Secondary somatic embryogenesis and applications in plant breeding

Summary Secondary somatic embryogenesis is the phenomenon whereby new somatic embryos are initiated from somatic embryos. Such cultures have been described in at least 80 Gymnosperm and Angiosperm species. In the initial step (primary somatic embryogenesis) such cultures have to be started from plant explants. In general, primary somatic embryogenesis from vegetative plant explants is, indirect and mostly driven by auxin (AUX) or auxin and cytokinin (AUX/CYT) supplemented media, whereas, from zygotic embryos it is direct and driven, to a larger extent, by CYT or growth regulator free media. Primary somatic embryogenesis from floral plant explants is between these two extremes. Indirect and direct somatic embryogenesis should be seen as two extremes of one continuum: in indirect somatic embryogenesis the embryos develop up to the (pre)-globular stage and in direct somatic embryogenesis to mature stages before they are subjected to secondary embryogenesis. In general, secondary embryogenesis requires no growth regulators in species with CYT driven primary embryogenesis. Whereas, continuous exposure to growth regulators is needed in species with CYT/AUX or AUX driven primary embryogenesis.In most species somatic embryos can be converted into shoots, although the frequencies are mostly low. In general, somatic embryos induced by growth regulator free or CYT supplemented media meet more difficulties in shoot development than embryos induced by AUX supplemented media. Applications of secondary somatic embryogenesis for plant breeding are discussed.     

Micropropagation of mother plants of lucerne (Medicago sativa L.) for somatic embryogenesis

Summary A reliable and standard method was established for micropropagation of the A70-34 selected genotype of lecerne (Medicago sativa L., genotype A70-34), aimed at reducing contamination problems, seasonal and phenological influences on regeneration and phytosanitary problems of the mother plants, while maintaining the regenerative potential for somatic embryogenesis of the plant tissues. Mother plants were routinely maintained for several subcultures and somatic embryogenesis was regularly obtained from the subcultured explants. Proliferation, rooting and embryogenetic ability of plants cultured for 30 days was greater than those cultured for 20 days. The regenerative potential of tissues from different organs and of triturated and intact whole plants was also tested. Petioles were confirmed as the best source for embryogenesis as far as efficiency and repeatability were concerned, even though regeneration from other explant types was also achieved. Production of somatic embryos through mechanical trituration of the in vitro cultured plants was obtained; the regeneration ability of the triturated plants was greater than that observed in the intact plants.     

Somatic embryogenesis from floral tissue of cassava (Manihot esculenta Crantz)

The aim of this study was to examine the embryogenic potential of floral material of the cassava cultivar MCOL 1505. Macerated immature inflorescences were found to be highly embryogenic, with almost 78% of the explants producing somatic embryos. Somatic embryos were also produced from whole male florets and half florets although at much lower rates. No regeneration was obtained from anther, microspore or floret wall tissue. Somatic embryos derived from immature inflorescences were regenerated via organogenesis and the plants derived from this process were assessed in terms of phenotype and ploidy level. If haploid plants could be produced by this method, this would have significant implications in assisting traditional cassava breeding, as this would allow homozygosity to be reached more rapidly. In a crop such as cassava, which is highly heterozygous in nature, the use of haploids in a breeding programme could considerably shorten the time taken to produce new desirable cultivars. This is the first report on plant regeneration through somatic embryogenesis from floral tissue of cassava. This revised version was published online in July 2006 with corrections to the Cover Date.     

Direct as well as indirect somatic embryogenesis from immature (unemerged) inflorescence of a minor millet Paspalum scrobiculatum L.

Somatic embryos differentiated directly on the rachis of immature inflorescences of Paspalum scrobiculatum L. cv. PSC 1 on culture to MS or N₆ medium supplemented with different concentrations (4.5–22.5 μM) of 2,4-dichlorophenoxyacetic acid (2,4-D). Direct embryogenesis on the rachis of inflorescence explants forms the first instance in graminaceous plants. Highest frequency of direct embryogenesis (34%and 30% cultures, respectively) was possible on N₆ medium supplemented with 4.5 μM of 2,4-D and MS medium fortified with9.0 μM of 2,4-D. Other tissues of the explant, floral-primordia, only after an initial phase of callusing differentiated into somatic embryos; indirect embryogenesis. Somatic embryogenesis, direct as well as indirect, was resolved by scanning electron microscopy. The somatic embryos germinated and developed into plantlets on regeneration medium. Interestingly, one week incubation of somatic embryos on activated charcoal (0.5%) fortified basal medium, supported high potential for ‘germination’ on transfer to charcoal-free basal medium. This beneficial effect of activated charcoal on regeneration of somatic embryos into plantlets is the first record in the Gramineae. This revised version was published online in July 2006 with corrections to the Cover Date.     

两种常用激素组合下棉花体细胞胚胎发生过程的组织学观察

 MSB培养基添加两种常用植物激素组合，Indole-3-butyric acid (IBA) + kinetin (KT)和2, 4-dichlorophenoxyacetic acid (2, 4-D) + KT，分别命名为IK和DK处理用来诱导陆地棉体细胞胚胎发生。陆地棉品系YZ1下胚轴切段作为外植体在诱导培养基上培养4周后，继代到分化培养基上促进体细胞胚胎发生。结果表明，两种处理均有体细胞胚胎发生，其中IK处理上外植体嫁接33 d后就可见体细胞胚出现，分化率高达97.6%，大多数外植体表现为体细胞胚较胚性愈伤早出现。DK处理中体细胞胚胎发生慢，体细胞胚都是经过明显的胚性愈伤发育而来，胚性愈伤分化率明显低于IK处理，仅为28.6%。此外，IK处理中的外植体在培养过程中大部分都出现了须根，而DK处理中则没有。两种处理中出现了两种不同的体细胞胚胎发生过程，组织学观察结果表明DK和IK处理中的体细胞胚分别主要起源于初生形成层和皮层。     

棉花优良品种中棉所19高频体细胞胚胎发生细胞系筛选与植株再生

选用我国优良棉花品种中棉所19进行组织培养, 研究了该品种的体细胞胚胎发生和植株再生能力。 中棉所19的体细胞胚胎发生有两种情况： 一是由外植体直接诱导获得胚性愈伤组织和体细胞胚; 二是先诱导获得愈伤组织, 再经继代培养获得胚胎发生。 单独使用ZT可在早期诱导胚胎发生, 在该情况下, 对子叶的诱导能力最强, 胚根     

The HMW Glutenin Subunit and Gliadin Compositions of German-Grown Wheat Varieties and their Relationship with Bread-Making Quality

An in vitro system for the initiation of somatic embryogenesis and plant regeneration of spring- and winter-type rye has been developed. The optimal developmental stage of immature embryos to be used as explants is the late stage of spherical coleoptile when the tissue turns milky in colour and the coleoptile is enlarged but spherical. The type and concentration of auxin in the induction medium is of importance for obtaining high efficiency of somatic embryogenesis. CC-medium high 30 μM Dicamba resulted in well developed somatic embryos in large umbers. Positive effects on the efficiency, intensity and speed of development of somatic embryos have been observed by the addition of coconut water to CC-medium, replacing agar by agarose and culturing the explants in low light intensity. Following a “step by step” optimization, culture conditions have been defined giving rise to a 90—100% efficiency of somatic embryogenesis and a high number of regenerated plants.     

几种抗生素对离体培养中植物形态发生的影响

抗生素羧苄青霉素(Carbenicillin),头孢霉素Ⅱ(Keflodin)和Claforan影响甘蓝型油菜(Brassica napus L.)游离小孢子培养胚胎发生,其浓度越高,影响越大.羧苄青霉素促进辣椒(Capsicum annuum L.)子叶外植体愈伤组织发生,同时,还影响油菜小孢子胚再生植株及辣椒子叶再生株的生根,产生粗短根甚或发育为瘤状物.     

低酚陆地棉直接体细胞胚胎发生和植株再生

选用低酚陆地棉无菌苗下胚轴为材料进行全固体组织培养,直接诱导获得了胚性愈伤组织,并进一步分化为再生植株.结果表明,激素是影响棉花直接体细胞胚胎发生的重要因素.MSB培养基中添加2,4-D有利于愈伤组织的形成,却不能直接诱导获得胚性愈伤组织.MSB培养基中添加IBA和BA也不能直接诱导获得胚性愈伤组织.MSB培养基附加适当浓度的IBA和KT能直接诱导出胚性愈伤组织.最适激素组合(1.0 mg/L IBA,0.5 mg/L KT)能使诱导棉花下胚轴产生大量胚性愈伤组织,并且在3个月内就可肉眼观察到不同发育时期的胚.MSB培养基中附加1.0 g/L谷氨酰胺和0.5 g/L天门冬酰胺有利于胚萌发成苗.本研究建立了简便高效的棉花直接体细胞胚胎发生和植株再生培养体系,从胚性愈伤组织诱导到植株再生约需5～6个月时间.     

不同成熟度茶籽外植体诱导发生体胚的研究

为了探究不同成熟度的茶籽作为外植体诱导体细胞胚胎发生的差异,本文选择成熟度依次递增的茶籽E1、E2、E3和E4作为外植体进行培养研究,结果表明：E4外植体接种成活率最高,但是诱导体细胞胚发生率较低。E1外植体在灭菌时耗损率相对较高,而且体胚诱导发生能力并非最佳。E2和E3外植体诱导分化体细胞胚的频率以及诱导获得的二级体胚的频率都较高,E最佳,E3次之。以上结果说明E2茶籽外植体可以作为诱导体胚发生的优良材料。     

Genetic analysis of in vitro wheat somatic embryogenesis

Summary A spring wheat genotype which produces somatic embryos in vitro, after short and long-term culture, was tested for its ability to sexually transmit this embryogenic trait. Reciprocal crosses were performed between a embryogenic line and a nonembryogenic variety.Immature embryos were cultured on Murashige and Skoog medium plus 2 mg/l 2,4-dichlorophenoxyacetic acid, gelled with 5.5 g/l agarose. Somatic embryogenesis was not expressed in the F₁'s. In contrast, from several hundred immature embryos of the F₂ generation of one cross, 10.7% and 1.6% expressed somatic embryogenesis in short and long-term cultures respectively. These percentages of embryogenic: non-embryogenic fits a model of a few complementary genes. The embryogenic capacity of the F₂ genotypes depends on the presence of recessive alleles at these gene loci. The long-term wheat somatic embryogenesis capacity requires a more complex mechanism than the short-term one.Abbreviations CS Chinese Spring - Aq Aquila - E Embryogenic - NE Nonembryogenic - SC Subculture     

Increasing embryogenesis and doubling efficiency by immediate colchicine treatment of isolated microspores in spring Brassica napus

The effect of colchicine on induction of embryogenesis andchromosome doubling during microspore culture was evaluated in twoF₁ hybrids of spring oilseed rape (Brassica napus L.). Immediatecolchicine treatment of isolated microspores with the concentrations 50 and500 mg/L for 15 h stimulated embryogenesis and produced largeamounts of healthy-looking embryos. These normal embryos germinatedwell at 24 ^°C after being transferred to solid regeneration mediumand an initial period of low temperature (2 ^°C) for 10 days, andcould directly and rapidly regenerate vigorous plants. A high doublingefficiency of 83–91% was obtained from 500 mg/L colchicinetreatment for 15 h with low frequency of polyploid and chimeric plants.The present experiment showed that a treatment duration of 30 h revealedless positive effects on embryogenesis and doubling efficiency, especially athigher colchicine concentration (1000 mg/L). Poor embryogenesis andembryo germination were observed from ordinary microspore culturewithout change of induction medium and colchicine treatment, and severalsubcultures were required for induction of secondary embryogenesis andplant regeneration.     

花生体细胞胚诱导及植株再生研究

为了建立花生体细胞胚诱导及植株再生体系,为花生分子育种提供技术支撑,以花生品种‘桂花30’和‘桂花771’为材料,采用预培养3天的种子胚小叶、下胚轴、子叶节为外植体,在添加外源激素的MS培养基中使体细胞脱分化形成体细胞胚,再分化成植株。结果表明,在所设定2,4-D浓度（3,5,10,15,20 mg/L）范围内,胚小叶最容易诱导形成体细胞胚,2,4-D的适宜浓度为10 mg/L,经过约30天培养,可产生大量体细胞胚,‘桂花30’和‘桂花771’的平均诱导率分别为55.37%和36.72%。平均每个外植体产胚量分别为5.68个和4.27个。将诱导形成的体细胞胚转接到6-BA浓度由5 mg/L逐渐降低到1.5 mg/L的MS培养基中,体细胞胚萌发再生成无根小苗,正常植株再生率‘桂花30’为32.6%,‘桂花771’为23.5%。将无根苗转接到生根培养基中可获得完整植株。花生是较难诱导体细胞胚形成的作物之一,筛选合适的基因型、外植体和激素浓度是获得较高体细胞胚发生率和植株再生率的关键技术。     

农杆菌介导陆地棉转化和再生体系的研究进展

陆地棉通过体细胞胚胎发生的农杆菌介导转化体系的主要瓶颈在于:基因型限制、转化周期长、胚胎发生率低、易出现畸形胚等问题.针对这些问题,研究者对传统转化体系的各个环节进行了优化,并在拓展受体基因型、筛选高频再生品(种)系或株系、选择不同的组织或器官做受体和建立新的不依赖于组织培养和基因型限制的农杆菌转化体系等方面进行了探索...     

Study of the variation in the somatic embryogenesis ability of some pea lines (Pisum sativum L. and Pisum arvense L.)

Summary Thirty lines of pea were tested in vitro to evaluate their ability to produce somatic embryos. Three distinct genotypic classes were detected (strong, medium and weak). The best responses were obtained in Pisum sativum. Abnormal somatic embryos and secondary embryogenesis seem to constitute the principal obstacle to the development of these structures.     

Effect of genotype and light intensity on somatic embryogenesis and plant regeneration in melon (Cucumis melo L.)

The efficiency of 14 commercial cultivars of melon (Cucumis melo L.) for callus induction, plant regeneration and somatic embryogenesis under different photosynthetic photon flux densities (PPFDs) (150 or 50μmol m^?2 s^?1) was investigated. Cotyledonal explants were cultured on a Murashige and Skoog (MS) medium supplemented either with 9.0 μM 2,4-dichlorophenoxyacetic acid and 23.2μM kinetin or with 0.05 μM 2,4-dichlorophenoxyacetic acid and 0.26 μM 6-benzyladenine for the induction of somatic embryogenesis and shoots, respectively. For embryo maturation and root induction, growing callus tissues were transferred on growth regulator-free MS medium. Both genotype and the intensity of light significantly affected the rate of somatic embryo-genesis, embryo maturation and plant regeneration. On average, 12–47 primary globular-stage embryos were produced per mm² of explant surface. Fully developed, cotyledonary-stage somatic embryos were obtained from only three cultivars. Relatively high root induction rates were observed both on the shoot induction medium (11 cultivars) and on growth regulator-free medium (seven cultivars). In contrast, only six cultivars responded positively to the shoot induction treatment. Callus growth and somatic embryogenesis were significantly improved if cultures were incubated under higher PPFD values, although plant regeneration from all cultivars was significantly reduced under the same conditions.     

Interspecific hybrids of Cucumis metuliferus × C. anguria obtained through embryo culture and somatic embryogenesis

Summary Interspecific crosses between Cucumis metuliferus Naud. and C. anguria L. were obtained through embryo culture. Embryos in the rabbit-ear to advanced fluke-shaped stages were rescued 34–99 days after pollination. Plants were obtained through direct embryo culture, and through somatic embryogenesis from immature embryos. For direct embryo culture, fluke-shaped embryos were stored in sterile water in darkness for three days at 25C prior to transfer on Murashige and Skoog (MS) culture medium plus 1.0 M 6-benzylamino-purine. Multiple plants were obtained from single embryos through somatic embryogenesis of rabbit-ear stages on MS plus 10 M indole-3-acetic acid and 5 M 6-benzylamino-purine. Evidence of hybridization included leaf shape intermediate between the two parents, penduncle shape prior to fertilization which resembled the male parent, low pollen viability and isoelectric focussing of protein bands for acid phosphatase of leaf extracts.     

杉木子叶和下胚轴的器官发生与体胚发生

以杉木实生苗的子叶和下胚轴为起始外植体,研究了基本培养基、6-BA、TDZ、苗龄等因素对器官发生和体胚发生的影响.研究结果表明：基本培养基、6-BA、TDZ及苗龄对子叶和下胚轴不定芽发生和体胚发生均产生显著影响.DCR＋6-BA 1.0mg/L＋TDZ 0.003mg/L＋NAA 0.1mg/L是子叶和下胚轴诱导不定芽发生及下胚轴诱导体胚发生的最佳培养基;DCR＋6-BA 1.0mg/L＋TDZ 0.002mg/L＋NAA 0.1mg/L对子叶诱导体胚最有效;不定芽在DCR基本培养基中可有效伸长;1/4MS＋IBA 0.2mg/L＋NAA 0.1mg/L可有效诱导不定芽生根.     

诱导不同基因型大豆未成熟子叶体细胞胚胎发生的研究

以未成熟子叶为外植体,研究25种基因型对大豆愈伤组织形成与体细胞胚胎发生的影响。结果表明,大豆不同基因型间愈伤组织诱导率变化较小,而基因型对体细胞胚胎发生的影响较大,依基因型不同体细胞胚诱导率变化在3.33%～83.33%。体细胞胚诱导率、平均胚数在基因型间的差异均达极显著水平,且两者间存在极显著相关关系。     

BA和NAA对水曲柳(Fraxinus mandshurica)未成熟胚子叶外植体褐化及体胚发生的影响

为了解析植物生长调节剂与水曲柳体细胞胚胎发生普遍伴随外植体褐化现象的关系,本研究以水曲柳未成熟合子胚子叶为外植体进行体胚发生,对细胞分裂素BA和生长素NAA的影响效应进行了探讨。结果表明,(1)当培养基中添加0.5mg/LBA和1.5mg/LNAA时,产生体胚的外植体褐化率达到91.5%;褐化外植体的体胚发生率达50.8%,而未褐化外植体的体胚发生率仅为9.6%;(2)添加NAA是水曲柳未成熟合子胚子叶外植体褐化的充分必要条件,即:无NAA存在时,未成熟合子胚子叶外植体不发生褐化,有NAA存在时才会发生褐化;只添加NAA外植体会产生褐化,但褐化程度低于同时添加BA,说明BA是促进褐化的条件;(3)添加NAA与BA是水曲柳未成熟合子胚外植体发生体胚的必要条件,即不添加生长素和细胞分裂素时无体胚发生;(4)NAA与BA的交互作用对褐化率的影响差异显著,但对体胚发生率的影响差异不显著,说明BA和NAA处理对外植体褐化的作用与对体胚发生的作用不同。由此可知:水曲柳未成熟合子胚子叶外植体的褐化和体细胞胚胎发生均受细胞分裂素BA和生长素NAA的影响,但BA和NAA分别调控了外植体的褐化和体胚的发生,与二者伴随的现象没有关系。本研究结果对进一步深入解析水曲柳体胚发生伴随外植体褐化的生物学机理提供了科学依据。