Regeneration Of Rice Mutants Harbouring Ac/Ds Elements Via Tissue Culture And The Analysis Of Ds Insertion

The rice seeds, which were confirmed to harbour both Ac and Ds elements, were used as experimental materials to perform tissue culture. High quality calli were generated on an induction culture media from dehusked seeds after treatment at low temperature. The effects of the ratio of different plant growth regulators, subculture strategy, desiccation treatment, additives and different regeneration method on the callus redifferentiation process were investigated. The optimum differentiation medium was screened, and regenerated plants (R0) were obtained. After transfer to soil and growing, mature seeds (R1) were harvested from regenerated plants. Leaf sampling was carried out when the regenerated plants grew vigorously at tillering stage, and the genomic DNA extraction was conducted from these sampled leaves. The extracted genomic DNA was adopted as template in a PCR using specific primers to check the Ac/Ds insertion, and the TAIL-PCR was also performed using genomic DNAs containing both Ac and Ds as templates to amplify the DNA fragments flanking Ds insertion sites. A simulation analysis for the Ds insertion site was executed by using a previously amplified Ds-flanking sequence as query sequence in a BLAST search. The main results were summarized as follows:1. The quality of calli was improved by supplementing 1% mannitol into the induction medium, thereby the proliferation rate of calli was also increased significantly. The regeneration ability was enhanced under different regeneration conditions, such as desiccation treatment, or different concentration agar. The addition of iP and Na2SiO3 into differentiation medium could promote the formation of plantlets. The regeneration frequency reached 53% when three-step culture procedure was adopted, which was obviously better than two-step and direct culture method.2. Among the five methods tested for genomic DNA extraction, SDS extraction procedure was more reliable with respect to concentration and purification of genomic DNA than other methods using CTAB or urea in extraction buffer.3. Ac/Ds insertion pattern was screened by PCR from a population of 111 plants regenerated from seeds harbouring both Ac and Ds. The result showed that 94% carried both Ac and Ds elements, while 6% Ac alone. The Ac plants were devoid of Ds element, the cause of which remains unclear and needs further work. Thus, it is suggested that enlarging the mutant library via tissue culture should be feasible in rice.4. The potential factors, which may impose effects on the TAIL-PCR performance, were put forward based on pilot experiments.