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An improved method for transformation of Actinidia arguta utilized to demonstrate a central role for MYB110 in regulating anthocyanin accumulation in kiwiberry
Plant Cell, Tissue and Organ Culture ( IF 2.3 ) Pub Date : 2020-08-20 , DOI: 10.1007/s11240-020-01915-1
Dinum Herath , Tianchi Wang , Yongyan Peng , Andrew C. Allan , Joanna Putterill , Erika Varkonyi-Gasic

Actinidia arguta and related species produce small edible kiwifruit (kiwiberry) with attractive consumer features and high nutritional value. As a recently developed crop, kiwiberry can be improved further by classical breeding or genetic manipulation. The currently available Agrobacterium-mediated transformation protocol is only applicable to a limited number of kiwiberry genotypes such as the female cultivar ‘Hortgem Tahi’. We developed protocols for regeneration and Agrobacterium-mediated transformation of two A. arguta genotypes, female AA06-01 and male AA05-06, also applicable to ‘Hortgem Tahi’. Altered composition of basal salts in the callus-induction media was sufficient to prevent callus browning in AA06-01 and ‘Hortgem Tahi’, but not in AA05-06. Altering the hormone composition to avoid a prolonged callus stage was successfully used to prevent browning and induce shoot development in all three genotypes. Using our improved protocols, Agrobacterium-mediated transformation of a binary vector carrying the neomycin phosphotransferase II (nptII) expression cassette gave rise to kanamycin-resistant plants of both AA06-01 and AA05-06 and the presence of the nptII transgene was confirmed by genomic PCR. The improved protocols were also used to ectopically overexpress the anthocyanin-related transcription factor AcMYB110 in ‘Hortgem Tahi’ giving rise to purple callus with elevated anthocyanin accumulation. AcMYB110 expression and increased levels of anthocyanins were detected in mature leaves of established transgenic plants compared to controls, clearly demonstrating the important role for MYB110 in regulation of anthocyanin accumulation in kiwiberry. The protocols developed during this study provide tools for further functional analyses and genetic manipulation of kiwiberry genotypes.



中文翻译:

一种改良的猕猴桃转化方法,用于证明MYB110在调节猕猴桃花色苷积累中的核心作用

猕猴桃和相关物种产生具有吸引力的消费特性和高营养价值的小型食用奇异果(奇异果)。作为一种新近发展的作物,可以通过经典育种或基因操作进一步改善奇异果。当前可用的农杆菌介导的转化方案仅适用于有限数量的猕猴桃基因型,例如雌性品种“ Hortgem Tahi”。我们开发了两种曲霉农杆菌的再生和农杆菌介导转化的方案基因型,雌性AA06-01和雄性AA05-06,也适用于'Hortgem Tahi'。愈伤组织诱导培养基中基础盐成分的改变足以防止AA06-01和'Hortgem Tahi'中的愈伤组织褐变,但不能防止AA05-06中的愈伤组织褐变。改变激素组成以避免延长的愈伤组织阶段已成功用于防止褐变并诱导所有三种基因型的芽发育。使用我们改进的方案,农杆菌介导的携带新霉素磷酸转移酶II(nptII)表达盒的二元载体的转化产生了抗AA06-01和AA05-06的卡那霉素植物以及nptII的存在通过基因组PCR证实了转基因。改进的协议还用于在'Hortgem Tahi'中异位过表达花色素苷相关的转录因子AcMYB110,从而导致紫色愈伤组织的花色素苷积累增加。与对照相比,在已建立的转基因植物的成熟叶片中检测到AcMYB110表达和花色苷水平升高,清楚地证明了MYB110在调节猕猴桃花色苷积累中的重要作用。在这项研究中开发的协议为进一步的猕猴桃基因型功能分析和遗传操作提供了工具。

更新日期:2020-08-20
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