Development of an efficient protocol for anther culture of selected indica genotypes (Oryza sativa) and selection of lines for iron toxicity tolerance.

Anther culture take part in key role in rapid development of homozygous lines and genetic  manipulation, especially in isolation of desirable individuals from segregating population in the early generation. The production of new varieties via anther culture shortens the breeding cycle, raises the efficiency of selection and saves space and labor in the experimental field. Intensive research on rice anther culture has been carried out. Extent of success of haploid induction by anther culture depends on a number of factors that include genotype,  developmental stage, cultivated conditions of plants, components of culture medium, pre  treatments etc. The goal of this study was to observe the anther culture response of selected  indica rice varieties and to develop an efficient protocol for callus induction and plant  regeneration by considering genotype, development stage of pollen, pollen density, growth condition of the anther donor plant,culture medium, pre-treatment,culture condition and in-vitro selection of iron toxicity tolerance rice line. Genotypes of BG358, BG 379-2, BW364, BG 357, BW 267-3, AT 306, BW267-3, Kahata wee, BW 272-6b and AT 362 varieties were selected for the study. Uncontaminated cultures were the established and maintained to use for further trials. The callus formation ability were tested using different concentration of plant growth regulators NAA , 2, 4-D and  Kinetin. N6 and  L8 media with 6% maltose were supplemented(2,0,0.5),(2,0,1) (0,2,0.5),(2,2,0.5),(2.5,2,0.5),(0,2.5,0.5) (2,2.5,0.5),(2.5,2.5,0.5),(0,2.5,1),(2,2.5,1) and (2.5,2.5,1) as respectively. L8 medium with 2, 4 D, NAA and Kinetin (2, 0, 0.5) were used for callus induction. To select the best panicle length 5-6 cm, 6-7cm, 7-8 cm, 8-9cm, 9-10cm, 10 12cm, 12-14cm and 14-16cm lengths of panicles were used with best media selected. Most suitable panicle length for each variety using earlier experiment and they were stored in cold temperatures of 6,7,8,9 and 10⁰C for 5 -14 days to find the best cold pretreatment temperature and duration.Callus induction medium were then supplemented with 6% maltose, 6% sucrose and 6% fructose  for callus induction to determine suitable sugar type. All rice varieties were used to find the effect of anther density. Using hundred, two hundred, three hundred and four hundred anthers were inoculated in Petri dishes separately. Furthermore the induced calli were transplanted into test tubes containing MS medium supplemented with different concentrations of growth regulators. MS medium was supplemented with Kinetin,NAA,BAP (2,1,2), (2,1,1),(0.5,1,2) and 3% sucrose. Induced calli were transferred to MS medium supplemented with different iron concentrations consisting with 4, 4.5, 5, 5.5,6 pH levels and each pH level with 50, 75,100,200,300 Fe ppm to find in vitro tolerance of iron toxicity. Highly  responsivegenotypes are AT 362, BG 379-2 and BW 267-3 with respect to callus initiation  frequency. BG 357 and BG 358   are less responsive genotypes. L8 medium supplemented with 2.0 mg l^-1 2, 4-D,Kinetin 0.5 mg l^-1 and 6% maltose  can be used to gain higher callus induction of AT 362, BG 379-2 and BW 267-3 varieties and most  appropriate panicle lengths were 5-6 cm,7-8 cm and 7-8 cm respectively. N6 medium supplemented with 2.0mgl^-1 2, 4-D, 0.5 mg l^-1 Kinetin and 6% maltose is suited for AT306, AT364, BW 272-6b, BW 364 and Kahata wee varieties to highest callus induction and most appropriate panicle lengths were 8-10 cm, 8-9 cm, 7-8 cm, 7-8 cm, 8-9 cm and 8-9 cm respectively. Cold pre treatment at 8 ºC for 8 days was found to be effective in AT 362 and BW 272-6b. 8 ºC for 7 days was effective in AT 364  AT 306, BG 379-2, BW 267-3 and Kahata wee 10ºC for 7days was effective in BW 364.Time taken for callus initiation varies according to the rice variety. AT 306, AT 362 and Kahata wee were taken four weeks and others were initiation within five weeks. Among the regeneration media tested MS medium containing 0.5 mg l^-1 Kinetin, 1.0 mg l^-1NAA  and 2.0 mg l^-1 BAP can be used to achieve higher regeneration rate of tested all rice varieties. Variety BW 272-6b, Kahata wee and AT 362   were regenerated green plants in iron medium.