Generation of hermaphrodite transgenic papaya lines with virus resistance via transformation of somatic embryos derived from adventitious roots of in vitro shoots

Abstract

Papaya production is seriously limited by Papaya ringspot virus (PRSV) worldwide and Papaya leaf-distortion mosaic virus (PLDMV) in Eastern Asia. An efficient transformation method for developing papaya lines with transgenic resistance to these viruses and commercially desirable traits, such as hermaphroditism, is crucial to shorten the breeding program for this fruit crop. In this investigation, an untranslatable chimeric construct pYP08 containing truncated PRSV coat protein (CP) and PLDMV CP genes coupled with the 3' untranslational region of PLDMV, was generated. Root segments from different portions of adventitious roots of in vitro multiple shoots of hermaphroditic plants of papaya cultivars 'Tainung No. 2', 'Sunrise', and 'Thailand' were cultured on induction medium for regeneration into somatic embryos. The highest frequency of somatic embryogenesis was from the root-tip segments of adventitious roots developed 2-4 weeks after rooting in perlite medium. After proliferation, embryogenic tissues derived from somatic embryos were wounded in liquid-phase by carborundum and transformed by Agrobacterium carrying pYP08. Similarly, another construct pBG-PLDMVstop containing untranslatable CP gene of PLDMV was also transferred to 'Sunrise' and 'Thailand', the parental cultivars of 'Tainung No. 2'. Among 107 transgenic lines regenerated from 349 root-tip segments, nine lines of Tainung No. 2 carrying YP08 were highly resistant to PRSV and PLDMV, and 9 lines (8 'Sunrise' and 1 'Thailand') carrying PLDMV CP highly resistant to PLDMV, by a mechanism of post-transcriptional gene silencing. The hermaphroditic characteristics of the transgenic lines were confirmed by PCR with sex-linked primers and phenotypes of flower and fruit. Our approach has generated transgenic resistance to both PRSV and PLDMV with commercially desirable characters and can significantly shorten the time-consuming breeding programs for the generation of elite cultivars of papaya hybrids.

The Uses of Somatic Embryogenesis for Genetic Transformation

“…An untranslatable chimeric construct (pYP08) with the truncated PRSV and PLSMV coat protein genes coupled with the 3' nontranslated region of PLDMV was used to transform embryogenic cultures of papaya cultivars 'Tainung No 2,' 'Sunrise,' and 'Thailand' with A. tumefaciens. Shoot tips from 1-year-old hermaphrodite papaya plants were surface sterilized and cultured to induce multiple shoots, which were dissected and grown on MS medium with 0.5 mg L −1 IBA for 1 week in dark conditions to induce adventitious rooting (Kung et al 2010). Treated shoots were transferred to perlite with half the volume of MS basal medium to stimulate the development of adventitious roots, which were the source of explants for somatic embryogenesis.…”

Biotechnology and Plant Disease Control-Role of RNA Interference

“…For the effective control of PRSV and Papaya leaf-distortion mosaic virus (PLDMV), an untranslatable chimeric construct containing truncated PRSV YK CP and PLDMV P-TW-WF CP genes has been transferred into papaya (Carica papaya cv. 'Thailand') by Agrobacterium-mediated transformation via embryogenic tissues derived from immature zygotic embryos of papaya [96]. Based on sequence profile of silencing suppressor protein, HcPro, it was that PRSV-HcPro acts as a suppressor of RNA silencing through micro RNA binding in a dose dependent manner.…”

Genetic Engineering for Viral Disease Management in Plants

“…For the effective control of PRSV and Papaya leaf-distortion mosaic virus (PLDMV), an untranslatable chimeric construct containing truncated PRSV YK CP and PLDMV P-TW-WF CP genes has been transferred into papaya (Carica papaya cv. 'Thailand') by Agrobacterium-mediated transformation via embryogenic tissues derived from immature zygotic embryos of papaya (Kung et al, 2009). Based on sequence profile of silencing suppressor protein, HcPro, it was that PRSV-HcPro, acts as a suppressor of RNA silencing through micro RNA binding in a dose-dependent manner.…”