Somatic Embryogenesis and Plant Regeneration from Protoplasts of Cowpea (Vigna sinensis)

Immature cotyledons of cowpea (Vigna sinensis Endlo) were used for protoplast isolation. Enzyme solution for protoplast isolation contained 40% cellulase Onozuka R-10,0.30% Macerozyme R-10 and 2% hemicellulase. The purified protoplasts were cultured in Bs,MS or KM8p liquid medium in dark (25℃) at a density of 1 × 105–5 × 105/ml. The protoplasts started cell division in 3–5 days . Sustained cell divisions resulted ill formation of cell clusters and small calli,with cell division frequency reaching 23%–28% in MS medium . Calli of 2 mm in size were transferred onto MSB (MS salts+B5 vitamins) medium with 2 mg/L 2,4-D, 0. 5mg /L BA forfurther growth. Embryogenic calli appeared on this medium. After passage to fresh medium with the same composition, the embryogenic calli were transferred into MSB liquid medium to establish suspension culture. When the suspended calli were transferred back onto MSB agar medium with 0. 1 mg /L IAA, 0.5mg/L KT, 5% mannitol (cultured in light,2000 lx,12h/d), a lot of adventitious roots formed in 7–10 days, and then somatic embryos formed from the protoplast derived calli. But only a few embryoids developed further into the cotyledonary stage ,and the others died at globular, heart-shaped, or torpeto stage . Finally, some cotyledonary embryoids germinated and developed into plantlets or shoots with leaves.     

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。     

茶（Camellia sinensis Kuntze）离体培养体细胞胚胎发生的组织细胞学研究

以茶树叶片为外植体．以ＭＳ培养基附加４ｍｇ·Ｌ－１６－ＢＡ,２ｍｇ·Ｌ－１ＩＡＡ．３ｍｇ·Ｌ－１ＧＡ３和０．２—０．３％活性碳诱导出愈伤组织和胚状体,进一步形成小植株．切片观察表明．茶叶愈伤组织胚状体的发生,起源于愈伤组织表层及其内部的单个细胞和细胞团,胚状体发育顺序与合子胚大致相似．经球形胚、心形胚、鱼雷胚和子叶胚阶段．但发育过程中常有畸形胚出现．     

Embryogenic Suspension Culture and Plant Regeneration from Suspension-Derived Proto-plasts of Cucumber (Cucumis sativus L.)

Fast growing embryogenic cell suspension culture was established when embryogenic callus derived from cotyledon protoplasts of cucumber was transferred into a liquid culture. So far the cell line has been subcultured for two years and retained the ability of embryogenesis and plant regeneration. Experimental data showed that the concentration of ABA or sucrose had a dramatic effect on embryogenesis and synchronization of embryoid development. Low level of sucrose concentration (1%) facilitated the precocious germination of the embryoids while 1 mg/l of ABA or 7–9% of sucrose was found to be effective for reducing callusing of the cultures and synchronisticly controlling the embryoids at globular or late globular stage. Embryogenic cells taken from 3–5 days after subculture were enzymatically digested. A large amount of viable protoplasts was isolated. Protoplasts were cultured in a DPDK1 medium either by means of drop or thin layer liquid culture or by means of sodium alginate encapsulation culture. Actively dividing cells formed cell colonies and globular embryoids which were transferred onto a solidified agar medium or directly into a liquid medium to form a shaken culture. The embryoids would proliferated continuously. Embryoids eventually developed into plantlets when they were transferred onto a 1/2 MSO medium devoid of phytohormones.     

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。     

Control of Embryoid Development in Tissue Cultures of Celery

Scanning electron microscope photographs of the embryoids showedglobular embryoids attached to the surface of aggregates inliquid medium and also some free floating. The surface structureof the unattached embryoids was very irregular, but, with thechange to polarized growth in the heart and torpedo forms, thesurface of the embryoid became smoother. The stage of developmentof the embryoids could be controlled by modifying the compositionof the medium to the extent that the majority of the embryoidsin the culture were either globular or torpedo forms. One ofthe most effective compounds in controlling development was2,4–dichlorophenoxy acetic acid (2,4–D). At high2,4–D concentrations, embryogenesis in the callus wasrestricted to the globular stage and after two subcultures itwas totally repressed, while after ten subcultures the potentialfor embryogenesis was lost and could not be regained even aftersubculture on a normal medium. On the normal agar medium thecallus always continued to show embryogenesis, but when it wastransferred to liquid medium of the same composition, embryoidswere produced in the first subculture but the potential haddeclined by the third subculture, when only roots were produced,and after ten subcultures cell growth and all differentiationwas totally it hibited. However, in the first subculture inliquid medium, embryogenesis was sequential with the whole cultureprogressing from globular to torpedo forms. This was particularlyeffective when the callus inoculum had been maintained on ahigh 2,4–D concentration for the two subcultures priorto inoculation of the liquid medium. By making use of this sequentialchange in embryoid development, a large number of embryoidscould be obtained at any particular stage. Apium graveolens, celery, tissue culture, embryoids, 2,4–D     

Fatty acid β-oxidation and glyoxylate cycle enzyme activities of induced glyoxysomes from anise suspension cultures

Homogenates of dedifferentiated anise (Pimpinella anisum L.) suspension cultures grown in B-5 medium with sucrose as source of carbon show all but 3 glyoxysomal enzyme activities: NAD-dependent oxidation of palmitoyl-CoA, isocitrate lyase, and malate synthase are lacking. Substitution of 20 mmol/l acetate for sucrose leads to the appearance of these enzyme activities. Only then glyoxysomes with a buoyant density of 1.23 kg/l in sucrose gradients are formed showing the enzyme activities for both ß-oxidation of fatty acids and glyoxylate cycle. Quantitatively and qualitatively they resemble glyoxysomes isolated from endosperm of 4 d old anise seedlings. Therefore, the suspension cultures constitute a valuable system for the study of both mechanisms and regulation of glyoxysome formation in anise.     

Somatic embryogenesis and plant regeneration in tissue cultures of sweet potato (Ipomea batatas Poir.)

Leaf, shoot-tip, stem, and root explants of sweet potato (Ipomea batatas Poir.) gave rise to two kinds of callus on nutrient agar medium containing 0.5 to 2.0 mg/l 2,4-D. One callus, bright- to pale-yellow, was compact and organized, while the other was dull-yellow and friable. The former callus gave rise to numerous globular and heart-shaped embryoids. When transferred onto hormone-free medium, the embryoids readily developed into a torpedo-shape before germination. The plantlets were transplanted to soil where they flowered and formed storage roots at maturity.     

A Scanning Electron Microscope Study of Theobroma cacao Somatic Embryogenesis

This study describes the somatic embryogenesis of Theobromacacao L. with a scanning electron microscope. It revealed earlydevelopmental stages as globular and incipient heart-shaped.Morphological abnormalities, such as the occurrence of threeand four cotyledons and round or long forms of embryoids witha long and thin or short and thick stalk-like structure whichseems to equate to a suspensor, were also observed. A suspensorwas found in some embryoids. cacao, Theobroma cacao, embryoids, somatic embryogenesis, SEM     

The Origin and Development of Embryoids in Suspension Cultures of Carrot (Daucus carota)

In suspension cultures of carrot, embryoids are initiated ator near the surface of characteristic cellular aggregates (embryogenicclumps) and are released from these aggregates as free-floatingstructures capable of further development into plantlets. Embryoiddevelopment is promoted by transfer from a medium containing2,4-D to one from which it is omitted. The structure of theembryogenic clumps, in these two media, has been studied bythe thin section technique involving fixation in glutaraldehydeand embedding in glycol methacrylate. The superficial cellsof the clumps are clearly marked off from the central cellsand contain large starch grains, a large central nucleus, abundantcytoplasm and a number of vacuoles. It is from individual superficialcells that the embryoids are seen to arise in the 2,4-D omittedmedium but it has not been possible to distinguish, in advanceof embryogeny, such cells from within the superficial cell layers.The fragmentation of the proliferating clumps in 2,4-D containingmedium and the release of embryoids into the 2,4-D omitted mediumis promoted by some of the superficial cells becoming free ofstarch and undergoing pronounced enlargement. A sequence of segmentations leading from single superficialcells of the clumps to globular embryoids has been traced. Thisdiffers from that reported to occur during early embryogenyfrom the egg cell of carrot. The resemblance to zygote embryogenyis closer from the late globular stage of embryoid development;embryoids then differ from zygotic embryos in their shortersuspensors. Attention is drawn to the initiation of additionalembryoids from the epidermis of some of the embryoids developingin culture.     

Enhanced embryogenesis and plant regeneration from cucumber (Cucumis sativus L.) callus by activated charcoal in solid/liquid double-layer cultures

Enhanced embryogenesis and plant regeneration methods were established in cucumber (Cucumis sativus L. cv. ‘Delilah’) using hypocotyl segments as explants. Callus formation, followed by pro-embryogenic aggregates and globular embryoids required liquid shake cultures. In liquid medium, however, many of the embryoids developed into abnormal structures — ‘neomorphs’ or succulent plantlets. Embryoids subcultured to stationary liquid or agar cultures dedifferentiated and underwent secondary embryogenesis. Neither increased osmolarity nor adding abscisic acid (ABA), zeatin or activated charcoal to the liquid medium inhibited abnormal morphogenesis. The use of double layer cultures containing activated charcoal in the lower agar layer and ABA with elevated calcium in the upper liquid phase prevented dedifferentiation and secondary embryogenesis and allowed normal organized growth of the embryoids. Hardening in vitro by partial desiccation with CaSO₄ under aseptic conditions improved the cucumber plantlet's leaf growth and their survival after transplanting to soil.     

Plant regeneration from protoplasts of immature Vigna sinensis cotyledons via somatic embryogenesis

Summary Protoplasts were isolated from immature cotyledons of Vigna sinensis and cultured in a modified MS Liquid medium containing 0. 2 mg/l 2, 4-dichlorophenoxyacetic acid (2, 4-D), 1 mg/l naphthaleneacetic acid (NAA) and 0. 5 mg/l 6-benzylaminopurine (BAP) in the dark at a density of 1 × 10⁵/ml. The protoplasts began to divide in 3–5 days. Sustained cell division resulted in formation of cell clusters and small calli, with the cell division frequency and plating efficiency of cell colonies reaching 27. 7% and 1. 7% respectively. When calli of 2 mm in size were transferred onto MSB medium (MS salts and B5 vitamins) containing 500mg/l NaCl, 500 mg/ 1 casein hydrolysate (CH), 2 mg/l 2,4-D and 0. 5 mg/l BAP for further growth, approximately 5% of the calli developed embryogenically. The embryogenic calli were selected and subcultured on the same composition of MSB medium and were able to maintain somatic embryogenesis capacity in subculture for a long time. When the calli were moved to MSB medium with 0. 1 mg/l indole-3-acetic acid (IAA), 0. 5mg/l kinetin(KT), 3–5% mannitol and 2% sucrose in the light, many somatic embryos formed from the calli. Only part of the embryoids developed further to the cotyledonary stage, and the others died at the globular, heart-shaped or torpedo stages. Finally, some cotyledonary embryoids germinated and developed into plants or shoots. The shoots were readily rooted on 1/2 strength MS medium with 0. 1–0.3 mg/l indole-3-butyric acid (IBA). The plants grew well in soil and were fertile.Abbreviations 2, 4-D dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - BAP 6-benzylaminopurine - IAA indole-3-acetic acid - KT kinetin - IBA indole-3-butyric acid - CH casein hydrolysate - CM coconut milk - ZT zeatin     

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.     

Induction of accumulation and degradation of the 18.4-kDa oleosin in a triacylglycerol-storing cell culture of anise (Pimpinella anisum L.)

Two closely related anise cell-culture lines, Pa15 and Pa19, differ considerably in growth rate, potential to form somatic embryoids, triacylglycerol (TAG) storage and pattern of lipid-body proteins. Line Pa15 grows very fast (doubling rate: 3 d), mainly as single cells, exhibits a low potential for somatic embryogenesis and its TAG content is relatively low (5–20 mg TAG per g dry weight). In contrast, the line Pa19 shows lower growth rates (doubling rate: 8 d), tends to form clusters of somatic cells, has a higher TAG content (100–150 mg TAG per g dry weight), and somatic embryoids are easily induced. Under defined culture conditions, the TAG content of the line Pa19 can be increased to approximately 70% of that of ripe anise seeds (150 and 220 mg TAG per g dry weight, respectively). Polyclonal antibodies prepared against the most abundant protein (relative molecular mass 18.4 kDa) from the lipid-body fraction of anise seeds (Radetzky et al. 1993, Planta 191, 166–172) react also with a 18.4-kDa protein from the lipid-body fraction of cells of the Pa19 culture. In contrast, only fairly low levels of the 18.4-kDa oleosin were detected in Pal5. Limited sucrose supply in the medium resulted in TAG degradation and the concomitant decrease in the amount of immunodetectible 18.4-kDa protein in the Pa19 cell culture. Treatment with sorbitol, or abscisic acid and sorbitol in combination, enhanced TAG contents and also the amount of immunostained 18.4-kDa protein in the cell culture Pa19, whereas no effect was found on either TAG content or 18.4-kDa protein in the cell-culture line Pa15. The 18.4-kDa protein can be classified as an oleosin, a proposal which is supported by the similarity in molecular mass compared with other known oleosins, its occurrence in the lipid-body fraction and the fact that its amount correlates with the TAG content. The results of this study indicate that the Pa19 cell culture provides a valid model system for investigations of lipid storage and mobilization in higher-plant cells.Abbreviations ABA cis-abscisic acid - TAG triacylglycerol(s) - 2,4-D 2,4-dichlorophenoxyacetic acid The authors thank Christiane Bernshausen for kind technical assistance.     

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.     

Regulation ofin vitro androgenesis in tobacco through iron-free media

Globular embryoids were continually produced in anther cultures of tobacco (Nicotiana tabacum L. cv. Samsun) from the pool of resting microspores if the iron-free medium was used. The supplement of iron stopped the development of fresh early embryoids still inducing continual conversion of the resting globular embryoids into torpedo-shaped embryoids, and into haploid plants. Globular embryoids in the anthers responded to the iron supply even after eight months’ cultivation on iron-free media. Isolated embryoids showed the same response. Haploid plants were regenerated from the anthers on the minimal medium consisting of agar, sucrose, iron and distilled water. Iron requirements of preglobular, globular and postglobular embryoids are discussed.     

2,4-D诱导的花生体细胞胚发生的组织学研究

花生成熟胚胚叶在佃附加20mg/L 2,4-D的培养基上诱导20d后,转移至无激素培养基MS0继续培养,可获高频体细胞胚发生。组织学观察表明,体细胞胚起源于胚叶上表皮及表皮下数层细胞,这些细胞脱分化形成细胞质浓厚、细胞核大的胚性细胞团,胚性细胞团继续分裂形成体细胞胚。体细胞胚的发育过程经历球形胚、心形胚、鱼雷胚、子叶胚四个时期。     

Xyloglucan Endotransglycosylase Activity in Carrot Cell Suspensions during cell Elongation and Somatic Embryogenesis

Xyloglucan endotransglycosylase (XET) has been proposed to contribute to cell elongation through wall loosening. To explore this relationship further, we assayed this enzyme activity in suspensions of carrot (Daucus carota L.) cells exhibiting various rates of cell elongation. In one cell line, elongation was induced by dilution into dichlorophenoxyacetic acid (2,4-D)-free medium. During this elongation, 93% of the XET activity was found in the culture medium; in nonelongating controls, by contrast, 68% was found in the cell extracts even though the specific activity of these extracts was lower than in the elongating cells. By far the highest rates of XET secretion per cell were in the elongating cells. A second cell line was induced to undergo somatic embryogenesis by dilution into 2,4-D-free medium. During the first 6 d, numerous globular embryoids composed of small, isodiametric cells were formed in the absence of cell elongation; extracellular XET activity was almost undetectable, and intracellular specific activity markedly declined. After 6 d, heart, torpedo, and cotyledonary embryoids began to appear (i.e. cell elongation resumed); the intracellular specific activity of XET rose rapidly and >80% of the XET activity accumulated in the medium. Thus, nonexpanding cell suspensions (whether or not they were rapidly dividing) produced and secreted less XET activity than did expanding cells. We propose that a XET molecule has an ephemeral wall-loosening role while it passes through the load-bearing layer of the wall on its way from the protoplast into the culture medium.     

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.     

Somatic embryogenesis and plant regeneration from cell suspension culture of niger (Guizotia abyssinica Cass.)

Somatic embryogenesis was achieved from cell suspension cultures of niger (Guizotia abyssinica Cass.). Initially, friable embryogenic calluses were induced from cotyledonary leaves of niger on Murashige and Skoog (MS) agar medium containing 5 μM 2,4-Dichlorophenoxyacetic acid (2,4-D) and 0.5 μM kinetin (KIN). Cell suspension cultures were established by using embryogenic calluses in MS liquid medium containing 5 μM 2,4-D and 0.5 μM KIN. Initiation of somatic embryogenesis and development up to globular stage from embryogenic cell clumps occurred in the liquid medium itself. Thereafter embryogenic cell aggregates were transferred to MS agar medium supplemented with 3 μM KIN for embryo differentiation, whereas maturation of somatic embryos occurred in MS agar medium containing 10 μM abscisic acid.