| Summary: | 博士 === 國立中興大學 === 植物學系 === 89 === Papaya ringspot virus (PRSV) is a major limiting factor for papaya production in tropical and subtropical areas through the world. Resistant commercial cultivars are still not available by conventional breeding, crop improvement to solve disease problems of papaya can be enhanced by plant biotechnology. This study attempted to establish a low cost and fast micropropagation protocol to produce the papaya plantlets with high quality of cultivar traits and desired sex type. In addition, an easy and highly stable gene transfer method by Agrobacterium-mediated transformation targeting on lateral bulbils of multiple shoots in vitro to produce transgenic papaya was developed.
In our results found that root induction in low-concentration IBA agar medium followed by root development in vermiculite containing half strength MS medium under aerated conditions results in efficient rooting of in vitro papaya shoots.
Carbenicillin and cefotaxime, two antibiotics commonly used for excluding Agrobacterium tumefaciens during plant transformation. A washing step with sterilized distilled water two days after co-culture enhanced the bacteria -suppressing effects of antibiotics. Proliferation of Agrobacterium was completely suppressed in the medium containing 125 mgl-1 carbenicillin or cefotaxime. Callus fresh weight increase was apparently enhanced in the media with higher concentrations of carbenicillin (250-500 mgl-1), but was extremely inhibited in media with the same concentrations of cefotaxime. Higher percentages of somatic embryos were found in the medium with 125 mgl-1 carbenicillin or 250 mgl-1 cefotaxime; however larger numbers of somatic embryos from the individual callus were obtained in the medium with 125 mgl-1 carbenicillin, than in the medium with 250 mgl-1 cefotaxime. Percentages of abnormal somatic embryos were lower in the medium with lower concentrations of carbenicillin (125-250 mgl-1).
Kanamycin and geneticin is frequently used for selection of nptII-transformed plants. In this investigation, it was found that the development of somatic embryos-forming callus of nptII-transformed tissue was strongly inhibited and very fewer numbers of somatic embryos occurred among media containing all the kanamycin concentrations. Less adverse effects during regeneration process of nptII-transformed tissue was noticed in the medium containing lower concentrations of geneticin (< 25 mgl-1) and the numbers and traits of somatic-forming callus were similar as the control treatment. Conversion rate of npt II transformed somatic embryos to shoots of transformed tissue was not significantly different among all kanamycin or geneticin treatments.
In our study, a method of Agrobacterium-mediated transformation of papaya using multiple shoots in vitro was developed to circumvent these obstacles and greatly shorten trnasformation times (only 3-4 months). Twenty-seven transgenic lines (17 PRSV CP lines and 10 GUS lines) were obtained from 1259 shoot explants of cv. Sunrise and 4 PRSV CP transgenic lines was obtained from 201 explants of cv. Thailand. The expression of the gus gene and the CP gene of PRSV in the putative transgenic lines was verified by GUS activity assay, polymerase chain reaction (PCR), Southern blotting, northern blotting and western blotting assays.
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