红豆草体细胞胚胎发生的激素调控及L—脯氨酸对它的促进作用

在 LS 附加1mg/1 BA+1mg/l KT 的培养基上,红豆草(Onobrychis viciaefolia Scop.)无菌苗的下胚轴切段产生淡黄色的愈伤组织。愈伤组织转移到 LS 附加1mg/l BA 的培养基上,诱导体细胞胚胎发生,而在 LS 附加1mg/l KT 的培养基上抑制体细胞胚胎发生。同时,发现红豆草胚性愈伤组织中游离脯氨酸的含量仅为非胚性愈伤组织的2/5。向培养基中加入L-脯氨酸可以促进红豆草体细胞胚胎发生。最适浓度为1000mg/l。     

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。     

盐肤木体细胞胚胎发生及植株再生

以盐肤木(Rhus chinensis Mill.)幼胚为外植体,研究不同植物生长调节剂组合对其愈伤组织诱导及体细胞胚胎发生的影响,以建立盐肤木体细胞胚胎发生及植株再生体系。结果表明,最适愈伤组织诱导培养基为MS+6-BA 0.2 mg/L+2,4-D 1.0 mg/L,诱导率为84.57%,诱导出的初代愈伤组织白色或淡黄色,质地疏松,表面光滑,为非胚性愈伤。初代愈伤组织转移到1/2 MS+6-BA 2 mg/L+NAA 0.5 mg/L培养基上培养1个月后,长出淡黄色质地紧密的胚性愈伤组织,诱导率高达100%,在此培养基上胚性愈伤组织增殖倍数为854.73%。所获得的胚性愈伤组织转接到1/2 MS+6-BA 2 mg/L+NAA 0.5 mg/L+蔗糖4%的培养基上培养1个月后可诱导体细胞胚胎发生,诱导率可达32.67%。诱导得到的体细胞胚胎经历球形胚、心形胚、鱼雷胚、子叶胚进一步分化发育成苗。无菌苗炼苗后栽种到泥炭土∶蛭石∶珍珠岩为2∶1∶1的生长基质上,能100%稳定成活。经过细胞学观察分析,体细胞胚的发育与合子胚相似。     

不同分化状态的白百利烟草愈伤组织在生长过程中核酸蛋白质的变化

本文报道了在正常分化芽和根、诱导芽或恨定向发生的白百利烟草(Nico-tiana tabacum Baibaili)愈伤组织在生长过程中DNA、RNA和蛋白质变化的结果。MS+0.2mg/1NAA+0.2mg/1 KT诱导白百利烟草愈伤组织正常分化出芽和根,MS+0.05mg/1NAA+2mg/1KT诱导愈防组织定向地芽发生,MS+0.5mg/1NAA+0.05mg/1KT诱导愈伤组织定向地根发生。在定向诱导芽或根发生愈伤组织里的RNA和蛋白质合成的第一个高峰出现,比正常发生芽和根的愈伤组织里DNA、RNA和蛋白质的第一个高峰迟5天,在芽发生的愈伤组织里DNA峰出现也迟5天,在根发生的愈伤组织里DNA蜂,则相同于正常分化的愈伤组织DNA峰出现。外源的植物生长物质诱导器官定向发生的作用表现在RNA水平上。在三种分化状态的愈伤组织里,蛋白质组成在第8天表现出明显的差异。41KD和46KD蛋白质在器官的定向发生中可能起着相当重要的作用。     

枸杞髓组织培养中体细胞胚胎发生与过氧化物酶同工酶分析

枸杞髓组织在 MS+6 - BA0 .1mg/ L+NAA0 .5mg/ L培养基上诱导愈伤组织发生。在 MS+6 - BA0 .1mg/ L+NAA0 .5mg/ L+CH50 0 mg/ L培养基上继代培养 ,再转入 MS+6 - BA2 mg/L +NAA 0 .5mg/ L的分化培养基上进行分化培养。显微观察表明 ,在培养过程中愈伤组织细胞由非胚性细胞转变为胚性细胞 ,直至发育成体细胞胚胎和完整植株 ;电泳结果显示 ,体细胞胚胎发生的各阶段 ,其过氧化物酶同工酶发生相应的变化。     

放线菌素D和环己亚胺对伊贝母胚性愈伤组织中胚性细胞团和球形胚发生及大分

伊贝母（F,pallidiflora Schrenk)胚性愈伤组织接种于NAA1．0mg/L 6=BA2.0mg/L的MS培养基上,在培养10天前可产生大量单细胞到多细胞胚性细胞团,培养10至15天,逐渐形成大量球形胚,利用这样一个实验体系,在培养0,1,2,3和4天后加入放线菌素D（AMD,20ug/ml)和环己亚胺（CHM,20ug/ml),继续培养至第6天,分析大分子代谢动态和观察胚性细胞团的形成情况;培养6和10天后加入同样浓度的AMD和CHM,继续培养至第15天,分析大分子代谢动态及观察球形胚形成情况,结果表明：（1）培养0,1,2,3和4天加入AMD的分别抑制胚性细胞团的100％,63％和45％,加入CHM的抑制100％,85％和75％,培养6和10天后加入CHM抑制球形胚的100％和75％;（2）DNA,RNA和蛋白质在胚性细胞团和球形胚形成时出现两个峰值,其中RNA变化剧烈,最早出现峰值,AMD和CHM分别抑制RNA和蛋白质的合成;（3）过氧化物酶同工酶带对胚性细胞团和球形胚形成过程中顺序表达。AMD和CHM分别在转录和转泽水平上对其进行规律性抑制,根据以上结果,本文对伊贝母体细胞胚胎发生的机制进行了初步讨论。     

红豆草组织培养中体细胞胚的形成及其胚胎学观察

红豆草(Onobrychis viciaefolia Scop)幼苗的茎或根放入含有Kinetin(1mg/1)和6-BA(1mg/1)的LS固体培养基上,4周后可产生愈伤组织。当愈伤组织被转移到含有6-BA(1mg/1)的LS固体培养基上3—4周后,发现在愈伤组织上形成了体细胞胚,这种胚在不含任何激素的LS液体培养基浸湿的滤纸上会发育为完整植株。在愈伤组织分化的过程中进行胚胎学显微观察,发现了从单个的胚性细胞到高级阶段胚的体细胞胚发育的各个阶段,看来十分明显,红豆草的分化植株最初来源于单个的胚性细胞,这就使得在红豆草的组织培养中通过体细胞胚发生的途径在细胞水平上进行遗传选择成为可能。     

放线菌素D和环已亚胺对伊贝母胚性愈伤组织中胚性细胞团和球形胚发生及大分子代谢动态的影响

伊贝母(F.pallidiflora Schrenk)胚性愈伤组织接种于NAA 1.0mg/L+6-BA2.0 mg/L的MS培养基上,在培养10天前可产生大量单细胞到多细胞胚性细胞团,培养10至15天,逐渐形成大量球形胚。利用这样一个实验体系,在培养0、1、2、3和4天后加入放线菌素D(AMD,20μg/ml)和环己亚胺(CHM,20μg/ml),继续培养至第6天,分析大分子代谢动态和观察胚性细胞团的形成情况;培养6和10天后加入同样浓度的AMD和CHM。继续培养至第15天,分析大分子代谢动态及观察球形胚形成情况。结果表明:(1)培养0、1、2、3和4天加入AMD的分别抑制胚性细胞团的100%、63%和45%,加入CHM的抑制100%、85%和75%,培养6和10天后加入CHM抑制球形胚的100%和75%;(2)DNA、RNA和蛋白质在胚性细胞团和球形胚形成时出现两个峰值,其中RNA变化剧烈,最早出现峰值。AMD和CHM分别抑制RNA和蛋白质的合成;(3)过氧化物酶同工酶带在胚性细胞团和球形胚形成过程中顺序表达,AMD和CHM分别在转录和转译水平上对其进行规律性抑制。根据以上结果,本文对伊贝母体细胞胚胎发生的机制进行了初步讨论。     

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。     

小麦幼胚培养中的体细胞胚胎发生

小麦品种崇阳红麦和鄂思一号杂种一代幼胚培养具有再生植株的潜力。从一个幼胚经200天左右的连续培养获得530多株再生植株,并从中获得了典型的具有两极性的与愈伤组织块仅局部相连的胚状体。体细胞胚胎发生是小麦幼胚培养的主要途径,但受培养条件的影响,以MS培养基作基本培养基,低浓度2,4-D(0.4mg/1)和水解酪蛋白(1000mg/l)有利于体细胞胚胎发生。     

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.     

Plant regeneration by somatic embryogenesis from callus cultures of sweet sorghum

Tissue culture methods were developed for the induction, maintenance, and regeneration of embryogenic callus in sweet sorghum (Sorghum bicolor) cultivars Keller, Rio, and Wray. No significant differences were observed in production of embryogenic callus in cultures established from developmentally immature or mature embryo explants cultured on LS medium with 2 mg/1 2,4-D plus 0.5 mg/1 kinetin. Prolific callus production did not occur until the third four-week culture period. Long-term maintenance of embryogenic callus was dependent upon the selective transfer of embryogenic callus, with other callus types discarded. High-frequency plant regeneration was achieved and quantified on a fresh weight basis of embryogenic callus.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BA benzyladenine - IAA indoleacetic acid - IBA indolebutyric acid - LS Linsmaier and Skoog basal medium (Linsmaier and Skoog, 1965)     

Somatic embryogenesis from stem and leaf explants of Quercus robur L.

Internodal and leaf segments from pedunculate oak (Quercus robur L.) seedlings were used as explant source to induce somatic embryogenesis. Auxin treatment influenced embryogenic response, which only occurred in explants initially cultured on media containing 4 mg/l naphthaleneacetic acid (NAA) and different benzyladenine (BA) concentrations. After 6 weeks of culture on induction medium, the explants were transferred to medium supplemented with 0.1 mg/l BA and 0.1 mg/l NAA, and 4 weeks later, they were subcultured in a growth-regulator-free medium, in which somatic embryos arose through indirect regeneration on the surface of a nodular callus. Somatic embryos were induced in explants of two out of four seedling provenances. The induction frequency ranged from 16% in leaf explants to 4% in internodal explants. Somatic embryos developed two cotyledons, which were translucent or opaque-white in appearance, but anomalous morphologies were also observed. Different embryogenic lines were established and maintained by repetitive embryogenesis in multiplication medium containing 0.1 mg/l BA plus 0.05 mg/l NAA. These results indicate that tissues from explants other than Q. robur zygotic embryos are able to produce embryogenic cultures. Received: 14 July 1998 / Revision received: 2 November 1998 / Accepted: 6 November 1998     

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。     

High efficiency plant regeneration by somatic embryogenesis from callus of mature embryo explants of bread wheat (Triticum aestivum) and grain sorghum (Sorghum bicolor)

Summary Tissue culture methods were developed for reproducible induction and maintenance of embryogenic (E) callus established from developmentally mature embryo explants of bread wheat (Triticum aestivum) and grain sorghum (Sorghum bicolor). Embryogenic callus was obtained by culturing seeds and mature embryos of wheat on Linsmaier and Skoog’s (LS) medium containing 5 or 2 mg/liter 2,4-dichlorophenoxyacetic acid (2,4-D), respectively, and for sorghum mature embryos on LS medium containing 2 mg/1 2,4-D plus 0.5 mg/liter kinetin. Plant regeneration from E callus was achieved for several months and quantified on a fresh-weight basis of E callus. Phenotypically normal plants were regenerated from E callus cultured on LS medium supplemented with 0.1 mg/liter IAA plus 0.5 mg/liter benzyladenine (BA) for wheat and 1.0 mg/liter IAA plus 0.5 mg/1BA for sorghum. Wheat research was funded by the United States Agency for International Development, Washington, DC, cooperative agreement DNA-4137-A-00-4-53-00. Sorghum research was supported by the Gas Research Institute, Chicago, IL, contract 5084-260-0973. Expert technical asistance was provided by Nitschka S. ter Kuile, Barbara J. Ashton, Laurie Osborne, Erin Scott, and Kathleen M. Petersen.     

Direct somatic embryogenesis and plantlet regeneration from seedling leaves of winged bean,Psophocarpus tetragonolobus (L.) DC

Excised seedling leaf segments of winged bean [Psophocarpus tetragonolobus (L.) DC.] underwent direct somatic embryogenesis under appropriate incubation conditions. Initiation and development of the somatic embryos occurred using a two-step culture method. The culture procedure involved incubation for 28 days on MS basal medium supplemented with 0.1–0.5 mg/l NAA and 1.0–2.0 mg/l BA (induction medium) before transfer to MS medium supplemented with 0.1 mg/l IAA and 2.0 mg/l BA (embryo development medium). The initial exposure to low levels of NAA coincident with high levels of BA in the induction medium was essential for embryogenic induction. Maximum embryogenesis (43.3%) was obtained with 0.2 mg/l NAA and 2.0 mg/l BA, and at least 14 days on induction medium were required prior to transfer to the embryo development medium. The conversion frequency of cotyledonary embryos was 53.3% upon culture on MS medium containing 0.1 mg/l ABA for 7 days followed by transfer to MS medium supplemented with 0.1 mg/l IBA and 0.2 mg/l BA. Following conversion, the regenerated plantlets were transferred to soil and showed normal morphological characteristics.Abbreviations MS Murashige and Skoog (1962) medium - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - BA 6-benzylaminopurine - ABA abscisic acid     

Production of solasodine by Solanum laciniatum using plant tissue culture technique

Leaf and hypocotyl explants of 15 days old aseptically grown seedlings of Solanum laciniatum were cultured on MS medium supplemented with NAA (2 mg/l) and kinetin (0.5 mg/l) for callus initiation. For maintenance and proliferation of callus MS medium supplemented with 2,4-D (1 mg/l) and kinetin (0.5 mg/l) was used. The growth of the calli derived from hypocotyls increased with time of incubation and remained almost constant after 45 days. The solasodine content in callus culture was maximum after 30 days of incubation. Addition of L-arginine in the medium (50-150 mg/l) increased growth as well as chlorophyll content in the callus culture. The solasodine content also increased up to 1.2 to 1.4 times in these cultures. High frequency shoot regeneration was obtained in MS medium having BA (4 mg/l) and IBA (0.25 mg/l). For shoot multiplication, MS medium having BA (4 mg/l) was used. Shoots rooted on the same medium. Organogenesis promoted solasodine accumulation in the cultures. Regenerated shoots yielded higher solasodine content than undifferentiated as well as organogenic callus. Solasodine contents in the regenerated shoots was found to be 10 times higher than the callus culture and approached towards the field grown plants. Thin layer chromatography revealed the presence of three compounds. The most predominant spot (Rf 0.789) corresponded to the reference solasodine.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of niger [<Emphasis Type="Italic">Guizotia abyssinica</Emphasis> (L. f.) Cass.] using seedling explants

A protocol was developed for Agrobacterium-mediated genetic transformation of niger [ Guizotia abyssinica (L.f.) Cass.] using hypocotyl and cotyledon explants. Hypocotyls and cotyledons obtained from 7-day-old seedlings were co-cultivated with Agrobacterium tumefaciens strain EHA101/pIG121Hm that harbored genes for beta-glucuronidase (GUS), kanamycin, and hygromycin resistance. Following co-cultivation, the hypocotyl and cotyledon explants were cultivated on MS medium containing 1 mg/l 6-benzylaminopurine (BA) for 3 days in darkness. Subsequently, hypocotyl and cotyledon explants were transferred to selective MS medium containing 1 mg/l BA, 10 mg/l hygromycin, 10 mg/l kanamycin, and 500 mg/l cefotaxime. After 6 weeks, hypocotyls and cotyledons produced multiple adventitious shoot buds, and these explants were subcultured to MS medium containing 1 mg/l BA, 30 mg/l hygromycin, and 30 mg/l kanamycin. After a further 3 weeks, the explants (along with developing shoot buds) were subcultured to MS medium containing 1 mg/l BA, 50 mg/l kanamycin, and 50 mg/l hygromycin for further selection. Transgenic plants were obtained after rooting on half-strength MS medium supplemented with 0.1 mg/l alpha-naphthaleneacetic acid, 50 mg/l kanamycin, and 50 mg/l hygromycin and were confirmed by GUS histochemical assay and polymerase chain reaction analysis. Genomic Southern blot hybridization confirmed the incorporation of the neomycin phosphotransferase II gene into the host genome.