Establishment And Optimization Of Regeneration And Transformation Systems Of Gerbera Hybrida

Gerbera hybrida is one of the six major cut flowers in the world and is an important potted flower. However, Gerbera is vulnerable to viruses, microorganisms and pests, and it is sensitive to heat or cold, which greatly limits its wide application to the market. There are increasing studies on the improvement of Gerbera by all kinds of means, one of which is tissue culture combined with gene engineering. In the past years, great achievements in tissue culture of Gerbera have been made. And the regeneration frequency of shoots differs greatly among different varieties. Shenzhen 5 (S5) is one of the varieties which are difficult to establish callus induction and regeneration systems.Although there are growing studies on the gene transformation of Gerbera, problems still remain to be solved.The aim of this study is to establish efficient and stable shoot regeneration systems of S5 and to develop the optimal transformation system through screening the factors and conditions involved in gene transformation.In this study, two shoot regeneration systems of S5 were established through tissue culture. The results showed that the optimal medium for callus and shoot induction from receptacle was 1/2 MS2 containing BA, NAA and IAA in higher levels. The other medium for shoot regeneration from single bud was YY10 and a rapid Agrobacterium-mediated transformation of S5 by using this shoot-regeneration system was developed.Major factors involved in Agrobacterium-mediated transformation of S5 were studied. The transformation frequency was detected with transient expression of GUS gene (containing an intron) and the regeneration of hygromycin (Hyg)-resistant shoot. The results showed that the optimal protocol include Agrobacterium concentration at 0.4-0.5 (O.D=600) for bacterium inoculation, 10 minutes for inoculation of the explant and 3 days for coculture. The Agrobacterium preculture in plant induction suspension for 2 hours was efficient for transformation. It was a good way for keeping activity ofAgrobacterium when they were diluted and transferred into another fresh medium for 12 h incubation after culture for 24h in YEB medium. In addition, the plant induction medium and the coculture medium containing 20 mg/L acetosyringone(AS) were perfect for activation of Agrobacterium, which increased the transformation frequency.Transformants showed stable expression of GUS gene in the leaves of plants and the hygromycin phosphotransferase gene (HPT) was integrated into the Gerbera genome, which was confirmed by histochemical GUS assay, PCR and Southern blot respectively. Nine individuals of the transformants were developed from the same optimal protocol. And the average transformation frequency reached 7.43%.