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近年来,苹果遗传转化研究取得了较大的进展,已有多种标记基因和目的基因成功转入苹果中并且得到稳定表达和遗传。农杆菌介导法是苹果上应用的主要转化方法,叶片再生不定芽途径是被广泛应用的受体系统。影响农杆菌介导成功与否的因素很多,文章从以下几方面综述了目前的有关研究成果:①转化方法。因农杆菌介导法简便、高效,应用最广泛。但在转化结束后除菌不彻底易造成假阳性植株,应用直接转化法可以减少假阳性植株,提高转化率。发展苹果直接转化法,一方面需要深入研究原生质体培养,另一方面可以选用不需要原生质体制备和再生的方法,如基因枪法。②叶片再生能力。影响叶片高效再生因素包括试材的基因型,基本培养基类型,培养基中植物生长调节物质的种类及配比,光照条件和接种前叶片的生理生化状态(包括苗龄、叶片成熟度、叶片不同部位以及外植体的来源等),其中试材基因型是最重要的因子。③菌株类型。应根据不同的受体基因型选择不同的菌株类型。④农杆菌侵染条件。繁殖速度快的菌株侵菌时间宜短,繁殖速度较慢的菌株可适当延长侵菌时间。对农杆菌敏感的植株,可采用较低的OD值和较短的浸泡时间。一般菌液浓度范围是OD600=0.05～0.70,侵染时间不超过30min;共培养时间必须长于16h,但要以不对叶片造成伤害又最大限度提高转化效率为标准;植物酚类化合物乙酰丁香酮(AS)、渗透保护剂磷酸甜菜碱(BP)能提高农杆菌的毒力,从而提高其侵染能力。最后,指出目前依然存在转化效率不高、外源基因表达强度不够、对转化植株的选择不够重视等诸多问题。
In recent years, great progress has been made in the research of genetic transformation of apple. A variety of marker genes and target genes have been successfully transferred into apple and have been stably expressed and inherited. Agrobacterium-mediated method is the main transformation method applied on apple, adventitious bud regeneration pathway is a widely used receptor system. Many factors affect the success of Agrobacterium -mediated mediated, the article summarizes the current research results from the following aspects: ① conversion method. Because Agrobacterium-mediated method is simple, efficient, the most widely used. But after the end of the transformation sterilization is not completely easy to cause false-positive plants, the application of direct transformation method can reduce false-positive plants and improve the conversion rate. The development of apple direct transformation method, on the one hand need to study in-depth protoplast culture, on the other hand can choose not protoplast preparation and regeneration methods, such as the gene gun method. ② leaf regeneration capacity. Factors that affect the efficient regeneration of leaves include the genotype of the test material, the type of basic medium, the type and proportion of plant growth regulating substances in the medium, the light conditions and the physiological and biochemical status of the leaves before inoculation (including seedling age, leaf maturity, Different parts and explants, etc.), of which the test material genotype is the most important factor. ③ strain type. Different strains should be selected according to different receptor genotypes. ④ Agrobacterium infection conditions. Infertility time should be short for strains that breed faster, and strains for slower propagation may be extended for invading bacteria. Agrobacterium-sensitive plants, the lower the OD value can be used and a shorter soaking time. The general bacterial concentration range is OD600 = 0.05 ~ 0.70, infection time does not exceed 30min; co-culture time must be longer than 16h, but to not damage the leaves and maximize the conversion efficiency as the standard; plant phenols acetosyringone AS), the osmoprotectant BPO can increase the virulence of Agrobacterium so as to improve its infectivity. Finally, it is pointed out that there are still many problems such as low transformation efficiency, insufficient expression of exogenous gene and insufficient attention on the selection of transformed plants.