Plantlet regeneration through somatic embryogenesis in Nordmann's fir (Abies nordmanniana)

I studied the influence of various combinations of auxin and cytokinin concentrations, and the increased content of zinc and enzymatic casein hydrolizate in SH medium on initiation and proliferation of embryogenic callus of Abies nordmanniana(Steven) Spach. Additionally, the effect of ABA, PEG-4000 and different wavelengths on the maturation of somatic embryos was tested.The use of optimum composition of modified SH medium with BA, KIN and 2.4-D while simultaneously ensuring appropriate external conditions resulted in 15.5 % embryogenesis. Finally, satisfactory results of micropropagation of A. nordmanniana by somatic embryogenesis were obtained providing seven lines of embryogenic callus with high proliferation capacity. Those lines gave properly developed seedlings in white LED light with a wavelength of 400-700 nm, preceded by eight-week vernalization treatment of the callus. This paper may provide a protocol by which all stages of somatic embryogenesis of A. nordmanniana can be carried out, including the preceding 24-h seed disinfection with Na OCl and PVP, which resulted in 100 % frequency of uninfected zygotic embryos that were capable of starting embryogenesis.     

水曲柳体细胞胚与合子胚发生的细胞学研究

水曲柳(Fraxinus mandshurica)属木犀科(Oleaceae)白蜡树属,是我国东北重要珍贵硬阔树种之一,主要分布于小兴安岭、长白山、辽宁东部山地等地区, 以材质优良而著称.由于长期不合理的采伐利用,目前可利用的资源急剧减少,已被列为国家三级保护植物(傅立国,1992).进行水曲柳体细胞胚胎发生的研究,在资源保护、树种快繁和基因工程育种上有其重要的现实意义.     

Plantlet regeneration through somatic embryogenesis in Nordmann’s fir (<Emphasis Type="Italic">Abies nordmanniana</Emphasis>)

I studied the influence of various combinations of auxin and cytokinin concentrations, and the increased content of zinc and enzymatic casein hydrolizate in SH medium on initiation and proliferation of embryogenic callus of Abies nordmanniana (Steven) Spach. Additionally, the effect of ABA, PEG-4000 and different wavelengths on the maturation of somatic embryos was tested. The use of optimum composition of modified SH medium with BA, KIN and 2.4-D while simultaneously ensuring appropriate external conditions resulted in 15.5 % embryogenesis. Finally, satisfactory results of micropropagation of A. nordmanniana by somatic embryogenesis were obtained providing seven lines of embryogenic callus with high proliferation capacity. Those lines gave properly developed seedlings in white LED light with a wavelength of 400-700 nm, preceded by eight-week vernalization treatment of the callus. This paper may provide a protocol by which all stages of somatic embryogenesis of A. nordmanniana can be carried out, including the preceding 24-h seed disinfection with NaOCl and PVP, which resulted in 100 % frequency of uninfected zygotic embryos that were capable of starting embryogenesis.     

核桃体细胞胚发生与转基因研究进展

总结了核桃体细胞胚发生的研究进展,列表统计已报道的核桃５个种和３个杂种体细胞胚发生的外植体与诱导条件,重点论述了影响核桃体细胞胚发生与次生胚发生的因素,介绍了核桃体细胞胚萌发与转化的方法。还总结核桃转基因研究的进展,提出了用核桃体细胞胚发生系统进行外源基因转移的操作模式。     

天山云杉体细胞胚的成熟及萌发条件

【目的】建立天山云杉体细胞胚成熟及萌发阶段的适宜培养条件,为该树种的体细胞胚大规模繁殖和工厂化生产提供基础。【方法】以天山云杉成熟合子胚为外植体所诱导形成的胚性愈伤组织为材料,通过14个株系、4种成熟培养基、接种量(100、150、200mg的新鲜胚性愈伤组织)、ABA浓度(1.9、3.8、7.6、19、38μmol·L^-1)等体细胞胚成熟培养条件,以及对不同起源(体细胞胚、合子胚和种子)的幼苗形态特征的比较,筛选出最优的天山云杉体细胞胚成熟与萌发条件。采用SPSS18.0等统计软件对体细胞胚进行统计分析。【结果】1)天山云杉14个株系诱导体细胞胚的结果表明,不同株系在同一成熟培养基上诱导体细胞胚的数量存在差异;其中4个株系及其接种量对体细胞胚形成数量影响的结果表明,不同株系及其接种量诱导体细胞胚的数量也存在差异,当接种量为100mg时,T36株系的体细胞胚诱导数量最高,为3470±546个,显著高于其他接种量和株系(P<0.05)。2)在4种不同成熟培养基上,均形成了成熟的体细胞胚,在1/2BLG+30g·L^-1麦芽糖培养基上形成的体细胞胚数量高于其他培养基,分别为2044个(T6株系)和2282个(T36株系),且与其他培养基差异显著(P<0.05)。3)不同ABA浓度对天山云杉体细胞胚诱导数量的影响无显著性差异(P>0.05),在ABA浓度为1.9~38μmol·L^-1时,其体细胞胚平均诱导数量为2850~2913个,其中以7.6μmol·L^-1ABA的浓度处理效果较好,平均获得2913个正常体细胞胚,高于其他处理。4)将不同起源(体细胞胚、合子胚和成熟种子)萌发的小植株进行对比,结果表明,体细胞胚和合子胚幼苗发育阶段基本一致,但合子胚的幼苗比体细胞胚的幼苗约大1倍,但二者间差异不显著(P>0.05);体细胞胚与合子胚子叶的平均数量一致,二者无显著差异(P>0.05),多数幼苗子叶数量为7~8个。【结论】天山云杉体细胞胚成熟阶段的适宜培养条件为胚性愈伤组织接种量100mg,1/2BLG培养基添加7.6μmol·L^-1的ABA及75%PEG4000、30g·L^-1麦芽糖,并添加750mg·L^-1的L-谷氨酰胺或50mg·L^-1L-天冬酰胺。不同株系在诱导体细胞胚的数量上存在差异。体细胞胚再生植株与合子胚植株在形态上相似。研究结果可为天山云杉体细胞胚的大规模繁殖和工厂化生产提供基础。     

Organogenesis and embryogenesis in mature zygotic embryos of Picea sitchensis

Differentiation of adventitious buds and somatic embryos from mature zygotic embryos of Picea sitchensis (Bong.) Carr. is described. Adventitious buds were formed on embryos pulse-treated with 250 microM benzyladenine for 2 h and cultured on medium lacking growth regulators. Buds were initiated at different frequencies and sites depending on when the BA-pulsed embryos were transferred to fresh culture medium. Embryogenic callus was formed when the zygotic embryos were cultured on medium containing 10 microM 2,4-dichlorophenoxyacetic acid and 5 microM benzyladenine. Although 50% of the embryos gave rise to embryogenic callus, only 20% of the callus continued to proliferate after subculture. Proliferation of new somatic embryos occurred from both the embryonic region and the suspensor region on previously formed somatic embryos. The pattern for development of adventitious buds and somatic embryos in Picea sitchensis is compared to that in Picea abies under similar culture conditions.     

火炬松胚性愈伤组织诱导和植株再生的研究

火炬松胚性愈伤组织诱导和植株再生的研究唐巍欧阳藩（中国科学院化工冶金研究所生化工程国家重点实验室北京１０００８０）郭仲琛（中国科学院植物研究所北京１０００９３关键词火炬松,体细胞胚胎发生,植株再生本文于１９９６年１０月２８日收到。国家“８６３”资...     

火炬松体细胞胚胎发生和干化体细胞的氧化物酶活性(英文)

培养于附加2,4-D、BA和KT的愈伤组织诱导培养基上的火炬松成熟合子胚在培养3-9周后形成白色、半透明、有光泽的粘性愈伤组织。这类愈伤组织形成于成熟合子胚的子叶,但当用NAA或者IBA代替愈伤组织诱导培养基中的2,4-D时,它的诱导频率明显降低。这种粘性愈伤组织在分化培养基上形成体细胞胚。体细胞胚经过去50μm ABA和8.5%PEG600处理后成为耐干化胚。扫描电镜观察表明,萌发处理36小时后,耐干化胚恢复到干化处理之前的状态且大小和形态正常,而不耐干化胚不能恢复到干化处理之前的状态且表面撕裂。过氧化物酶活性的分析结果表明,耐干化胚有更高的过氧化物酶活性。耐干化胚的高过氧化物酶活性可能与催化H2O2的分解和保护体细胞胚免受氧化的伤害有关。     

黑松合子胚和体细胞胚发育阶段的形态特征比较

为掌握黑松合子胚和体细胞胚发育进程,以未成熟合子胚为外植体,建立了黑松体细胞胚胎发生体系。对合子胚和体细胞胚发育阶段的形态特征进行观察和比较,结果表明,合子胚和体细胞胚的发育进程均可划分为8个阶段,两者同一阶段的形态特征大致相同,只有第2阶段和第4阶段有细微差别。     

白刺花胚性愈伤组织诱导及体细胞胚发生

【目的】探讨不同植物生长调节剂对白刺花胚性愈伤组织诱导的作用,以及培养基中氮源和无机盐浓度对白刺花体细胞胚发生和植株再生的影响,以期建立白刺花体细胞胚发生、发育及调控技术体系,为白刺花种苗快速繁殖体系建立及遗传转化研究提供参考。【方法】以白刺花叶片为外植体,研究生长调节剂2,4-D(1.0、2.0、3.0、4.0 mg ·L -1 )、NAA(0、0.5、0.8、1.0 mg ·L -1 )、6-BA(0.2、0.5、1.0、2.0 mg ·L -1 )和TDZ(0、0.2、0.5、1.0 mg ·L -1 )组合对胚性愈伤组织诱导,及NAA(0、0.2、0.5 mg ·L -1 )、6-BA(0、0.5、1.0 mg ·L -1 )和TDZ(0、0.2、0.5 mg ·L -1 )组合对体细胞胚发生的调控作用,筛选最优生长调节剂组合;并研究培养基中KNO 3和NH 4NO 3比例对体细胞胚发生的作用,及MS培养基中无机盐浓度(1/5MS 、1/4MS、1/3MS、1/2MS)对体细胞胚萌发的影响,筛选最佳的体细胞胚发育及成熟萌发条件。【结果】白刺花叶片外植体胚性愈伤组织诱导适宜培养基为MS + 2,4-D 3.0 mg ·L -1 + NAA 0.5 mg ·L -1 + 6-BA 0.2 mg ·L -1 + TDZ 1.0 mg ·L -1 +蔗糖40 g ·L -1 +琼脂7.0 g ·L -1 ,诱导率为42.0%。采用MS基本培养基时,最佳的体细胞胚发生培养基为MS + NAA 0.5 mg ·L -1 + 6-BA 1.0 mg ·L -1 + TDZ 0.5 mg ·L -1 +蔗糖40 g ·L -1 +谷氨酰胺100 mg ·L -1 +琼脂7.0 g ·L -1 ,体细胞胚发生率为78.46%,总胚数为对照的3.6倍;MS培养基中,KNO 3浓度提高1倍、NH 4NO 3降至1/2时,体细胞胚发生率可提高至91.33%,总胚数为采用MS基本培养基时的1.4倍;1/3MS培养基有利于体细胞胚的萌发,萌发率为82.75%,幼苗长势良好,单株平均鲜质量为76 mg,幼苗驯化移栽1个月后成活率达90%以上。【结论】白刺花叶片接种于添加2,4-D、NAA、6-BA和TDZ不同组合的诱导培养基上,可脱分化形成愈伤组织或胚性愈伤组织,2,4-D浓度对愈伤组织形态和质地有较大影响。TDZ有利于体细胞胚的形成,适宜浓度的生长素与细胞分裂素组合及硝态氮和铵态氮的比例对体细胞胚的形成和发育具有调控作用,降低MS无机盐浓度可提高体细胞胚萌发率,本试验体系的再生植株移栽成活率达90%以上。     

杂交落叶松体细胞胚胎发生的初步研究

本文报道杂交落叶松体细胞胚胎发生的初步结果。该实验系统和系列培养基在西加云杉、日本落叶松和马尾松的种胚和幼嫩组织的体细胞胚胎发生试验中有效。种胚和来源于２８￣４２天无菌实生苗下胚轴、子叶和胚根幼嫩组织不同位置切段接种到ＳＥＩＭ培养基，除ＣＴ和ＨＴ外，所有外植体愈伤组织（Ｃ）的诱导率为１００％；胚性愈伤组织（ＥＣ）的诱导率为３．３０％￣５８．２０％，胚性愈伤组织接种到ＳＥＭＭ培养基，经３￣５次继代培     

Storage lipid dynamics in somatic embryos of Norway spruce (Picea abies): histochemical and quantitative analyses

Adequate storage compounds are a prerequisite for successful development during the later stages of somatic embryogenesis; however, the critical amount of reserves below which somatic embryos fail to mature and germinate has not been determined. We analyzed storage lipids during Norway spruce (Picea abies (L.) Karst.) somatic embryogenesis. As maturation progressed, lipids, which were stored as lipid bodies in the cytoplasm, were localized first in suspensor cells of the early embryos, and later in the embryonic root pole, superficial layers of the hypocotyl and in cotyledons. The concentration of total lipids exhibited marked variation, with values peaking during cotyledon development and then decreasing during maturation. Linoleic (18:2), oleic (18:1), palmitic (16:0) and 5,9-octadecenic (5,9-18:2) acids were the most abundant fatty acids in embryos. As embryos developed, linoleic acid concentration increased slightly, whereas oleic acid concentration decreased. Oleic acid was the most prominent component of the fatty acid spectrum in isolated dormant zygotic embryos and megagametophytes. Addition of 5% polyethylene glycol to the medium during somatic embryo maturation caused a shift in the fatty acid spectrum toward that of zygotic embryos. During maturation, changes in the exogenous carbohydrate supply had no significant effect on total lipid concentration in mature embryos. A marked decrease in lipid concentration was detected during desiccation, indicating the importance of adequate lipid reserves during this developmental stage. The lipid content of zygotic embryos differed considerably with harvest year and location, suggesting that zygotic embryo data cannot be an indicator of somatic embryo quality.     

刺槐未成熟合子胚的体细胞胚胎发生和植株再生

以刺槐不同胚龄的未成熟合子胚为外植体,采用混合正交试验设计,研究幼胚胚龄和不同外源激素种类及质量浓度对刺槐胚性愈伤组织的诱导和体细胞胚分化、萌发的影响.结果表明:开花后8周(55天左右)是胚性愈伤组织和体胚诱导的最佳外植体取材时期;MS+2,4-D 5.0 mg·L-1 +BA0.5 mg·L-1是诱导胚性愈伤组织的最佳培养基,出愈率最高为95.42％±0.02％;MS +NAA0.5 mg·L-1 +BA0.5 mg·L-1+谷氨酰胺250 mg·L-1+水解酪蛋白500 mg·L-1是体细胞胚诱导和分化的最佳培养基,直接体细胞胚发生率最高为92.40％±0.12％,通过愈伤组织诱导体细胞胚发生的频率最高为90.73％ ±0.49％.一旦形成球形胚,将培养物转接到不含任何生长调节剂的MS培养基上,体细胞胚经成熟萌发可进一步形成完整小植株.     

Decreased morphogenetic potential in peach palm stem-like cells in long-term in vitro conditions

Peach palm(Bactris gasipaes Kunth)has been micropropagated from pre-procambial cells that provide stem-like cell niches,(i.e.,pre-procambial cells),multipotent,pluripotent and totipotent for direct vascularization,adventitious buds and somatic embryogenesis,respectively.The direct induction of adventitious buds and somatic embryogenesis reduces the frequency of mutations when compared to indirect morphogenesis.Long-term in vitro cultivation of perennial species such as peach palm cause the clones to age and deteriorate;however,the consequences for morphogenesis potential are not fully clear.The morphogenic potential of peach palm clones established and in vitro cultivated for 8 years(regeneration of adventitious buds without callus formation)was investigated in leaves,roots and stem bases using histological and histochemical analyses.Data from long-term cultures(8-years-old)was compared to data from short-term cultures(1-year-old).Morphogenic pathways monitoring for direct induction of somatic embryos and adventitious buds revealed a strong morphogenic reduction potential in the pre-procambial cells,parenchyma cells in the proximal region of stem bases,and external cells of leaf sheaths.Initial cells of shoot apical meristems and pre-procambial cells commit cell reprogramming to the undifferentiated state and subsequent acquisition of cellular competence.These results are applicable in the micropropagation of peach palm,with consideration to obtaining clones and their long-term in vitro culture.     

利用悬浮培养和培养基选择改善火炬松的体细胞胚胎发生和植株再生(英文)

三个基固型的火炬松成熟合子胚被培养在附加 8mg·L-12 ,4 D ,4mg·L-1BA ,4mg·L-1KT ,5 0 0mg·L-1水解酪蛋白和 5 0 0mg·L-1谷氨酰胺的愈伤组织诱导培养基上诱导愈伤组织 .来自于子叶、胚轴和胚根的愈伤组织在附加 1 6mg·L-12 ,4 D ,0 8mg·L-1BA和 0 8mg·L-1KT的愈伤组织增殖培养基上培养 9周后 ,可获得 16 9%的胚性愈伤组织 .通过建立胚性细胞悬浮系和研究ABA、PEG和活性炭对体细胞胚成熟的促进作用 ,优化的体细胞胚胎发生体系被建立 .71棵再生小苗被用于移栽试验 ,2 3棵小苗在田间移栽成活     

Zygotic and somatic embryo morphogenesis in Pinus pinaster: comparative histological and histochemical study

We compared morphogenesis and accumulation of storage proteins and starch in Pinus pinaster Ait. zygotic embryos with those in somatic embryos grown with different carbohydrate sources. The maturation medium for somatic embryos included 80 microM abscisic acid (ABA), 9 g l(-1) gellam gum and either glucose, sucrose or maltose at 44, 88, 175 or 263 mM in the presence or absence of 6% (w/v) polyethylene glycol (PEG) 4000 MW. Maturation medium containing 44 or 88 mM of a carbohydrate source produced only one or no cotyledonary somatic embryos per 0.6 g fresh mass of culture. The addition of PEG to the basal maturation medium resulted in a low yield of cotyledonary somatic embryos that generally showed incomplete development and anatomical abnormalities such as large intercellular spaces and large vacuoles. High concentrations of maltose also induced large intercellular spaces in the somatic embryonic cells, and 263 mM sucrose produced fewer and less developed cotyledonary somatic embryos compared with 175 mM sucrose, indicating that the effect of carbohydrate source is partially osmotic. Zygotic embryos had a lower dry mass than somatic embryos at the same stage of development. Starch granules followed a similar accumulation pattern in zygotic and somatic embryos. A low starch content was found in cotyledonary zygotic embryos and in somatic embryos developed in the presence of 175 mM maltose or 263 mM glucose. In zygotic embryos and in PEG-treated somatic embryos, protein bodies appeared later and were smaller and fewer than in well-developed somatic embryos grown without PEG. We propose that storage protein concentration might be a marker of embryo quality.     

Using synergistic exogenous phytohormones to enhance somatic embryogenesis from leaf explants of a Eucalyptus grandis clone

Somatic embryogenesis (SE) in Eucalyptus spp. has been limited to germinated seeds, flowers, lignotubers or zygotic embryos. The low yield of somatic embryos from leaf explants has hampered progress, even though leaves offer a more viable source of clonal explants from superior selected genotypes. It was hypothesised that SE from leaf explants could be enhanced through pairing of synergistic exogenous plant growth regulators, such as natural auxins with natural cytokinins. Callus and embryo induction using 2,4-dichlorophenoxyacetic acid (2,4-D), α-naphthalene acetic acid (NAA), indole acetic acid (IAA), and indole butyric acid (IBA), each at either 1.0 or 3.0?mg L^?1, indicated that IAA and IBA favoured significantly higher numbers of embryos compared with 2,4-D or NAA. Hence, IAA and IBA were used for subsequent experiments, combining them (at 1.0?mg L^?1) with either the synthetic cytokinin, kinetin, or the natural cytokinin, trans-zeatin, both at 0.1?mg L?1. The combination of trans-zeatin and either IAA or IBA resulted in a significant increase in SE (e.g. 86 ± 17.2% and 23 ± 3.2% for IAA with trans-zeatin and kinetin, respectively), compared with kinetin, or with these auxins alone. Embryo maturation and plantlet regeneration was highest in those calli that were induced with IAA and trans-zeatin, indicating that maturation was dependent on auxin depletion, based on the stability of the analogue used for induction. For the E. grandis clone under study, the use of synergistic plant growth regulators significantly enhanced SE from leaf explants, thus presenting the opportunity to benefit from the advantages that SE offers over conventional organogenesis.     

In vitro somatic embryogenesis in callus cultures of Azadirachta indica A. Juss.—a multipurpose tree

Plant regeneration via somatic embryogenesis was achieved in embryogenic callus cultures derived from immature zygotic embryos 40 days after anthesis of Azadirachta indica A. Juss. on semisolid basal Murashige and Skoog (MS) salts and vitamins supplemented with 1.11 µM 6-benzylaminopurine (BA) and 4.52–6.78 µM 2,4-dichlorophenoxyacetic acid (2,4-D). The globular-stage embryos were induced when the callus was transferred to medium with 1.11 µM BA and 0.45 µM 2,4-D. The highest average number of somatic embryos per 200 mg of callus was 152.8 after 8 weeks of culture on the medium. Maturation and germination of the somatic embryos were achieved on half-strength MS salts and vitamins supplemented 0.38–0.94 µM abscisic acid (ABA) and 2% (w/v) sucrose. The maximum percentage (64.2%) of germination was obtained with 0.94 µM ABA within 2 weeks of culture. Somatic embryo-derived plantlets were acclimatized in a greenhouse and subsequently showed normal growth. This efficient protocol will be helpful for propagating elite clones on a mass scale and will also be useful for genetic transformation study.