基于响应面设计茎段形成层培养猕猴桃幼苗的优化研究

Optimized study for plantlet inducing from cambium stem cell of young stem based on response surface design in Actinidia chinensis

为降低猕猴桃组培快繁中的污染率,提高其繁殖效率,该文以猕猴桃的幼嫩茎段为外植体,采用两步培养法进行茎段形成层的愈伤及成苗诱导研究,并利用响应面设计软件对NAA浓度、6-BA浓度、低渗处理时间进行了各条件的优化,同时通过组织切片确定愈伤的来源及幼苗的形成方式。结果表明:(1)培养过程中撕除茎段周皮能显著降低污染率,用200~400 mg·L^(-1)的PVP处理猕猴桃茎段可有效防止去皮茎段的褐化。(2)愈伤诱导的最佳条件为预培养28.3 h、NAA 4.45 mg·L^(-1)、6-BA 0.28 mg·L^(-1),而幼苗形成的最佳条件为预培养26.4 h、NAA 4.84 mg·L^(-1)、6-BA 0.42 mg·L^(-1)。这表明形成层愈伤诱导需较长低渗处理时间和较高生长素,而成苗诱导则需较高生长素、激动素及较短的低渗处理时间。(3)组织切片观察结果表明猕猴桃愈伤组织源于形成层干细胞的分裂,且幼苗株源于胚状体的发育。综上结果表明,通过除去猕猴桃嫩茎周皮,外加抗氧化、低渗处理,可有效降低猕猴桃组培快繁中的污染率,提高繁殖系数和胚状体发生率,为猕猴桃种苗的规模化生产提供技术支撑。

In order to reduce the contamination rate and improve the propagation efficiency during the Aitinidia chinensis tissue rapid propagation,the young stem segments of Aitinidia chinensis were used as explants,using the two-step culture method.The response surface design software had been used to optimize the condition or influencing factor for callus inducing and its differentiation in this experiment.The condition was designed at three levels for the concentration of NAA and 6-BA,and hypotonic treatment time,respectively.Meanwhile,the origin of callus and the way of seedling formation were determined by tissue section.The results were as follows:(1)The periderm of young stem was removed using tweezers under aseptic conditions can reduce the contamination rate,and the stems without periderm were treated with 200-400 mg·L^(-1) PVP to prevent stem browning.(2)The optimum condition for callus induction was 28.3 h for hypotonic treatment time,NAA and 6-BA concentration was 4.45 mg·L^(-1) and 0.28 mg·L^(-1),respectively.On the other hand,the optimum condition for callus differentiation or seedling formation was 26.4 h for hypotonic treatment time,NAA and 6-BA concentration was 4.84 mg·L^(-1) and 0.42 mg·L^(-1),respectively.That suggested that the formation of callus induction required a longer hypotonic treatment time and a higher content of auxin,and a higher content of auxin and kinetin and shorter hypotonic treatment time was required for plantlets inducing.(3)Furthermore,the slice observation during culture indicated that the inducing callus would be derived from division of cambium stem cell and the plantlets would be originated from embryoid development.To sum up,lower contaminated rate,higher propagation coefficient and somatic embryogenesis which had been obtained in this experiment would be based the foundation for the stable propagation system for kiwi fruit industrial culture.