A refined protocol for embryogenesis to transfer PRSV-P resistance genes from Vasconcellea pubescens to Carica papaya
2014
Razali, R.M. | Drew, R.
Papaya ringspot virus form P (PRSV-P) is continuing to spread and devastate Carica papaya (papaya) crops worldwide. Recently, we have identified and sequenced putative PRSV-P resistance genes in Vasconcellea pubescens. This study documents experiments that were designed to optimize a protocol for papaya embryogenesis and regeneration so that it could be used for efficient transformation of these putative resistance genes into papaya. Three media were compared as induction media for embryogenesis: Drew and Smith (DS) (1986), Murashige and Skoog (MS) (1962) and modified MS (mMS). Zygotic embryos produced embryogenic callus after two to three weeks of culture on mMS medium. They developed as cream white callus initially then increased in size continuously on mMS medium and started to produce embryos after week-week incubation. The addition of 250 mg L-1 casein hydrolysate (CH) and 143 mg L-1 adenine hemisulfate to mMS medium produced the highest frequency of embryogenic callus formation. The use of high DS mineral concentrations plus 3% sucrose in DS maturation medium increased the proliferation of embryos. Late stage embryos with normally formed leaves germinated in medium that contained 1 µM 6-Benzylaminopurine (BAP) and 1 µM Naphthalene acetic acid (NAA). Subsequently, germinated plants were transferred to regeneration medium without plant growth regulators to enhance shoot and root growth. Future research will use this protocol to evaluate both the efficiency of transformation of papaya and the efficacy of the putative resistance genes when transferred from V. pubescens into papaya.
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