贡蕉胚性细胞悬浮系的建立和植株再生

鲜食蕉品种的高度不育性和多倍性制约了用传统育种方法培育生产实践中所需的新品种 ,建立稳定的胚性细胞悬浮系是香蕉生物技术育种的前提。以目前国内尚未建立该体系的鲜食蕉品种贡蕉 (AA)未成熟雄花序的第 1～ 15位花梳为外植体 ,对胚性细胞悬浮系的建立和植株再生体系进行了优化。结果表明 ,5～ 6个月的培养后可获得分生小球体和浅黄色、松散易碎的胚性愈伤组织。 9μmol/L 2,4 D对外植体愈伤组织的诱导效果最好 ,诱导率为 40.96 % ,胚性愈伤组织诱导率可达7.45 % ,其中5.79%的胚性愈伤组织来源于第 6～12号位置的花梳。胚性愈伤组织悬浮培养后 ,通过 3个月的筛选和继代培养 ,可得到均质的胚性细胞悬浮系。该培养体系合适继代周期为 15d ,继代时合适的起始接种量为每 30mL培养基加 2mLPCVECS。培养 6个月的胚性细胞在体细胞胚诱导培养基中培养15d后可见到白色半透明体细胞胚的发生 ,体细胞胚诱导率为 2 80× 10³个 mLPCV。成熟体细胞胚的萌发率为 17 2 8% ,其中发育成正常的再生植株的百分率为 14 16 %。     

Inflorescence proliferation for somatic embryogenesis induction and suspension-derived plant regeneration from banana (<Emphasis Type="Italic">Musa</Emphasis> AAA,cv. ‘Dwarf Cavendish’) male flowers

Availability of explants with adequate embryogenic competence is one of the most important limitations for the development of regenerable cell suspensions in banana. To increase the number and ease of accessibility to potentially embryogenic explants, a novel methodology is described by which young male flower clusters isolated from adult plants are induced to form new flower buds and proliferate in vitro. Different concentrations of the plant growth regulator thidiazuron (TDZ) induced inflorescence proliferation, which could be maintained over time as a continuous source of young flower buds. Intensity of proliferation was evaluated during successive subcultures. At the third cycle of proliferation, the highest multiplication rate (2.89) was obtained on the medium containing 5 microM TDZ. Newly generated floral tissues were assessed for embryogenic competence, resulting in an average embryogenic frequency of 12.5%. The observed embryogenic capacity, together with the recurrent availability of immature flowers, allowed for the direct initiation of cell suspensions from bulked explant cultures. Regular observation and regeneration tests during the development of suspended cell cultures confirmed their embryogenic condition. Produced embryos successfully matured and germinated to regenerate hundreds of somatic in vitro plants.     

Characterization and regeneration of wheat (Triticum aestivum L.) embryogenic cell suspension cultures

Summary Stable cell suspension cultures were established from two types of calli (one compact, nodular and embryogenic, the other friable and embryogenic) derived from cultured immature embryos of wheat (cv FLA302). Only aged calli, which had been subcultured for at least 5–8 months, formed suspensions comprised mainly of groups of small, round, densely cytoplasmic, starch-containing cells. Only the embryogenic suspension derived from the aged, compact and nodular callus formed distinct somatic embryos when plated on regeneration media containing IAA and zeatin. Upon subsequent transfer to fresh regeneration medium more than 200 green rooted plants were obtained.Abbreviations 6-BA 6-benzylaminopurine - CH casein hydrolysate - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MS Murashige and Skoog (1962) basal medium - NAA naphthaleneacetic acid - PCV packed cell volume     

Establishment of oil palm cell suspensions and plant regeneration

Primary globular callus from immature zygotic embryos and friable embryogenic tissue derived from mature zygotic embryos were used to establish suspension cultures. Callus cultures were established either on modified Y3 or MS medium containing 475–500 M 2,4-D or 250 M picloram and 0.3% (w/v) activated charcoal. Suspension cultures of both cell lines were established in modified Y3 medium containing 10 M 2,4-D. The establishment of cell suspensions from friable embryogenic tissue took only 2 months, in contrast with suspensions from primary globular callus which took 3–5 months to establish. Embryo differentiation was observed only in cell suspensions derived from the friable embryogenic tissue after plating aliquots on regeneration medium. Germinated embryos were recovered and plantlets were successfully established under greenhouse conditions.Abbreviations CET compact embryogenic tissue - FET friable embryogenic tissue - CIM callus induction medium - PGC primary globular callus - 2,3-D 2,4-dichlorphenoxyacetic acid Y3-Eeuwens' medium - MS Murashige & Skoog medium - PVP-40 polyvinylpyrrolidone - KM Kao & Michayluk vitamins - ABA abscisic acid     

Somatic embryogenesis and plant regeneration from immature embryo cultures of onion (Allium cepa L.)

Somatic embryos were obtained and plants regenerated from immature embryos of onion following culture on embryogenic induction media. Highest rates of somatic embrogenesis resulted from 0.5- to 1.5-mm immature embryos cultured on media containing 5 mg/l of picloram. Somatic embryos formed either directly on the surface of embryos or developed from compact cultures. The production of somatic embryos was significantly affected by the addition of auxin, embryo size and cultivar. The potential of somatic embryogenic cultures for plantlet regeneration has been maintained for over 1 year in some lines. Three types of immature-embryo-derived cultures were characterized by histology. Some cultures were morphologically similar to immature-embryo-derived embryogenic cultures of other monocotyledonous species. Cultures such as these have proven to be useful target tissues in transformation studies. Received: 16 December 1997 / Revision received: 23 February 1998 / Accepted: 13 March 1998     

The somatic embryogenesis and establishment of transformation experiment system in Larix principis-Rupprechtii]

Larix principis-Rupprechtii is one of the superior afforestation forest trees growing in north China. Embryogenic cultures were initiated from immature zygotic embryos of Larix principis-Rupprechtii on S culture medium containing 2, 4-D 0-2.2 mg/L, KT and BA each at 0-0. 8 mg/L. Embryogenic calli were subcultured and multiplicated on S + B culture medium containing dropping off each hormone concentration. We set up 33 steady-going embryogenic cell lines; We studied on the growth stage and genotype differences of every embryogenic cell lines; and Finded more than 10 high-frequency somatic embryogenesis cell lines such as 2K, 2T, 2I, 2J, 3C etc.. The number of 2T somatic embryos reaches 314/per gram of embryogenic tissue and the number of 3C somatic embryos is 185/per gram of embryogenic tissue. The re-induction method of Larix principis-Rupprechtii from somatic embryos was used to produce renewable embryogenic cultures and steady-going embryogenic cell lines effectively. Mature somatic embryos can germinate and develop further into plantlets when they are isolated and cultured on a hormone-free WPM culture medium. The regeneration plantlets were obtained. Furthermore, the transformation with a truncated gene of Bacillus thuringensis (B. t) were carried out, the PCR showed positive results, because of this, embryogenic cell line of Larix principis-Rupprechtii can be used for transformation experiments to support further breeding in forestry.     

华北落叶松体细胞胚胎发生及遗传转化实验系统的建立(简报)

华北落叶松(Larix principis-Rupprechtii)是我国北方中高山地区重要的针叶速生用材树种,进行其体细胞胚胎发生和植株再生的研究,在针叶树无性快速繁殖及基因工程育种上有其特殊的用途,既可为针叶树无性系林业提供产业化途径,也可作为目的基因遗传转化实验系统。针叶树的基因转化相对较难,再生更属不易,Lelu等报道过杂种落叶松与欧洲落叶松体细胞胚胎发生方面的研究;而我国尚未见有落叶松体细胞胚胎发生的研究报道。我们     

Plant regeneration from embryogenic suspension cultures ofCamellia Japonica

Summary Two methods (I and II) for somatic embryo production from embryogenic suspension cultures ofCamellia japonica are presented. Method I, embryogenic suspension cultures, was established from suspension cultures initiated from leaf-derived callus. These cultures were maintained by reducing agitation and increasing subculture interval. Induction of somatic embryogenesis was achieved in MS28 medium, 6, 12, 24, and 36 mo. after culture establishment. Embryo production decreased after 1 yr of culture. Method II, suspensions of single embryogenic cells and proembryos, was obtained from leaves cultured in liquid MS13 medium 6 wk after culture initiation. Embryo production was 23 embryos/ml. Germination of cell suspension-derived embryos on MS56 medium was 16.7 % (&#x00B1;4.2%) for method I, and 35.4% (&#x00B1;5.1%) for method II. The embryos germinated into plantlets with 0 to 7 axillary shoots.     

Somatic embryogenesis and plant regeneration from protoplasts isolated from embryogenic cell suspensions of wheat (Triticum aestivum L.)

We describe the early formation of somatic embryos followed by plant regeneration from protoplasts isolated from an embryogenic wheat cell suspension, which was initiated from small granular (0.2 to 1 mm in size) embryogenic calli. These granular calli formed embryogenic cell suspensions within 20 days in liquid culture, and were selected gradually from young inflorescence-derived nodular embryogenic calli of the winter wheat cv. Kehong 1041. The division frequency of protoplasts was 11 to 16%, and the frequency of differentiation into plants was about 0.001% (number of plants formed divided by the total number of protoplasts plated). About 20% of somatic embryos present in the culture formed directly from protoplast-derived cells within 15 days of cultures.     

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 protoplasts of dessert banana cv. Grande Naine (Musa spp., Cavendish sub-group AAA) via somatic embryogenesis

Protoplast culture and plant regeneration of the dessert banana cultivar Grande Naine (Musa spp., Cavendish sub-group AAA) were achieved through somatic embryogenesis. Protoplasts were isolated from cell suspensions at a yield of 3᎒⁷ protoplasts/ml packed cell volume (0.5 g). For the induction of cell divisions, two banana cell suspensions, SF265 (AA) and IRFA903 (AA), were used as feeder layers. SF265 (AA) was found to be more efficient for inducing cell divisions than IRFA903 (AA). The first embryogenic cell suspensions were established from protoplast-derived microcalli. The transfer of microcalli and protoplast-derived cell suspensions onto regeneration medium containing plant growth regulators slightly increased the number of embryos relative to those maintained on a feeder layer with growth regulators. Plant regeneration was achieved in the same regeneration medium.     

Somatic embryogenesis from immature zygotic embryos of oil palm

Immature zygotic embryos at different developmental stages were used for callus induction and regeneration studies. Immature embryos excised from fruits 77, 91, 100, 114, 128, 140 and 193 days after pollination and mature embryos were cultured on modified Y3 medium containing 500 mgl^–1 cysteine, 0.5% (w/v) PVP-40, 500 M 2,4-d and 0.3% (w/v) charcoal. Compact embryogenic tissue began differentiating directly from embryo explants after 2 weeks of culture. The percentage of embryos forming compact embryogenic tissue ranged from 28.6% for 91-day-old embryos to 0% for 140-day-old and older embryos. Friable embryogenic tissue was observed in callus cultures derived from 100-day-old embryos. Although both compact and friable embryogenic tissues were successfully isolated, normal embryo and plantlet development was observed only from friable embryogenic tissue.Abbreviations ABA abscisic acid - 2,4-d 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - PVP polyvinylpyrollidone     

Embryogenic and non-embryogenic cell lines of Daucus carota cloned from meristematic cell clusters: relation with cell ploidy determined by flow cytometry

The presence of totipotent and non-totipotent cells in embryogenic carrot cell suspension cultures was examined by cloning of cell microclusters. Forty clones were isolated and the distribution of their embryogenic potential was studied. Nonembryogenic, weakly and highly embryogenic cell lines were selected. After one year of subculture a second cloning round showed that the highly embryogenic and the non-embryogenic cell lines were homogenous and stable. A measurement of ploidy levels of clones by flow cytometry showed that the embryogenic clones were all diploid whereas the non-embryogenic were diploid or tetraploid. Hence, for our strain, there was a strict relationship between the tetraploid state and the inability to produce somatic embryos.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - PCV packed cell volume - MS Murashige and Skoog medium - MES 2(N-morpholino) ethanesulfonic acid - a.u. arbitrary units     

Plant regeneration from embryogenic suspension cultures of Chinese yam (Dioscorea opposita thunb.)

A competent, embryogenic suspension culture of Chinese yam (Dioscorea opposita Thunb. cv. ‘Nagaimo’) has been obtained. Embryogenic callus was induced from stem segments cultured on an agar-solidified MS medium containing 2,4-dichlorophenoxyacetic acid (2,4-D). One month following placement of the embryogenic callus in a liquid medium containing 2,4-D, the embryogenic tissue began to proliferate rapidly. Established suspension cultures consisted almost entirely of early-stage pro-embryos with very little contamination from non-embryogenic tissues. Under optimum conditions, suspension culture packed cell volume increased 2.5-fold per week. Following transfer of the tissue to a hormone-free medium, the embryogenic tissue developed. Globular embryos were formed within 4 weeks and addition of benzyl adenine further enhanced development and germination. Plantlets were regenerated by culturing embryos on a hormone-free agar-solidified medium.     

Acquisition of embryogenic potential in carrot cell-suspension cultures

Embryogenic suspension cultures of domesticated carrot (Daucus carota L.) are characterized by the presence of proembryogenic masses (PEMs) from which somatic embryos develop under conditions of low cell density in the absence of phytohormones. A culture system, referred to as starting cultures, was developed that allowed analysis of the emergence of PEMs in newly initiated hypocotyl-derived suspension cultures. Embryogenic potential, reflected by the number of FEMs present, slowly increased in starting cultures over a period of six weeks. Addition of excreted, high-molecular-weight, heat-labile cell factors from an established embryogenic culture considerably accelerated the acquisition of embryogenic potential in starting cultures. Analysis of [³⁵S]methionine-labeled proteins excreted into the medium revealed distinct changes concomitant with the acquisition of embryogenic potential in these cultures. Analysis of the pattern of gene expression by in-vitro translation of total cellular mRNA from starting cultures with different embryogenic potential and subsequent separation of the [³⁵S]methionine-labeled products by two-dimensional polyacrylamide gel electrophoresis demonstrated a small number of abundant in-vitro-translation products to be present in somatic embryos and in embryogenic cells but absent in nonembryogenic cells. Several other in-vitro-translation products were present in explants, non-embryogenic and embryogenic cells but were absent in somatic embryos. Hybridization of an embryoregulated complementary-DNA sequence, Dc3, to RNA extracted from starting cultures showed that the corresponding gene is expressed in somatic embryos and PEMs but not in non-embryogenic cells.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - cDNA complementary DNA - PAGE polyacrylamide gel electrophoresis - PEM proembryogenic mass     

Differences in compounds released by embryogenic and non-embryogenic suspension cultures of Euphorbia pulcherrima

Media from embryogenic and non-embryogenic cell suspension cultures were analysed for protein content, electrophoretic protein patterns, glycoproteins and activity of peroxidases and β-glucosidases in order to characterize the physiological status of the cultures. On a dry mass basis the amount of extracellular proteins per cell was greater in embryogenic suspensions than in non-embryogenic suspensions. Non-embryogenic suspensions contained unidentified slimy compounds which were not present inembryogenic cultures. The extracellular Concanavalin A-specific glycoproteins gave different isoelectric focussing patterns and thus enabled embryogenic and non-embryogenic cultures to be differentiated. The extracellular peroxidase activity per cell dry mass was far greater in embryogenic than in non-embryogenic cultures. The isoenzymes differed in number and composition of the anionic bands. β-glucosidases were found in the same range of activity in both culture types, but the time course of enzyme activity during cultivation was significantly different. In the embryogenic culture the activity was correlated with dry mass increase, whereas in the non-embryogenic suspension the activity reached maximum during the linear growth phase. Polyphenoloxidase which was recently recognized as an intracellular marker for embryogenic stages was not released into culture media. This revised version was published online in July 2006 with corrections to the Cover Date.     

Patterns of somatic embryo formation in Siberian larch: embryological aspects

Somatic embryogenesis was induced in Siberian larch by in vitro culturing zygotic embryos at different developmental stages. Cultures were grown in modified Murashige and Skoog medium supplemented with hormones 2,4-dichlorophenoxyacetic acid (2 mg/l) and 6-benzylaminopurine (0.5-1 mg/l). The success of somatic embryogenesis in this species depended on the tree genotype and developmental stage of embryos used for culturing. Somatic embryogenesis from immature zygotic embryos at the stage of cotyledon initiation was most active. After 5-10 days, such embryos formed the embryogenic tissue including two cell types--elongated highly vacuolated embryonic tubes and small embryonic cells. Somatic embryos were isolated from proliferating embryogenic tissues after 2 months of culture.     

Development and characterization of embryogenic suspension cultures of pecan

A method for the establishment and proliferation of developmentally stable, embryogenic suspension cultures in pecan is described, and the growth and development of cultures characterized. Suspension cultures were generated from somatic embryos derived from zygotic embryo cotyledon explants induced on a solidified medium with naphthaleneacetic acid. Cultures were repetitively embryogenic and proliferated in growth-regulator-free medium. The suspensions consisted of a mixture of globular stage embryo-aggregates, freely suspended globular embryos and pre-globular stage embryo masses. Culture growth and proembryo production were evaluated with respect to several liquid media and pH conditions. Significant differences in growth and productivity were observed between cultures. Pre-globular stage embryo masses collected on filter paper and overlaid on solidified medium continued ontological development and converted into plants. Thus a method has been developed for pecan suspension culture, which presents a major improvement in embryogenic tissue culture within the Juglandaceae. This revised version was published online in June 2006 with corrections to the Cover Date.     

Plant regeneration from protoplasts of embryogenic suspension cultures of orchardgrass (Dactylis glomerata L.)

An embryogenic suspension culture of orchardgrass (Dactylis glomerata L.) consisting of small, embryogenic cell clusters was obtained from callus formed on basal sections of young leaves through a process of selective enrichment. These suspensions were used as a source of protoplasts. The isolated protoplasts divided at a frequency of 0.5–10% when plated in an agarose solidified culture medium. Conditioned medium, in which embryogenic Dactylis suspension cultures had been grown, was found to increase the rate of cell colony formation. Protoplast-derived colonies grew rapidly in a bead-type culture system of floating agarose slabs in liquid medium. New suspension cultures formed as the colonies grew out of the agarose. These cultures were embryogenic and formed green plantlets when plated on a solid medium lacking auxin. The plantlets were established in soil and grown to mature plants.Abbreviations B5 medium according to Gamborg et al. (1968) - SH-x medium according to Schenk and Hildebrandt (1972) supplemented with x M dicamba - dicamba 3,6-dichloro-o-anisic acid - KM-8p medium 8p of Kao and Michayluk (1975)     

Long-term suspension cultures of cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.) with high embryogenic potential

Cucumber (Cucumis sativus L.) cytokinin-independent embryogenic cell suspension cultures were derived and maintained for more than 3.5 years without losing the embryogenic potential. The preparation and the characteristics of the cucumber embryogenic cell suspension possess many similarities to that of carrot. The cultures were induced from hypocotyl explants of in vitro grown cucumber plants in liquid MS media containing 2,4-dichlorophenoxyacetic acid as the sole growth regulator during 6 weeks and they contained a heterogeneous array of several different types of single cells and cell clusters (PEMs). The established cell suspensions were subcultured in 1-week interval, while the inoculation density was optimized to 2.0 × 10⁵ cells ml⁻¹ using cell viability as a marker. Somatic embryos were obtained after the transfer of the proembryogenic masses to a hormone-free semisolid MS medium with a frequency of 388 ± 57 somatic embryos per 1 ml of packed cell volume of the established cucumber embryogenic culture within 7 days. The frequency of normal somatic embryos with two cotyledons was found to be 78%. Such embryos possessed the potential of spontaneous maturation and the embryo conversion rates were 87%. The yield of normally growing plants was much higher compared with that previously described for cucumber systems. Somatic embryo-derived plants were successfully transferred to the greenhouse where they flowered and fruited.