Research progress in plant somatic embryogenesis and its molecular regulation mechanism

WANG Shiyi; HUANG Yizi; LI Zhouyang; HUANG Huahong; LIN Erpei

doi:10.11833/j.issn.2095-0756.20210141

Volume 39 Issue 1

Feb. 2022

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WANG Shiyi, HUANG Yizi, LI Zhouyang, HUANG Huahong, LIN Erpei. Research progress in plant somatic embryogenesis and its molecular regulation mechanism[J]. Journal of Zhejiang A&F University, 2022, 39(1): 223-232. doi: 10.11833/j.issn.2095-0756.20210141

Citation:

WANG Shiyi, HUANG Yizi, LI Zhouyang, HUANG Huahong, LIN Erpei. Research progress in plant somatic embryogenesis and its molecular regulation mechanism[J]. Journal of Zhejiang A&F University, 2022, 39(1): 223-232. doi: 10.11833/j.issn.2095-0756.20210141

Research progress in plant somatic embryogenesis and its molecular regulation mechanism

doi: 10.11833/j.issn.2095-0756.20210141

State Key Laboratory of Subtropical Silviculture, Zhejiang A&F University, Hangzhou 311300, Zhejiang, China

Received Date: 2021-01-25
Rev Recd Date: 2021-09-24

Available Online: 2022-02-14

Publish Date: 2022-02-14

Abstract

Each plant cell harboring all the genetic information of the species has the genetic potential to develop into a whole plant, which is termed plant cell totipotency. Somatic embryogenesis is a form of induced plant cell totipotency, by means of which embryos develop from somatic or vegetative cells in the absence of fertilization. Somatic embryogenesis, an increasingly important tool of plant biotechnology, has been widely applied in germplasm reservation, seedling propagation, molecular breeding and basic research of many plants and it has been implied in previous studies on molecular genetics that somatic embryogenesis is subject to the regulation by a complex network composed of transcription factors, hormone signaling pathways and epigenetic modifications. Therefore, this review, with a summary of the development routes of plant somatic embryogenesis, is aimed to give a comprehensice overview of the research progress achieved in the functions of key genes and epigenetic modification in the process of somatic embryogenesis along with an introduction to the applications of several key genes in genetic engineering. The development of new technologies is conducive to better and more profound insigts into the dynamic changes of of metabolic components, transcriptional regulation, phytohormone signal transduction and epigenetic modification during plant somatic embryogenesis, which will promote the understanding of the underlying molecular mechanism of somatic embryogenesis. Besides, by using those key genes of plant somatic embryogenesis, it is possible to development new methods and technologies to improve the efficiency of somatic embryogenesis induction and genetic transformation. [Ch, 81 ref.]
- somatic embryogenesis,
- transcription factor,
- regulatory mechanism,
- epigenetic modification

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植物体细胞胚胎(体胚)发生是指已分化的体细胞不经过性细胞融合，经历类似合子胚发育的途径直接形成植株的过程^[1]，是除合子胚发育途径之外另一种获得完整植株的重要手段，也是植物细胞全能性的一种体现。自首次在胡萝卜Daucus carota中发现体胚发生现象以来，人们在大量不同植物的组织培养、单细胞悬浮培养、原生质体培养和花粉培养的过程中都观察到或实现了体胚发生^[2-3]。因具有相对的遗传稳定性、可重复性和高效性等优点，体胚发生已经成为了重要的生物技术工具，在种质资源保存、优质种苗生产、人工种子、分子及细胞工程育种和基础研究等方面都有着广泛的应用。

植物体胚发生是一个涉及生长素、细胞分裂素等激素信号和复杂基因调控网络的过程。近年来的研究表明：一些关键的转录因子是体胚发生的主要介导者，其在生长素和细胞分裂素等激素信号刺激下启动和调控下游基因的表达，从而启动体胎发生，调控体胚发育^[2]。此外，基因组的表观修饰也被认为是影响植物体胚发生的重要因素，DNA甲基化、组蛋白去乙酰化等表观修饰都是植物体胚发生调控途径的重要组成部分^[4-6]。随着研究的深入，一些体胚发生的关键基因(如Baby Boom、Wuschel等)也被用于提高体胚发生的频率和转基因效率，已在玉米Zea mays等较难转化的植物中获得了成功^[7-8]。本研究将对体胚发生的途径、体胚发生关键基因及其调控机制、表观遗传修饰对体胚发生的调控及体胚发生关键基因的利用等进行综述，以期为后续的研究和技术开发提供科学依据。

1. 植物体胚发生的途径

体胚发生的过程极其复杂，从体细胞向胚性细胞转变是体胚发生的前提，也是其中最关键的一步。这个过程需经过脱分化，激活细胞分裂周期和重新组织生理、代谢与基因表达等多个步骤。外植体经过诱导产生体胚的途径主要有2种，即间接体胚发生和直接体胚发生。

间接体胚发生是最常见的途径，即在离体条件下细胞经过愈伤组织诱导再分化形成体细胞胚。间接体胚发生需要经过3个阶段：第1阶段是愈伤组织的形成，愈伤组织是一种看似无组织的原始空泡细胞团，由活的薄壁细胞组成，表现不同程度的紧实度和致密性；第2阶段是愈伤组织的胚性化，胚性的愈伤组织由周细胞和非周细胞共同发育而来^[9-10]，其质地一般较为坚实，颜色呈乳白色或黄色，表面具球形颗粒，由直径细胞组成，细胞较小，无液泡，且常富含淀粉粒；第3阶段是体细胞胚的形成，胚性愈伤组织形成之后，在愈伤组织的表面或内部产生原胚团，单个细胞或细胞团再从中发育成胚^[11]。在以幼胚、胚或子叶为外植体时，通常可以直接诱导胚性愈伤组织产生，进而产生体细胞胚。因此，就经过愈伤组织的间接体胚发生而言，胚性愈伤组织的形成是体胚发生的关键。

直接体胚发生，即从培养中的器官、组织、细胞或原生质体直接分化成胚，不经过愈伤组织阶段。这种途径中，外植体增殖较少，且致密细胞分裂更规则^[2]。单个或多个细胞层中的单个或多个细胞无须进一步处理即可分裂并膨大，形成形态可识别的胚胎^[1]。直接体胚发生主要可分为2个阶段：第1阶段为诱导期，在此阶段，细胞进入分裂状态；第2阶段为胚胎发育期，前一阶段形成的瘤状物继续发育，经过球形胚、心形胚等发育过程，最终形成体细胞胚。下胚轴、子叶、茎表皮等外植体体细胞脱分化后，由表皮细胞或亚表皮细胞经过不等分裂，产生1个胚细胞和1个胚柄细胞，后者发育类似胚柄，前者进一步分裂，由原胚发育为成熟胚。

直接途径和间接途径产生的体胚在形态学上相似，但由于间接途径的组织培养时间较长，产生的体胚更容易发生基因组水平上的变化(体细胞变异)^[12]。体胚也可以用来诱导新一轮的胚胎发生，称为次生或重复体胚发生^[13]。次生胚可以直接从原胚诱导，也可以在胚性愈伤组织形成后间接诱导。另外，体胚发生经历直接途径或间接途径往往取决于外植体的年龄：外植体离合子胚胎阶段越远，就需要越多的重编程过程将外植体重新转化为体胚^[14]。尽管从发育成熟或较老的组织和器官中诱导获得体胚通常比较困难，但无论组织的年龄如何，它们都可以通过直接途径或间接途径产生体胚^{[9, 15]}。因此，在确定体胚发生是通过直接途径还是间接途径时，细胞或组织与培养环境相结合的发育背景会比其距离胚胎阶段的时间更为重要。

2. 植物体胚发生相关的关键基因及其调控机制

2.1. Somatic Embryogenesis Receptor Kinase (SERK)

SERK基因是在研究胡萝卜体细胞向胚性细胞转变的过程中被发现的。SCHMIDT等^[16]从胡萝卜下胚轴悬浮培养的胚性细胞中分离出了第1个SERK基因(即DcSERK)，其只在胚性细胞内表达且只表达到体胚发育的球形期，而在非胚性细胞及球形期后的体胚中均不表达。随后在许多其他植物的研究中也鉴定到了不同的SERK同源基因，如椰子Cocos nucifera (CnSERK)、柑橘Citrus reticulata (CrSERK1)、鸭茅Dactylis glomerata (DgSERK)、蒺藜苜蓿Medicago truncatula (MtSERK)、水稻Oryza sativa (OsSERK)、小麦Triticum aestivum (TaSERK)和葡萄Vitis vinifera (VvSERK)等^[17]。在拟南芥Arabidopsis thaliana中过量表达SERK基因，能使体细胞胚胎发生的能力增加3~4倍，表明它能够促进植物体胚的发生^[18]。进一步研究发现：当SERK在细胞表面过表达时，可以通过识别分子信号介导其蛋白LRR区与胞外蛋白结合，这种结合能诱导细胞内部的信号级联放大(如油菜素甾醇信号通路)^[19]。这些信号可以识别不同的靶点，并通过染色质重塑增强体胚发生早期基因的表达(例如Leafy Cotyledon和Baby Boom)，进而诱导细胞或组织向胚胎发生转变^[18-19]。因此，SERK可能是体胚发生过程中促使体细胞向胚性细胞转变的关键。

2.2. Leafy Cotyledon (LEC)

LEC属于nuclear factor Y(NF-Y)转录因子家族，也是涉及许多功能的B3结构域蛋白大家族的成员，诸如LEC1、LEC2和FUSCA3(FUS3)等LEC基因都被认为是调节植物胚胎发生的转录因子^[20]。LEC基因(LEC1和LEC2)最先在拟南芥中发现，lec突变体具有胚胎发育不正常、缺少胚胎特异性蛋白、胚胎提早萌发等特征，表明LEC基因对于维持植物胚胎特性具有重要的功能^[21-22]。与其他调控因子只在胚胎发育特定阶段起作用不同，LEC基因在早期的胚胎形态发生阶段及后期的胚胎成熟阶段都起着重要的作用。在胚胎发育早期，LEC基因决定胚柄细胞命运，控制子叶特性，对维持体胚发生和促进球形胚的产生也具有重要作用；而在后期的胚胎成熟阶段，LEC基因的表达与种子成熟过程相关，包括储藏物质的积累和胚抗脱水性的获得等^[23]。

研究发现：LEC2基因的过表达会表现出异常发育的现象，如异位愈伤组织的产生等，但无法进一步分化为体胚^[24]，这表明LEC2基因可能提供了实现体胚发生所需的条件，但仅在体胚发生的诱导阶段起着重要作用^[25-26]。而LEC1基因在转基因植物中的异位表达会诱导体细胞胚样结构的形成^[27]，并在拟南芥体胚发生的整个过程中表现出差异表达模式。这表明LEC1基因可能参与了体胚的分化和发育，而不是体胚的诱导^[28]。FUS3和LEC2有43%的同源性，均为VP1/ABI3-like B3家族转录因子^[29]。FUS3基因在顶端分生组织中的特异表达，可以促使转基因植物顶端分生组织产生体胚^[30]。

2.3. Baby Boom(BBM)

BBM是AP2/ERF转录因子家族AINTEGUMENTA-LIKE(AIL)进化枝的成员。最初是在利用甘蓝型油菜Brassica napus未成熟花粉诱导单倍体胚胎时，发现的1个花粉体胚发生调节基因(BnBBM)，在甘蓝型油菜和拟南芥中异位表达时可诱导体胚的发生，该基因被认为在体胚发生过程中能促进细胞分裂和形态发生^[31]。后续研究表明：BBM基因是植物细胞全能性的关键调控因子，在没有外源植物生长调节剂或胁迫的情况下也能诱导体细胞胚的形成^[8]。比如BnBBM的异位表达可以在无需施加植物生长调节剂的条件下诱导拟南芥幼苗叶片和子叶上的体胚发生^[31]。利用BnBBM基因还可促进毛白杨Populus tomentosa愈伤组织形成体胚^[32]，而在再生困难的辣椒Capsicum annuum中异源表达BnBBM基因能有效克服辣椒遗传转化过程中再生及转化困难的瓶颈问题^[33]。BBM基因过表达还可以诱导其他类型的再生，包括愈伤组织、不定芽和根的形成，它的这种特性已被用于改善作物和模式植物的遗传转化效率^{[32, 34-35]}。

2.4. Wuschel (WUS)

WUS基因编码1个同源盒转录因子，它具有1个高度保守的同源盒结构域和保守的C末端区(包含3个功能性结构域：1个酸性结构域、1个WUS-box和1个EAR-like元件)^[36]。WUS基因的1个重要特征是它具有可移动性，它会从其生物合成的位置(干细胞龛中央区)移动到周边区的子细胞中，并在周边区激活1个负调控因子CLAVATA3(CVL3)的转录^[37]。CLV3移动到胞外与CLV1结合，反过来抑制WUS的转录，这种WUS-CLV反馈系统维持了干细胞库的稳定和顶端分生组织的发育^[38-39]。因此，WUS基因被认为是体胚发生和芽再生过程中所必需的^[39-40]。

与LEC2类似，WUS基因会对生长素作出响应，生长素能促发1个调控营养组织向胚性组织转变的信号级联途径，而这种转变是受WUS基因调控的^[41]。大量研究表明：在蒺藜苜蓿、陆地棉Gossypium hirsutum和中粒咖啡Coffea canephora等不同植物体胚发生过程中，WUS同源基因的表达都会明显上调^[42-45]。WUS基因在拟南芥中的过表达可以诱导体胚发生以及芽和根尖的器官发生^[46]。拟南芥WUS基因经过雌二醇诱导，在中粒咖啡中异源表达，能够诱导愈伤组织的产生，并促进中粒咖啡体胚的循环发生^[42]，而在陆地棉中则可通过启动生长素运输和信号转导途径提高胚性愈伤组织的分化效率^[45]。

2.5. 体胚发生的转录调控网络

近年来，大量的染色质免疫共沉淀和基因表达研究表明：植物体胚发生存在复杂的转录调控网络和信号转导途径(尤其是生长素途径)，不同的转录因子之间存在转录交互(cross-talk)调控^[2]。

BRAYBROOK等^[47]以过表达LEC2的拟南芥幼苗为材料，利用基因芯片分析确定了LEC2的靶基因，其中包括生长素途径基因和AGAMOUS-LIKE 15(AGL15)转录因子。LEC2能够激活生长素生物合成途径中的关键酶TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS 1 (TAA1)以及YUCCA2和YUCCA4 (YUC)的表达。而LEC2过量表达可以补偿不足量2,4-二氯苯氧乙酸(2,4-D)或效率较低的生长素[如吲哚乙酸(IAA)或萘乙酸(NAA)]在体胚诱导中的作用^[48]。反之，在正常的2,4-D含量下，LEC2的异位过表达不利于体胚的发生，往往会产生愈伤组织而非体胚^{[26, 48]}。LEC2与AGL15间存在转录交互调控作用，即LEC2能够上调AGL15的表达，AGL15也可以上调LEC2的表达。AGL15的过量表达会促进未成熟胚的体胚形成，但不会诱导种子苗自发的体胚发生，这表明AGL15只是增强了胚性组织的体胚发生能力^[49]。值得注意的是，LEC2和AGL15均能激活INDOLE-3-ACETIC ACID INDUCIBLE 30 (IAA30)的表达，该基因编码一种非典型的Aux/IAA蛋白质，且AGL15促进的体胚发生在iaa30突变体中明显受到削弱^{[47, 50]}。这暗示LEC2和AGL15可能共同通过生长素途径调控体胚发生，但目前AGL15和IAA30在LEC2诱导的体胚发生中作用尚不明确。

通过染色质免疫沉淀，在经2,4-D和BBM诱导的体胚中都鉴定出了BBM的下游靶基因，发现BBM能够结合LAFL (LEC1-ABI3-FUS3-LEC2)和AGL15基因的启动子区域^[2]。在lec1和fus3突变体中，BBM不能诱导体胚的发生，而在lec2和agl15突变体中，BBM诱导的体胚明显减少，说明BBM调控的LAFL/AGL15表达是BBM途径关键的下游路径^[2]。反过来BBM的表达也受到LAFL蛋白质的调节：BBM的表达量在lafl突变体种子中降低，说明LAFL转录因子正向调节BBM的表达^[2]。这些发现表明：BBM和LAFL基因间也存在转录交互调控作用，是生长素信号通路的一部分。

WOUND INDUCED DEDIFFERENTIATION1(WIND1)是AP2/ERF转录因子家族的另一成员，其过表达也能诱导体胚发生^[51]。WIND1及其同源基因WIND2~4由伤口诱导，并在组织损伤后刺激愈伤组织增殖^[52]。与BBM/LAFL蛋白不同，WUS和WOUND INDUCED DEDIFFERENTIATION1 (WIND1)蛋白通过细胞分裂素信号通路调控体胚发生。WUS通过抑制A型ARR基因来控制茎分生组织的生长，该基因是细胞分裂素应答的负调节因子^[53]，而WIND1通过B型ARR基因刺激愈伤组织的形成，该基因是细胞分裂素应答的正调节因子。WUS和WIND1也与LEC途径相互作用。比起单独激活WIND1或LEC2，按顺序激活WIND1和LEC2在外植体中能诱导更多的胚性愈伤组织^[54]。WIND1的过表达增加了外植体中胚性细胞的数量，这些细胞对LEC2作出响应后就形成体胚^[54]。反之，WUS被诱导表达后会降低其诱导的体胚组织中LEC1的表达水平，这表明WUS抑制了LEC途径^[41]。综上所述，在植物体胚发生过程中，LEC、BBM和WUS等重要的转录因子构成了1个复杂的转录调控网络。

3. 体胚发生的表观遗传调控

表观遗传调控也是植物体胚发生的关键因素。近年来，DNA甲基化、组蛋白去乙酰化/甲基化等重要的表观遗传机制均已被证实可以控制植物体胚发生的过程^[55-56]。

3.1. DNA甲基化

DNA甲基化是涉及生物学过程的重要表观遗传机制，它是表观基因组调节和维持基因表达程序的关键因素^[57]。DNA甲基化对体胚发育具有重要作用。通常，非胚性组织的基因组甲基化水平更高，而胚性组织的基因组甲基化水平则较低^[58-60]。在白橡Quercus alba中，体胚诱导时基因组DNA会被去甲基化，但随着胚胎发育，甲基化水平逐渐提高^[61]。在进行中粒咖啡体胚诱导时，原胚组织的DNA甲基化水平较低，而随着体胚的成熟，甲基化水平逐渐升高^[62]。在拟南芥中研究发现：DNA甲基化及其维持对体胚发生的调控是必需的^[63]，类似的结果在挪威云杉Picea abies中也有发现^[64]。

DNA甲基化也可通过引起特定基因的沉默，从而在体胚发生中发挥重要作用。如LEC1基因的启动子区域在体胚发生开始之前发生低甲基化，随后在胚胎成熟及营养生长期中甲基化水平增加；利用RNA导向的DNA甲基化对LEC1基因的启动子区域进行超甲基化会下调其转录，表明LEC1基因的转录受其启动子的甲基化调节^[65]。此外，还发现甲基化抑制剂5-azacitidine的应用可抑制或阻断胡萝卜培养物中的体细胞胚发生^[66]，而药物5-aza-2′脱氧胞苷可以通过抑制甲基转移酶1的活性来促进体胚发生，并且它还增加了体胚发生的关键调控因子STM的转录。这些证据表明：基因组DNA的甲基化水平和特定基因区域的DNA甲基化修饰与体胚发生有直接的联系。

3.2. 组蛋白甲基化

组蛋白甲基化是由组蛋白甲基化转移酶完成的。组蛋白甲基化转移酶在决定细胞命运中的重要性首先在动物上得到了证实，研究发现Polycomb抑制复合体2 (PRC2)成员进行组蛋白H3K27me3标记，是干细胞多能性所必需的^[67]。在拟南芥中，PRC2基因CURLY LEAF (CLF)和SWINGER (SWN)或VERNALIZATION 2 (VRN2)和EMBRYONIC FLOWER 2 (EMF2)双突变体在茎尖上形成愈伤组织，最终会引起间接的体胚发生和异位根^[68]。CLF还抑制成熟胚胎中的大量基因，包括AGL15、FUS3、ABI3、AIL5和AIL6/PLT3等与体胚发生相关的转录因子^[69]。IKEUCHI等^[70]也发现：PRC2突变体的根毛无法维持其已分化的状态，反而会形成无组织的细胞团，并且最终通过愈伤组织形成体胚，其部分原因是由于PRC2基因的突变导致其靶基因WIND3和LEC2的表达抑制被解除，进而诱导根毛细胞脱分化。这些研究说明：PRC2通过组蛋白甲基化抑制相关基因的表达来促进细胞分化的过程，反之则会引起细胞的脱分化，进而诱导体胚的发生。

3.3. 组蛋白去乙酰化

组蛋白H3和H4的乙酰化对基因表达有正向的调控作用，组蛋白乙酰化的水平和位置受到组蛋白乙酰转移酶(HATs)和组蛋白去乙酰化酶(HDACs)的严格控制。组蛋白去乙酰化也是与体胚发生密切相关的表观遗传修饰。TANAKA等^[71]提供了组蛋白去乙酰化在体胚发生过程中起主要作用的第1个证据。他们研究表明：HDAC抑制剂曲古抑菌素A(TSA)能使拟南芥种子苗的萌发生长停滞，并诱导胚胎标记基因LEC1、FUS3和ABI3的表达上调，导致胚胎不能完成向幼苗生长的过渡。类似现象在2个HDACs-hda6/hda19双突变体中也可以观察到，且该双突变体能在拟南芥叶片上产生体胚结构^[71]。后续的研究进一步揭示了这2个HDACs抑制胚胎基因表达的机制。其中，HDA19会特异性地结合VAL2^[72]，HDA6则特异性结合VAL1^[73]，VAL1和VAL2又会与转录中介复合体(mediator complex)的抑制性亚基CDK8结合，进而招募这2个HDACs和抑制形态的转录中介复合体抑制LAFL基因的表达^[73]。

组蛋白去乙酰化促进体胚发生的作用在其他植物的体胚诱导实验中也获得了证实。萌动的云杉Picea asperata体胚经TSA处理后会维持其体胚的状态，而不会转化为幼苗^[74]。在小麦中，TSA和丁酸钠(另一种HDACs抑制剂)的处理可以增加胚性愈伤的诱导率和芽的分化率，但TSA有广谱的效果，丁酸钠对不同基因型的效果有差异^{[6, 75]}。TSA处理还可显著提高热胁迫后的甘蓝型油菜小孢子单倍体体胚发生的效率，表明热激处理和组蛋白去乙酰化可能共同作用于体胚发生调控因子的上游，从而启动体胚发生的程序^[76]。与H3K27me3类似，组蛋白乙酰化水平和HDACs的活性在激素诱导的间接体胚发生中会发生变化^[77-78]，暗示在间接体胚发生的早期阶段，组蛋白去乙酰化作用可能参与了体细胞的重编程。这些研究表明：在植物中组蛋白去乙酰化是调控体胚发生的保守途径。

4. 关键基因在体胚发生和遗传转化中的应用

目前，植物的高效遗传转化仍然是一个巨大挑战，其主要原因是转基因细胞往往难以发育成完整的植株。虽然，通过组培方法、农杆菌侵染等条件的优化，水稻、拟南芥、杨树Populus等少数植物的遗传转化获得了成功，但仍存在基因型依赖严重、转化效率低等问题。随着体胚发生机制研究的深入，一些关键的体胚发生调控基因被逐渐用于提高植物遗传转化和再生的效率^[79]。

BBM和WUS是2个最常用于植物遗传转化的体胚发生关键基因。例如，甜椒Capsicum frutescens是具有重要营养和经济价值的蔬菜，但也是公认的难以转化的顽拗材料。HEIDMANN等^[35]研究发现：在甜椒中瞬时表达BnBBM基因可以高效地诱导细胞再生，并产生大量可发育成植株的体胚，认为利用该基因可以为难转化的植物开发一种有效的遗传转化和再生的体系。在玉米中，ZmBBM和ZmWUS2基因共转化未成熟胚时，转基因愈伤的比例显著提升，且在多个难转化的玉米近交系中均有明显效果^[80]。此外，ZmBBM和ZmWUS2的共转化还可以在高粱Sorghum bicolor、甘蔗Saccharum officinarum和水稻中提高转化效率^[80]。因此，BBM和WUS被认为是单子叶作物基因工程中具有重要潜在利用价值的关键基因^[79]。而在双子叶植物中，MAHER等^[81]将WUS2和ipt或WUS2和STM共转化烟草Nicotiana tabacum无菌种子苗，在烟草叶片上实现了芽的原位诱导，避免了繁琐的组培过程；且利用该策略，成功地对PDS基因进行了基因编辑，获得了基因编辑的后代。针对栽培的烟草植株，MAHER等^[81]通过注射携带WUS2和ipt或WUS2和STM基因的农杆菌在伤口处直接诱导了芽的形成，并且也可以实现对PDS基因的编辑获得后代。该方法在番茄Solanum lycopersicum、葡萄中也获得了成功试验，证明了WUS等基因在双子叶植物基因工程中的应用潜力。

5. 总结与展望

体胚发生是体现植物细胞全能性的一种重要形式，一直是植物学研究的热点。对于植物体胚发生的研究，从最初的激素含量和组合方式等诱导条件的优化，到在多种植物中鉴定和表征了许多参与体胚发生的基因，并通过操纵关键基因的表达来启动及调控体胚的发生发育，再到全面研究体胚发生的通路和基因调控网络，观察再生过程中的动态表观遗传变化，标志着对体胚发生规律的认识一直在不断深入和完善。

细胞和分子生物学的进步以及各种新技术的出现，为进一步研究植物体胚发生过程涉及的更深层次细胞和分子生物学机制提供了条件和机遇。深入探究体胚发生过程中激素信号与基因表达之间，以及调控基因间复杂的网络关系，解析体胚发生的分子机制及信号途径，将为阐明体胚发生的内在规律提供更多的证据。表观遗传修饰在体胚发生调控中的重要性已得到证实，但表观遗传修饰如何参与体胚发生，在体胚发生过程中如何维持和建立DNA甲基化，以及基因不同区域的甲基化如何参与植物体胚发生中的基因转录调控等问题仍没有答案，需深入的研究来解开表观遗传修饰调控体胚发生的谜团。

此外，将关键基因的表达与遗传转化技术有机结合起来，在更多的植物中实现体胚发生，进而提高植物的再生能力和遗传转化的效率，可为更多植物实现基因编辑等高效和精细的遗传操作提供新的途径，这对加快优良品种的繁育和分子育种平台的建立，促进转基因植物的产业化开发都具有十分重要的意义。

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