小麦幼胚与成熟胚遗传转化体系研究进展

doi:10.13560/j.cnki.biotech.bull.1985.2026-0095

摘要/Abstract

摘要：

小麦遗传转化长期受限于显著的基因型依赖性，愈伤组织再生效率偏低，核心瓶颈在于受体细胞全能性重获机制及基因重编程的分子调控机制尚未完全明确。本文聚焦小麦遗传转化领域核心科学问题，系统阐述当前主流转化技术的特征与应用局限，通过对比小麦幼胚与成熟胚转化体系差异，明确两类体系的优势与短板，剖析限制转化的分子屏障及潜在突破路径。在此基础上，分析农杆菌菌株类型、受体材料生长状态，以及筛选标记与启动子元件等关键因子对农杆菌介导小麦转化效率的调控效应，同时围绕小麦愈伤组织分化再生过程，从激素信号转导调控、形态建成关键基因功能解析、细胞壁结构重塑机制及染色质可及性调控等维度，系统阐明遗传转化中植株再生的分子调控网络。基于上述分析，最后提出挖掘并利用具有广谱调控效应的再生关键因子、精细优化组织培养与转化体系参数以及开发不依赖组织培养的新型转化技术，是突破小麦遗传转化基因型限制与提升转化效率的核心策略。本文旨在为构建“基因型非依赖性”小麦通用转化平台提供理论支撑，助推小麦分子育种技术的规模化与精准化应用。

关键词: 小麦, 遗传转化, 再生体系, 生长素, 形态发生基因

Abstract:

Wheat genetic transformation has been hampered by marked genotype dependence and low callus regeneration efficiency, with the core bottleneck being the incomplete elucidation of molecular regulatory mechanisms governing totipotency reacquisition and genetic reprogramming in recipient cells. Focusing on the core scientific issues in this field, this review systematically elaborates on the characteristics and application limitations of current mainstream transformation technologies. By comparing immature and mature embryo-based transformation systems in wheat, it clarifies their respective strengths and shortcomings, and further analyzes the molecular barriers restricting transformation as well as potential breakthrough pathways. On this basis, the regulatory effects of key factors, including Agrobacterium strain type, growth status of receptor materials, selection markers, and promoter elements, on Agrobacterium-mediated wheat transformation efficiency are analyzed. Meanwhile, centering on the differentiation and regeneration of wheat calli, it systematically illustrates the molecular regulatory network of plantlet regeneration during genetic transformation from the perspectives of hormone signal transduction, functional characterization of morphogenesis-related genes, cell wall remodeling mechanisms, and chromatin accessibility modulation. Based on the above discussion, this review proposes that the core strategies for overcoming genotype limitation and improving transformation efficiency in wheat involve mining and utilizing key regeneration regulators with broad-spectrum effects, finely optimizing parameters of tissue culture and transformation systems, and developing novel tissue culture-independent transformation technologies. This review aims to provide theoretical support for constructing a genotype-independent universal transformation platform for bread wheat, thereby facilitating the large-scale and precise application of wheat molecular breeding technologies.

Key words: wheat, genetic transformation, regeneration system, auxin, morphogenetic genes

黄效贤, 何轶洋, 刘梦月, 石佳, 李文阳, 任兰天, 王冠军, 王东, 郑甲成. 小麦幼胚与成熟胚遗传转化体系研究进展[J]. 生物技术通报, doi: 10.13560/j.cnki.biotech.bull.1985.2026-0095.

HUANG Xiao-xian, HE Yi-yang, LIU Meng-yue, SHI Jia, LI Wen-yang, REN Lan-tian, WANG Guan-jun, WANG Dong, ZHENG Jia-cheng. Progress in Genetic Transformation Systems Using Immature versus Mature Embryos in Bread Wheat[J]. Biotechnology Bulletin, doi: 10.13560/j.cnki.biotech.bull.1985.2026-0095.

图/表 4

图1 小麦遗传转化主流递送系统的演进与比较分析A：农杆菌介导法。共转GRF4-GIF1、TaWOX5等形态发生基因可克服小麦转化的基因型依赖性；B：基因枪介导法。递送RNP复合物可实现无外源DNA的编辑，但假阳性率高；C：HI-Edit单倍体诱导法。携带基因编辑元件的诱导系与目标品种杂交，完成编辑，获得无转基因成分的编辑单倍体；D：病毒与纳米载体递送。病毒载体受限于装载量，存在生物安全逃逸风险，纳米颗粒递送难以获得稳定后代

Fig. 1 Evolution and comparative analysis of mainstream delivery systems for wheat genetic transformationA: Agrobacterium-mediated transformation, co-transformation of morphogenic genes could overcome genotype dependence in wheat transformation, such as GRF4-GIF1 and TaWOX5. B: Particle bombardment transformation, delivery of RNP complexes enables DNA-free genome editing, but suffers from a high false-positive rate. C: HI-Edit haploid induction. An inducer line, carrying gene-editing elements, is crossed with a target variety, to accomplish editing and obtain edited haploids free of transgenic components. D: Viral and nanocarrier-mediated delivery. Viral vectors are limited by cargo capacity and pose a biosafety risk of escape, nanoparticle-mediated delivery struggles to achieve stable progeny

表1 幼胚与成熟胚转化体系比较

Table 1 Comparison of transformation systems between immature embryos and mature embryos

比较维度 Comparison dimension	幼胚转化 Immature embryo transformation	成熟胚转化 Mature embryo transformation
取材便利性	受季节、发育期（花后12-15 d）严格限制，需田间或温室种植	干种子全年可用，易储存，来源极其广泛，无需特殊种植管理
生理状态	代谢旺盛，分化程度低，细胞全能性高	处于休眠状态，高度分化，需打破休眠并逆转分化状态
再生潜力	易诱导产生致密的胚性愈伤组织，分化率高	传统路径诱导愈伤困难，多为非胚性，新型直接器官发生率可达5%-10%以上
转化效率	模式品种可达40%-70%，最新优化可达66.8%^［27］	传统方法低于2%，直接器官发生路径有望新突破^［34］
基因型依赖性	仅少数模式品种转化高效	新型物理处理和直接再生方法可有效缓解基因型限制
主要应用场景	基因功能验证、高通量功能基因组学研究	规模化、工厂化转基因育种，商业化品种直接改良
最新技术进展	形态基因辅助，如BBM/WUS2、TaLAX1等	离心辅助直接器官发生^［34］

表2 农杆菌介导转化的关键影响因素及优化

Table 2 Key influencing factors and optimization of Agrobacterium-mediated transformation

关键因素 Key factors	作用机制 Mechanism of action	优化建议 Optimization strategies	代表性研究成效 Representative research outcomes
农杆菌菌株	决定T-DNA转移效率及Vir基因诱导能力	首选菌株C58C1，其次为EHA105，并优化菌液密度	菌株C58C1的GFP瞬时表达率达12%，显著优于GV3101
愈伤生理状态	影响T-DNA整合及细胞再生能力	诱导时间3周左右，继代次数小于2次	避免胚性退化，保证基因整合发生在细胞分裂期
预处理措施	破坏细胞壁屏障，抑制防御反应	辅助离心、真空渗透、热激等方法，并添加甘露醇及抗氧化剂防止褐化	显著提高侵染率，将褐化率降至10%
筛选标记系统	区分转化与非转化细胞组织	Bar优于HPT，因后者毒性过大易致细胞死亡	Bar筛选再生率达43%，而HPT筛选仅20%

图2 调控小麦体细胞再生与转化效率的多维分子框架A：激素平衡与TaLAX1基因，TaLAX1响应上游生长素信号并激活细胞分裂素合成基因TaIPT1，从而促进芽再生；B：基因调控网络，TaLAX1和TaWOX5因子激活BBM-WUS模块以促进体细胞胚发生，同时激活GRF4-GIF1模块以提升器官发生效率；C：细胞壁重塑，过表达TaLAX1基因或经化学处理可使细胞壁疏松化，增加T-DNA的穿透率；D：染色质重塑，生长素促进组蛋白乙酰化使染色质保持开放状态，增加Cas9对靶点的可及性

Fig. 2 Multidimensional molecular framework regulating somatic cell regeneration and transformation efficiency of wheatA: Hormone balance and TaLAX1 gene. TaLAX1 responds to upstream auxin signaling and activates the cytokinin biosynthesis gene TaIPT1, thereby promoting shoot regeneration. B: Gene regulatory network. TaLAX1 and TaWOX5 factors activate the BBM-WUS module to promote somatic embryogenesis, while simultaneously activating the GRF4-GIF1 module to enhance organogenesis efficiency. C: Cell wall remodeling. Overexpression of the TaLAX1 gene or chemical treatment loosens the cell wall, increasing the penetration efficiency of T-DNA. D: Chromatin remodeling. Auxin promotes histone acetylation to maintain an open chromatin state, increasing the accessibility of Cas9 to its target sites

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