福禄考的组织培养研究：Ⅲ 体细胞胚胎发生的细胞学观察

福禄考叶片直接和间接地由单细胞形成体细胞胚。直接形成的体细胞胚来自叶表皮细胞、紧邻表皮的第一层栅栏细胞及维管束鞘内部和外部的薄壁细胞。间接形成的体细胞胚来自愈伤组织的表层或较深层细胞。体细胞胚有典型胚柄、多细胞胚柄和无胚柄3种,均经类似于合子胚的发育过程形成无根小植株。     

伏令夏橙愈伤组织体细胞胚发生中多胺水平的变化

以继代培养8年的伏令夏橙愈伤组织为材料,研究了不同类型愈伤组织体细胞胚发生能力的差异和多胺水平的变化及两者之间的关系.结果表明,胚性愈伤组织的多胺含量高于非胚性愈伤组织,体细胞胚发生能力与多胺水平呈正相关.体细胞胚发生早期Put含量的增加有利于体细胞胚发生.球形胚大量形成时,Spd达到最高值;球形胚发育后期并有少量心形胚形成时Spm达到峰值.随着倍性的增加,伏令夏橙体细胞胚发生能力降低.精氨酸脱羧酶的活性变化与Put水平呈正相关,表明它是调节伏令夏橙体细胞胚发生中多胺水平的重要因子.     

香果树体细胞胚胎发生过程中4种同工酶的研究

用非变性聚丙烯凝胶电泳技术对香果树体细胞胚胎发生及形态建成过程中过氧化物酶(POD)、酯酶(EST)、淀粉酶(AMY)和超氧化物歧化酶(SOD)4种同工酶进行分析.结果表明:香果树体细胞胚胎发生及形态建成过程中,POD、EST、AMY和SOD活性变化与胚性愈伤组织的诱导及体细胞胚的发生发育密切相关.非胚性愈伤组织和胚性愈伤组织酶谱差异明显,胚性愈伤组织中EST和AMY同工酶酶带多且活性高,非胚性愈伤组织中缺乏EST和AMY同工酶表达,AMY同工酶可作为胚性细胞分化和发育的重要标志.香果树体细胞胚形态建成过程中,球形胚时期的AMY、POD、EST同_T酶活性最强,表明这一时期生理代谢旺盛,是体细胞胚形态建成的关键时期;POD、AMY和SOD 3种同工酶的酶谱及表达强弱在形态建成的不同时期呈现有规律的变化,可作为香果树体细胞胚发生发育特定时期的参考标记. 与胚性愈伤组织的诱导及体细胞胚的发生发育密切相关.非胚性愈伤组织和胚性愈伤组织酶谱差异明显,胚性愈伤组织中EST和AMY同工酶酶带多且活性高,非胚性愈伤组织中缺乏EST和AMY同工酶表达,AMY同工酶町作为胚性细胞分化和发育的重要标志.香果树体细胞胚形态建成过程 ,球形胚时期的AMY、POD、EST同_T酶活性最强,表明这一时期生理代谢旺盛,是体细胞胚形态建成的关键时期;POD、AMY和SOD 3种同工酶的酶谱及表达强弱在形态建成的不同时期呈现有规律的变化,可作为香果树体细胞胚发生发育特定时期的参考标记. 与胚性愈伤组织的诱导及体细胞胚的发生发育密切相关.非胚性愈伤组织和胚性愈伤组织     

白Qian体细胞胚胎发生的细胞组织学和淀粉积累动态的研究

以白Ｑｉａｎ的成熟种胚为外植体,诱导体细胞胚胎发生。整体染色封片和组织切片的观察结果表明,白Ｑｉａｎ体细胞胚起源于胚性愈伤组织的单个细胞。胚性细胞经过一次不均等分裂产生两个细胞,即胚细胞和胚柄细胞。然后依次经过胚性胚柄团、球形胚、心形胚及鱼雷形胚阶段,最后发育成具有子叶的成熟胚。通过ＰＡＳ反应研究后发现,在体细胞胚发育过程中,淀粉粒在胚性胚柄团时期开始积累,至心形胚时期达到积累高峰,且淀粉粒的分布     

大蕉(Musa paradisiaca ABB Linn.)花序愈伤组织的诱导及其体细胞胚发生

大蕉未成熟雄花接种到胚性愈伤组织诱导培养基中,4～5个月后可诱导出胚性愈伤组织,并可在继代培养基上增殖.胚性愈伤组织转移到体细胞胚诱导培养基中可诱导出体细胞胚.体细胞胚在成熟培养基上培养2个月后转移到含有0.2mg·L-1 6-BA的分化培养基上可以萌发,进而形成再生植株.组织学切片证明所诱导的愈伤组织是胚性组织,其所产生的体胚具有典型的单子叶植物体细胞胚的组织结构.     

黄山栾树体细胞胚的发生和组织学观察

黄山栾树无菌苗的节间和叶柄离体培养后,其体细胞胚发生的结果表明：节间愈伤组织可诱导产生体细胞胚,而叶柄愈伤组织则生根：节间愈伤组织诱导培养基为MS＋3．0mg．L～2,4．D＋0．5～3．0mg．L-1NAA;节间胚性愈伤组织诱导培养基为MS＋2．0nag．L-2,4-D;胚性愈伤组织转移到无植物生长调节剂的MS培养基上可发育成正常植株。组织学观察表明,体细胞胚在胚性愈伤组织中有的发生于愈伤组织表层细胞,有的发生在愈伤组织内部。黄山栾树体细胞胚的形成经历球形胚、心形胚、鱼雷胚和子叶胚几个阶段,这与合子胚的发育途径相似。     

秦艽(Gentiana macrophylla Pall.)离体培养再生过程中体细胞胚的发生(简报)

本文以秦艽叶片和茎段作为外植体,通过离体培养对秦艽植株再生途径进行研究。愈伤组织在添加2mg/L 2,4-D和0.5mg/L BA的MS培养基上诱导,两周内可出现愈伤组织。愈伤组织在相同激素配比并附加500mg/L LH的MS培养基上继代。愈伤组织的分化在添加有0.1mg/L 2,4-D和0.5mg/L BA的MB培养基上进行。通过显微观测,疑似体细胞胚可以在叶片和茎段的愈伤组织上产生。形态学和组织学的分析进一步证实了秦艽离体再生过程中体细胞胚发生的现象。体细胞胚和合子胚一样,也经历球形、心形、鱼雷和子叶胚等发育时期。相对独立的结构说明秦艽的体细胞胚可能是单细胞来源。体细胞胚在愈伤组织的表面和内部都有出现。在本实验中,体细胞胚发生途径是在秦艽愈伤组织形成后观察到的唯一再生途径。     

石刁柏组织培养中体细胞胚发生的组织细胞学观察

以石刁柏(Asparagus officinalis)无菌苗的嫩茎切段为外植体,在含有1 mg/LNAA+0.5 mg/L BA的MS培养基上可100%地被诱导形成愈伤组织,在此条件下可长期继代,将继代的愈伤组织转入含有2 mg/L 2,4-D 0.5mg/L NAA的MS培养基上后,约有70%的愈伤组织块转变为胚性愈伤组织,这些胚性愈伤组织在3,4-D浓度进一步降低为0.5mg/L的条件下发育形成体细胞胚。切片观察表明:这些胚性愈伤组织是从愈伤组织的表层或近表层产生的。这些细胞核大,多核仁,细胞质浓、染色深的胚性细胞中的一些单个细胞处于与邻近细胞隔离状态,细胞壁也明显加厚。这些单细胞开始分裂,第一次分裂多为不均等分裂,形成一个大的基细胞和一个小的顶细胞,进一步分裂形成三细胞、四细胞、五细胞和具胚柄的多细胞原胚。原胚发育形成球形胚、梨形胚、香蕉形胚,由于在胚的一侧细胞分裂旺盛形成单子叶突起,最后形成子叶胚。其发育过程类似于单子叶植物合子胚的发育过程。     

白杄体细胞胚胎发生的细胞组织学和淀粉积累动态的研究

以白木千（ＰｉｃｅａｍｅｙｅｒｉＲｅｈｄ．ｅｔＷｉｌｓ．）的成熟种胚为外植体,诱导体细胞胚胎发生。整体染色封片和组织切片的观察结果表明,白木千体细胞胚起源于胚性愈伤组织的单个细胞。胚性细胞经过一次不均等分裂产生两个细胞,即胚细胞和胚柄细胞。然后依次经过胚性胚柄团、球形胚、心形胚及鱼雷形胚阶段,最后发育成具有子叶的成熟胚。通过ＰＡＳ反应研究后发现,在体细胞胚发育过程中,淀粉粒在胚性胚柄团时期开始积累,至心形胚时期达到积累高峰,且淀粉粒的分布主要集中于胚柄细胞、分裂旺盛的胚细胞、器官原基及其附近细胞。据此结果推测淀粉的消长与体细胞胚发生的能量供应有关。     

逆境处理和DNA甲基化影响柑橘体细胞胚发生

对 1 5种柑橘胚性愈伤组织进行体细胞胚诱导 ,发现逆境处理有利于体细胞胚发生 ,并可以恢复部分品种的体细胞胚发生能力。对具有和失去体细胞胚发生能力的两种纽荷尔脐橙 (CitrussinensisOsb .)愈伤组织进行随机扩增多态性DNA (RAPD)分析没有检测到带型的差异 ,而对它们的甲基化敏感扩增多态性 (MSAP)进行分析则发现两种愈伤组织间具有明显的DNA甲基化差异 ,具体细胞胚发生能力的愈伤组织的甲基化水平较失去体细胞胚发生能力的低     

Direct and indirect somatic embryogenesis in Freesia refracta]

Somatic embryogenesis can be induced in tissue cultures of Freesia refracta either directly from the epidermal cells of explant, or indirectly via intervening callus. In direct pathway, somatic embryos were in contact with maternal tissue in a suspensor-like structure. In indirect pathway, the explants first proliferacted to give rise to calluses before embryoids were induced. The two sorts of calluses were defined to embryogenic callus and non-embryogenic callus according to producing of somatic embryos. An indirect somatic embryo is developed from a pre-embryogenically determined cell. This kind of somatic embryo has no suspensor structure instead of a complex with maternal tissue. Somatic embryos have their own vascular tissues, and can develop new plantlets independently.     

Somatic embryogenesis and plant regeneration of neem (Azadirachta indica A. Juss.)

Somatic embryos were initiated with mature seeds of neem (Azadirachta indica A. Juss.) when cultured on Murashige and Skoog's medium supplemented with thidiazuron (TDZ). Regeneration occurred via somatic embryogenesis: direct embryo formation and through an intermediary callus phase. TDZ was very effective and induced somatic embryogenesis across a wide range of concentrations (1–50 μm). However, somatic embryogenesis was accompanied by callus formation at concentrations of 20 μm and above. Cell suspension cultures were established with the TDZ-induced callus and groups of large cell clumps were formed within 2–3 weeks. Plants were regenerated from both directly formed somatic embryos and somatic embryos derived from cell suspensions plated on semisolid medium devoid of growth regulators. Regenerated plantlets continued to grow after transfer to a greenhouse environment and were similar phenotypically to zygotic seedlings. This simple regeneration system may be beneficial for mass propagation of selected elite clones of neem. Received: 13 May 1997 / Revision received: 13 November 1997 / Accepted: 2 December 1997     

Somatic embryogenesis from callus cultures of <Emphasis Type="Italic">Terminalia chebula</Emphasis> Retz.: an important medicinal tree

Somatic embryogenesis was obtained from cotyledon and mature zygotic embryo callus cultures of Terminalia chebula Retz. Callus cultures of cotyledon and mature zygotic embryo were initiated on induction medium containing Murashige and Skoog (MS) nutrients with 1.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) either 0.01 or 0.1 mg/l Kinetin and 30 g/l sucrose. Induction of somatic embryogenesis, proliferation and development was obtained through different culture passages. Embryogenic cotyledon callus with globular somatic embryos was obtained on MS basal medium supplemented with 50 g/l sucrose. Globular somatic embryos were observed from mature zygotic embryo callus on induction medium. Different stages of somatic embryo development from cotyledon and mature zygotic embryo calluses were observed on MS basal medium supplemented with 50 g/l sucrose after 4 weeks of culture. Histological studies have revealed the developmental stages of somatic embryos. A maximum of 40.3±1.45 cotyledonary somatic embryos/callus was obtained from mature zygotic embryo compared to 7.70±0.37 cotyledonary somatic embryos/callus initiated from cotyledons. Germination of somatic embryos and conversion to plants were achieved. Highest frequency of germination (46.66±0.88) of somatic embryos was obtained on MS basal medium containing benzyladenine (0.5 mg/l) with 30 g/l sucrose.     

Regeneration of Acacia mangium through somatic embryogenesis

 Somatic embryogenesis and whole plant regeneration were achieved in callus cultures derived from immature zygotic embryos of Acacia mangium. Embryogenic callus was induced on MS medium containing combinations of TDZ (1–2 mg/l), IAA (0.25–2 mg/l) and a mixture of amino acids. Globular embryos developed on embryogenic callus cultured on the induction medium. Nearly 42% of embryogenic cultures with globular embryos produced torpedo- and cotyledonary-stage embryos by a two-step maturation phase. The first stage occurred on 1/2-strength MS basal medium containing 30 g/l sucrose and 5 mg/l GA₃ followed by the second stage on 1/2-strength MS basal medium containing 50 g/l sucrose. Of the cotyledonary-stage somatic embryos, 11% germinated into seedlings that could be successfully transferred to pots. Light- and scanning electron microscopy showed that the somatic embryos originated from single cells of the embryogenic callus. Further, a single cell layer could be detected beneath the developing somatic embryos that appeared to be a demarcation layer isolating the somatic proembryonic structure from the rest of the maternal callus. A suspensor-like structure connected the globular embryos to the demarcation layer. This is the first successful report of plant regeneration through somatic embryogenesis for this economically important tropical forest species. Received: 20 January 2000 / Revision received: 28 September 2000 / Accepted: 29 September     

The histological analysis of indirect somatic embryogenesis on <Emphasis Type="Italic">Drosera spathulata</Emphasis> Labill

We studied indirect somatic embryogenesis in the callus tissue of Drosera spathulata Labill. originated from isolated leaves. Callogenesis was induced on MS medium (Murashige and Skoog 1962), supplemented with various concentrations of NAA and BA. Somatic embryos regenerated on half-strength MS medium supplemented with 20 μM of NAA or without growth regulators. The highest efficiency of somatic embryo production was achieved on hormone-free medium. Globular, heart-, torpedo- and cotyledonary-shaped embryos were observed in embryogenic clusters. Histological and scanning electron microscopy analysis verifies somatic embryogenesis. Regenerated plants were transferred to soil and were grown to maturity.     

Growth regulators affect primary and secondary somatic embryogenesis in Madagaskar periwinkle (<Emphasis Type="Italic">Catharanthus roseus</Emphasis> (L.) G. Don) at morphological and biochemical levels

An efficient somatic embryogenesis system has been established in Catharanthus roseus (L.) G. Don in which primary and secondary embryogenic calluses were developed from hypocotyls and primary cotyledonary somatic embryos (PCSEs), respectively. Two types of calluses were different in morphology and growth behaviour. Hypocotyl-derived embryogenic callus (HEC) was friable and fast-growing, while secondary callus derived from PCSE was compact and slow-growing. HEC differentiated into somatic embryos which proliferated quickly on medium supplemented with NAA (1.0 mg l⁻¹) and BA (1.5 mg l⁻¹). Although differentiation and proliferation of somatic embryos were faster in primary HEC, maturation and germination efficiency were better in somatic embryos developed from primary cotyledonary somatic embryo-derived secondary embryogenic callus (PCSEC). At the biochemical level, two somatic embryogenesis systems were different. Both primary and secondary/adventive somatic embryogenesis and the role of plant growth regulators in two modes of somatic embryo formation have been discussed.     

Regeneration of somatic embryos in Theobroma cacao L. in temporary immersion bioreactor and analyses of free amino acids in different tissues

The present study aimed at developing temporary immersion bioreactor techniques for multiplication of cacao somatic embryos. Temporary Immersion System (TIS), i.e. flooding of plant tissue at regular time intervals provides an efficient way to propagate plants. Somatic embryos were regenerated in twin flask bioreactors. The TIS proved to be suitable for mass regeneration of somatic embryos and for their subsequent direct sowing. The number of embryos after 3 months of culture was significantly higher in TIS cultures than in the solid medium variant. TIS also improved embryo development regarding the conversion to torpedo shaped forms. Matured embryos derived from TIS and pre-treated with 6% sucrose were converted into plants after direct sowing. Additionally to the influence of culture conditions on the development of somatic embryogenesis the content and composition of free amino acids were analysed. The content of free amino acids in somatic embryos rose as immersion frequency increased. The endogenous free GABA content in embryogenic callus was significantly higher than in non-embryogenic callus.     

栓皮栎体细胞胚胎发生的细胞组织学观察

以栓皮栎未成熟合子胚为外植体,在添加0.25mg/L 2,4-D和0.5mg/L 6-BA的MS培养基上6周可诱导产生2种类型的胚性愈伤组织,一种表面具光泽、白色;另一种表面光滑湿润具光泽,色泽淡黄或无色透明。组织切片表明,胚性愈伤组织的细胞体积小,细胞核大,细胞质浓,细胞排列紧密;非胚性愈伤组织细胞的体积大,细胞核小,细胞质稀薄。胚性细胞团培养在不含激素的培养基上可诱导产生体细胞胚。体细胞胚直接起源于胚性细胞团表皮或近表皮的单细胞,经历与合子胚相似的球形胚、心形胚、鱼雷胚和子叶胚发育阶段。所有发育时期的体细胞胚的胚轴、子叶均产生次生体胚,它们起源于细胞质较浓的表皮单细胞。     

Structure and development of somatic embryos formed inArabidopsis thaliana pt mutant callus cultures derived from seedlings

Summary Seeds of theArabidopsis thaliana mutant primordia timing (pt) were germinated in 2,4-dichlorophenoxyacetic acidcontaining liquid medium. The seedlings formed somatic embryos and nonembryogenic and embryogenic callus in vitro in a time period of approximately two to three weeks. Embryogenesis and callus formation were monitored with respect to origin, structure, and development. Ten days after germination globular structures appeared in close vicinity of and on the shoot apical meristem (SAM). Somatic embryos formed either directly on the SAM region of the seedling or indirectly on embryogenic callus that developed at the SAM zone. Globular structures developed along the vascular tissue of the cotyledons as well, but only incidentally they formed embryos. Upon deterioration, the cotyledons formed callus. Regular subculture of the embryogenic callus gave rise to high numbers of somatic embryos. Such primary somatic embryos, grown on callus, originated from meristematic cell clusters located under the surface of the callus. Embryos at the globular and heart-shape stage were mostly hidden within the callus. Embryos at torpedo stage appeared at the surface of the callus because their axis elongated. Secondary somatic embryos frequently formed directly on primary ones. They preferentially emerged from the SAM region of the primary somatic embryos, from the edge of the cotyledons, and from the hypocotyl. We conclude that the strong regeneration capacity of thept mutant is based on both recurrent and indirect embryogenesis.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - DIC days in culture - SAM shoot apical meristem