Cytology and Histology in Somatic Embryogenesis of Indica Rice

The somatic embryogenesis was established from mature dehulled seeds. The histological research showed that embryogenic calli were initiated first from absorbed cells of scutellum of mature seed. And then the embryoids derived from the surface of embryogenic callus. Having been the same structure like a zygotic embryo of rice, the embryoids possessed the major parts of scutellum, coleoptile and coleorhiza. In an embryoid, several developmental stages of pro-embryoid, including single embryogenic cells, two, four and multiple cell stage pro-embryeids and some abnormal embryoids were observed. It could be concluded from this experiment that the embryoid from somatic cell culture in Indica rice possessed an original form of a plant in structure like a zygotic did and derived from a single cell.     

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。     

苜蓿胚状体的分离及其发育过程中三种同工酶酶谱分析(简报)

目前,对胚状体发生过程中的生理生化研究表明,这一过程伴随有核酸、蛋白质等大分子物质合成速度的增加及与胚胎发生有关的特异性蛋白的合成;一些同工酶,如过氧化物酶、脂酶、细胞色素氧化酶和谷氨酸脱氢酶     

石防风细胞悬浮培养中的体细胞胚发生

石防风试管苗的根经2,4-D诱导可形成具有发生体细胞胚潜能的愈伤组织,用愈伤组织制备悬浮细胞。细胞及组织学的观察表明,体细胞胚发生经历了单细胞、丝状体、细胞团、愈伤组织及胚性细胞团的出现及类胚体的各个发育阶段。丝状体可以经过不同的分裂途径发育为细胞团。愈伤组织表面或者内部的某些细胞演变为胚性细胞,它们不断分裂形成了体细胞胚,一个愈伤组织可形成一个或几个体细胞胚。     

Observations on Morphology and Cytology of Embryoids Derived from Callus of Maize

Observations on the callus sections showed that most embryoids were produced from surface layer cells of the callus and a few embryoids from inside layers of the callus. The initial cell of the embryoids possessed denser protoplasm and larger nucleus than the others cells did. The developmental sequence of the initial cell was similar to that of the zygotic embryos. First division gave rise to two daughter cells, the basal cell and terminal cell. The basal cell was able to divide or not divide again and changed into the suspensor, The terminal cell first divided longitudinally and then transversely into four cells. As the four cells further divided the embryo was formed properly. The embryoids possessed monody or polycotyledons. The production of the embryoids from a callus was not synchronous. So embryoids of different development stages could be found on the same callus.     

Studies on the Induction of Pollen Sporophyte of Coix lacryma Jobic L.

The developmental pathways of pollen sporophyte in anther culture of Coix were observed. The types of androgenesis are different, and are relative to the degree of the differentiation in pollen cells. Pollen-inductors develop via multicellular mass into embryoids or calli. Both of them can develop into plantlets, but the frequency of the regeneration of the plantlets in calli is higher than that of in embryoids, because there are a lot of aberrant embryoids in the latter, which cannot develop further. It was found that the induction frequency of the polleninductors can be increased apparently by the pretreatment in a short time with a hypertonic sucrose solution. The chromosomes of the somatic cells in l0 plantlets were examined. It was found that all the plantlets derived from the embryoids are haploids, while there are haploid, diploid and mixoploid in the plantlets from the calli. The effects of anther wall cells and the stability of haploid cells were discussed.     

Change of Superoxide Dismutase Activities During Somatic Embryogenesis in Asparagus officinalis L.

The superoxide dismutase activities in callus and somatic embryoids at different developmental stages of Asparagus officinalis L. were determined by means light of induced oxidation-reduction of nitro blue tetrazolium. It was shown that the superoxide dismutase activity was higher in callus than in somatic embryoids at different developmental stages. The activity increased with growth, differentiation and maturation of somatic embryoids during somatic embryogenesis. The relations between superoxide dismutade activity and somatic embryogenesis were discussed.     

Microscopical Observations on the Embryoid Formation in Cultured Unfertilized Ovules of Helianthus annuus L.

A recent advance in plant experimental embryology is the induction of haploid plants via in vitro culture of unfertilized ovules. Using float culture method on liquid media, we have raised haploid as well as diploid embryoids in sunflower cultivars by ovule culture. The present investigation was aimed to know the origin and developmental processes of these embryoids. Young flowers 1–4 days before anthesis were dissected and ovules were inoculated on N6 medium supplemented with 0.5–2 ppm MCPA and 6% sucrose. During culture period, samples were collected at intervals, fixed, stained and sectioned by paraffin method. Fifty one gynogenie embryoids of various sizes were observed among Ca. 2000 ovules. They were located at the micropylar end of the embryo sacs and proved to be originated from the unfertilized egg cells. At the early stages, they bore a strong resemblance to the zygotic proembryos in vivo, but after a considerable enlargement, they grew into globular, ovoid or elongated big bodies without polarized organ differentiation. Chromosome counts on some mitotic figures in these embyoids revealed their haploid nature. Embryoids were also produced from the endothelial tissue, which proliferated markedly after inoculation, especially at the chalazal parts, resulting in massive multilayered irregular folds and then degenerated. In some eases, cell divisions at one or several places led to embryoid or callus formation. The problems of how to regulate the growth of in vitro ovules in order to promote the gynogenic embryoids and inhibit the somatic embryoids or calli are left for future research.     

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。     

Induction of androgenesis and callus formation inin vitro cultured anthers of a myrtaceous fruit tree (Psidium guajava L.)

Anthers ofPsidium guajava cultivated on either Murashige-Skoog's or Nitsch's basal medium (BM) or the BM supplemented with 10⁻⁶ M benzylaminopurine (BAP) were observed to contain microspores undergoing androgenic segmentations as well as a few multicellular microscopic embryoids. However, final morphogenic response from such cultured anthers was the development of calli. These calli had restricted growth accompanied by their early browning. Suspecting the browning to be due to accumulation of polyphenols, the culture medium was fortified with polyvinylpyrrolidone (PVP). PVP, increased sucrose concentration in the medium and cold pretreatment of anthers decreased the proportion of anthers turning brown as well as delayed the browning of calli, but it was not possible to maintain the calli for differentiation. Cold pretreatment significantly increased the percentage of callusing anthers and also resulted in the early emergence of calli.     

辣椒游离小孢子细胞团培养的胚状体形成

从预培养15天后的花药中机械游离小孢子及其细胞团,经28℃液体悬浮暗培养．30天后,获得了自球形期胚到子叶期胚发育程度不等的各类胚状体。从12个花药中可以形成高达22个胚状体,且子叶期胚的比例约为23％。显微镜检表明,这些胚状体来自游离的小孢子细胞．经核的对称分裂形成多核细胞或者早期形成多细胞团,最后经细胞的分裂分化形成。胚状体体表具毛,活力有差异。在适当培养基上,具活力的鱼雷期及子叶期胚状体均能发育成正常植株。7℃、32℃、35℃8天的胁迫处理均能诱导小孢子胚状体发生。但花药培养中7℃、35℃处理下的出胚率较32℃下高,而游离小孢子细胞团培养中以35℃、32℃下较好。7℃处理下获得的胚状体数很少．对产生这种现象的原因进行了探讨。出胚率在基因型间,不同胁迫处理温度间表现明显差异。而在温度处理的不同天数间差异不明显。流式细胞仪对再生株真叶的DNA含量分析表明．获得的再生株中具有单倍体、双单倍体以及单倍一双倍嵌合体植株。本结果为进一步开展辣椒雄性生殖途径的胚状体发育研究。提高辣椒成熟胚状体的频率提供了实验体系。     

Regulation of In Vitro Parthenogenesis and Somatic Proliferation in Sunflower by Several Factors

Culture of unfertilized ovules in sunflower (Helianthus annuus L.) could be induced to form parthenogenetic embryoids, endothelial embryoids and integumentary calli. Various factors exerted regulatory influences on the production of these structures. The key factors were exogenous hormone, sucrose concentration and cold-pretreatment. In hormone-free condition, parthenogenesis was promoted without any occurence of somatic embryoids or calli. Addition of exogenous hormone MCPA induced proliferation of sporophytic tissue and meanwhile, reduced the induction frequency of parthenogenesis. Relatively high sucrose level favored the Froduction of parthenogenetic embryoids. Reducing sucrose concentration favored the integumentary calli. The optimal sucrose concentration for endothelial embryoids was intermediate between them. Cold-pretreatment also played an effective role in promoting parthenogenesis and inhibiting integumentary calli. By combining these three factors, we were able to. work out a culture technique which significantly enhanced the inductionfrequency and growth. of parthenogenetic embryoids and completely eliminated somatic embryoids and calli.     

Callus and embryoid formation from protoplasts of Helianthus annuus

Sunflower hypocotyl protoplasts have been isolated and cultured. Optimum plating density for cell division and colony formation was in the range of 5 to 7×10⁴ cells/mi in an agarose medium supplemented with BAP (1 mg/l) and NAA (1 mg/l). Plating efficiency was 60% after 21 days of culture. In the resultant culture a mixed population of calli and embryoids was observed. Thirty seven percent of the cell clusters exhibited a developmental pattern similar to an embryoid. Many stages of embryogenesis were observed in the same cultures.Abbreviations 2,4-D 2,4 dichlorophenoxyacetic acid - NAA 1-naphtaleneacetic acid - IAA Indole-3-acetic - BAP 6-benzylamino purine - GA₃ Gibberellic acid     

Direct somatic embryogenesis from protoplasts of Foeniculum vulgare

Protoplasts prepared from an embryogenic cell suspension culture of fennel gave rise to somatic embryoids directly through unequal cell divisions of enlarged, ellipsoidal cells, when embedded in hormone-free LS agarose medium. On the other hand, protoplasts embedded in LS agarose medium containing 2,4-D and kinetin proliferated through unpolarized cell divisions to form calli, which gave somatic embryoids on the surface upon transfer onto the same medium. In either case, somatic embryoids germinated to develop into normal plantlets when cultured on hormone-free LS agar medium under illumination.     

蛇床幼茎离体培养中体细胞胚胎形成的观察

蛇床幼茎外植体经诱导产生了愈伤组织。在ＭＳ＋２,４－Ｄ,０．２ｍｇ／Ｌ＋ＺＴ０．４ｍｇ／Ｌ培养基中,愈伤组织转变成胚性愈伤组织。转入ＭＳ＋ＮＡＡ０．２ｍｇ／Ｌ＋ＺＴ０．８ｍｇ／Ｌ培养基以后,胚性愈伤组织分化出体细胞胚胎。体细胞胚胎在ＭＳ＋ＮＡＡ０．５ｍｇ／Ｌ培养基中可直接发育成为完整植析。显微观察表明,体细胞胚胎产生于愈伤组织的表层细胞或内部细胞。在鱼雷胚期已有螺纹导管的分化。子叶期的维管组织从两     

Developmental SEM observations on an extracellular matrix in embryogenic calli ofDrosera rotundifolia andZea mays

Summary Primary embryogenic callus ofDrosera rotundifolia and long-term cultured embryogenic callus ofZea mays possess a conspicuous extracellular matrix (ECM) around and between embryogenic cells. The structural arrangement of ECM depends on the developmental stage of the embryogenic cells. Single embryoid cells were covered with, and connected by net-like material. However, surface cells of young globular embryoids were covered with a coherent layer of ECM which forms bridges with net-like material between the cells which was gradually reduced to coarse strands. When protodermis was formed on the surface of globular embryoids, the ECM disappeared completely. The ECM network was never observed on the surface of heart- and torpedo-shaped embryoids. Safranine (especially 0.1%) stabilized the structure of ECM. Digestion with pronase E and proteinase K indicated that the ECM contains proteinaceous components. Similar developmental patterns of ECM were observed in dicotyledonous and monocotyledonous examples. The ECM represents a stable morphological structure even during long-term embryogenic culture in maize.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - Dicamba 3,6-dichloro-o-anisic acid - ECM extracellular matrix - KIN kinetin - SEM scanning electron microscopy - TEM transmission electron microscopy     

Somatic embryogenesis and plantlet regeneration in the soybean Glycine max

A tissue culture procedure for the regeneration of somatic embryos and plantlets from somatic cells of the soybean Glycine max is described. Bean pods of soybean cv. TGM119 were immersed in liquid nitrogen for 20 minutes. Young embryos were excised from the immature seeds and cultured to form calli. Calli grown from the young embryos were incubated in liquid culture for two weeks. The liquid suspension culture was filtered to obtain single cells. The soybean cells were cultured for one month in a liquid medium in hanging drop cultures for development into proembryoids. The proembryoids were maintained on a solid growth medium for 40 days. The resultant callus tissue was transferred into MS media containing selected combinations and concentrations of 2,4-Dichlorophenoxyacetic acid, Naphthaleneacetic acid, Kinetin, Benzyladenine and Indoleacetic acid. In the presence of Benzyladenine (0.2 mg/l) and Indoleacetic acid (0.01 mg/l), globular and heart shaped somatic embryos were formed on the surface of the calli. Calli containing somatic embryos were transferred into liquid medium and incubated under low light conditions. After six months further incubation, more than 1,000 plantlets and a large number of somatic embryoids at various developmental stages were obtained per flask.Abbreviations KT kinetin - CM coconut milk - BA benzyladenine - NAA napthalene acetic acid - IAA indole acetic acid - 2,4-D 2,4 dichlorophenoxy acetic acid - MS Murashige and Skoog medium     

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。     

Production of Doubled Haploids from Androgenic Embryoids and Plantlets of Tobacco

In four experiments, the chromosome doubling effect of acenaphthene vapour was tested on androgenic embryoids and/or plantlets of tobacco. The maximum rate of plants with doubled chromosome numbers (41.3 % diploids plus 2.7 % tetraploids) was recorded when approx. 100 mg acenaphthene in small and sterilized cheese-cloth bags were hanged for 72 h, into the culture jars (6.0 × 11.5 cm), containing 4 – 6 androgenic plantlets newly transferred from culture tubes. Acenaphthene treatments performed at the earlier stages of culture resulted in lower percentages (from 0.0 to 6.3 %). This revised version was published online in July 2006 with corrections to the Cover Date.     

Phytohormone content in microstrobiles and androgenic callus of Siberian larch

Androgenesis in vitro in plants is a phenomenon of developmental switching of male generative cells, microspores, from their normal gametophytic to sporophytic pathway. We obtained androgenic callus and embryoids (embryo-like structures derived from microspores) of the conifer plant, Siberian larch (Larix sibirica Ledeb.) in the in vitro culture. The immune-enzyme analysis of the hormonal balance of larch androgenic cultures showed a substantial increase in the content of all phytohormones, especially cytokinins and ABA, as compared with initial explants (microstrobiles). This was evidently related to active cell divisions and embryoid formation. A comparison of androgenic cultures derived from trees nonifested and infested with larch gall midges (Dasineura rozhkovi Mam. et Nik.) revealed a cytokinin content increase (by two times) and an ABA content decrease (by two times), which indicates more intense growth of cultures derived from healthy trees. Phytohormone content in the androgenic callus was compared with their accumulation in the embryos of larch seeds harvested from noninfested trees. We concluded that successful growth of androgenic cultures and embryoid formation demand an additional medium supplement with auxins (not more than 0.5 mg/l).