松柏类植物体细胞胚胎发生的研究进展

松柏类植物的体细胞胚胎发生既是繁育的一种手段,又是研究胚胎发育过程中结构、生理和分子事件的一种重要的模式系统.整个体细胞胚胎发生过程主要包括3个步骤:胚性组织的诱导和增殖、体细胞胚的成熟以及体细胞胚的萌发和转换.过去为了提高胚胎发育过程所做的努力主要都集中在胚的成熟阶段,这是因为一直认为能否成功再生的关键在于胚发育成熟阶段的处理.然而,在过去几年里,结合生理生化以及分子生物学的研究发现,胚胎发生的早期阶段对于完成整个发育过程也是至关重要的,早期阶段培养条件的优化可以显著提高培养过程中体细胞胚的数量和质量.此外,萌发过程培养条件的调节对于提高成熟体细胞胚的萌发率和转换率也很重要.因此,这些新的研究成果对于改善松柏类植物体细胞胚胎发生中的胚的诱导率和转换率低的现象具有重要的意义.     

松杉类植物体细胞胚发育机理的研究进展

植物体细胞胚胎发生不仅可作为其繁育的重要手段,而且也是研究胚胎发育过程的一种重要模式系统.体细胞胚在形态和生理上的成熟,直接影响到植株的萌发和再生频率.本文综述了近年来国内外有关裸子植物中几种松杉类植物体细胞胚发育过程的研究报道,其中主要涉及培养基成分和脱落酸(ABA)对体细胞胚发育的影响,以及体细胞胚发育在细胞学、细胞程序性死亡、相关基因和蛋白质组学等方面的研究进展,并进一步讨论了松杉类植物体细胞胚的发育机理,以及体细胞胚在遗传转化系统中的作用.     

胁迫诱导植物体细胞胚发生中相关基因及蛋白的研究进展

植物体细胞胚发生是一个复杂的发育过程,体细胞胚发生已成为研究植物胚胎发育过程中生理、生化、分子生物学等方面分子机理的模式系统。胁迫被认为是对体细胞胚的诱导有重要作用的因素。植物生长调节物质如2,4-D、ABA等目前认为是与胚性能力获得有关的胁迫物质。在蛋白和转录水平上对基因表达的分析中已鉴定出一些与体细胞胚发生相关的基因和蛋白。该文主要对近年来国内外有关胁迫诱导体细胞胚发生的相关基因及蛋白的研究进展进行综述。     

植物体细胞胚发生过程中基因表达的研究进展

植物体细胞胚胎发生是一个复杂的发育过程,研究者们通过分析植物体细胞胚发生过程中的基因表达或胚性组织和非胚性组织中基因的差异表达,获得了在体细胞胚发生过程不同时期表达的基因,并分析了这些基因在胚胎发生途径中可能的作用。综述了在植物体细胞胚发生过程中细胞周期相关基因、胁迫和激素应答相关基因、信号转导相关基因、晚期胚胎丰富蛋白基因及与体细胞胚发生相关的胞外蛋白基因表达的研究进展。     

栎属植物体细胞胚胎发生研究现状

总结了影响栎属植物体细胞胚胎发生的主要可控因素及体细胞胚的遗传变异,组织学研究现状。目前,已能从成年组织上诱导出体细胞胚,但诱导率较低。重复性体胚发生系统已被认为是一种可资利用的繁殖途径,倍受关注。应用DNA分子标记分析表明：体胚细胞系内存在遗传变异。成熟和较低的萌发率是这一技术广泛应用的瓶颈。     

云南大叶茶体细胞胚发生及体细胞胚苗形成体系的建立

利用云南大叶茶(Camellia sinensis var.assamica Kitamura)胚性细胞系(CL_1)中悬浮培养物,建立了高频率同步化体细胞胚发生及体胚苗形成体系。以改良的MS为基本培养基,将CL_1中培养物由液体保持培养基(0.1mg/L 2,4-D 0.5mg/L 6-BA)继代转入液体诱导培养基(0.05mg/L 2,4-D 0.50mg/L6-BA),暗培养诱导28d,转入不含任何激素的液体分化培养基中再培养28d,获得了不同发育时期的体细胞胚,其发生频率为81.5％。不同发育时期的体细胞胚用不同目的细胞筛收集,在液体生长培养基(1/2 MS 1.0mg/L GA_3 0.5mg/L 6-BA)中培养发育成熟。ABA有利于高质量体细胞胚的形成。20～70月大小的体细胞胚在固体生长培养基中成苗转换率为75％。在液体悬浮培养条件下观察记录了体细胞胚发育过程,证实其过程与合子胚的形态发生过程相似。     

马尾松幼胚体细胞胚胎发生研究

本论文首次报道了马尾松（Pinus massoniana Lamb.）幼胚体细胞胚胎发生的完整发育过程,并对影响马尾松胚性愈伤组织诱导的因素如球果采种期、球果冷藏处理时间、外植体处理方式等进行了探讨,统计胚性愈伤组织诱导率,进行增殖评价,探讨ABA浓度梯度对马尾松体细胞胚分化成熟的影响,试验数据用SPSS16统计分析软件进行方差分析、差异显著性检验。结果表明：1）2008~2009连续2年内15个采种期得到的幼胚,胚性愈伤组织诱导和增殖有显著性差异,最适宜的马尾松球果采种期是6月下旬至7月下旬,诱导率在9.66%~22.59%之间;2）球果冷藏处理时间,对胚性愈伤组织诱导有显著性差异,其中4℃冷藏球果15d有利于幼胚胚性愈伤组织诱导;3）雌配子体包含幼胚的接种处理方式是可取的;4）胚性愈伤组织经稳定增殖培养后,转入分化成熟培养基,得到体细胞胚状体＂爆发式＂分化成熟,数量多,质量好。适宜体胚成熟转化的培养基为：成熟LP培养基添加ABA5.0mg·L-1＋60.0g·L-1蔗糖,并附加L-谷氨酰胺和水解酪蛋白;5）成熟体细胞胚在无激素萌发型LP培养基上正常萌发,并转化为结构完整的小植株。本研究首次建立了马尾松幼胚体细胞胚胎发生技术平台,为马尾松遗传改良种质创新、缩短育种周期奠定了研究基础。     

枸杞体细胞胚发生中蛋白质代谢动态的立体计量

以宁夏枸杞无菌苗叶片为材料,离体培养并诱导体细胞胚胎发生。根据细胞形态计量学原理,应用数字图像处理软件计量由光学底片经Ａ／Ｄ转换成的数字图像中的蛋白质大分子,对于枸杞体细胞胚发生过程中蛋白质分子的代谢动态进行了量化处理,并对量化结构分析了蛋白质代谢动态与体细胞胚发生、发育的关系。     

渗透剂对白刺花体细胞胚成熟及萌发的影响

该研究以蔗糖、麦芽糖、山梨醇及PEG(6000)为渗透剂,探讨了不同渗透剂对白刺花体细胞胚发育、胚成熟及萌发的影响。结果表明:白刺花下胚轴形成的胚性愈伤组织接种至MS+2,4-D 0.2 mg·L~(-1)+NAA 1.0 mg·L~(-1)+6-BA 2.0 mg·L~(-1)+TDZ 1.0 mg·L~(-1)+蔗糖40 g·L~(-1)+谷氨酰胺100 mg·L~(-1)+植物凝胶3g·L~(-1)的培养基上,体细胞胚发生率高达66. 21%,总胚数为79个; 7%蔗糖可使体细胞胚成熟率高达64.36%,同时也可提高多子叶畸形胚形成; 2%麦芽糖+2%山梨醇+4%蔗糖组合使体细胞胚成熟率最高达88.89%,畸形胚比例最低; 30 g·L~(-1)PEG培养时,体细胞成熟率最高,为82.35%;鱼雷期的体细胞胚最合适转接,可使体胚萌发率达90.58%,复合糖上培养得到的成熟体细胞胚生根率最高,为87.47%。这为实现白刺花体细胞胚育苗奠定了理论基础,并提供了可行的方案。     

芸芥体细胞胚胎发生及植株再生体系的建立

以芸芥子叶为外植体,诱导芸芥体细胞胚胎发生并建立植株再生体系.结果表明:基因型及植物生长调节剂对芸芥体细胞胚胎发生均有一定的影响,其中以含有1.0mg·L-12,4-D的MS培养基诱导芸芥体细胞胚胎发生的效果最优.在MS 0.2mg·L-12,4-D培养基上,胚性愈伤组织可大量增殖.对芸芥体细胞胚胎成熟的研究表明,体胚在N6培养基上成熟最佳,且45.2%的成熟体胚可在1/2MS 0.1mg·L-1IBA培养基上萌发生长.     

Recent advances in conifer somatic embryogenesis: improving somatic embryo quality

Somatic embryogenesis of coniferous species was first reported more than 20 years ago. Since then, there has been an explosion of research aimed at developing and optimizing protocols for efficient regeneration of plantlets. Although routinely used both as a means of propagation, as well as a valuable model system for investigating the structural, physiological, and molecular events occurring during embryo development, in vitro embryogenesis is still problematic for some coniferous species. Major problems include: low number of embryos generated; and low frequency of mature embryos able to convert into viable plantlets. Until recent years, despite the fact that embryogenesis is comprised of a sequence of defined steps which include proliferation of embryogenic tissue, embryo maturation, and germination, attempts at improving the whole procedure have been made almost exclusively during the maturation stage. This strategy was based on the assumption that successful regeneration is related to treatments provided during the development of the embryos. Major optimizations of the maturation medium have involved judicious selections of type and concentration of growth regulators, namely abscisic acid, and adjustments of the osmoticum of the culture medium. Extensive work has been conducted in defining the effects of plasmolysing and non-plasmolysing osmoticum agents during maturation, as well as in improving desiccation techniques required for the completion of the maturation program. In the last 2 years, however, work on spruce has clearly demonstrated that the early events in embryogenesis are crucial for the successful completion of the overall embryogenic program. The use of cell tracking techniques, implemented by physiological and molecular studies, has revealed that manipulations of the culture conditions early in the process can increase both number and quality of embryos produced in culture. Additional manipulations of the germination medium can also enhance germination and conversion frequency of somatic embryos matured in a sub-optimal environment. These new findings, together with the unraveling of molecular mechanisms involved in the control/regulation of embryo development hold considerable promise for clonal propagation in conifers.     

Maturation of somatic embryos in conifers: Morphogenesis,physiology, biochemistry,and molecular biology

Summary In the past 15 years tremendons progress has been made towards the development of systems for the induction and development of somatic embryos of coniferous species. Since the first report in 1985, several species have been induced to produce somatic embryos. This has been rendered possible by the development of rational media and improvement of culture conditions, which have resulted in increased embryo quality and higher conversion frequency. Understanding the physiological and biochemical events occurring during in vivo embryogenesis has been fundamental in the design of new protocols for improving the somatic embryogenic process. Specifically, the inclusions of abscisic acid (ABA) and osmotic agents, such as polyethylene glycol (PEG), have been shown to be necessary for the functional development of somatic embryos. In the past few years, physiological and biochemical investigations have been useful in increasing our knowledge on the mode of action of ABA and PEG during embryo development. In comparison with the flowering plants, our understanding on the molecular mechanisms regulating the embryogenic process in coniferous species is still very limited. The application of new molecular techniques is therefore fundamental towards this end. The emphasis of this review is on recent information dealing with the maturation of conifer somatic embryos.     

Non-Zygotic Embryogenesis in Hardwood Species

Hardwood species are valuable biological resources that have an important role in the economy and ecology of ecosystems worldwide. Non-zygotic or somatic embryogenesis (SE) is a powerful tool in plant biotechnology as it is a form of clonal propagation, amenable to cryopreservation of valuable germplasm and genetic transformation including gene editing. The SE process involves five steps and includes somatic embryo induction, proliferation, maturation, plantlet conversion, and subsequent plant acclimatization. This review aims to provide a general overview of these steps in different SE systems developed for hardwood species. Factors that influence the induction stage such as the age of the donor plant, genotype and culture media are discussed. The role of different explant types, i.e. zygotic embryos and non-zygotic tissues, such as roots, flower tissues, nodes, internodes, leaves or shoot apices, in SE induction are especially emphasized. Histological studies of the origin of somatic embryos and the sequence of events leading to their development from initial explants are assessed. Maintenance of embryogenic capacity carried out by subculture of embryogenic inocula on semisolid or liquid media through cell suspension cultures or by temporary immersion systems is described. At present, the main concerns associated with the application of SE for large-scale propagation of elite hardwoods are related to the embryo maturation, germination, and plantlet conversion steps, and these are highlighted in this review. Finally, molecular aspects associated with somatic embryo induction and development are also described. Attempts to overcome the hurdles identified in the embryogenic process, and future lines of research are proposed.     

Factors Affecting Somatic Embryogenesis from Cotyledonary Explants of Safflower

Frequency of safflower (Carthamus tinctorius L.) somatic embryogenesis, number of somatic embryos per responding explant and somatic embryo maturation and germination were affected by genotype, explant age, carbon source, and ethylene. Among 8 cultivars tested, 7 were embryogenic with varying frequencies. The best response was obtained with cv. Girna. Whole cotyledonary explant from 10-d-old plants was best responding compared to 5- or 15-d-old ones. Among different carbon sources, sucrose at 87.6 mM concentration was most suitable for embryo induction, maturation and germination. Of the different ethylene inhibitors, silver nitrate at 50 [micro ]M concentration significantly increased the embryogenic frequency and also the number of embryos per responding explant. Silver nitrate has pronounced effect on embryo maturation but had no effect on germination.     

Induction of somatic embryogenesis from female flower buds of elite <Emphasis Type="Italic">Schisandra chinensis</Emphasis>

Somatic embryogenesis (SE) was induced in female flower buds from mature Schisandra chinensis cultivar ‘Hongzhenzhu’. Somatic embryo structures were induced at a low frequency from unopened female flower buds and excised unopened on Murashige and Skoog (MS) agar medium containing 4.0 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D). Friable embryogenic calli were induced from somatic embryo structures after three to four subcultures on initiation medium. The frequencies of mature somatic embryo germination and plantlet conversion were low, but increased in the presence of gibberellic acid (GA₃). Some germinated somatic embryos could form friable embryogenic calli on medium without plant growth regulators (PGRs). The germination and conversion frequencies of somatic embryos from embryogenic calli induced using PGR-free medium were higher than for somatic embryos from embryogenic calli induced on medium containing 2,4-D. Most somatic embryos from 2,4-D-induced embryogenic calli had trumpet-shaped embryos, and most somatic embryos from PGR-free medium–induced embryogenic calli had two or three cotyledons. Histological observation indicated that two- and three-cotyledon embryos had defined shoot primordia, but most of the trumpet-shaped embryos yielded plantlets that lacked or had poorly developed meristem tissue. Cytological and random amplification of polymorphic DNA (RAPD) analyses indicated no evidence of genetic variation in the plantlets of somatic embryo origin.     

Enhanced production of recombinant human gastric lipase in turnip hairy roots

Somatic embryogenesis is a reliable and important tool, and the relevant genes controlling this process act as vital roles through the whole development of somatic embryos. However, regeneration via somatic embryogenesis in Chinese chestnut has been impeded and its molecular mechanism is not known. Therefore, firstly we described a protocol for somatic embryo initiation, development, maturation and germination. Embryogenic calli were obtained in embryo initiation medium containing 1.8 μM 2,4-D and 1.1 μM 6-BA, and then were transferred to embryo development medium without any hormones for at least 4 weeks, until cotyledonary embryos appeared. Next, the somatic embryos were transferred to embryo maturation medium containing Gamborg’s B-5 Basal Salt Mixture with 0.5 μM NAA and 0.5 μM 6-BA for 3 weeks. Finally, these mature embryos were germinated in embryo germination medium consisting of WPM with 0.5 μM NAA and 0.5 μM 6-BA, resulting in shoot regeneration with a 2.1% conversion rate. Additionally, eight embryogenesis-related genes were identified, and the expression profiles of these genes during embryogenesis were analyzed via quantitative real-time RT-PCR (qRT-PCR). The CmSERK, CmLEC1, CmWUS and CmAGL15 genes exhibited high expression in the initial embryo stages, which inferred that these genes played key roles during the initiation of embryogenesis. Studies on embryogenesis-related genes will provide an insight for further elucidating molecular mechanism during somatic embryogenesis of Chinese chestnut. Furthermore, the successful establishment of a somatic embryo regeneration system for Chinese chestnut will lay a significant foundation for a stable genetic transformation system and genetic improvement.     

Improvement of somatic embryogenesis in Hevea brasiliensis (Müll. Arg.) using the temporary immersion technique

Summary A culture procedure using temporary immersion in a liquid medium was tested for somatic embryogenesis of Hevea brasiliensis (Müll. Arg.). Embryogenic callus was placed under regeneration conditions, either on a gelled medium (Phytagel, Sigma, St. Louis, MO) or in a container designed for temporary immersion. The latter technique has some advantages over the use of a gelled medium during both the early steps of somatic embryogenesis, i.e., embryo development, and later on, i.e., during maturation, desiccation and germination. Somatic embryo production in a liquid medium was three to four times greater than on a semi-solid medium: 400 embryos/g fresh weight under the best embryogenesis induction conditions. Somatic embryogenesis had to be initiated on a gelled medium before the embryogenic callus was transferred to temporary immersion, and the amounts of 3,4- dichlorophenoxyacetic acid and N⁶-benzyladenine had to be reduced. Temporary immersion resulted in substantially more consistent, synchronized somatic embryo development, reducing the number of abnormal embryos by half and stimulating germination. All of the late events could be carried out in the temporary immersion container. Effective drying conditions were achieved after 12 wk without immersion and without selection of the embryos. Temporary immersion during germination greatly stimulated root development (+60%) and epicotyl emergency (+35%), combined with increased synchronization and a substantially reduced workload.     

Comparative Efficacy of Abscisic Acid and Methyl Jasmonate for Indirect Somatic Embryogenesis in Medicago sativa L.

The effects of methyl jasmonate (MeJA) in relation to abscisic acid (ABA) on different phases of somatic embryogenesis were studied in Medicago sativa L. Different concentrations of both the growth inhibitors (0.0, 0.5, 5.0, 50.0 and 500.0 μM) were tested in five distinct phases of somatic embryogenesis, viz., induction, proliferation, differentiation, maturation and regeneration. Like ABA, MeJA also inhibited callus induction, callus growth, proliferation of embryogenic suspension as well as germination and conversion of somatic embryos. However, its inhibitory effects on various phases of somatic embryogenesis were less pronounced as compared to that due to ABA. In contrast to ABA, MeJA did not have any significant influence on the development of somatic embryos when applied in the differentiation phase. The study showed that ABA used routinely as an inducer of somatic embryo maturation in M. sativa could not be replaced by MeJA.     

Enhancement of American chestnut somatic seedling production

Somatic embryogenesis holds promise for mass propagation of American chestnut trees bred or genetically engineered for resistance to chestnut blight. However, low germination frequency of chestnut somatic embryos has limited somatic seedling production for this forest tree. We tested the effects of culture regime (semi-solid versus liquid), cold treatment, AC and somatic embryo morphology (i.e., cotyledon number) on germination and conversion of the somatic embryos. Cold treatment for 12 weeks was critical for conversion of chestnut somatic embryos to somatic seedlings, raising conversion frequencies for one line to 47%, compared to 7% with no cold treatment. AC improved germination and conversion frequency for one line to 77% and 59%, respectively, and kept roots from darkening. For two lines that produced embryos with one, two or three-plus cotyledons, cotyledon number did not affect germination or conversion frequency. We also established embryogenic American chestnut suspension cultures and adapted a fractionation/plating system that allowed us to produce populations of relatively synchronous somatic embryos for multiple lines. Embryos derived from suspension cultures of two lines tested had higher conversion frequencies (46% and 48%) than those from cultures maintained on semi-solid medium (7% and 30%). The improvements in manipulation of American chestnut embryogenic cultures described in this study have allowed over a 100-fold increase in somatic seedling production efficiency over what we reported previously and thus constitute a substantial advance toward the application of somatic embryogenesis for mass clonal propagation of the tree.