楸树体细胞胚胎发生过程中4种同工酶分析

采用不连续聚丙烯酰胺凝胶电泳技术对楸树体细胞胚胎发生过程中的酯酶(EST)、过氧化物酶(POD)、淀粉酶(AMY)及ATP酶4种同工酶进行分析。结果表明,EST及POD同工酶酶带在楸树体细胞胚胎发生不同时期呈现规律性变化,胚性愈伤组织中EST、POD同工酶酶带较非胚性愈伤组织多且表达活跃,子叶胚时期活性最强,表明这一时期细胞内代谢旺盛。EST、POD、AMY及ATP同工酶在楸树胚性与非胚性愈伤组织中谱带差异明显,表明这4种同工酶与体胚发生具有密切关系,可以作为楸树胚性愈伤组织和体胚发生的重要标志。     

香果树体细胞胚胎发生过程中4种同工酶的研究

用非变性聚丙烯凝胶电泳技术对香果树体细胞胚胎发生及形态建成过程中过氧化物酶(POD)、酯酶(EST)、淀粉酶(AMY)和超氧化物歧化酶(SOD)4种同工酶进行分析.结果表明:香果树体细胞胚胎发生及形态建成过程中,POD、EST、AMY和SOD活性变化与胚性愈伤组织的诱导及体细胞胚的发生发育密切相关.非胚性愈伤组织和胚性愈伤组织酶谱差异明显,胚性愈伤组织中EST和AMY同工酶酶带多且活性高,非胚性愈伤组织中缺乏EST和AMY同工酶表达,AMY同工酶可作为胚性细胞分化和发育的重要标志.香果树体细胞胚形态建成过程中,球形胚时期的AMY、POD、EST同_T酶活性最强,表明这一时期生理代谢旺盛,是体细胞胚形态建成的关键时期;POD、AMY和SOD 3种同工酶的酶谱及表达强弱在形态建成的不同时期呈现有规律的变化,可作为香果树体细胞胚发生发育特定时期的参考标记. 与胚性愈伤组织的诱导及体细胞胚的发生发育密切相关.非胚性愈伤组织和胚性愈伤组织酶谱差异明显,胚性愈伤组织中EST和AMY同工酶酶带多且活性高,非胚性愈伤组织中缺乏EST和AMY同工酶表达,AMY同工酶町作为胚性细胞分化和发育的重要标志.香果树体细胞胚形态建成过程 ,球形胚时期的AMY、POD、EST同_T酶活性最强,表明这一时期生理代谢旺盛,是体细胞胚形态建成的关键时期;POD、AMY和SOD 3种同工酶的酶谱及表达强弱在形态建成的不同时期呈现有规律的变化,可作为香果树体细胞胚发生发育特定时期的参考标记. 与胚性愈伤组织的诱导及体细胞胚的发生发育密切相关.非胚性愈伤组织和胚性愈伤组织     

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。     

连香树胚性与非胚性愈伤组织生理生化差异及同工酶分析

本研究以连香树未成熟种子子叶胚诱导产生的胚性愈伤组织与非胚性愈伤组织为原材料,测定了两者的可溶性蛋白质、可溶性糖及游离脯氨酸的含量,并利用非变性聚丙烯酰胺凝胶电泳技术(polyacrylamide gelelectrophoresis, PAGE)对两者的过氧化物酶(peroxidase, POD)、超氧化物歧化酶(superoxide dismutase,SOD)、淀粉酶(amylase, AMY)同工酶进行了分析。比较发现胚性愈伤组织在可溶性蛋白质和游离脯氨酸的含量上均极显著高于非胚性愈伤组织。在可溶性糖含量上,胚性愈伤组织也显著地高于非胚性愈伤组织。胚性愈伤组织具有较高的代谢活性,为进一步胚性分化提供物质和能量基础。POD、SOD和AMY同工酶在连香树胚性愈伤组织与非胚性愈伤组织中酶带差异明显,表明这3种酶的活性与胚性愈伤组织的诱导密切相关,酶谱的差异可作为体细胞胚胎发生的参考标记。     

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。     

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。     

金鱼草愈伤组织过氧化物酶活性及其同工酶变化

在不同激素比例的MS培养基上 ,金鱼草 (Antirrhinummajus)茎愈伤组织主要有三种分化状态 :a愈伤组织不分化 ;b以器官发生途径分化出不定芽和不定根 ;c通过胚胎发生途径形成胚状体。过氧化物酶活性与愈伤组织分化程度呈正相关。未分化愈伤组织与分化芽、分胚状体及分化芽和根的愈伤组织酶活性呈线性递增关系 ,相对酶活力为 1:3:5 :6。在其同工酶谱中 ,三种状态的愈伤组织都具有酶带I和II,表明这两种同工酶与细胞的增殖生长有联系。分化的愈伤组织比未分化愈伤组织多出现酶带Ⅲ、Ⅳ和Ⅴ ,说明这三条酶带是影响分化的特异蛋白质。在分化芽的愈伤组织中 ,还存在酶带Ⅳ ,说明它是催化器官发生途径的特有同工酶。     

红豆草体细胞胚胎发生早期过氧化物酶和酯酶同工酶酶谱变化

红豆草下胚轴切段接种于含1mg/l BA,1mg/l KT的LS培养基上,通过筛选和繁殖由一块外植体而来的淡黄色愈伤组织,而得到生理状态比较一致具有较高胚性发生能力的非胚性愈伤组织,将其转移到含1mg/l BA的LS培养基上后可诱导体细胞胚眙发生。在体细胞胚胎发生早期发现过氧化物酶同工酶和酯酶同工酶酶谱均有规律性变化。过氧化物酶同工酶酶谱在胚性培养的第10天,两条明显的A_1、A_2带消失。酯酶同工酶各酶带之间酶活性比例在胚性培养过程中变化很大,培养后期酶带变得不明显或酶带数下降。说明胚性发生过程遗传信息的表达有选择性并为激素所调控。     

苜蓿组织培养中球形胚发生时特异蛋白质和同工酶分析

试验在苜蓿组织培养中,对球形胚形成过程中特异蛋白质表达的模式、过氧化物酶及酯酶同工酶酶谱变化进行研究,结果表明：苜蓿组织培养中从胚性愈伤组织到球形胚发育的进程中,顺序消失和出现了11种中小分子量多肽;过氧化物酶同工酶酶谱发生了显著的变化;酯酶同工酶酶谱变化不大,但其总活力对于维持体细胞胚胎发生是必须的。     

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。     

Embryogenic Callus Formation and Plantlet Regeneration of Piragmites communis

The present paper reports the embryogenic callus formation and plantlet regeneration of Phragmites communis. The results have been obtained as follows: The efficiency of callus induction was much higher, if reed seeds were used as explants. No dedifferentiation was observed by using leaf sheath and leaf blade as explants. The optim, um concentration of sucrose was 4% in medium. VB group and inositol had beneficial effects on callus growth. But yeast extract inhibited callus induction and callus growth markedly. For this inhibited reaction, the higher concentration, the more obviously the callus growth was inhibited. Higher levels of 2,4-D had unfavourable effects on callus growth in callus subculture. The concentration of 2,4-D in dedifferentiation medium had relation to embryogenic callus formation. Embryogenic callus had higher frequency of differentiation for long-term subculture. On the other hand, nonembryogenic callus most often lost their morphogenetic competence. Authors found that the surface structure of the two types of calluses was different by means of observation by scanning electron microscope. The peroxidase and the esterase isoenzyme pat- terns, as well as the soluble protein of both types of calluses were different too.     

Morphohistobiochemical characteristics of embryogenic and nonembryogenic callus cultures of sweet potato (Ipomoea batatas L.)

Ipomoea batatas callus culture raised in a medium supplemented with 2,4-D (2,4-dichlorophenoxy acetic acid) alone or 2,4-D in combination with benzyl adenine, were found to be embryogenic. Supplementation of exogenous chemicals, such as 5 g/l NaCI or 0.7 g/l proline together with a mild dose of 0.2 mg/l 2,4-D, enhanced somatic embryogenesis significantly in all the genotypes tested. Morphological, growth, physiological, histological, and biochemical characteristics of the embryogenic callus were different from the nonembryogenic callus. The former was compact, slow growing, and nodular compared with the fast growing, fragile, nonembryogenic callus. The embryogenic callus tissue had more dry matter, protein and reducing sugar contents compared with the less embryogenic callus. The somatic embryogenic response remained steady in the cultures for up to 96 weeks.     

Indole-3-acetic acid metabolism in hormone-autotrophic, embryogenic callus of Inmil® cherry rootstock (Prunus incisa×serrula'GM 9') and in hormone-dependent, nonembryogenic calli of Prunus incisa×serrula and Prunus domestica

Callus derived from the roots of Inmil® cherry rootstock ( Prunus incisa × serrula ) proliferates in a hormone-free solid medium. When transferred to a hormone-free liquid medium, such callus forms somatic embryos. On the other hand, leaf-derived callus of P. incisa × serrula and leaf- and root-derived calli of P. domestica require exogenous auxin for sustained growth and never form embryos. Levels of free and esterified indole-3-acetic acid (IAA) were similar in both types of calli grown on a solid medium, whereas the amide-conjugated IAA was higher in the root-derived embryogenic one. Transfer to a liquid medium did not affect the level of both free and conjugated IAA in the nonembryogenic callus, but in the embryogenic callus it decreased the level of amide-conjugated IAA. Isotopic dilution of 13 C-IAA taken up from a medium was faster in the embryoegenic than in the nonembryogenic calli, which shows that the rate of IAA metabolism was higher in embryogenic callus. Besides IAA, indole-3-ethanol and indole-3-acetyl-N-aspartate were detected in nonembryogenic calli and in in vitro-grown shoots of P. domestica , whereas in embryogenic callus and in in vitro - grown shoots of P. incisa × serrula indole-3-acetamide was detected.     

Sorbitol as the Primary Carbon Source for the Growth of Embryogenic Callus of Maize

The effects of various carbon sources on initiation and maintenance of embryogenic callus of maize (Zea mays L.) and on the regeneration of plants from embryogenic callus were studied. Growth of embryogenic callus tissue on media containing sucrose was typified by the subsequent growth of both embryogenic (regenerable) and nonembryogenic (nonregenerable) callus. Growth of embryogenic callus on sorbitol was unique among the carbon sources tested in that sorbitol supported the subsequent growth of only embryogenic callus. Further experiments demonstrated that embryogenic callus grown on sorbitol had a greater regenerative capacity (more plants produced per gram fresh weight of callus) than callus grown on sucrose. Sorbitol dehydrogenase was detected in embryogenic callus of maize at a specific activity roughly equivalent to that found in zygotic embryos of developing seeds. Nonembryogenic callus did not contain significant levels of sorbitol dehydrogenase activity.     

Analysis of sweet orange (Citrus sinensis (L.) Osbeck) callus cultures for volatile compounds by gas chromatography with mass selective detector

Volatile constituents of embryogenic and nonembryogenic sweet orange (Citrus sinensis (L.) Osbeck) callus cultures were analyzed by gas chromatography-mass spectrometry to determine if sweet orange flavor essences were produced. Fifteen compounds were identified from the embryogenic callus methylene chloride extracts, with 10 previously reported as volatile constituents of orange juice or peel essential oil, 3 are known fermentation products, 2 have no reported aroma, and 2 were unknown. No volatile compounds were detected from nonembryogenic callus methylene chloride extracts. This revised version was published online in June 2006 with corrections to the Cover Date.     

DNA Methylation and Embryogenic Competence in Leaves and Callus of Napiergrass (Pennisetum purpureum Schum.)

Quantitative and qualitative levels of DNA methylation were evaluated in leaves and callus of Pennisetum purpureum Schum. The level of methylation did not change during leaf differentiation or aging and similar levels of methylation were found in embryogenic and nonembryogenic callus.     

Biochemical differences between embryogenic and nonembryogenic callus of Picea abies (L.) Karst

Both embryogenic and nonembryogenic calli of Picea abies (L.) Karst. were initiated from the hypocotyl region of immature embryos. The two callus phenotypes were manually separated and subsequently maintained independently, but under identical culture conditions. Biochemical analysis of the two phenotypes revealed significant differences in ethylene evolution rate and in concentrations of glutathione and total reductants. Due to the constancy of the genetic background, age and growth conditions of the two callus types, differences in the measured quantities are not likely to be traceable to the genetic origin of the callus and serve to highlight biochemical changes associated with somatic embryogenesis in Norway spruce.Abbreviations GSH glutathione - 2,4-D 2,4-dichlorophenoxyacetic acid - BA N⁶-benzyl adenine - E embryogenic - NE nonembryogenic - NS Norway spruce     

Somatic embryogenesis and mass spectrometric identification of proteins related to somatic embryogenesis in Eruca sativa

Several different proteins expressed in embryogenic and nonembryogenic Eruca sativa calli were identified by combining one-dimensional SDS-PAGE protein mapping with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis. By querying the widely recognized MASCOT search engine, it was found that three of the proteins that were particularly strongly expressed in the embryogenic callus represented sucrose synthase, phospholipase D, and enolase, respectively. RT-PCR analysis also confirmed that the gene coding for enolase was transcribed especially strongly in the embryogenic callus but not in nonembyogenic callus. Finally, the relationship between the three proteins and somatic embryogenesis is discussed.     

Proteins related with embryogenic potential in callus and cell suspensions of sugarcane (Saccharum sp.)

Summary Previous results have shown that some proteins secreted in the culture medium are involved with the formation of embryogenic cells and can modify somatic embryo differentiation. Undifferentiated cell suspensions grown in the presence of 13 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and obtained from embryogenic and non-embryogenic callus were used to study these events in sugarcane plants (cv.PR-62258). The cell suspension growth curves were determined and soluble proteins were extracted from embryogenic and non-embryogenic callus and culture medium from cell suspensions. In embryogenic callus we detected 1.43 times more protein than in non-embryogenic callus and the electrophoretic protein patterns show specific polypeptides for both callus types. In embryogenic callus we detected a cluster of four polypeptides in the range of 38–44 kDa and another polypeptide of 23 kDa that were not observed in non-embryogenic callus. In nonembryogenic callus there is a 35-kDa polypeptide that was not detected in embryogenic callus. In the case of extracellular proteins, the medium from embryogenic cell suspensions contained four polypeptides of 41, 38, 34 and 28 kDa that were slightly detected in the medium from non-embryogenic cell cultures; we also detected a band at 15 kDa that could not be observed in the medium from non-embryogenic cell suspensions. These results suggest that the development of embryogenic callus and cell suspensions is related to the type and amount of intracellular proteins in the callus cells and to the secreted proteins from these cells into the medium.     

Biochemical differences between embryogenic and nonembryogenic calli of conifers

Summary Embryogenic and nonembryogenic calli of loblolly pine (Pinus taeda), Eastern white pine (P. strobus), pond pine (P. serotina), white spruce (Picea glauca), and European larch (Larix decidua) were analyzed for biochemical parameters previously shown to be indicative of an embryogenic state in Norway spruce (Picea abies). Concentrations of glutathione and total reductants as well as rates of ethylene evolution and incorporation of radioactive leucine into protein in the two callus types were consistent with the Norway spruce observations. Embryogenic potential of loblolly pine and pond pine callus was predicted by biochemical analysis in advance of the appearance of somatic embryos. Other parameters such as isozyme patterns and SDS-PAGE of soluble proteins could also be used to distinguish embryogenic from nonembryogenic conifer callus. Among the species investigated, white spruce was the most difficult to sort by these methods.