on the suitability of MS media for callus induction, growth and regeneration of plantlets. The reason for the superiority of standard MS medium may be due to its composition. It is having higher concentrations of nutrients especially macronutrients like MgSO₄. 7H₂0 (370 mg l^-1) and CaCl₂. 2H₂O (440 mg l^-1) when compared to N₆ medium. The concentration of micronutrients viz., MnSO₄. 4H₂O, ZnSO₄, 7H₂ O, H₃BO₃ is also higher in MS medium. In N₆ medium minor elements like Na₂ MO O₄. 2H₂O, CuSO₄. 5H₂) and COCl₂.6H₂O are absent. Similar findings were observed by Sudha (2000). Concentration of carbon source in the medium has a determining role in callus induction, organogenesis and embryogenesis (Gupta 1997). This study indicated that the callus induction per cent and embryogenic callus formation was higher at 30 g l^-1 sucrose when compared to 60 g l^-1 sucrose. The above finding is in conformity with those of Sudha (2000), Saharan et al. (2004), Monirul Islam et al. (2005), Agrawal et al. (2006) and Hoque et al. (2007) also concluded that there was no callus induction without sucrose and callus induction and generation was maximum when the sucrose level was 30 g l^-1 and it decreased with the increased concentration of sucrose. Apart from finding the optimum concentration of sucrose, the effect of sucrose and maltose was studied using seed as well as immature embryo explants. In the present study, the embryogenic callus formation was higher on media with maltose in both seed and immature embryo culture (69.12 and 79.78). In accordance to this findings, Kumar et al. (2005) and Zaidi et al. (2006) also reported that formation of embryogenic calli was enhanced with the use of maltose as a carbon source and maltose proved to be the most effective, resulting in the highest frequencies of callus induction, embryogenic calli and regeneration.

In this investigation the seed and immature embryo explants taken from the same genotype did not respond similarly in culture, i.e. immature embryo of Pusa Sugandh 4 recorded the highest per cent of callus induction, while the seed explant gave lowest per cent of callus induction on same growth media. Sudha (2000) and Monirul Islam et al. (2005) also evidenced these findings (Table 2 and 3).

There was a wide difference in embryogenic callus formation exhibited by white ponni in seed culture. But in immature embryo culture Pusa Sugandh 5 showed maximum per cent of embryogenic callus formation in MS medium with 2,4-D and maltose (Table 5 and 6). These genotypic variations may be attributed to differences in the components and the concentration of the endogenous plant hormones and sensitivity to 2, 4-D between varieties as reported earlier by Agrawal et al. (2006) Hoque et al. (2007) and Monirul Islam et al. (2005). Katiyar et al. (1999) also concluded that callus induction and plant regeneration were found to be greatly influenced by the genotypes and 2, 4-D concentration in media, it was also noted there was reduction in regeneration in the genotypes White ponni, and ASD 16. In accordance to this finding, Sumathi (1992) concluded that conditions optimal for plant regeneration in one cultivar fail to produce plants in another cultivar of the same species (Table 6).

Plant growth regulators are the critical media components in determining the developmental pathway of plant cells. Addition of 2, 4-D induced callus development and also stated that the presence of 2, 4-D in the culture medium is critical for rice callus induction from mature seeds and formation of embryogenic callus. In this experiment, the addition of 2, 4-D (2.0 mgl^-1) was found to induce good callus growth in MS medium and percentage of embryogenic callus formation was higher when 2, 4- D (2 mg l^-1) was combined with maltose (30 g l^-1) than with sucrose (30 g l^-1). This findings was in confirmation with earlier reports by Azhakanandam et al. (2000), Lin and Zhang (2005), Al-Forkan et al. (2004), Agrawal et al. (2006) and Zaidi et al. (2006). The effect of kinetin on callus differentiation could be promotive or inhibitory depending upon the kind and concentration of auxin in the medium, it was observed that addition of 0.5 mg l^-1 K to the culture media promoted callus induction and embryogenic callus formation and this was in conformation with the results of Brar et al. (1985), Chaliha et al. (1993), Sudha (2000), Khaleda and Al-Forkan (2006), Agrawal et al. (2006). In this study it was seen that maximum percentage of callus induction in MS + 2 mg l^-1 2,4-D + 0.5 mg l^-1 K + 30 gl^-1 maltose compared to MS + 2.0 mg l-1 2, 4-D + 0.5 mg l-1 K + 30 gl-1 sucrose (Table 4) and addition of 100 ml^-1 coconut milk as organic supplement gave higher callus induction percent with early days (Table 7 and 8).

Many factors such as culture medium, growth regulators, culture environment, explant and genotype of donor are known to influence regeneration of plants. In the present study, regeneration was obtained through callus formation in explants and subsequent formation of shoots and roots. The present study also noted that for embryogenesis, callus induction and root induction the requisite ratio of auxin and cytokinin is high, This was in accordance with Yamada (1967), Sumathi (1992), Sudha (2000) and Lin and Zhang (2005), they reported that cereals require the strong action of auxin to induce callus and to maintain their growth in subcultures. However, for regeneration, it was essential that there can be a decrease in the 2,4-D concentration or the substitution of a weaker auxin such as IAA and NAA. Highest regeneration frequency of (0.33%) was observed in regeneration media containing 0.5 mg⁴ BAP, 0.5 mg⁴ Kinetin and 80 mg-l adenine sulphate in Azucena variety (Dalpat Lal, et al. 2014). Agrawal et al. (2006) reported that green islets formation was more in MS + 1.0 mg l^-1 BAP + 1.0 mg l ¹ K + 0.1 mg l^-1 NAA with the maximum of 57.14 per cent (Table 9). Among the hardening methods studied, hardening in water followed by mist chamber recorded the highest results (Table 10).