青扦胚性细胞悬浮培养中影响体细胞胚发生因素的研究

试验以青扦（Ｐｉｃｅａｗｉｌｓｏｎｉ）的胚性愈伤组织为材料,以改良５９基本成分附加２４－Ｄ１ｍｇ／Ｌ及ＫＴ１ｍｇ／Ｌ为培养介质,比较了液体悬浮与半固体二种培养方式对胚性愈伤组织增殖和体细胞发生的影响,研究了液体悬浮培养过程中影响体细胞胚发生的因素。结果表明：液体悬浮培养好于半固体培养,它的胚性愈伤组织的生长率为２６８％,是半固体培养的１２４倍;体细胞胚的分化率为９３％,是半固体培养的２２倍;悬浮培养较佳的培养条件为：初始细胞密度为２％（鲜重）,蔗糖浓度为２０ｇ／Ｌ,摇床转速为１００ｒ／ｍｉｎ,ｐＨ为５８。经过两个月悬浮培养,将培养物转至１／２改良５９附加ＡＢＡ１ｍｇ／Ｌ的分化培养基上,３个月后每ｇ培养物上可获得２８５个正常的子叶期体细胞胚。     

Somatic Embryogenesis in Cell Suspension Culture of Saposhnikovia divaricata

Calli of Saposhnikovia divaricata (Turcz.) Schischk. were induced from the roots of test tube seedlings cultured on MS medium containing 0.5 mg/L 2,4-D. Cell suspension was established by shaking the caul in liquid medium of the same components supplemented with 10% coconut milk. After the formation of embryogenic clusters, 2,4-D was omitted promoting the transformation of the embryogenic clusters to somatic embryos. Micro and submicroscopic: structural changes during the single cell to globular embryonic stage were observed. It was noticed that cortical endoplasmic reticulum appeared in the cells at the stage of embryogenic clump formation but was absent in other stages. Perhaps this was related to the metabolic specification leading to embryo formation. Spherosomes were observed of embryogenesis but remarkably increased in number at proglobular embryo stage. Meanwhile, the central electric dense matrix became progressively smaller and paler, while the outer part became enlarged and more transparent. This implied that the spherosomes took part in the storage of proteins or lipidproteins in the early stages of embryogenesis and transformation to lipid dror)s when the proteins were exhausted in the development of embryo, vacuolar protein bodies could be risualized in many cells in the proglobular embryo stage. This together with the existance and changes of spherosomes was similar to that observed in Peucedanum terebmthaceum. Further studies are meritted to approach, whether these are general phenomena in Umbelliferae. This work also revealed that the aggregation of single cells in suspension culture stimulated the initiation of embryogenesis.     

海岛棉悬浮培养体细胞胚胎发生

取海岛棉无菌苗下胚轴作外植体,接种在含有激素２,４－Ｄ０．１ｍｇ／Ｌ和ＫＴ０．５ｍｇ／Ｌ的改良ＭＳ培养基上。经近２个月的诱导培养,获得淡黄色或淡灰色、松软、湿润、颗粒状的愈伤组织。将此类愈伤组织接入液体培养基进行悬浮培养,３～４个月后产生体细胞胚,获得各个时期的体细胞胚胎。胚性细胞悬浮系经适当的继代培养可长期保持成胚性。     

悬浮培养中旱芹体细胞胚发生的工艺条件研究

以旱芹 (Apium graveolens L.)下胚轴切段 (5～ 1 0 mm )为外植体 ,在 MS 1 .0 mg/L 2 ,4- D培养基中诱导愈伤组织 ,经几次固体—液体交替培养筛选后 ,将此胚性愈伤组织接种于 MS液体分化培养基(MS 0 .5mg/L KT 50 0 mg/L CH 50 0 mg/L Prolin)中 ,就摇床转速、接种量、初始 p H的调控等工艺条件对体细胞胚诱导的最佳条件进行了研究。结果表明 ,较佳的培养条件为 :摇床转速 1 0 0～ 1 50r/min;初始细胞密度和 p H分别为 2 .0 % (鲜重 )及 5.5左右。最后获得正常子叶期体细胞胚数为 1 30个 /m L。     

Somatic Embryogenesis and Plantlet Formation of Picea wilsonii Mast. in Ditterent Conditions

Embryogenic callus (translucent callus) was produced from immature zygotic embryos of Picea wilsonii Mast. Subsequently somatic embryogenesis occurred on the brown callus. The somatic embryos could be stimulated to developinto plantlets on the medium without hormone. Young somatic embryos were produced from embryogenic callus in liquid suspension culture, in which suspensor was several or more than ten times the size of the somatic embryo. The somatic embryo showed very similar to zygotic embryos in micro-section and living material.     

2，4_D对甘薯体细胞胚胎发生的调控

将来源于‘徐薯１８’叶片的胚性愈伤组织,接种在含有不同２,４Ｄ浓度的液体ＭＳ培养基中进行悬浮培养,悬浮细胞表现出不同的形态结构、分裂方式和发育途径：２,４Ｄ浓度为１ｍｇ／Ｌ时,细胞均等分裂,增殖迅速;不含２,４Ｄ时,细胞多进行不均等分裂,并发育成体细胞胚。不同２,４Ｄ浓度中培养的悬浮细胞,其胞外过氧化物同工酶谱及其随时间变化的方式有很大差异,并与细胞的生长、发育过程密切相关     

Ultrastructural Studies of Somatic Embryogenesis in Wheat

Electron microscopic observation of somatic embryogenesis from cultured immature wheat embryos revealed the presence of a lot of small vacuoles, a large nucleus, clear nucleolus and polynucleoli. The electron density of cytoplasm was strengthened during somatic embryogenesis. Quantity and type of organelles—plastid, ribosome and mitochondrion were increased; thickened cell wall, disappeared plasmodesmata, increased organelles andstarch accumulation in the embryogenic cells. Nucleolus vacuoles, autophagic vacuoles and secretory vesicles were present in the embryogenic cells with thickened cell walls. The multicellular proembryos, globular embryoid and pear-shaped embryoid were surrounded by an envelop, but plasmodesmata existed extensively between cells of somatic embryoid. The membranous structures appeared in the plastid which underwent transformation into chloroplast in the cells of growing point in almost mature embryoid. The relation of the above-mentioned structureal changes to somatic embryogenesis is also discussed.     

Ultrastructure of the Cell of Peucedanum terebinthaceum in Different Stages of Embryogenesis in Suspension Cell Culture

The ultrastructure of the cells from single cell to globular embryo stage in suspension culture were investigated. The amount and morphology of the organelles, especially the plastids spherosomes and protein bodies changed obviously during the embryogenesis. In embryogenetic clump stage there occured in the vacuolated cells direct nucleus division and peripheral cytoplasmic swelling. The vaeuolated cells returned to embryonic state quickly in this way. In this paper the physiological and biological significance of the ultrastructural changes of cells in different stages of somatic embryogenesis are discussed.     

Studies on Cell Suspension Culture of Panax notoginseng

The optimum period of harvesting in cell suspension culture of Panax notoginseng was 30 days. The time course of sap.nih formation proceeded almost in parallel with the cell growth. An appropriate concentration of oligosaccharms from Panax ginseng, precursor fames.l, mannffol and lysozymum which were added into tbe culture broth 10 days before harvesting, all induced saponin biosynthesis significantly. Oligosaecharins at a concentration of 15ppm(it increased 1 fold of saponin yield, and increased 22.7%(of cell growth rate compared with those of the control) and farnesol at 200ppm(it increased 70.5% of sap.nih yield and stimulated cell growth compared with those of the control) were more effective.     

白蜡树属植物体细胞胚胎发生进展

综述了近年来国内外对白蜡树属植物体细胞胚胎发生的研究概况,对影响体细胞胚胎发生的主要可控因素以及体细胞胚胎发生过程、机理、组织学方面作了介绍,并提出了白蜡树属体细胞胚胎发生研究的应用前景。     

松柏类植物体细胞胚胎发生的研究进展

松柏类植物的体细胞胚胎发生既是繁育的一种手段,又是研究胚胎发育过程中结构、生理和分子事件的一种重要的模式系统.整个体细胞胚胎发生过程主要包括3个步骤:胚性组织的诱导和增殖、体细胞胚的成熟以及体细胞胚的萌发和转换.过去为了提高胚胎发育过程所做的努力主要都集中在胚的成熟阶段,这是因为一直认为能否成功再生的关键在于胚发育成熟阶段的处理.然而,在过去几年里,结合生理生化以及分子生物学的研究发现,胚胎发生的早期阶段对于完成整个发育过程也是至关重要的,早期阶段培养条件的优化可以显著提高培养过程中体细胞胚的数量和质量.此外,萌发过程培养条件的调节对于提高成熟体细胞胚的萌发率和转换率也很重要.因此,这些新的研究成果对于改善松柏类植物体细胞胚胎发生中的胚的诱导率和转换率低的现象具有重要的意义.     

High Frequency Induction of Somatic Embryoid of Rice in Suspension Culture

On MS and B5 basic media the mature seed of rice (Oryza sativa L.) was induced to produce somatic embryoid. The results showed that the rate of occurrence of somatic embryoid varied with the variety of rice and the concentration of 2,4-D was important to the occurrence of somatic embryoid. For those varieties of rice suitable for induction, the induction factors such as KT, zeatin, BAP, ABA, osmotic pressure and so forth were stringently required at different stages of somatic embryoid development, and they also influenced the formation of somatic embryoid. The results of various layered cultures of embryonic calli proved that the somatic embryoid was derived from the surface layer callus containing embryonic cells. At the induction stage no somatic embryoid was formed in callus. The rate of somatic embryoid formation was over seventy per cent on liquid differentiation medium I.     

Direct Differentiation of Plants from Small Cell Clusters of Indica Rice in Suspension Culture

The major factors affecting plant regeneration in suspension culture were investigated.The resultsshow that,in order for cell clusters to differentiate directly in liquid medium,it is essential toestablish an embryogenic callus line with high potential for plant regeneration.Embryogenic calliwere suspended in AA basic medium supplemented with 2,4-D 1 mg/L,6-benzylaminopurine 0.5mg/L,CH 300 mg/L,sucrose 3％ and mannitol 3％ and subcultured 7 days for each passage.Aftermore than 6 months of culturing,a fine suspension culture of small cell clusters(SCC)wasestablished.The SCC,80-270μin diameter,were then transferred to a liquid medium(MSsupplemented with NAA 0.01 mg/L and 4-pyridylurae(4-PU)0.5 mg/L)and allowed to grow instationary culture.Finally direct differentiation from SCC was observed.Several factors in suspension-subculture exerted strong after-effects on differentiation ofSCC.Basicmedia,kinds and concentration combinations of auxin and cytokinin in the subculture media,duration of shaking at each subculture passage,and amount of packed cell volume transferred to newmedium at each subculture passage and so on,all these affected the frequency of differentiation ofSCC.Higher concentrations of NAA and kinetin in the differentiation medium inhibited the directdifferentiation of SCC.Low concentrations(0.01-0.1 mg/L)of NAA with 4-PU in thedifferentiation medium were helpful for the direct differentiation of SCC.The differentiated clusterspossessed typical embryogenic structure from which normal plantlets could develop after transferringto agar medium.     

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.     

油茶优良无性系子叶体细胞胚植株再生

以油茶优良无性系‘湘林4号’子叶为外植体,采用附加不同种类激素的MS培养基对其进行组织培养实验。研究结果表明:子叶形成胚性愈伤组织的最适合培养基为MS+2.0mg.L-12,4-D+1.0mg.L-1KT;经胚状体诱导产生不定芽分化的最适合培养基为MS+2.5mg.L-16-BA+1.5mg.L-1IAA;油茶优良无性系的生根培养基以MS+7.0mg.L-1NAA最适;通过对植株再生过程中各阶段的组培材料进行RAPD鉴定分析表明,DNA水平上未发现变异,说明通过组织培养建立的油茶优良无性系再生植株同原无性系无明显差别,最终获得的组培苗木能够保持原无性系的优良特性,其遗传是稳定的。     

Studies on Cell Suspension Culture and Fermentation Culture in Arnebia euchroma

This paper reports some characteristics of cell suspension and fermentation culture in Arnebia euchroma (Royle) Johnst. The yield of suspension culture reached 22.0g dry wt/L per month when inoculum quantity was 2.50 g dry wt/L. Time-course study showed that cell growith lagged in 0–3 days and enhanced greatly in 3–12 days, and almost ceased after 12 days of culture, pH value changed during the culture period and peaked on the 12th day after inoculation. When cells were cultured in liquid production medium, the contents of shikonin derivatives increased quickly and reached to the maximum about the 25th day. The cell yield of 9.47 and 9.34 g dry wt/L per month was obtained in fermentation culture. Timecourse of cell growth in fermentation culture was similar to that in suspension culture. The total content of shikonin derivatives in fermentation culture was 14.26% dry weight from 10 L bioreactor. The yield of shikonin derivatives was 1.93 g/L.     

Somatic Embryogenesis and Plant Regeneration from Papaya Suspension Cultures

Compact embryogenetic calli were obtained from explants on P3 medium after 4 weeks of culture and high-frequency somatic embryogenesis occurred after these calli were transferred into suspension culture. Experimental data showed that low level (0.2%W/V) of activated charcoal had beneficial effects on somatic embryogenesis. Abundant calli on P4 medium however, showed no embryogenesis. On the other hand, callus induction and somatic embryogenesis varied with different rarities of exptants. The efficiency of somatic embryogenesis was much higher, if roots were used as explants, whereas stems were more suitable for callus formation Mature somatic embryos with cotyledons were cultured on MS medium containing different plant hormones. The optimum medium for germination and growth of entire plantlet was Mso medium. The somatic embryos on MS2, MS and MS3 media germinated rapidly, but formed excessive callus from the surface of germinating embryos.     

木本植物体细胞胚胎发生技术

体细胞胚胎发生技术是植物规模化、产业化快速繁殖和基因转化再生植株的重要手段。对近年来进行体细胞胚胎诱导并再生植株的木本双子叶植物、单子叶植物及裸子植物等树种进行了综述 ,并探讨体细胞胚胎发生中的技术影响因素及其基因表达与调控等研究进展 ,最后提出今后应该加强研究的关键问题。     

大蒜体细胞胚胎发生研究进展

大蒜生产主要靠无性繁殖 ,因此 ,进行大蒜体细胞胚发育研究具有重要意义。本文对大蒜体细胞胚发育的影响因子进行了综述 ,其中较高浓度的维生素B1 及还原态氮源可能有利于胚胎发生 ,而大蒜体细胞内含物则不利于胚胎发生。此外 ,对大蒜体细胞胚培养中存在的主要问题进行了讨论 ,并认为系统开展体细胞胚发生的细胞分子生物学机理研究、建立悬浮培养体系以及进行大蒜体细胞胚无性系变异的研究等 ,具有广阔的前景。