Regeneracija biljaka in vitro i genetička transformacija Viola cornuta (Violaceae) genom za kapsantin-kapsorubin-sintazu
Plant regeneration in vitro and genetic transformation of Viola cornuta (Violaceae) using capsanthin-capsorubin-synthase gene
Doctoral thesis (Published version)
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A protocol for the plant regeneration of Viola cornuta L. cv. ’Lutea Splendens’, horned pansy with yellow flowers and the genetic transformation with the capsanthin-capsorubin-synthase gene in order to change flower color by metabolic modification of carotenoid biosynthesis were developed. The most efficient induction of adventitious shoots was obtained from hypocotyl explants grown on ½MS medium with 0,1 mg/l 2,4-D and 2 mg/l BAP under 16h light/8h dark conditions. The induction of adventitious shoots was achieved through a process of indirect and direct organogenesis.Transformed shoots were obtained by genetic transformation using ”empty” (Llccs-) and pWBVec10a/CaMV 35S::Llccs::TNos (35S-Llccs) vectors, while using pWBVec10a/PchsA-Llccs::TNos (PchsA-Llccs) vectors transformed calli were obtained. The presence of the Llccs, hpt and uidA genes in the V. cornuta transformed plants were confirmed by PCR analysis, while qPCR analysis showed an unequal level of Llccs gene expression in the petals and leaves of 35S-Llccs transformed lines. The color of the flower petals and the stigma changed from yellow or green to shades of orange, due to the accumulation of capsanthin, a newly synthesized red pigment in the chromoplasts of 35S-Llccs transformed plants. The phenotype of transformed plants differed significantly from the phenotype of untransformed plants. A protocol for the long-term storage (cryopreservation) of untransformed and transformed lines using the vitrification method with PVS3 solution has been developed. The recovery of shoot tips and regeneration of transformed lines (Llccs- and 35S-Llccs) after cryopreservation was significantly lower compared to the recovery of the shoot tips of the untransformed shoots. The ploidy levels of V. cornuta plants regenerated in culture in vitro, transformed lines and plants recovered after cryopreservation were similar.
Keywords:Agrobacterium tumefaciens; Horned pansy; Hypocotyl; de novo organogenesis; Carotenoids; Llccs gene; Capsanthin; Flower colour change; Cryopreservation
Source:University of Belgrade, Faculty of Biology, 2020, 1-143
- Development and application of biotechnological methods for obtaining healthy plants of ornamental species (RS-31019)