柑橘愈伤组织倍性变化及其与体细胞胚胎发生能力的关系

柑橘愈伤组织是细胞融合、遗传转化等生物技术研究的良好材料。本实验室通过近20年的工作,先后诱导和保存了近80份不同种类和品种的柑橘愈伤组织。这些愈伤组织在MT基本培养基上继代保存。在长期保存过程中,部分材料仍然保持较好的胚胎发生能力。为研究愈伤组织在继代过程中的染色体变异趋势以及这种变异对胚胎发生能力的影响,本文选择有代表性的35份材料作为试材,在4年中连续3次使用流式细胞仪,对细胞DNA含量进行测定。结果发现,待测的35种愈伤组织均存在部分细胞DNA含量加倍的现象,而且,随着时间的推移,除佩奇橘柚、沙漠蒂甜橙、鲁斯脐橙、印度酸橘等4种愈伤组织的DNA含量加倍的细胞有减少的趋势外,其他愈伤组织的DNA含量加倍的细胞均有增加的趋势。数据分析表明,在待测的35种愈伤组织中,有71．4％的基因型的愈伤组织细胞出现了多倍体细胞增加的趋势。对这35种柑橘愈伤组织进行体细胞胚诱导,发现只有9种愈伤组织具有不同程度的胚胎发生能力,其他26种愈伤组织不能再生。相关性分析表明,柑橘愈伤组织细胞DNA含量增加的细胞比例与体细胞胚胎发生能力之间的相关性较小,相关系数为忙-0．10（P〈0．01）,未达到显著水平。另外,柑橘胚性愈伤组织继代时间与体细胞胚胎发生能力之间的相关性也不显著。     

高粱不同类型胚性和非胚性愈伤组织的生理生化差异

植物体细胞胚胎发生过程中伴随着复杂的生理生化变化,为进一步揭示胚性愈伤组织的再生潜力,该研究以高粱Sb19未成熟胚诱导产生的两种胚性愈伤组织和一种非胚性愈伤组织为材料,通过测定各愈伤组织中可溶性蛋白、游离脯氨酸和可溶性糖的含量,采用方差分析法对高粱体细胞胚胎发生过程中不同类型愈伤组织的生理生化指标进行了差异比较研究。结果表明:(1)高粱两种胚性愈伤组织中可溶性蛋白、游离脯氨酸和可溶性糖的含量均显著高于非胚性愈伤组织,表明胚性愈伤组织中的代谢活性高于非胚性愈伤组织,能够为体细胞胚胎发生提供更多的物质能量基础。(2)两种类型胚性愈伤组织之间生理生化差异显著,其中,Ⅱ型胚性愈伤组织中可溶性蛋白和游离脯氨酸含量均显著高于Ⅰ型胚性愈伤组织,相反,Ⅱ型胚性愈伤组织中可溶性糖含量显著低于Ⅰ型胚性愈伤组织,这种生理生化差异在一定程度上影响了后期的分化。该研究结果为愈伤组织的胚胎发生能力与生化代谢的关系提供理论依据。     

逆境处理和DNA甲基化影响柑橘体细胞胚发生

对 1 5种柑橘胚性愈伤组织进行体细胞胚诱导 ,发现逆境处理有利于体细胞胚发生 ,并可以恢复部分品种的体细胞胚发生能力。对具有和失去体细胞胚发生能力的两种纽荷尔脐橙 (CitrussinensisOsb .)愈伤组织进行随机扩增多态性DNA (RAPD)分析没有检测到带型的差异 ,而对它们的甲基化敏感扩增多态性 (MSAP)进行分析则发现两种愈伤组织间具有明显的DNA甲基化差异 ,具体细胞胚发生能力的愈伤组织的甲基化水平较失去体细胞胚发生能力的低     

逆境处理和DNA甲基化影响柑橘体细胞胚发生

对15种柑橘胚性愈伤组织进行体细胞胚诱导,发现逆境处理有利于体细胞胚发生,并可以恢复部分品种的体细胞胚发生能力.对具有和失去体细胞胚发生能力的两种纽荷尔脐橙( Citrus sinensis Osb.)愈伤组织进行随机扩增多态性DNA (RAPD) 分析没有检测到带型的差异,而对它们的甲基化敏感扩增多态性 (MSAP) 进行分析则发现两种愈伤组织间具有明显的DNA甲基化差异,具体细胞胚发生能力的愈伤组织的甲基化水平较失去体细胞胚发生能力的低.     

高粱体胚发生的组织细胞学观察

以Sb33高粱非胚性、胚性愈伤组织和体胚为材料,用传统石蜡切片法对各组织材料进行组织化学染色,对高粱胚性与非胚性愈伤组织以及体胚进行组织细胞学观察。结果表明：高粱非胚性愈伤组织无淀粉粒积累,高粱胚性愈伤组织淀粉粒积累较多,而与胚性愈伤组织相比,高粱体胚淀粉粒积累更多,这说明淀粉粒的积累与高粱体细胞的胚胎发生密切相关。此外,高粱可通过鱼雷胚基部产生球形胚的方式实现体胚的增殖,高粱离体再生途径以体细胞胚发生为主,并同时存在少量器官发生途径。在高粱体细胞胚胎发生中,外起源和内起源同时存在。本研究为高粱体细胞胚胎发生提供细胞学理论基础。     

棉花体细胞胚胎发生的研究进展

经过30多年的发展,已经在多个不同的棉花品种中获得了体细胞胚,并再生成苗。但由于体细胞胚胎发生往往受多种因素影响,如何从愈伤组织高效率地转化为胚性愈伤组织依然是限制棉花组织培养与遗传转化的关键问题之一。本文概述了棉花体细胞胚胎发生的研究进展,分别从棉花体细胞胚胎发生的起源、影响棉花体细胞胚胎发生的内外因素以及寻找棉花体细胞胚胎发生特异表达基因等几方面进行综述,并对研究中存在的问题进行了讨论。     

棉花体细胞胚胎发生的研究进展

经过30多年的发展, 已经在多个不同的棉花品种中获得了体细胞胚, 并再生成苗。但由于体细胞胚胎发生往往受多种因素影响, 如何从愈伤组织高效率地转化为胚性愈伤组织依然是限制棉花组织培养与遗传转化的关键问题之一。本文概述了棉花体细胞胚胎发生的研究进展, 分别从棉花体细胞胚胎发生的起源、影响棉花体细胞胚胎发生的内外因素以及寻找棉花体细胞胚胎发生特异表达基因等几方面进行综述, 并对研究中存在的问题进行了讨论。     

影响水稻幼穗培养体细胞胚胎发生因素的研究

用水稻幼穗为外植体进行组织培养,研究了影响其体细胞胚胎发生的有关因素,建立了高频率体细胞胚胎发生的培养程序。结果表明不同基因型之间体细胞胚胎发生率的差异达到61．2％;生长素2,4－D对诱导水稻体细胞胚起重要的调控作用,细胞分裂素BA有一定的协同促进作用。干燥处理可提高愈伤组织体细胞胚胎发生率,愈伤组织含水量在60％～80％范围的培养效果较好;外源DNA溶液浸泡发育早期的心状体细胞胚．其进一步发育时异常胚的频率显著增加．成苗率降低。     

香雪兰的直接与间接体细胞胚胎发生

香雪兰的体细胞胚胎发生可通过两种途径进行,即直接发生与间接发生。在直接发生方式中,体细胞胚直接来源于尚未完全分化的外植体表皮细胞;体细胞胚与母体组织以一种类似胚柄的结构相联系。间接发生方式中,体细胞胚的形成要经过一个愈伤组织阶段。以是否能形成体细胞胚分类,可将愈伤组织分为胚性和非胚性愈伤组织。以间接方式形成的体细胞胚是由胚性愈伤组织中的一种决定细胞发育来的。这种体细胞胚不具有类似胚柄的结构,而与母体组织共同形成一个复合体。体细胞胚具有自己独立的维管束系统,在脱离母体组织后能够独立发育成株。     

棉花高频体细胞胚胎发生及再生体系建立

棉花的体细胞胚胎发生率低,限制了转基因棉花的发展。为了扩大可再生棉花的基因型,发展新疆优质棉花的特点,以新疆的主要栽培品种新陆早33为材料,以下胚轴为外植体,使用葡萄糖,麦芽糖作为主要碳源,用Phytagel固化,对不同激素的浓度组合和培养条件进行探索,对棉花胚性愈伤的诱导到体细胞胚胎的发生阶段进行优化,体细胞胚胎成熟以后进行萌发培养可以获得正常的植株。通过试验证明,有利于棉花的愈伤组织生长的激素浓度为KT 0.1 mg/L,2,4-D 0.1 mg/L,下胚轴的中部诱导愈伤形态最好。体细胞胚胎发生阶段以KT,IBA组合,以低盐培养基进行子叶胚的成苗,建立了再生体系。     

GFP expression as an indicator of somatic hybrids between transgenic Satsuma mandarin and calamondin at embryoid stage

Somatic hybridization was performed via electrofusion between embryogenic suspension-derived protoplasts of transgenic green fluorescent protein (GFP) Satsuma mandarin (Citrus unshiu Marc. cv. Guoqing No. 1) (G1) callus and mesophyll protoplasts of calamondin (Citrus microcarpa Bunge), and three embryoids expressing GFP under UV light were obtained after 60 days of culture. The three embryoids were considered not as diploid cybrids but true allotetraploid somatic hybrids, as it was based on: (1) citrus heterokaryons are generally more vigorous and have higher capacity for embryogenesis as compared with unfused and homo-fused embryogenic callus protoplasts; (2) the callus line of G1 Satsuma mandarin has lost the embryogenesis capacity; and (3) citrus diploid cybrids produced by symmetric fusion always possess nuclear genome of mesophyll parent, and calamondin without GFP gene was used as leaf parent in this study. Subsequent flow cytometry, simple sequence repeat and cleaved amplified polymorphic sequence analysis of one regenerated callus mass and three resulting plants validated this supposition, i.e., the callus was derived from transgenic G1 callus protoplasts, and the three plants were true allotetraploid somatic hybrids possessing nuclear genomic DNA of both parents and cytoplasmic DNA from callus parent. The potential of transgenic GFP citrus callus as suspension parent in citrus somatic fusion to study the mechanism of cybrid formation, create new citrus cybrids, and transfer organelle-encoded agronomic traits was also discussed.     

Change of Superoxide Dismutase Activities During Somatic Embryogenesis in Asparagus officinalis L.

The superoxide dismutase activities in callus and somatic embryoids at different developmental stages of Asparagus officinalis L. were determined by means light of induced oxidation-reduction of nitro blue tetrazolium. It was shown that the superoxide dismutase activity was higher in callus than in somatic embryoids at different developmental stages. The activity increased with growth, differentiation and maturation of somatic embryoids during somatic embryogenesis. The relations between superoxide dismutade activity and somatic embryogenesis were discussed.     

Identification of 2,4-D-responsive proteins in embryogenic callus of Valencia sweet orange (<Emphasis Type="Italic">Citrus sinensis</Emphasis> Osbeck) following osmotic stress

The compound 2,4-Dicholorophenoxyacetic acid (2,4-D) is an important growth regulator which is used in the majority of embryogenic cell and tissue culture systems. However, 2,4-D also appears to have a negative effect on growth and development of plant tissues and organs cultured in vitro. For example, 2,4-D exerts inhibition on in vitro somatic embryo initiation and/or development of most citrus species. To understand the molecular mechanism by which 2,4-D inhibits somatic embryogenesis (SE), proteomic changes of Valencia sweet orange (Citrus sinensis) embryogenic callus induced by treatments with a high concentration of 2,4-D (6 mg l⁻¹) was investigated. Nine 2,4-D-responsive proteins were identified, of which eight were up-regulated and one was down-regulated. Interestingly, three of the eight up-regulated proteins were osmotic stress-associated, suggesting that 2,4-D induced osmotic stress in Valencia embryogenic callus. This speculation was supported by results from our physiological studies: 2,4-D treated callus cells exhibited increased cytoplasm concentration with a significant reduction in relative water content (RWC) and an obvious increase in levels of two osmolytes (proline and soluble sugar). Taken together, our results suggested that 2,4-D could inhibit somatic embryo initiation by, at least in part, inducing osmotic stress to citrus callus cells.     

Production of Virus-tree Nucellar Plantlets from Unfertilized Ovules of Citrus in vitro

On a series of Murashige and Tucker (MT) media supplemented with different growth regulators, the 8-week-old unfertilized ovules of Washington navel orange (Citrus sinensis) were able to regenerate perfect plantlets via somatic embryogenesis or organogenesis. The sorts and combinations of exogenous hormones had remarkable effects on the induction, growth and differentiation of its callus. It was found that the most suitable induction medium was MT medium supplemented with 1.0 mg/l BA and 0.5mg/l 1AA. The most suitable differentiation medium was MT medium supplemented with 1.0 mg/l IBA. It was proved by indicator plant examination that the nucellar plantlets free of citrus exocortis virus (CEV) and citrus tristeza virus (CTV) had been obtained from infected trees.     

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.     

Requirement of Ethylene for Growth of Callus and Somatic Embryogenesis in Medicago sativa L.

The role of ethylene in the growth of callus and somatic embryogenesisin Medicago sativa was examined. The application of 2,5-norbornadiene,a competitive inhibitor of ethylene action, during a 10 d inductionperiod to medium containing 2,4-D and kinetin inhibited thegrowth of callus but did not affect somatic embryogenesis, nordid it affect ethylene production during the induction stage.The exposure of tissue, incubated on differentiation medium,without hormones, to an atmosphere of 2,5-norbornadiene, inhibitedboth growth and embryo maturation and stimulated pigmentation.The inhibition of embryo maturation was observed even in thepresence of norbornadiene at a concentration which did not affectgrowth of tissue. It is suggested that the action of endogenous ethylene is necessaryfor the growth of the callus and embryo maturation. Key words: Medicago sativa, ethylene, callus growth, somatic embryogenesis     

Effect of MGBG on Growth of the Callus Tissue,Induction of Somatic Embryogenesis of Alfalfa and Their Ethylene Biosynthesis

MGBG [methylglyoxal bis(guanylhydrazone)], when added to the initial of callus subculture, promoted ethylene production in callus grawn in Bsh medium, and enhanced the ACC (1-aminocyclopropane-l-carboxylic acid) level and the ACC synthase activity both in the callus grown in Bsh medimn, and in the suspension cultures in Bsg liquid medium for induction of somatic embryogenesis. However, MGBG reduced the malonyl-ACC (MACC) level of the suspension tissues. The treatments of MGBG caused growth of the cultured tissues and induction efficiency of somatic embryogenesis to reduce. Moreover, the effects of MGBG was enhanced when it was added at induction medium of somatic embryogenesis. It could be concluded that this inhibitory effect of MGBG could be due to its promotion of ethylene, ACC levels and ACC synthase activity which has shown to inhibit somatic embryogenesis. The reduction of MACC level could be also involved in the MGBG effect on induction of the embryogenesis.     

IN VITRO SOMATIC EMBRYOGENESIS AND REGENERATION OF SOMATIC EMBRYOS FROM PIGMENTED CALLUS OF KAPPAPHYCUS ALVAREZII (DOTY) DOTY (RHODOPHYTA,GIGARTINALES)1

In vitro somatic embryogenesis and regeneration of somatic embryos to whole plants through micropropagules was successfully demonstrated from pigmented uniseriate filamentous callus of Kappaphycus alvarezii (Doty) Doty in axenic cultures. More than 80% of the explants cultured on 1.5% (w/v) agar‐solidified Provasoli enriched seawater (PES) medium showed callus development. The callus induction rate was consistently higher for laboratory‐adapted plants. The excised callus grew well in subcultures and maintained its growth for prolonged periods if transferred to fresh medium in regular intervals. Some subcultured calli (<10%) did undergo transformation and produced densely pigmented spherical or oval‐shaped micropropagules (1–5 mm in diameter) that subsequently developed into young plantlets in liquid PES medium. The micropropagule production was further improved through somatic embryogenesis by a novel method of culturing thin slices of pigmented callus with naphthaleneacetic acid (NAA) or a mixture of NAA and 6‐benzylaminopurine. Transfer of embryogenic callus along with tiny somatic embryos to liquid medium and swirling on orbital shaker facilitated rapid growth and morphogenesis of somatic embryos into micropropagules that grew into whole plants in subsequent cultivation in the sea. The daily growth rate of one tissue cultured plant was monitored for seven generations in field and found to be as high as 1.5–1.8 times over farmed plants. The prolific somatic embryogenesis together with high germination potential of somatic embryos observed in this study offers a promising tool for rapid and mass clonal production of seed stock of Kappaphycus for commercial farming.     

ABA对枸杞体细胞胚发生的调节作用

Using Enzyme Linked Immunosorbent Assay (ELISA) method, we determined the ABA contents of different stages in somatic embryogenesis. The results showed that endogenous ABA contents increased to maximum value twice during somatic embryogenesis. After first maximum value of ABA contents embryogenic cells were observed in callus, and simultaneously, there was a specific protein of somatic embryogenesis investigated by SDS-PAGE. This protein accumulates preferentially in embryogenic callus but not in transferred callus. So it is suggested that ABA could promote the expression of specific genes and the synthesis of embryogenic protein during somatic embryogenesis in Lycium barbarum L. and ABA play an important role in globular stage as well. In addition, treatment of non-embryogenic activity callus with 4 mumol/L exogenous ABA could stimulate somatic embryogenesis. And the ABA function mechanism in relation to somatic embryogenesis was discussed.