红豆草体细胞胚胎发生早期DNA,RNA和蛋白质的合成动态变化

红豆草(Onebrychis viciaefolia Scop.)下胚轴切段在含有1mg/IBA、1mg/1 KT 的 LS培养基上培养,两周后产生愈伤组织,通过筛选、克隆得到大量的具有胚胎发生潜力的非胚性愈伤组织,当将其转移到含1mg/1BA 的 LS 培养基上后可诱导体细胞胚胎发生。应用放射性同位素液体闪烁技术测得在胚性培养的前2天,RNA 合成速度迅速上升,随后下降,第五天后又呈缓慢上升趋势,尔后平稳。蛋白质合成速度在胚性培养的第三天达到高峰,升高很快。而 DNA 合成速度变化平缓,只是在胚性培养的第五天出现一较小的峰。胚性培养过程的 DNA、RNA 蛋白质合成速度均高于非胚性培养。     

体细胞胚发生的生化基础

在胚性细胞分化和分裂过程中ATP酶活性和分布的动态变化表明,这些胚性细胞进行着旺盛的主动物质吸收和活跃的新陈代谢过程。在多种植物的体细胞胚发生中过氧化物酶的活性与同工酶的种类都高于对照,而且在大麦中发现过氧化物酶、酯酶和酸性磷酸酶同工酶的结合应用可以作为体细胞胚发生的标志酶。胚性愈伤组织中可溶性蛋白质含量与组分远高于或多于非胚性愈伤组织。大多数材料中都存在45kD-55kD的胚胎发生特异性蛋白质组分。而且在体细胞胚发生中蛋白质和核酸代谢动态呈规律性变化,首先是RNA合成速率增加,继而是蛋白质的迅速合成,并在胚性细胞分化和发育过程中一直保持相对较高水平,其中mRNA种类丰富,不同发育时期mRNA种类不同,因此转译形成多种蛋白质。DNA的代谢相对较稳定,但在胚性细胞系中DNA合成量仍高于非胚性细胞系。加入蛋白质或核酸合成抑制剂,不仅抑制了蛋白质和核酸的合成,同时也抑制了体细胞胚的发生与发育,而且抑制剂加和时间愈早,影响愈严重。由此表明,蛋白质与核酸的合成为体细胞胚的分化和发育奠定了分子基础。     

宁夏枸杞器官发生和体细胞胚胎发生过程中DNA、RNA和蛋白质合成动态的比较研究

宁夏枸杞(Lycium barbarum L.)叶外植体来源的愈伤组织经筛选、繁殖后,将来源相同、状态较为一致的淡黄色愈伤组织转移至O型或E型培养基上,可以诱导出器官发生和体细胞胚胎发生。利用该体系,对两条离体再生途径进行了比较研究。结果表明:(1)在拟分生组织和胚性细胞形成之前,RNA合成首先被激活,随后DNA、蛋白质合成加速;而球形胚形成期间,先是DNA合成的加快,接着RNA、蛋白质的合成高峰出现,在不定芽形成期间却正好相反;(2)可溶性蛋白组分发生规律性变化;器官发生和体细胞胚胎发生的启动阶段都有-153.6kD多肽出现,一些多肽分子在分化早期逐渐消失,而随芽原基或球形胚的形成又重新合成;与形态发生相对应,两种再生体系都有作为各自分子标记的特异多肽(84.9kD、46.3kD和44kD、36.2kD)的表达。此外,还对两种离体再生体系之间的关系和发生机制进行了讨论。     

莲胚发育与核酸,蛋白质含量的变化

莲胚发育达到最大鲜重(开花后21d)前,胚轴和子叶的DNA,RNA都持续增长。开花13d后,蛋白、淀粉等贮藏物质显著积累,核酸增长速度加快。成熟胚轴的DNA和RNA含量很高,而子叶中积累大量的淀粉、可溶性糖和蛋白质。发育前期胚乳的生长速度较快,开花后16d左右鲜重和物质积累达到高峰。胚生长后期胚乳逐渐败育,贮藏物质和结构物质都减少,膨大的子叶逐步取代了胚乳的地位。 莲胚生物大分子物质含量的模式属于双子叶植物类型。讨论了莲胚细胞多倍化的问题。     

玉米花粉胚状体发育过程中DNA,RNA蛋白质含量及合成动态变化

继代培养的玉米花粉胚状体的发育过程可划分为6个时期:胚性细胞团时期、球形胚时期、心形胚时期、梨形胚时期、子叶形胚时期以及分化期。我们应用微量生化分析技术以及放射性同位素液体闪烁计数技术研究了玉米花粉胚状体发育过程的DNA、RNA、蛋白质含量及合成动态,发现DNA、RNA和蛋白质含量在胚性细胞团期较高,然后下降,但到了分化期时又有所升高。DNA合成速度在胚性细胞团时期较高,在以后的各时期降低并保持平稳。RNA和蛋白质的合成动态呈相似的变化规律。这个结果说明DNA、RNA和蛋白质在胚状体发育早期的活跃代谢,可能与胚性细胞的快速分裂以及胚性结构的形成有关,而后期的活跃代谢可能与胚状体的分化有关。     

石刁柏体细胞胚胎发生过程中蛋白质及同工酶变化的研究

本文报道石刁柏胚性愈伤组织的可溶性蛋白质含量与组分、过氧化物酶和酯酶的活力及同工酶带均比其体细胞胚的要少。而在体细胞胚胎发生过程中,过氧化物酶和酯酶活力、可溶性蛋白质含量均以球形胚为最低,子叶分化期胚为最高而呈递增趋势;可溶性蛋白质组分以子叶分化期胚、成熟胚为最多,球形胚、香蕉形胚为最少;过氧化物酶同工酶带以梨形胚为最多,子叶分化期胚、成熟胚为最少;酯酶同工酶则以子叶分化期胚为最多,成熟胚为最少。胚性愈伤组织与体细胞胚均有其特异性可溶性蛋白质及同工酶带,可作为体细胞胚胎发生的分子标记。     

杉木雌配子体与后胚发育过程中核酸、蛋白质和类脂的消长

在杉木胚胎分化期至成熟期,每个雌配子体总核酸和DNA,RNA含量在初期增加,后期则随着胚的分化发育逐渐下降,而蛋白质和类脂则一直上升。每胚总核酸、DNA,RNA则相应增加,而蛋白质、类脂的含量和干重亦逐渐增加。在胚分化早期RNA的迅速合成与细胞的分化及器官形成有关。但以胚干重为单位的DNA、RNA含量却随着胚的发育而有所减少;蛋白质含量先增加,至成熟后才下降。授粉前的胚珠,以及雌配子体、胚中都发现有凝集素存在。     

小麦胚胎发育过程中蛋白质、核酸的动态变化

小麦每胚总核酸、RNA 和每细胞 RNA 含量均随胚的发育而增加,直到开花后21天达到高峰。每胚蛋白质和每细胞蛋白质含量也相应增加,但每胚蛋白质含量的增加持续到开花后24天。每毫克胚干重为单位的 RNA 含量在胚发育早期有下降,在后期有增加,每毫克胚干重的蛋白质含量也相应变化。每胚 DNA 含量在胚发育早期有增加,但之后即相对稳定。作者对 RNA 含量的变化与细胞蛋白质的合成和胚胎发育的关系进行了讨论。     

白Qian体细胞胚胎发生的细胞组织学和淀粉积累动态的研究

以白Ｑｉａｎ的成熟种胚为外植体,诱导体细胞胚胎发生。整体染色封片和组织切片的观察结果表明,白Ｑｉａｎ体细胞胚起源于胚性愈伤组织的单个细胞。胚性细胞经过一次不均等分裂产生两个细胞,即胚细胞和胚柄细胞。然后依次经过胚性胚柄团、球形胚、心形胚及鱼雷形胚阶段,最后发育成具有子叶的成熟胚。通过ＰＡＳ反应研究后发现,在体细胞胚发育过程中,淀粉粒在胚性胚柄团时期开始积累,至心形胚时期达到积累高峰,且淀粉粒的分布     

马唐体细胞胚胎发生过程中生理变化的研究

本文通过对马唐体细胞胚胎发生过程中生理变化的研究后发现,球形胚游离氨基酸种类最少、浓度最低;其过氧化物酶、酯酶和淀粉酶同工酶活性较高、种类较多;其可溶性蛋白质相对浓度最高,并且出现至少两种新带。这说明球形胚已开始分子水平的分化;在这一时期合成的蛋白质(酶),对于马唐体细胞胚胎发生中胚体细胞水平的分化有着重要作用。     

Kinetic Changes of Nucleic Acids and Protein During Embryogenesis of Wheat (Triticun vulgaris L.)

Per embryonic total nucleic acid, RNA content and per cell RNA content increased during embryogenesis, reached maximun at 21 day after anthersis. The per embryo and per cell protein content also increased concomitantly. But the protein content continued to increase up to 24 days after anthersis. On the basis of dry weight, RNA content decreased in the early stage of embryogenesis, but then increased over the period of later developmental stage. The protein content on the basis of dry weight also changed in similar way. It was likely the protein and RNA content changes concomitantly during the developmental process of wheat embryo. As to per embryo DNA content, it increased in early developmental stage, but then remained in a similar level during the later stage. The relationship between the changes of RNA content and protein synthesis, embryonie develope is also discussed in present paper.     

Storage protein dynamics in zygotic and somatic embryos of white fir

Composition and accumulation patterns of storage proteins in female gametophyte and embryos of the white fir (Abies concolor) were investigated during embryogenesis and germination of mature seeds using SDS-PAGE and immunological approach. Altogether 9 major and minor protein components with molecular masses of 14, 16, 22, 24, 27, 30, 35, 38, and 43 kDa were detected in female gametophytes and 9 protein bands in the embryos with the molecular sizes of 14, 16, 22, 24, 25, 27, 34, 38, and 43 kDa. The species seems to deviate in this respect from other representatives of Pinaceae. A conspicuous increase of storage protein synthesis was observed at the stage of fully cellularized female gametophytes and at the cotyledonary stage of embryo development. There exists a high degree of similarity between storage protein profiles of white fir zygotic and somatic embryos. Successive stages of somatic embryogenesis exhibited a high degree of similarity of storage proteins except for cotyledonary stage when a noticeable increase in storage protein synthesis was registered. Conversely, during germination of somatic embryos, an overwhelming majority of storage proteins was depleted.     

Changes in the Syntheses of DNA,RNA and Protein during Early Somatic Embrogenesis in Sainfoin(Onobrychis viciaefolia Sccp.)

Hypocotylar explants of Onobrychis viciaefolia Scop. were cultured on LS basal medium supplemented with 1 mg/l BA and 1 mg/l KT. After two weeks of culture, calli were initiated on the surface of sections. Light-Yellow callus from .one of the explants was selected and proliferated on the medium above. Then it was transfered to LS medium with 1 mg/l BA to initiate somatic embryogenesis. The activity of RNA synthesis increased rapidly during the first two days. Of embryogenic culture and then decreased, but on the 5th day increased gradually. The activity of protein synthesis increased during the first three days and was the highest on the 3rd day. The activity of DNA synthesis had no mark change and emerged, a small peak on the 5th day. All the activities of syntheses of DNA, RNA and protein were higher on embryogenic culture than on nonembryogenic culture.     

Aspects of DNA, RNA and protein synthesis during somatic embryogenesis in a carrot cell suspension culture

Changes in DNA, RNA and protein content, incorporation of ³H-thymidine, ¹⁴C-uridine and ³H-leucine and template activity of chromatin were investigated in the early process of somatic embryogenesis in a carrot (Daucus carota L. cv. Kurodagosun) cell suspension culture using a synchronous system. An embryogenetic culture in a medium containing 10^-7M zeatin was compared with a non-embryogenetic culture in a medium containing 10^-7M zeatin and 5 x 10^-7M 2,4-D. DNA was synthesized very actively prior to and during the formation of globular embryos in the embryogenetic culture. The RNA and protein content per tube increased at an almost constant rate in both cultures, while the rate of incorporation of labelled precursors of RNA and protein rose much more prior to active DNA synthesis in the embryogenetic culture than in the non-embryogenetic culture. Template activity of chromatin was high in the early stage of embryogenesis in the embryogenetic culture. The results obtained here showed that synthesis and turnover of RNA and protein became active prior to active DNA synthesis in the early stage of embryogenesis, and that these changes at macromolecular levels may play important roles in embryogenesis.     

In vitro plant regeneration from immature zygotic embryos and repetitive somatic embryogenesis in kohlrabi (Brassica oleracea var. gongylodes)

A simple and efficient protocol for direct somatic embryogenesis and plant regeneration of kohlrabi (Brassica oleracea var. gongylodes) was developed. Somatic embryos were induced from immature zygotic embryos at different developmental stages cultured on Murashige and Skoog medium supplemented with 0, 0.5, 1.0, or 1.5 mg/l 2,4-dichlorophenoxyacetic acid. Zygotic embryos at the early cotyledonary stage, which were cultured for 4 wk on plant growth regulator-free (PGR-free) medium, displayed the highest percentage of somatic embryogenesis (80.7%). Embryogenic tissue could be subcultured on the same medium for over 1 yr. Embryogenic lines derived from early cotyledonary stage zygotic embryos displayed the highest intensity of secondary embryogenesis (highest mean number of new somatic embryos per responsive somatic embryo explant). Histological analyses confirmed the direct origin of the secondary somatic embryos. Prolonged culturing of embryogenic tissue on PGR-free medium led to somatic embryo development into plantlets that were successfully acclimated in the greenhouse with a survival rate of 72.5%. Flow cytometry analysis showed no ploidy variation in 96.7% of the acclimated plants.     

Peroxidase activity in non-embryogenic and embryogenic calli and in developing somatic embryos of white fir (Abies concolor Gord. et Glend)

ABSTRACT

Peroxidase activity was monitored during somatic embryogenesis of white fir (Abies concolor Gord. et Glend) starting from a non-embryogenic callus. Results revealed profound differences between non-embryogenic and embryogenic calli with an elevated level of enzyme activity in non-embryogenic ones. Precotyledonary, early cotyledonary and late cotyledonary stages of somatic embryogenesis were characterized by a substantially reduced peroxidase activity compared to callus tissues and regenerated plantlets. Changes in peroxidase activity are as a rule paralleled by variation in isoenzyme composition. The utility of the enzyme in the induction stage of somatic embryogenesis in white fir is proposed.     

苜蓿体细胞胚胎发生过程中DNA、RNA和蛋白质的合成动态

苜蓿(Medicago sativa L.)下胚轴切段产生的愈伤组织经2,4-D短时间诱导后,在无激素液体培养基中可形成大量体细胞胚胎。经2,4-D诱导后的愈伤组织在转入无激素培养基1天后,其DNA、RNA和蛋白质的合成即进入活跃合成状态,并在体细胞胚胎发育过程中保持逐步升高的趋势。在苜蓿体细胞胚胎发生过程中,有些蛋白质组分含量减少或消失,但绝大部分蛋白质组分的含量明显增加,并且有若干新蛋白的出现,其中24 KD和46 KD蛋白质为体细胞胚胎发生早期所特有。     

Somatic embryogenesis,encapsulation, and plant regeneration of <Emphasis Type="Italic">Rotula aquatica</Emphasis> lour., a rare rhoeophytic woody medicinal plant

Summary Indirect somatic embryogenesis, encapsulation, and plant regeneration was achieved with the rare rhoeophytic woody medicinal plant Rotula aquatica Lour. (Boraginaceae). Friable callus developed from leaf and internode explants on Murashige and Skoog (MS) medium with 0.45 μM 2,4-dichlorophenoxyacetic, acid (2,4-D) was most effective for the induction of somatic embryos. Subculture of the callus onto half-strength MS medium with the same concentration of 2,4-D resulted in highly embryogenic callus. Suspension culture was superior to solid medium culture for somatic embryogenesis. Embryogenic callus.during subsequent transfer to suspension cultures of half-strength MS medium having 0.23 μM 2,4-D induced the highest number of somatic embryos (a mean of 25.6 embryos per 100 mg callus) and the embryos were grown up to the torpedo stage. Transfer of embryos to half-strength MS basal solid medium allowed development, of 50% of the embryos to the cotyledonary stage. Of the cotyledonary embryos, 90% underwent conversion to plantlets on the same medium. Encapsulated cotyledonary embryos exhibited 100% conversion to plantlets. Ninety-five percent of the plantlets established in field conditions survived, and were morphologically identical to the mother plant.     

Expression of abundant mRNAs during somatic embryogenesis of white spruce [Picea glauca (Moench) Voss]

Embryogenic tissues of white spruce [Picea glauca (Moench) Voss] remain in an early developmental stage while cultured on 2,4-dichlorophenoxyacetic acid and N⁶-benzyladenine, but develop to cotyledonary embryos when these phytohormones are replaced by abscisic acid. Twenty-eight cDNAs were isolated from cotyledonary embryos by differential screening against immature embryo and non-embryonic tissues. Temporal expression patterns of these cDNAs during ABA-stimulated somatic embryo development were observed. This showed that clones could be allocated to various groups, including embryo-abundant, embryo-maturation-induced, and those whose expression was modulated during embryo development, germination or in non-embryogenic tissues. Expression corresponding to these cDNA clones showed that there were various responses to exogenous ABA or polyethylene glycol during a period of 48 h. Analyses of DNA and predicted amino acid sequence revealed that 12 of 28 cDNA clones had no known homologues, while others were predicted to encode different late-embryogenesis-abundant proteins, early methionine-labelled proteins, storage proteins, heat-shock proteins, glycine-rich cell wall protein, metallothionein-like protein and some other metabolic enzymes.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - ABA abscisic acid - BA N⁶-benzyladenine - cDNA complementary deoxyribonucleic acid - Em early methionine-labelled - HSP heat-shock protein - LEA late embryogenesis abundant - PEG polyethylene glycol The authors thank Mr. Terry Bethune for his assistance, and Dr. Larry Pelcher, Mr. Barry Panchuk and Mr. Don Schwab for DNA sequencing and primer synthesis. This is National Research Council of Canada publication number 38929.