石刁柏体细胞胚胎发生过程中蛋白质及同工酶变化的研究

本文报道石刁柏胚性愈伤组织的可溶性蛋白质含量与组分、过氧化物酶和酯酶的活力及同工酶带均比其体细胞胚的要少。而在体细胞胚胎发生过程中,过氧化物酶和酯酶活力、可溶性蛋白质含量均以球形胚为最低,子叶分化期胚为最高而呈递增趋势;可溶性蛋白质组分以子叶分化期胚、成熟胚为最多,球形胚、香蕉形胚为最少;过氧化物酶同工酶带以梨形胚为最多,子叶分化期胚、成熟胚为最少;酯酶同工酶则以子叶分化期胚为最多,成熟胚为最少。胚性愈伤组织与体细胞胚均有其特异性可溶性蛋白质及同工酶带,可作为体细胞胚胎发生的分子标记。     

Cytohistological Study on Somatic Embryogenesis in Asparagus officinalis L.

The single cell was the first cell of somatic embryoid in Asparagus officinalis L.. Electron microscopic observations revealed that early embryogenic cells and meristematic cells had the same characteristics in cell shape and structure. The embryogenic cell was small in size, with large nucleus, dense cytoplasm, thin wall, lots of small vacuoles, and rich in organelles. At later stage, the polarity of embryogenic cell appeared, i.e. nucleus situated at one end of the cell, while the other end was occupied by a large vacuole. The polar type of the embryc)genic cell was similar to that of a zygote. Light microscopic observations revealed :hat ihe embryoids were sequentially differentiated through 2-cells, 4-cells, 8-cells, multicellular proembryo, globularshaped, pear-shaped, rod-shaped, cotyledonary-differentiated and mature embryo stages. The heart-shaped and torpedo-shaped stages were observed during the early stage of embryogenesis. In addition, a typical embryoids were also found in vitro.     

Histological comparison of single somatic embryos of maize from suspension culture with somatic embryos attached to callus cells

An embryogenic suspension culture of Zea mays, genotype 4C1, was obtained from friable callus that was cultured on solid medium and had been obtained from zygotic embryos. The suspension contained non-dividing elongated cells, clusters of dividing isodiametric cells, and globular, ovoid, and polar stages of somatic embryos. The single somatic embryos were blocked in shoot meristem formation: when transferred to regeneration medium they developed a root and, at the shoot side, a green cap with meristematic cells, but a scutellum and leaf primordia were not formed. In medium containing 2,4-dichlorophenoxy acetic acid, somatic embryos formed embryogenic callus aggregates, consisting of globular stage somatic embryos attached to each other via undifferentiated callus cells. These somatic embryos developed into mature embryos with the zygotic histological characteristics, such as scutellum and leaf primordia, in maturation medium, and then regenerated into plants in regeneration medium. By omitting the maturation phase, regeneration occurred via organogenesis. Polyembryos, i. e. embryos attached to each other without callus tissue in between, behaved as single somatic embryos. It is concluded that the attached callus tissue provides a factor that stimulates scutellum and leaf primordia formation.Abbreviations CMM callus maintenance medium - 2,4D 2,4-dichlorophenoxy acetic acid - PCV packed cell volume - MS Murashige and Skoog medium     

香根草体细胞胚胎发生的细胞学特点与形成条件

香根草是一种优良的生态环境治理植物,但也存在着一些局限性。为了对香根草进行遗传改良,选育出性状更优、抗性更强的新品种,开展了香根草离体培养研究。离体培养采用了两种外植体,一是带腋芽的节,二是由器官发生方式所产生的无菌不定芽。基本培养基为MS,根据不同的目的附加不同种类或配比的生长素与细胞分裂素。观察到香根草的外植体的离体发育途径,有器官发生和体细胞胚胎发生两种,依培养基中所含细胞分裂素或生长素的种类和用量不同而异。结果表明,香根草的这两种离体发育途径的植株再生能力均可以长期保持。细胞学的研究显示,香根草离体发育的启动可在外植体的表皮细胞或薄壁细胞中进行,这些细胞逐渐发育成为胚性细胞。胚性细胞分裂活跃,经二细胞、四细胞而发育成为多细胞的胚性细胞团。由显微观察可知,香根草的体细胞胚胎发生是单细胞起源的,成熟的体细胞胚具有单子叶植物典型的胚胎结构。在分化培养基的作用下,体细胞胚组织上所有的胚状体可以出芽而形成再生植株。所建立的香根草体细胞胚胎发生的植株再生体系,完全适用于遗传转化等生物工程方法对离体培养要求。此外,还观察到一些一般只有在双子叶植物才出现的鱼雷形体细胞胚,这是体细胞胚胎发生中的异常现象。认为这种异常胚是离体培养所引起的。     

Histology of somatic embryogenesis in cultured immature embryos of maize (Zea mays L.)

Summary The developmental histology of somatic embryo (=embryoid) formation in cultured immature embryos of hybrid maize cultivars (Zea mays L.) is described. Embryos cultured on media containing 2% sucrose formed distinct globular embryoids. These embryoids arose either directly by divisions confined to the epidermal and the subepidermal cells at the coleorhizal end of the scutellum or from a soft and friable embryogenic callus produced by them. On media containing 6% sucrose divisions were initiated in the cells adjacent to the procambium of the cultured embryos. Subsequently, zones of meristematic cells also were observed in the region of the node and in the basal portion of the scutellum. Mature, well organized somatic embryos as well as a compact nodular type of embryogenic callus were produced as a result of localized meristematic activity along the tip of the scutellum toward the coleorhiza. Some embryos formed only the compact type of callus, and shoot primordia were organized later in the surface layers of this callus.Abbreviations CH casein hydrolysate - MS Murashige and Skoog's nutrient medium - 2,4-D 2,4-dichlorophenoxyacetic acid     

High Frequency Induction of Somatic Embryoid of Rice in Suspension Culture

On MS and B5 basic media the mature seed of rice (Oryza sativa L.) was induced to produce somatic embryoid. The results showed that the rate of occurrence of somatic embryoid varied with the variety of rice and the concentration of 2,4-D was important to the occurrence of somatic embryoid. For those varieties of rice suitable for induction, the induction factors such as KT, zeatin, BAP, ABA, osmotic pressure and so forth were stringently required at different stages of somatic embryoid development, and they also influenced the formation of somatic embryoid. The results of various layered cultures of embryonic calli proved that the somatic embryoid was derived from the surface layer callus containing embryonic cells. At the induction stage no somatic embryoid was formed in callus. The rate of somatic embryoid formation was over seventy per cent on liquid differentiation medium I.     

油菜小孢子胚发生的超微结构和胚状体形态

应用透射电镜和扫描电镜分别研究油菜游离小了包子培养后细胞的超微结构和胚状体的形态。单核晚期小孢子经培养后,具有胚状体发生能力的细胞中央液泡消失,积累淀粉,含丰富细胞器。     

辣椒花药培养的胚状体分化及其与成苗率的关系

12个辣椒品种的花药培养的结果表明,所有品种均可诱导出胚状体,其中9个品种可获得健壮的再生植株。每一辣椒品种的适宜植物生长调节利配比不同。不同品种的胚状体诱导率和成苗率有差异。成熟的胚状体均能分化成苗,根先分化或停止发育的胚状体很少成苗。     

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。     

石刁柏胚乳愈伤组织内胚胎发生的组织细胞学观察

石刁柏(Asparagus officinalis L.)的幼嫩胚乳接种于MS+NAA 5ppm+6-BA 1ppm和MS+NAA 1ppm+6-BA 0.5ppm的培养基上诱导产生愈伤组织。将愈伤组织转移到MS+6-BA 1ppm+NAA 0.5ppm的分化培养基上,培养30天后即可形成胚状体,组织细胞学观察表明:胚状体起源于愈伤组织内或近表层的单个胚性细胞。在胚状体发生的早期阶段,观察到与柳叶菜型和藜型胚胎发育大致相似的细胞分裂方式,从而出现了T型或线型的四个细胞的原胚。在多细胞原胚及球形胚期具有单列或不规则排列的多细胞胚柄。石刁柏胚乳愈伤组织中的胚胎发生是不同步的,在同一块愈伤组织的切片中可以观察到不同发育阶段的胚状体。在外形上还可以观察到一个子叶、两个子叶或四个子叶等多种不同形态的胚状体。一部份胚状体能发育成完整小植株。     

金花茶子叶在离体培养中胚状体的发生和小植株的形成

研究了金花茶(Camellia chrysantha(Hu)Tuyama)子叶在离体培养中体细胞胚状体发生的条件。在MS基本培养基中附加苄基嘌呤(BA)或苄基嘌呤与萘乙酸(NAA)组合,诱导了胚状体发生。组织学观察表明,胚状体起源于子叶的表皮细胞。在增添细胞分裂素和生长素的MS或改良B_5液体培养基里振荡培养,明显地促进了胚状体根的生长和茎的发育。胚状体在继代培养中能保持旺盛的再生能力。已得到两个繁殖率较高的胚状体无性系。在合适的条件下,胚状体能长成正常的小植株。     

CHARACTERIZATION OF AN EMBRYOGENIC CELL SUSPENSION CULTURE DERIVED FROM CULTURED INFLORESCENCES OF PENNISETUM AMERICANUM (PEARL MILLET,GRAMINEAE)

An embryogenic suspension culture was established from cultured inflorescence segments of Pennisetum americanum in Murashige and Skoog's medium supplemented with 2.5 mg/1 2,4-dichlorophenoxyacetic acid (2,4-D) and 5% coconut milk. The suspension was composed of two major cell types: 1) small, richly cytoplasmic and starch-containing cells, generally found in small, compact clumps, here termed embryogenic cells; and 2) elongated, thick-walled cells with large vacuoles. By manipulating the duration of culture and dilution ratios (cell suspension: fresh medium) at the time of subculture, suspensions consisting predominantly of embryogenic cells were obtained. Suspensions grown for 2-3 wks were transferred to agar media with reduced amounts of 2,4-D. This resulted in the production of hundreds of globular and early cotyledonary embryoids. Further development of the embryoids was promoted by their transfer to a medium containing abscisic acid. Many of the embryoids germinated and produced normal green plants. Atypical embryoids, some containing many shoot meristems and a leafy scutellum, were also observed. The relevance of such atypical embryoids in the interpretation of organogenesis and embryogenesis reported in tissue cultures of cereal species is discussed. It is also suggested that somatic embryogenesis occurs in tissue cultures of most, if not all, species of cereals and grasses.     

Ultrastructural Studies of Somatic Embryogenesis in Wheat

Electron microscopic observation of somatic embryogenesis from cultured immature wheat embryos revealed the presence of a lot of small vacuoles, a large nucleus, clear nucleolus and polynucleoli. The electron density of cytoplasm was strengthened during somatic embryogenesis. Quantity and type of organelles—plastid, ribosome and mitochondrion were increased; thickened cell wall, disappeared plasmodesmata, increased organelles andstarch accumulation in the embryogenic cells. Nucleolus vacuoles, autophagic vacuoles and secretory vesicles were present in the embryogenic cells with thickened cell walls. The multicellular proembryos, globular embryoid and pear-shaped embryoid were surrounded by an envelop, but plasmodesmata existed extensively between cells of somatic embryoid. The membranous structures appeared in the plastid which underwent transformation into chloroplast in the cells of growing point in almost mature embryoid. The relation of the above-mentioned structureal changes to somatic embryogenesis is also discussed.     

枣树体细胞胚发生和组织学研究

以临泽小枣子叶切块为外植体,在附加０．２mg/L IBA 1．0mg/L 6-BA的ＭＳ培养基上１周后切块边缘可诱导出白色胚愈伤组织,继续培养１个月后愈伤组织中产生体细胞胚。体细胞胚发生不同步,经历球形胚、心形胚、子叶胚等阶段,与合子胚发育途径相似。组织切片表面胚性愈伤组织细胞体积小,细胞核大、细胞质浓,细胞排列紧密;而非胚性愈伤组织细胞体积大、细胞核小、细胞质稀薄,子叶胚时期体细胞胚内部出现维管束,并观察到螺纹导管。     

Plant regeneration via somatic embryogenesis in mature embryo-derived callus culture of Sinocalamus latiflora (Munro) McClure

Somatic embryogenesis and subsequent formation of plantlets was achieved from callus cultures derived from mature zygotic embryos of Sinocalamus latiflora (Munro) McClure (Bamboo). Embryogenic callus was initiated on Murashige and Skoog's medium (MS) supplemented with 6 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), 3 mg/l kinetin, 250 mg/l polyvinylpyrrolidon and 5% sucrose. Prolonged culture of the embryogenic callus on the same medium resulted in embryoid formation. The embryoids developed further to yield whole plantlets when transferred to a medium containing lower concentrations of 2,4-D (3 mg/l) and kinetin (2 mg/l).     

HISTOLOGY OF SOMATIC EMBRYOGENESIS IN PANICUM MAXIMUM (GUINEA GRASS)

Immature embryos of Panicum maximum Jacq. (Guinea grass) were cultured on Murashige and Skoog's medium containing 2,4-dichlorophenoxyacetic acid (5 mg/1) and coconut milk (5%). The epidermal and subepidermal cells of the scutellum at the coleorhizal end either formed somatic embryos directly or gave rise to embryogenic callus at several loci. Somatic embryos were formed later also at the periphery of these embryogenic calli. The embryoids originated from single, densely staining, nonvacuolated and starch-containing cells with thickened walls and developed through characteristic stages of grass embryogeny.     

冬小麦原生质体培养的胚状体直接发生

冬小麦品种“京花一号”胚性愈伤组织在改良的N₆培养基(NBD培养基)上继代得到易碎型胚性愈伤组织,转入改良MS液体培养基(MSDL培养基)后得到胚性悬浮系,分离的原生质体在改良的MS培养基(MSDP培养基)上培养,再生细胞直接产生体细胞胚胎,并再生出完整植株。体细胞胚胎形成过程与小麦合子胚的形成过程十分相似。     

虎眼万年青的直接体细胞胚胎发生和植株再生

以虎眼万年青(Ornithogalum caudatum Jacq.)子鳞茎的鳞叶为外植体,不经过愈伤组织阶段,直接诱导体细胞胚胎发生并再生植株。组织切片和扫描电镜的观察结果表明:体细胞胚来自鳞叶表皮最外层的单个细胞。这个细胞经第一次平周分裂,成为2-细胞原胚,然后依次经过4-细胞原胚、球形胚、香蕉形胚等阶段发育成小植株,其形态学发育过程与合子胚相似。小植株移入土中,1周后即可成活。此外,还观察到直接体细胞胚只产生于鳞叶的近轴面,而远轴面无体细胞胚产生,这两个部位在细胞结构和形态上有显著差异。     

A statistical comparison of various factors on embryogenic proliferation,morphogenesis and regeneration in Lolium temulentum cell suspension colonies

Cell suspension colonies from four embryogenic Lolium temulentum lines were selected and plated individually in 25 embryoid maturation treatments which varied in various factors reported to stimulate embryogenesis or improve regeneration. Using a numerical scoring system to compare the cultures against a control, treatments were identified which increased growth, suppressed morphogenesis or encouraged premature shoot formation.No treatment significantly improved the proportion of colonies with globular or mature embryoids, but some prevented maturation and increased the proportion with translucent embryogenic proliferation. Other treatments accelerated maturation causing increased de-differentiation of embryogenic tissues. These treatments also tended to discourage the differentiation of discreet embryoids.Colonies were later transferred en masse to a regeneration medium and scored using another numerical system. Embryoid maturation conditions were then identified which increased or suppressed subsequent shoot regeneration. The two scoring systems enabled cultures of the four lines to be characterised in detail and identified somatic variation in embryogenic development, morphogenesis and de-differentiation.     

红江橙体胚发生及影响因素的研究

红江橙花后４周的幼果,直径约１ｃｍ左右,这时期的胚珠最适于胚性愈伤组织的诱导。以ＭＴ为基本培养基,加上适当浓度的ＩＡＡ、ＢＡ和ＭＥ可显著提高胚性愈伤组织诱导率。体胚发生,以ＭＴ＋ＩＡＡ０．１ｍｇ１－１＋ＢＡ（ＺＴ）１ｍｇ１－１培养基较好,能诱导产生正常体胚,且频率较高。胚性愈伤组织继代次数对体胚发生也有一定的影响,继代６次以内,胚性愈伤组织具有旺盛产生体胚能力;继代至第９次时,产生体胚的能力明显下降;至第１２代时,不能产生胚状体。成熟胚转换成小植株,以ＭＴ＋ＧＡ２ｍｇ１－１＋ＮＡＡ０．１ｍｇ１－１培养基成苗率最高。