| Calla lily(Zantedeschia spp.)have great aesthetic value due to their spathe-like appearance and richness of coloration.However,embryonic callus regeneration is absent from its current regeneration mechanism.As a result,constructing an adequate and stable genetic transformation system is hampered,severely hindering breeding efforts.In this research,the callus induction effectiveness of calla lily seed embryos of various maturities was evaluated.The findings indicated that mature seed embryos were more suitable for in vitro regeneration.Using orthogonal design experiments,the primary elements influencing in vitro regeneration,such as plant growth regulators,genotypes,and nanoscale materials,which was emergent uses for in vitro regeneration,were investigated.(1)Optimization of seed embryo callus induction conditions of calla lilyIn this study,the seed embryos of calla lily with different maturity after crosspollination were used as materials to evaluate the callus induction effect through morphological observation and calculation of callus induction rate.The results showed that fully mature hybrid seed embryos were more suitable for callus induction.Microscopic observation of the induced callus by paraffin section showed that the cell morphology was small and the nuclei were closely consistent with the characteristics of embryonic callus.Four main factors affecting callus induction including 6-BA,NAA,genotypes and nanomaterials were set up by orthogonal experimental design.The results showed that the hybrid seed embryos of ’4d’ and ’Liming’ had the highest callus induction efficiency when 6-BA 2 mg/L and NAA 0.1 mg/L were added in MS medium.(2)Regeneration of callus of calla lilyOrthogonal experiments were designed to optimize the culture conditions at the stage of germination and rooting.Three factors,6-BA,NAA and CNTs,were set respectively,and three levels were set for each factor.The experimental results showed that,the optimal medium for the germination stage was MS + 6-BA 2 mg/L + NAA 0.2mg/L + 1 mg/L CNTs,and the germination rate was 82%.The main and secondary effects of factors affecting the germination stage were NAA concentration > 6-BA concentration > CNTs concentration.The optimal rooting medium was MS + 6-BA 2mg/L + NAA 0.7 mg/L + 2 mg/L CNTs,and the average rooting length was 3.5cm.The main and secondary effects of factors affecting the rooting stage were 6-BA > CNTs >NAA.(3)Establishment of transient expression system mediated by Agrobacterium tumefaciensIn this study,a genetic transformation system was developed based on Agrobacterium-mediated transformation.In order to directly verify the transformation efficiency,Agrobacterium tumefaciens containing GUS gene p CAMBIA1304 vector was used to infect callus of calla lily.GUS staining was used to directly observe the transformation effect.The genetic transformation mediated by Agrobacterium tumefaciens was real and effective.PCR results showed that the gene was introduced into callus of calla lily,and GUS gene expression was not detected in the control test. |
