INTRODUCTION

Anther culture has proved to be a very useful tool in plant breeding. It elicits information in the quality of the selection process. Anther culture can be employed to evolve homozygous doubled haploid lines and can be used for simultaneous evaluation of their yield potential and resistance to pests and diseases. The genetic modulation through exploitation of androclonal variation is a feasible proposition in scented indica rice^[1]. Spontaneous regenerants from some of the haploidy lines is of significance to get homodiploid lines^[2]. However, the frequency of whole plantlet induction through androgenesis is still very low in indica rice, about 1 per cent based on anther number. In this study, an experiment was designed to standardize the protocol for anther culture and to identify the best rice line and hybrid suited for androgenesis.

MATERIALS AND METHODS

Anther culture was tried in three indica type parents, two hybrids and one japonica type parent of rice (Table 1). The experiment was conducted during 2001-2002 at Agricultural College and Research Institute, Killikulam. The basic N₆ medium of Chu et al.^[3] was modified to contain 0.8% agar, 5.0% glucose, 50% maltose, 1.5 mg L^-1 2, 4-D, 5% coconut water and used as callus induction medium for callusing anthers. For regeneration medium, the MS^[4] was used supplemented with Benzylaminopurine (BAP), Casein hydrosylate and coconut water.

After the pretreatment, panicles were surface sterilized with 70% ethanol for 3 min and then sterized with 0.1% HgCl₂ for 8 to 10 min, then rinsed with sterile distilled water for three times to remove the toxic substances. Panicles were removed aseptically and using a fine forceps, the spikelets were separated from the panicle. After this, a sharp cut was given at the base of the spikelets and the anthers were tapped gently on the media. The inoculated anthers were incubated in dark at 25±21C. To compare the callus induction frequency, fresh anthers were also inoculated without pretreatment. After the induction of callus, the calli were transferred to regeneration media and incubated in continuous lighting condition for organogenesis.

RESULTS AND DISCUSSION

The direct androgenesis will be useful in getting homozygous lines without much ploidy level variation in addition to the fact that time required for regeneration and plantlet development can be cut short. Deyao et al.^[5] reported a protocol for synthesizing homozygous diploids.

The hybrids IR 58025 A x IR 66 R and IR 58025 x C20R, two Assam cultures, ARC 15759, ARC 18023, one japonica type Taipei 309 and best performing local variety ASD16 was used for anther culture studies (Table 1). These parents and hybrids are having high per se performance. Thus by assuming that the hybrids are having high vigour and can be taken for anther culture induction.

Table 1:	Details of parentage of genotypes

Table 2:	Callus induction and callus differentiation under N6+2.5 mg L-1 of 2,4D +3 mg L-1 of kinetin

Table 3:	Callus induction and callus differentiation under N6+2 mg L-1 of 2, 4D + 1 mg of IAA +3 mg L-1 of kinetin

Table 4:	Impact of growth regulators upon callus induction
** Significant at 1% level

The hybrid, IR 58025 x IR 66 R performed better for calli induction. The hybrid, IR 58025A x IR 66R, responded well and produced more per cent calli in 2.5 mg L^-1 of 2, 4D +3 mg L^-1 of kinetin. This is in accordance with earlier findings of Raina^[6] and Narasimman^[7]. The combination of 2.5 mg L^-1 of 2, 4D+3 mg L^-1 of kinetin was observed earlier for calli induction when compared to the other combination (Table 2). Mandal^[8] reported similar results for transfer of submergence tolerance from wild species Oryza rufipogan to Oryza sativa. Asaduzzaman et al.^[9] reported that green plantlets appeared within 15-30 days of culture and highest number of regenerated green (33.32%) and albino (11.27%) plantlets were produced in BR-37 (Table 3).

The hybrid, IR 58025A x IR 66R was best hybrid for anther culture studies. The combination of 2.5 mg L^-1 of 2, 4D+3 mg L^-1 of kinetin was observed earlier for calli induction when compared to other combinations (Table 4). Among the parents, ARC 18023 and ASD 16 found to be anther culture friendly lines.

The anther culture technique helps to develop high yield lines with resistant to pest and diseases in a short span of time as compared to the conventional method.