Development of doubled haploids for iron toxicity tolerance in rice (Oryza sativa L.)

Abstract

Rice is the staple food crop of the people of Kerala. The production of rice in Kerala is impeded by many biotic and abiotic stresses. Iron toxicity is one of the major abiotic stresses of acidic soils in Kerala. The present study “Development of doubled haploids for iron toxicity tolerance in rice (Oryza sativa L.)” was taken up with the objective of developing doubled haploids with tolerance to iron toxicity through in vitro anther culture. Doubled haploids being homozygous stable lines, can be used both as immortal populations for QTL mapping as well as varieties, if suitable agronomic traits are present in them. Therefore, this study was taken up which comprised of three experiments. In experiment I, two tolerant genotypes (Swetha and Mangala Mahsuri) and two susceptible genotypes (Cul-90-03 and Cul 8709) were crossed with Tulasi. In the second experiment the parents along with their hybrids were evaluated for biometric characters and yield. The hybrids obtained from the crosses were significantly different for all the characters observed. Panicles per plant and grain yield per plant recorded highly significant and positive relative heterosis and heterobeltiosis. The anthers from the F1s produced in the previous experiment were used for anther culture studies in the third experiment. Sterilisation with various sterilants like 70 % ethanol, 0.1 % mercuric chloride, 5.25% sodium hypochlorite and their combinations were done. Explant sterilisation with 5.25% sodium hypochlorite for 5 to 20 minutes was effective in controlling contamination in the in vitro cultures. Study to know the best number of days of cold pre-treatment at 10oC was done (0, 3, 6, 9, 12, 15 days). Cold pre-treatment of anthers at 10oC for 9 days was found to be optimum for most of the genotypes studied. Two different media viz., N6 and B5 were tried with different combinations of 2,4 - D and Kn. N6 media responded better than B5 media for callus induction in all the growth hormone combinations. The effect of carbon source on callusing was studied using maltose and sucrose at various levels (30,40,50 mg/L) and it was found that maltose at 30g/L gave the best callus induction (7.95%). Among the auxins used in the present study,2,4-D was found to be better than NAA for callus induction while among cytokinins, Kinetin responded better than BAP for callus induction. The growth hormone combination 2,4- D (2mg/L) + Kn (0.5mg/L) was adjudged the best for callus induction. Additives like silver nitrate (AgNO3), casein hydrolysate (CH), yeast extract (YE), proline and activated charcoal were added to the basal media to improve callusing. when AgNO3 is applied from 0 to 1ml with 0.1 ml gradation, 0.5-0.6ml of 0.1N AgNO3 was found to be better in callus induction as well as the days to callus induction was reduced at these concentrations. 250 – 500 mg/L of CH and 250mg/L proline were found to induce significant response for callusing while activated charcoal and yeast extract did not have any considerable effect on callus induction. Hybrid H1 recorded good callus response when maltose at three levels was tried in the media (30, 40, 50g/L) whereas hybrid H2 gave uniform response at all the levels of maltose. There was significant variation among the genotypes in their response to all the factors studied. The best responses were as follows: - H1(Swetha x Tulasi) - N6+2,4-D (2mg/L) + Kn (0.5mg/L) + 30g/L maltose +0.5ml (0.1 N AgNO3) + 250mg/L proline + 250mg/L CH +2.5g/L gelrite gellan gum. H2(Mangala Mahsuri x Tulasi) - N6+2,4-D (2mg/L) +Kn (0.5mg/L) + 30g/L maltose +0.5ml (0.1N AgNO3) +250mg/L proline+250mg/L CH +2.5g/L gelrite gellan gum. H3(Cul-90-03 x Tulasi) - N6+2,4-D (2mg/L) +Kn (0.5mg/L) + 30g/L maltose + 0.5ml (AgNO3) + 250mg/L proline + 250mg/L CH + 2.5g/L gelrite gellan gum. H4(Cul 8709 x Tulasi)- B5 + 2,4-D (2mg/L) + Kn (0.5mg/L) + 30g/L maltose + 0.5ml (0.1N AgNO3) + 250mg/L proline + 250mg/L CH + 2.5g/L gelrite gellan gum. The calli obtained from the different genotypes were plated on two different callus regeneration media R1 (MS+ NAA(1mg/L) + Kn (2mg/L) + IAA (0.5mg/L) + CW (5%)) and R2 (MS+NAA (0.25mg/L) + BAP (0.75mg/L) + Kn(0.25mg/L)). There was no response in R1 media. Callus regenerated into plantlets in R2 but all the plantlets obtained were albinos leading to mortality and hence plantlet hardening and field planting could not be undertaken.