Formation of callus and somatic embryos from protoplasts of a commercial hybrid of maize (Zea mays L.)

Summary Protoplasts isolated from a totipotent embryogenic cell suspension culture of Zea mays L. (cultivar Dekalb XL82) underwent sustained cell divisions when cultured in liquid as well as agarose media. Optimal colony formation (5%) occurred in a liquid medium containing 0.5 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D). A soft and unorganized callus was formed when the protocolonies were transferred to agar solidified suspension maintenance medium. Compact, organized and yellow to pale green folded structures and somatic embryos were formed upon subsequent transfer of this callus to a low 2,4-D medium. Clusters of somatic embryos germinated precociously but no plants were recovered.     

Efficient microspore embryogenesis in wheat (Triticum aestivum L.) induced by starvation at high temperature

We have established an efficient method to induce embryo formation from isolated wheat (Triticum aestivum L.) microspores. Culture of excised anthers under starvation and heat shock conditions induced the formation of embryogenic microspores at high frequency in nine Austrian winter wheat genotypes, including cultivars that had been considered as recalcitrant in anther culture. Percoll gradient centrifugation of the mechanically isolated microspores allowed us to obtain homogeneous populations of embryogenic microspores in all genotypes which, after transfer to a rich medium containing immature ovaries for conditioning, divided and produced globular embryos. Thousands of embryos were produced in one petri dish. Many of these embryos developed into plantlets after transfer to a solid medium without ovaries.     

Characterization and manipulation of embryogenic response from in-vitro-cultured immature inflorescences of rice (Oryza sativa L.)

Characterization and optimization of the embryogenic response from in-vitro-cultured immature inflorescences of rice (Oryza sativa L. sub-species indica and japonica) are described. Histological and morphological analyses revealed that the parenchymatous ground tissue present in the region between the second whorl of sterile bracts and the base of the fertile bracts, the embryogenically competent region (ECR), was involved in the embryogenic response. Initial cell divisions within the ECR occurred in the vicinity of the pro-vascular regions of the spikelet. Continued cell divisions resulted in groups of proliferating units and each single proliferating unit was the product of a coordinated behavior of neighboring cells functioning as a morphogenic group. Further proliferation of this embryogenic tissue was due to the development of cambium-like tissue(s) often forming an embryogenic stratum which under optimal culture conditions produced plants at a high frequency. The morphogenic pathways governing plant regeneration from spikelets of the immature rice inflorescence were dependent upon the growth-regulator composition of the culture medium. Three different modes of plant regeneration were observed: (i) direct plant regeneration, (ii) plant regeneration with an intervening callus phase (prolific non-embryogenic growth associated with unorganized, loose and mucilaginous tissue), and (iii) plant regeneration without an intervening callus phase (compact embryogenie tissue with highly organized growth). The efficiency of plant regeneration, via somatic embryogenesis without an intervening callus phase, was increased by optimizing the culture conditions. In a two-step procedure, immature inflorescences of rice were first cultured on a conditioning medium supplemented with 2.0 mg · 1^–1 2,4-dichlorophenoxyacetic acid + 1.5 mg · 1^–1 kinetin + 0.75 mg · 1^–1 -naphthaleneacetic acid for a period of two weeks. The conditioning medium, with the appropriate culture conditions, allowed redirection of partially differentiated cells of the ECR into embryogenically competent pro-embryogenic groups. Maturation of these pro-embryogenic groups was achieved by transferring them to an embryo proliferation medium, and plants could then be regenerated at a high frequency upon their transfer to the regeneration medium.Abbreviations CM conditioning medium - 2,4-D 2,4-dichlorophenoxyacetic acid - ECR embryogenically competent region - NAA -naphthaleneacetic acid - SEM scanning electron micrograph Thanks are extended to Stephanie Lara, Barbara Bricks, Kay Robbinson-Beers, James Haudenshield, Dave Bayer and Gene Nester, for their invaluable help during the course of this research. The research was partly supported by the Rockefeller Foundation through the Rice Biotechnology program as a grant to W.J.L. and a Postdoctoral Fellowship to J.R.R.     

福录考体细胞胚胎发生的观察

福录考（ＰｈｌｏｘｄｒｕｍｍｏｎｄｉｉＨｏｏｋ）体细胞胚胎发生的方式有两种：一是由叶外植体具有一定功能的特化细胞脱化为胚性细胞,如叶表皮细胞,维管束鞘及韬皮薄壁细胞均可发生脱分化进行分裂,形成胚性细胞或分细胞团;二是叶外植体先脱分化形成愈伤组织。     

Fertile wheat (Triticum aestivum L.) regenerants from protoplasts of embryogenic suspension culture

We report regeneration of fertile, green plants from wheat (Triticum aestivum L. cv. Aura) protoplasts isolated from an embryogenic suspension initiated from somatic early-embryogenic callus. The present approach combines the optimization of protoplast culture conditions with screening for responsive genotypes. In addition to the dominant effect of the culture media, the increase in fresh mass and the embryogenic potential of somatic callus cultures varied considerably between the various genotypes tested. Establishment of suspension cultures with the required characters for protoplast isolation was improved by reduction of the ratio between cells and medium and by less frequent (monthly) transfer into fresh medium. A new washing solution was introduced to avoid the aggregation of protoplasts. However, the influence of the culture medium on cell division was variable in the different genotypes. We could identify cultures from cultivar Aura that showed approximately a 9% cell division frequency and morphogenic response. The protoplast-derived microcolonies formed both early and late-embryogenic callus on regeneration medium and green fertile plants were obtained through somatic embryogenesis. The reproducibility of plant regeneration from protoplast culture based on the cultivar Aura was demonstrated by several independent experiments. The maintenance of regeneration potential in Aura suspension cultures required establishment of new cultures within a 9-month period.     

牛体细胞核移植技术研究进展

对近年来牛体细胞核移植技术研究的进展作一综述。其中包括:供体细胞种类、传代次数和所处细胞周期的选择;对供体细胞的特殊处理;卵母细胞的采集;传统去核方法的优化、去透明带核移植技术的建立与发展;胚胎重构、激活和体外培养条件的比较与改进等内容。     

The Agrobacterium rhizogenes rolC-gene-induced somatic embryogenesis and shoot organogenesis in Panax ginseng transformed calluses

Expression of the Agrobacterium rhizogenes rolC gene in Panax ginseng callus cells results in formation of tumors that are capable to form roots. The selection of non-root forming tumor clusters yielded the embryogenic 2c3 callus line, which formed somatic embryos and shoots independently of external growth factors. Although the 2c3 somatic embryos developed through a typical embryogenesis process, they terminated prematurely and repeatedly formed adventitious shoot meristems and embryo-like structures. A part of the shoots and somatic embryos formed enlarged and fasciated meristems. This is the first indication of the rolC gene embryogenic effect and, to our knowledge, the first indication that a single gene of non-plant origin can induce somatic embryogenesis in plants.     

植物游离小孢子胚胎形成机理

植物小孢子胚胎的形成是基于具有单套染色体的小孢子在外界胁迫条件下通过脱分化和再生而形成一个完整植株的过程,是产生双单倍体的常用方式.它体现了植物细胞的全能性,为植物组织培养和胚胎发育的基础研究提供了一个独特的模式.从细胞学水平、代谢水平和分子水平3个方面综述在植物游离小孢子胚胎形成机制方面已取得的研究进展,并提出目前尚待解决的问题,以及对未来的展望,为进一步研究小孢子胚胎发生机制提供理论依据和奠定技术基础.     

百合体细胞胚胎发生和植株再生

以切花百合(Lilium)品种‘黄天霸’(‘Manissa’)花器官为外植体诱导体细胞胚胎发生与植株再生。结果表明,不同花器官、不同激素配比对愈伤组织形成均具有显著影响。花丝为最佳外植体,激素对愈伤组织诱导的影响效应为NAA>6-BA>2,4-D,最适培养基为MS+1.0 mg.L-1NAA+0.2 mg.L-16-BA;激素诱导体细胞胚胎发生的影响效应为2,4-D>KT>6-BA,最佳培养基配方为MS+1.0 mg.L-12,4-D+0.2 mg.L-1KT+1.0 mg.L-16-BA;MS培养基添加IBA可促进体细胞胚萌发成苗,体细胞胚芽成苗的最佳培养基为MS+0.2 mg.L-16-BA+1.0 mg.L-1IBA。