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。     

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.     

Origin of somatic embryos from cultured shoot tips ofSorghum bicolor (L.) moench

Summary Histologic examination of shoot-tip explants, 1 wk after culture initiation on Murashige and Skoog medium with 2.5 mg/liter 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.05 mg/liter kinetin, reveals active meristematic centers inside cultured tissue. Clusters of cells in these meristematic centers exhibit remarkable resemblance to the initial three divisions in the zygotic embryo. Several such meristematic groups of cells are observed in the cultured explant at this stage. Embryogenesis is obviously initiated very early in this tissue in the presence of 2,4-D. A well-defined, white globular embryogenic callus develops in culture in about 4 wk, and it consists of clusters of embryoids with large cells characterized by thick cell walls, numerous lipoidal vesicles, and localized areas of carbohydrate storage. These cells resemble the scutellar tissue of the embryo. However, there are cells within this tissue that themselves appear embryogenic. They undergo cell division giving rise to small clusters of cells. As long as 2,4-D is present in the medium, the cells apparently retain the capacity to proliferate and to produce more cells capable of embryogenesis. Embryogenesis seems to occur via two processes, initiation of somatic embryos early in culture and secondary embryogensis from the scutellar tissue that forms in vitro.     

Calcium increases the yield of somatic embryos in carrot embryogenic suspension cultures

An upward shift in the concentration of calcium present in the medium during somatic embryogenesis increased the number of embryos produced approximately two-fold. This was observed when embryogenic suspension cells grown in 2,4-D medium with the normal calcium concentration of 10^–3 M were transferred to hormone-free medium containing 10^–2 M calcium and when embryogenic suspension cells grown in 2,4-D medium containing 10^–4 M calcium were transferred to hormone-free medium with 10^–3 M calcium. At calcium concentrations between 6·10^–3 and 10^–2 M globular stage somatic embryos were found in cultures supplemented with 2·10^–6 M of 2,4-D indicating that elevated calcium counteracts the inhibitory effect of 2,4-D on somatic embryogenesis. No qualitative changes were found in the pattern of extracellular polypeptides as a result of growth and embryogenesis in media with different calcium concentrations.     

Studies on Somatic Embryogenesis and Histological Observation in Gossypium hirsutum L. cv. Coker 312

A reproducible 3-step procedure of somatic embryogenesis of Gossypium hirsutum L. cv. Coker 312 has been developed. Calli were initiated on LSC medium containing 0.1mg/L 2,4-D plus 0.1 mg/L KT from cotyledon tissue of 5-day-old-seedlings, and subcultured on the same medium with 4 mg/L NAA and 1 mg/L KT. Embryogenic calli and few globular embryos developed at a frequency of 67.5% after 55 days’ culture in the latter medium. When the embryogenic calli were transferred to growth regulator-free medium, embryogenesis occured and all stages of normal zygotic embryos, globular-, heart- and arrowhead- or torpedo-shaped embryo, ,were developed. Cyto-histological study showed that embryogenic calli were very easily distinguished from non-embryogenic calli. Embryoids were mainly initiated from the cells in the peripheral area of embryogenic calli. At the early stage the development of embryoid was limited in a boundary of thicken cell wail. There were 2 peaks of starch accumulation in the process of embryogenesis, one was at the early globular stage, and the other at the later torpedo-shaped stage.     

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。     

Relationship between auxin-induced cell proliferation and somatic embryogenesis in culture of carrot cotyledons

We elucidated the relationship between cell proliferation and somatic embryogenesis in the culture of carrot cotyledons. Fresh weights of the cotyledon expiants were determined every five days while being cultured on a medium containing 2,4-D. Callus production increased exponentially from Day 20 to Day 25, showing a two-fold rate of proliferation. To examine the embryogenic potential of the callus, we pre-cultured cotyledon explants on an MS medium with 2,4-D, then transferred them to an MS basal medium at five-day intervals. Somatic embryos formed most frequently when the cotyledons were pre-cultured for 20 days on an MS medium that contained 5 μ2,4-D. The frequency of somatic embryo formation was 81%, while that of normal embryos with two cotyledons was 51% among those formed on a hormone-free medium. We used FACScan analysis to relate the embryogenic potential of the callus to the S phase in the cell cycle of cultured cells. The S phase was high after 25 days of culture on the medium with 5 μM 2,4-D. In contrast, the frequency of normal embryogenesis was higher at Day 20 of the pre-culture period. Culturing embryogenic calli on a medium with 5 μM 2,4-D was most favorable for producing somatic embryos with two cotyledons. We verified that active somatic embryogenesis was apparently related to cell division activity; somatic embryos induced from actively dividing cells were apt to accompany cotyledonary abnormality.     

High Frequency of Plant Production via Somatic Embryogenesis from Callus or Cell Suspension Cultures inEleutherococcus senticosus

Hypocotyl segments ofEleutherococcus senticosuscultured on Murashigeand Skoog's (MS) medium with 4.5 µM2,4-D produced somaticembryos directly from the surface of explants without interveningcallus formation. When these somatic embryos were subculturedto the same MS medium with 4.5 µM2,4-D, friable embryogeniccalli were formed mainly from radicle tips of somatic embryos,but at a low frequency (5%). Selected embryogenic calli weremaintained on MS agar or liquid medium with 4.5 µM2,4-D.To induce somatic embryo development, embryogenic calli andcell clumps were transferred to MS medium lacking 2,4-D. Thefrequency of somatic embryo formation differed between culturetypes with 1570 embryos formed per Petri dish from callus cultureand 5514 embryos formed per flask from cell suspension cultures.Somatic embryos formed on agar medium had larger cotyledonsthan those of embryos formed in liquid medium. GA₃treatmentwas necessary to induce germination from somatic embryos. Therate of plant conversion was 97% in somatic embryos from callusculture and 76% in embryos from liquid culture. Regeneratedplantlets were successfully acclimatized in the glasshouse.Copyright1999 Annals of Botany Company Eleutherococcus senticosus, micro propagation, somatic embryogenesis.     

Proteins related with embryogenic potential in callus and cell suspensions of sugarcane (Saccharum sp.)

Summary Previous results have shown that some proteins secreted in the culture medium are involved with the formation of embryogenic cells and can modify somatic embryo differentiation. Undifferentiated cell suspensions grown in the presence of 13 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and obtained from embryogenic and non-embryogenic callus were used to study these events in sugarcane plants (cv.PR-62258). The cell suspension growth curves were determined and soluble proteins were extracted from embryogenic and non-embryogenic callus and culture medium from cell suspensions. In embryogenic callus we detected 1.43 times more protein than in non-embryogenic callus and the electrophoretic protein patterns show specific polypeptides for both callus types. In embryogenic callus we detected a cluster of four polypeptides in the range of 38–44 kDa and another polypeptide of 23 kDa that were not observed in non-embryogenic callus. In nonembryogenic callus there is a 35-kDa polypeptide that was not detected in embryogenic callus. In the case of extracellular proteins, the medium from embryogenic cell suspensions contained four polypeptides of 41, 38, 34 and 28 kDa that were slightly detected in the medium from non-embryogenic cell cultures; we also detected a band at 15 kDa that could not be observed in the medium from non-embryogenic cell suspensions. These results suggest that the development of embryogenic callus and cell suspensions is related to the type and amount of intracellular proteins in the callus cells and to the secreted proteins from these cells into the medium.     

Embryogenic suspensions of adult cork oak: the first step towards mass propagation

Protocols have been established to clone adult cork oak trees by somatic embryogenesis using semisolid medium. However, for economically viable mass propagation, embryogenic cultures in liquid medium need to be developed. In this study, suspension cultures were initiated from embryo clusters obtained by secondary embryogenesis on a gelled medium lacking plant growth regulators. After 6 days of culture, these embryo clusters generated high cell density suspensions that also contained small organized structures (embryos and embryogenic clumps). As the culture duration increased, tissue necrosis and fewer embryogenic structures were observed and the establishment of suspension cultures failed. An alternative method was found adequate for initiation of embryogenic suspensions: embryo clusters from gelled medium were briefly shaken in liquid medium and detached cells and embryogenic masses of 41–800 μm were used as inoculum. Maintenance of embryogenic suspensions was achieved using a low-density inoculum (43 mg l^?1) by subculturing four embryogenic clumps of 0.8–1.2 mm per 70 ml of medium. Proliferation ability was maintained for almost 1 year through ten consecutive subcultures. The initiation and maintenance protocols first developed for a single genotype were effective when tested on 11 cork oak genotypes.     

Plant regeneration from embryogenic cell suspension cultures of wild sorghum (Sorghum dimidiatum Stapf.)

A simple and efficient protocol is described for regeneration of wild sorghum (Sorghum dimidiatum) from cell suspension cultures. Fast-growing cell suspensions were established from shoot-meristem-derived callus. Plating of the suspension on Murashige and Skoog agar medium supplemented with 2.5 mg l^–1 2,4-dichlorophenoxyacetic acid (2,4-D) resulted in the formation of embryogenic calli. High-frequency (80%) somatic embryogenesis from small cell clusters (300–400 μm) was observed when the cultures were initially maintained in liquid medium with reduced levels of 2,4-D (0.25 mg l^–1), followed by transfer to regeneration medium. Direct plating of these small clusters on regeneration medium or transfer to liquid regeneration medium containing kinetin and 6-benzylaminopurine resulted in the development of mature somatic embryos and plantlets. The regenerants developed to maturity and were all phenotypically and cytologically normal. Received: 20 May 1998 / Revision received: 1 September 1998 / Accepted: 23 September 1998     

Somatic embryogenesis and morphogenesis in callus derived from the epiblast of immature embryos of wheat (Triticum aestivum)

Factors affecting the formation of embryogenic callus from the epiblast of immature embryos of wheat (Triticum aestivum L.) are described. Embryos were incubated on a modifed Murashige and Skoog medium with the scutellum in contact with medium. Callus formation from the epiblast was affected by the type of cultivar used, the stage of embryo development, and the concentration of 2,4-dichlorophenoxyacetic acid (2,4-D) in the culture medium. The number of embryos forming embryogenic callus ranged from 10% to 72%, depending on the cultivar. Embryo development was classified into five distinct morphological stages and higher yields of callus were produced using embryos excised at stages II and III. Concentrations of 2,4-D higher than 1 μM were required for the formation of embryogenic callus. Epiblast callus gave rise to embryoids which differentiated into multiple plants at a high frequency when placed on hormone-free 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.     

Repetitive somatic embryogenesis from peanut cultures in liquid medium

Summary A regeneration system based on repetitive somatic embryogenesis was developed for peanut (Arachis hypogaea L.). Embryogenic suspension cultures were initiated using individual somatic embryos induced from immature cotyledons cultured on a modified Murashige and Skoog medium containing 40 mg/l 2,4-D for 30 days. After transfer to a modified MS liquid medium, the somatic embryos produced masses of secondary and tertiary embryos which continued to proliferate following manual separation and subculture of the embryogenic clumps. The cultures exhibited exponential growth, and have been maintained for over one year without apparent loss of embryogenic potential. Further embryo development, germination, and conversion were achieved by placing embryo clumps onto hormone-free, solid medium. The inclusion of a desiccation period during embryo development enhanced conversion four-fold. Plants have been established in soil and appear to be phenotypically normal.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - BA 6-benzylaminopurine - MSO Modified Murashige and Skoog basal medium - EM embryogenic masses     

Somatic embryogenesis and plant regeneration from cell suspension and tissue cultures of mature himalayan poplar (Populus ciliata)

Somatic embryogenesis and plantlet formation were obtained from callus and cell suspension cultures of 40-year- old Himalayan Poplar (Populus ciliata Wall ex Royle). Callus and cell suspensions were obtained by transfer of inoculum of semiorganized leaf cultures, which were maintained on Murashige and Skoog (MS) medium supplemented with benzylaminopurine (BAP), to MS with 2,4-dichlorophenoxyacetic acid (2,4-D). Reduction of 2,4-D concentration during subsequent subculture of cell suspensions resulted in the formation of embryoids. These embryoids developed further only after being transferred to agar-based MS medium supplemented with BAP and naphthalene acetic acid. Loss of embryogenic potential was observed in cell suspensions after 6 subcultures. However, callus cultures retained the embryogenic potential even after repeated subcultures for more than a year. Plantlets could be successfully hardened and grown in natural outdoor conditions.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthalene acetic acid - MS Murashige and Skoog (1962) medium     

High frequency plant regeneration from immature ovule-derived embryogenic cell suspension cultures of Chelidonium majus var. asiaticum

Culture conditions for high frequency plant regeneration via somatic embryogenesis in cell suspension cultures of Chelidonium majus var. asiaticum are described. Immature ovules formed embryogenic calluses at a frequency of 40% when cultured on Murashige and Skoog (MS) medium supplemented with 4.52 μM 2,4-dichlorophenoxyacetic acid (2,4-D). The optimum ovule size for embryogenic callus formation ranged from 1 to 1.5 mm in length. Cell suspension cultures were established from embryogenic calluses using MS liquid medium containing 4.52 μM 2,4-D. Upon plating onto MS basal medium, cell aggregates from cell suspension cultures produced somatic embryos which then developed into plantlets. Regenerated plantlets were transplanted to potting soil and grown to maturity in a growth chamber. This revised version was published online in June 2006 with corrections to the Cover Date.     

Somatic embryogenesis and plant regeneration from suspension cultures of Acanthopanax koreanum Nakai

High-frequency somatic embryogenesis was achieved from an embryogenic cell suspension culture of Acanthopanax koreanum Nakai. Stem segments were cultured on Murashige and Skoog (MS) medium containing auxins and cytokinins. Opaque and friable embryogenic callus formed on MS medium with 4.5 μm 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.0 μm kinetin or zeatin, but was highest on medium containing 4.5 μm 2,4-D alone. Embryogenic calli were transferred to MS liquid medium containing 4.5 μm 2,4-D and maintained by subculture at 2-week intervals. Initiation of somatic embryogenesis and development up to the globular stage from embryogenic cell clumps occurred in medium containing 0.45 μm 2,4-D, whereas maturation and germination of somatic embryos occurred in MS medium lacking 2,4-D. Cytokinin treatment suppressed the normal growth of embryos, but stimulated secondary somatic embryogenesis from the surfaces of primary embryos. Plants from somatic embryos were acclimatized in a greenhouse. Received: 14 January 1997 / Revision received: 17 June 1997 / Accepted: 5 July 1997     

Organogenesis and somatic embryogenesis in Foeniculum vulgare: histological observations of developing embryogenic callus

Different NAA plus kinetin or BA combinations were tested on Francia Pernod fennel seedlings for callus induction and plant regeneration. Callogenesis from hypocotyls was obtained in all auxin/cytokinin-containing media. The organogenic response was observed especially in presence of NAA plus kinetin. The highest frequency of shoot regeneration was found when the auxin and kinetin were used at a 1:1 ratio. Moreover, a prolonged culture period increased shoot formation. Somatic embryogenesis was tested on several fennel populations. The results gave evidence of the genotypic importance. Two different protocols were used for somatic embryo induction. Using the first protocol among the different fennel genotypes tested, only Francia Pernod showed embryogenic capacity. In this case, from a primary non-embryogenic callus cultured for 12 months in presence of 2,4-D, an embryogenic secondary callus was produced. When transferred to the medium without 2,4-D (agarized or liquid), this gave embryogenic plants in high frequency. As far as the second embryogenic method is concerned, secondary embryogenic callus developed only in the presence of 2,4-D plus kinetin in Francia Pernod genotype. Thereafter, the replacement of those growth regulators by GA₃ into the medium greatly increased the somatic embryo development, especially in `Francia Pernod', but also in `Aboca erbe' callus, a population with a very poor embryogenic capacity. In Francia Pernod, the primary and secondary (embryogenic) calli showed different morphological and histological responses, either when the secondary callus was induced by 2,4-D alone or by 2,4-D plus kinetin. Ontogenetic processes leading to somatic embryo formation are described in this context. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of Plant Growth Regulators on Somatic Embryogenesis of Peucedanum terebinthaccum

The influence of different plant growth regulators including 2,4-D,ZT, 6-BA and ABA on somatic embryogenesis and the amount of endogenous ABA at different stages of embryogenesis was investigated. The effect of each plant growth regulator changed according to the stage of embryogenesis. The amount of endogenous ABA was rather high in single cell stage, decreased at cell clump and embryogenic cell clump stages and dramatically increased at globular embryo stage. It decreased again as the embryo developed. This change in amount of the endogenous ABA explained very well the difference in the effect of exogenous ABA when applied at different stages of embryogenesis.     

Establishment of a System with High Synchronous Frequency of Somatic Embryogenesis and Embryo Seedling Formation in Camellia sinensis var. assamica

The system of high synchronous frequency of somatic embryogenesis and somatic embryo seedling formation was established by means of embryonic cell hne 1 ( CL1 ) of Camellia sinensis var. assamica Kitamura. Modified MS was used as the basic medium. Cultures of CL1 was transferred to the aqueous induced medium (0.05 mg/L 2,4-D + 0.50 mg/L 6-BA) from the maintenance medium (0.1 mg/L 2,4-D + 0.5 mg/L 6-BA) for somatic embryos induction under dark condition. 28 days later, they were cultured in the liquid differentiation medium. Various kinds of somatic embryos were obtained after another 28 days. The frequency of somatic embryos was 81.5 %. Various mesh sizes of sieves were applied to collect the somatic embryos in different developmental stages which could develop to mature stage in the aqueous growth medium ( 1/2 MS + 1.0 mg/L GA3 + 0.5 mg/L 6-BA). ABA was effective to promote the formation of highly qualified somatic embryo. The mature somatic embryos sized 20 to 70 mesh had the conversion frequency 75 %. The development of somatic embryogenesis studied under a cell suspension culture system was similar to the zygotic embryogenesis.