茴香组织培养中体细胞胚胎发生的组织细胞学研究

将茴香幼茎或叶柄的愈伤组织转入附加6-BA和低浓度2,4-D的MS培养基以后,愈伤组织逐步由松软状转变成为颗粒状的胚性愈伤组织,胚状体起源于胚性愈伤组织中的单个细胞或胚性细胞团。在含NAA和6-BA的培养基中,胚状体发育成熟,并再生小植株。茴香的胚状体主要以单细胞内起源方式发生。首先由胚状体单个原始细胞分裂形成2-细胞原胚,2-细胞原胚以三种方式进行分裂:1.T- 形分裂;2.直线形分裂;3.田字形分裂。不同的分裂方式决定了胚柄的有无。茴香胚状体的发育过程与合子胚基本相同。由原胚发育成为球形胚,依次经过心形胚和鱼雷胚阶段,形成成熟的子叶胚。在胚状体发育的每一个阶段,都有其分生组织的活动中心。球形胚期,两团分生组织位于胚体中部对应的两点;心形胚期,位于两侧和中部;鱼雷胚期,分生组织的分布在子叶形成区域呈倒“U”形,在下胚轴部位呈中空的梭形。到子叶期,分生组织从两片子叶伸向胚根,呈“Y”形分布。两子叶间产生茎生长点,由生长点分化出叶原基。胚状体最终发育成为完整植株。     

枣合子胚和体细胞胚发育过程的观察与比较

本实验对临猗梨枣、壶瓶枣、晋矮1号等13个品种的枣胚的发育过程进行了观察,并诱导晋矮1号成熟胚的愈伤组织通过体细胞胚发生途径形成再生植株。结果表明:体细胞胚产生于愈伤组织的表层细胞或内部细胞。在鱼雷胚期已有导管的分化,子叶期的维管组织呈“Y”形。枣合子胚及体细胞胚的发育均经历了原胚、球形胚、心形胚、鱼雷胚和子叶胚五个时期。大多数品种的枣胚从球形胚期或心形胚期即开始败育,只有极少数品种可发育到成熟胚,而且合子胚形成的能力、胚败育时发育的程度等均存在着大的品种间差异,同一品种甚至同一子房内胚的发育进程也不同步。     

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     

渗透胁迫和外源ABA对芦苇愈伤组织中3种保护酶活性的影响

PEG-6000渗透胁迫下水生芦苇胚性愈伤组织（WREC）和沙生芦苇胚性愈伤组织（SREC）的质膜相对透性增大,前者增幅明显大于后者;过氧化氢酶CAT）和过氧化物酶（POD）的活性升高显著,超氧化物歧化酶（SOD）活性则呈小幅度升高,SREC的3种保护酶活性升幅较大,胁迫同时施加外源ABA时,WREC的质膜透性明显降低。CAT和POD的活性则显著提高,但对SREC的影响不大。     

花楸合子胚诱导体细胞胚胎发生研究

分别以完整成熟胚、切去一个子叶的成熟胚和切下的子叶为外植体,以MS为基本诱导培养基、1/2MS为基本分化培养基,进行了花楸体细胞胚胎发生研究。结果表明:以完整合子胚作为外植体的体胚诱导率最高,为100%,最佳植物生长调节剂组合为5 mg.L-1NAA+2 mg.L-16-BA;NAA和6-BA浓度及二者的交互作用对愈伤组织和体胚诱导率的影响极显著;光照配合延长继代间隔时间有利于体胚发生。实体观察结果表明,花楸体胚发生方式有直接发生和间接发生两种;体胚发育经历了球形期、心形期、鱼雷形期和子叶期。组织学观察结果表明,体胚具有两极性,子叶期体胚结构完整。     

槐树子叶组织培养中的形态发生研究

槐树子叶在MS附加0.1～5 mg/L BA 和0.1—5 mg/L 2,4-D的培养丛上培养两周,获得了灰色疏松型及白、绿色紧密型两种类型的愈伤组织。后者经培养产生不定芽并再生植株。子叶切块在MS 附加0.1～8 mg/L 2,4-D等培养基上培养3周,在切口处形成愈伤组织的同时,由其表面直接分化产生胚状体。组织学观察表明,胚状体来源于子叶的表皮及叶肉细胞,胚状体产生通过单细胞起源及多细胞出芽两种方式发生。胚状体发育依次通过球形胚、心形胚、鱼雷形胚和子叶胚等阶段形成再生植株,其发育过程与合子胚相似。     

渗透胁迫和外源ABA对芦苇愈伤组织中3种保护酶活性的影响

PEG-6000渗透胁迫下水生芦苇胚性愈伤组织(WREC)和沙生芦苇胚性愈伤组织(SREC)的质膜相对透性增大,前者增幅明显大于后者;过氧化氢酶(CAT)和过氧化物酶 (POD)的活性升高显著,超氧化物歧化酶(SOD)活性则呈小幅度升高,SREC的3种保护酶活性升幅较大.胁迫同时施加外源ABA时, WREC的质膜透性明显降低,CAT和POD的活性则显著提高,但对SREC的影响不大.     

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。     

大蒜体细胞胚胎发生过程中抗氧化酶活性变化及某些生理特征

以大蒜的发芽叶基(鳞茎)为外植体诱导体细胞胚胎发生,研究大蒜体胚发生过程中SOD、POD和CAT 3种抗氧化酶的活性以及可溶性糖和可溶性蛋白质含量变化.结果表明:在大蒜体胚发生过程中,SOD、POD和CAT活性变化与胚性愈伤组织的诱导及体胚的发育密切相关,POD对体胚的诱导起主导作用,而SOD和CAT在体胚的发育和成熟中起主导作用.可溶性糖和可溶性蛋白质累积与大蒜体细胞胚胎发生密切相关.     

黄连体细胞胚胎发生的研究

黄连(Coptis chinensis)叶片外植体在 MS 2,4-D 1 ppm 培养基上很容易产生愈伤组织。愈伤组织在转入分化培养基 MS 6-BA 0.5ppm NAA 1ppm 培养基上以后,能产生大量胚状体。胚状体可经过球形、心形、鱼雷形及子叶期等诸阶段发育成小植株。对胚状体用4%的藻酸钠和2%的氯化钙进行人工种皮包埋后,在无菌条件下,胚状体转变成苗。愈伤组织在分化培养基上经几次继代后,整个愈伤组织可转变为胚性愈伤组织并形成一个个胚性细胞团。胚状体可从其表面或愈伤组织内的任一细胞团产生。这一研究结果为获得大量分散的单个胚状体及人工种子的研制提供了良好的实验系统。     

Somatic embryogenesis in the medicinal legume <Emphasis Type="Italic">Desmodium motorium</Emphasis> (Houtt.) Merr.

An efficient protocol was established for regeneration of Desmodium motorium via somatic embryogenesis. Embryogenic calli were induced from cotyledon segments (6 mm, 16 days old) lacking embryo axis, excised from seedlings grown in vitro on Murashige and Skoog (MS) medium supplemented with indole-3-acetic acid (IAA) (2.9 μM) in combination with 6-benzyladenine (BA) (4.44 and 8.88 μM). Differentiation of embryogenic calli into globular and heart-shaped somatic embryos was achieved on transfer to hormone-free MS medium. When incubated for 4 days on MS medium supplemented with BA (8.88 μM), 95% of the globular and heart-shaped somatic embryos matured into torpedo and cotyledonary stages with minimum (10%) abnormalities. Modified MS basal medium without hormones and containing half-strength macronutrients and 0.88 M sucrose was suitable for germination of mature somatic embryos. Regenerated plantlets were successfully transferred to earthen pots with survival rate of 50%. Secondary embryogenesis was observed when pre-existing somatic embryos at globular and heart-shaped stages were cultured on MS medium supplemented with various concentrations of BA, adenine sulphate (AdS) and abscisic acid (ABA) individually.     

欧石楠体细胞胚发生和植株再生

以欧石楠茎段为外植体,研究其体细胞胚胎发生和植株再生。对影响茎段不定芽分化及胚性愈伤组织诱导的主导因子进行比较分析,并研究其体胚萌发、生根及移栽;同时,采用树脂切片法对茎段脱分化产生胚性愈伤组织及体胚发育过程进行组织细胞学观察。结果表明,接种在1／2WPM基本培养基上的茎段,胚性愈伤组织诱导率为88．7％,显著高于其他处理,不定芽诱导率可达90．6％,平均分化倍数为3．6个,平均分化苗高3．82cm;体细胞经过成熟培养后。在添加1．0mg·L-1 ZT和0．3mg·L-1 IBA的1／2WPM培养基上萌发,萌发的体胚在I／2WPM附加0．2mg·L-1 NAA和0．3mg·L-1 IBA的培养基上形成完整的体胚苗植株,体胚苗生根率达到87．4％,经炼苗后移栽到蛭石：珍珠岩=3：1（V／V）的栽培基质中,成活率可达63．7％。在显微镜下可观察到球形胚、心形胚、鱼雷形胚和子叶形胚;体细胞胚以间接方式发生,表现为愈伤组织外层细胞直接发生和愈伤组织组织内部细胞发生。     

Dynamics of morphological and anatomical changes in leaf tissues of an interspecific hybrid of oil palm during acquisition and development of somatic embryogenesis

Oil palm is an economically important plant species due to its high oil production per unit area. Large-scale clonal propagation of the species’s elite specimens is only possible through somatic embryogenesis, although methodology is partially still unknown and insufficiently understood. Current study characterizes in morphological and anatomical terms the acquisition and development stages of somatic embryogenesis of the oil palm’s immature leaves. The respective embryogenic stages were analyzed and characterized: immature leaves (initial explants); leaves with calli formation; leaves which failed to respond to calli formation; leaves with formation of root structures; primary calli; primary calli with differentiation of embryogenic calli; embryogenic calli; pro-embryogenic calli; calli with differentiated somatic embryos; somatic embryos at globular and torpedo stage; and mature fruit zygotic embryos. Cell masses emerged after approximately 60 days of cultivation through the proliferation of cells associated to initial explants´ vascular bundles. Consequently, the formation of two different types of calli was identified, namely, primary and embryogenic, respectively consisting partially and completely of meristematic cell clusters. After 420 days of cultivation, the propagules formed somatic embryos with no connection to source tissues, initially composed (globular stage) of a very characteristic ground meristem and protoderm. After 480 days of cultivation, as the cultures matured (torpedo stage), procambial strands, a structural characteristic also observed in mature zygotic embryos, were reported. The results provide an in-depth understanding of somatic embryogenesis of immature leaves of oil palm. Further, current analysis develops morphological markers at different stages of development obtained during the process.     

Somatic embryogenesis in saffron (<Emphasis Type="Italic">Crocus sativus</Emphasis> L.). Histological differentiation and implication of some components of the antioxidant enzymatic system

The ontogenetic developmental stages of saffron somatic embryogenesis have been studied and characterized using light microscopy and the biochemical determination of the antioxidant enzymatic system. The embryogenic callus underwent internal segmented divisions with the formation of globular embryos that were attached to the callus surface by a broad multicellular structure. Further development of the embryoids was characterized by the emergence of a shoot apical meristem and cotyledon (monopolar stage) with the subsequent differentiation of a minicorm at the basal part of the somatic embryo (dipolar stage). During the morphological differentiation of the somatic embryos changes in the antioxidant enzymatic system with increased superoxide dismutase (SOD) and catalase (CAT) activities were detected at the initial stages of somatic embryogenesis. The isoforms of SOD, including two Mn-SODs and four Cu, Zn-SODs, were also detected. Although all the isoforms were expressed during the successive stages of somatic embryogenesis, an increase in Mn-SODs and a decrease in Cu, Zn-SODs during the last two stages was observed. Significant changes were also detected in the antioxidant activities ascorbate peroxidase, dehydroascorbic acid reductase and glutathione reductase.     

Role of enzymes and identification of stage-specific proteins in developing somatic embryos of chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis> L.)

Accumulation of proline, activities of peroxidase (POX), catalase (CAT), phenylalanine ammonia lyase (PAL) and malate dehydrogenase (MDH) were studied during different developmental stages of somatic embryos in chickpea. Callus cultures that did not form somatic embryos served as control. While increased levels of proline and POX activity were noticed in globular stages of embryos, CAT activity increased during early and late heart-shaped embryo formation indicating tissue-specific activation of these enzymes. The activity of PAL reached a peak during torpedo and cotyledonary stages of embryo development. On the other hand, MDH activity enhanced during the germination of somatic embryos inferring more requirement of energy during this stage. Electrophoretic (sodium dodecyl sulfate polyacrylamide gel electrophoresis) pattern of proteins revealed that ten bands are associated with non-embryogenic tissues, whereas 11 bands with globular, heart, torpedo and cotyledonary stages of embryo development and nine bands during the germination stage of embryos. Two extra stage-specific protein bands with molecular masses of 16 and 18 kDa appeared during globular, heart, torpedo, and cotyledonary stages. But, these bands disappeared during germination of embryos and are absent in non-embryogenic cultures. This study thus may help in the identification of proteins and the role of above enzymes during different developmental stages of somatic embryo induction and their maturation in a recalcitrant leguminous crop plant chickpea.     

盐肤木体细胞胚胎发生及植株再生

以盐肤木(Rhus chinensis Mill.)幼胚为外植体,研究不同植物生长调节剂组合对其愈伤组织诱导及体细胞胚胎发生的影响,以建立盐肤木体细胞胚胎发生及植株再生体系。结果表明,最适愈伤组织诱导培养基为MS+6-BA 0.2 mg/L+2,4-D 1.0 mg/L,诱导率为84.57%,诱导出的初代愈伤组织白色或淡黄色,质地疏松,表面光滑,为非胚性愈伤。初代愈伤组织转移到1/2 MS+6-BA 2 mg/L+NAA 0.5 mg/L培养基上培养1个月后,长出淡黄色质地紧密的胚性愈伤组织,诱导率高达100%,在此培养基上胚性愈伤组织增殖倍数为854.73%。所获得的胚性愈伤组织转接到1/2 MS+6-BA 2 mg/L+NAA 0.5 mg/L+蔗糖4%的培养基上培养1个月后可诱导体细胞胚胎发生,诱导率可达32.67%。诱导得到的体细胞胚胎经历球形胚、心形胚、鱼雷胚、子叶胚进一步分化发育成苗。无菌苗炼苗后栽种到泥炭土∶蛭石∶珍珠岩为2∶1∶1的生长基质上,能100%稳定成活。经过细胞学观察分析,体细胞胚的发育与合子胚相似。     

Somatic embryogenesis in Vicia faba L.

The paper describes a method of somatic embryo induction in callus and suspension cultures of Vicia faba L. Callus was induced from immature cotyledons (green maturity stage) of white-flowering horse bean lines cultured on L2 medium (Phillips and Collins 1979) supplemented with 1% sucrose, 0.7% agar and different concentrations of 2,4-dichlorophenoxyacetic acid. The medium with 2.5 M 2,4-Dichlorophenoxyacetic acid was found optimum for embryogenic callus induction. Somatic embryos developed after transfer of the callus to media lower or zero 2,4-Dichlorophenoxyacetic acid and increased level of sucrose (2.5%). The release of somatic embryos from the callus was more apparent after transfer to liquid medium. There were various stages of somatic embryo development, i.e. globular, heart-shaped and torpedo ones.