扫描电镜下的器官发生——植物胚状体和不定芽的分化(封二说明)

在离体培养条件下,植物组织的器官分化基本可分为不定芽发生及胚状体(体细胞胚)发生这两大主要类型。它们既可以从外值体(即接种材料)的表面直接发生,也可以由外植体经脱分化后形成愈伤组织,由愈伤组织再分化而产生。 封二,图1、2,为刺五加(Acanthopanaxsenticosus)的胚接种在MS 2,4-D0.5ppm培养基上1—2个月后,由子叶及胚轴直接分化出大量胚状体的情况。胚状体的分化是不同步的,     

‘SK4—316’胡萝卜体胚的诱导和培养

以'SK4-316'胡萝卜无菌苗的下胚轴为外植体,研究不同培养基配方和培养条件对愈伤组织诱导、体细胞胚间接发生及其同步化培养的影响,以及不同脱分化时间、脱分化培养基及外植体续存时间对体细胞胚直接发生的诱导及其培养的影响.结果表明:含3%蔗糖、0.8%琼脂的1/2MS + 2,4-D 2.5 mg/L + 6-BA(或KT)0.5 mg/L + CH 300 mg/L是诱导愈伤组织的良好培养基;1/2MS + 2,4-D 1.25 mg/L + KT 0.25 mg/L + 6-BA 0.25 mg/L(含3%蔗糖)适于愈伤组织分化并诱导体胚发生,0.02% ABA对体胚的诱导有促进作用,0.06% ABA或15% PEG能促进体胚成熟;外植体在MS + 2,4-D 1.0 mg/L固体培养基上脱分化培养48 h,再转入MS + CH 300 g/L液体培养基中可诱导体胚直接发生,但随着外植体续存于诱导培养基中时间的延长,体胚发生变异的几率也渐增.     

橡胶树未成苗体细胞胚的茎芽分化及利用

以橡胶树未成苗体细胞胚为试材,选取未成苗双子叶胚、连体胚、多子叶胚、单子叶胚和子叶愈伤化胚作为外植体,研究不同体细胞胚类型的外植体分化茎芽的情况,并利用其进行幼态微型芽条的培育研究。结果表明:在植株诱导培养基中培养80d后,未成苗双子叶胚、连体胚、多子叶胚、单子叶胚、子叶愈伤化胚均能分化出芽,双子叶胚分化率最高(达90%),单子叶胚最低(20%),多子叶胚和连体胚最多可分化出芽3个,双子叶胚2个。幼态微型芽条增殖的最优培养基为MS+6-BA2mg·L-1或MS+6-BA2mg·L-1+KT1mg·L-1,而添加NAA对芽的伸长有抑制作用;适宜幼态微型芽条伸长培养的基本培养基为MS。     

花楸合子胚诱导体细胞胚胎发生研究

分别以完整成熟胚、切去一个子叶的成熟胚和切下的子叶为外植体,以MS为基本诱导培养基、1/2MS为基本分化培养基,进行了花楸体细胞胚胎发生研究。结果表明:以完整合子胚作为外植体的体胚诱导率最高,为100%,最佳植物生长调节剂组合为5 mg.L-1NAA+2 mg.L-16-BA;NAA和6-BA浓度及二者的交互作用对愈伤组织和体胚诱导率的影响极显著;光照配合延长继代间隔时间有利于体胚发生。实体观察结果表明,花楸体胚发生方式有直接发生和间接发生两种;体胚发育经历了球形期、心形期、鱼雷形期和子叶期。组织学观察结果表明,体胚具有两极性,子叶期体胚结构完整。     

马尾松幼胚体细胞胚胎发生研究

本论文首次报道了马尾松（Pinus massoniana Lamb.）幼胚体细胞胚胎发生的完整发育过程,并对影响马尾松胚性愈伤组织诱导的因素如球果采种期、球果冷藏处理时间、外植体处理方式等进行了探讨,统计胚性愈伤组织诱导率,进行增殖评价,探讨ABA浓度梯度对马尾松体细胞胚分化成熟的影响,试验数据用SPSS16统计分析软件进行方差分析、差异显著性检验。结果表明：1）2008~2009连续2年内15个采种期得到的幼胚,胚性愈伤组织诱导和增殖有显著性差异,最适宜的马尾松球果采种期是6月下旬至7月下旬,诱导率在9.66%~22.59%之间;2）球果冷藏处理时间,对胚性愈伤组织诱导有显著性差异,其中4℃冷藏球果15d有利于幼胚胚性愈伤组织诱导;3）雌配子体包含幼胚的接种处理方式是可取的;4）胚性愈伤组织经稳定增殖培养后,转入分化成熟培养基,得到体细胞胚状体＂爆发式＂分化成熟,数量多,质量好。适宜体胚成熟转化的培养基为：成熟LP培养基添加ABA5.0mg·L-1＋60.0g·L-1蔗糖,并附加L-谷氨酰胺和水解酪蛋白;5）成熟体细胞胚在无激素萌发型LP培养基上正常萌发,并转化为结构完整的小植株。本研究首次建立了马尾松幼胚体细胞胚胎发生技术平台,为马尾松遗传改良种质创新、缩短育种周期奠定了研究基础。     

绿巨人的组织培养及快速繁殖

绿巨人 ( Spathiphyllum floribundum)是天南星科苞叶芋属的一种多年生草本植物。组织培养多采用茎尖或茎段做外植体 ,接种材料有限 ,可能破坏整个植株 ;如以花序为外植体 ,则接种量大 ,污染率低 ,而且不经愈伤组织也不经胚状体直接出芽 ,增殖率高。1　材料　绿巨人的肉穗花序。2　培养条件　以 MS为基本培养基 ,诱导分化与生长培养基为 1MS BA2 IAA1(单位 :mg/ L,下同 ) ;生根培养基为 2 1/ 2 MS IBA0 .5～ 1,培养基 p H5.4～ 5.8之间 ,培养温度 2 3± 2℃ ,上方两支 4 0 W日光灯光照培养 ,光照时间 14h。3　培养与继代培养　取佛…     

甘露醇、蔗糖和低温预处理对花楸体细胞胚诱导的影响

用渗透剂甘露醇和蔗糖以及2～5℃低温分别预处理未成熟合子胚外植体,研究其对花楸体胚诱导率影响的结果表明,未成熟的合子胚经1.0 mol·L-1甘露醇预处理24 h后,接种在添加0.5 mg·L-1 6-BA、0.2 mg·L-1 2,4-D和4%蔗糖的MS培养基上培养可产生大量体胚,体胚诱导率可达84%,显著高于未经甘露醇预处理的(诱导率为38%),每个外植体产生的体胚数可达12.9个.体胚经成熟和萌发培养后形成再生植株.扫描电镜观察表明,甘露醇预处理可改变外植体表面细胞的有序排列,改善体胚发生状况,因而体胚诱导率增加.     

新疆天山雪莲体胚诱导与分化研究

以新疆天山雪莲的叶片为外植体,分别用不同配方培养基诱导愈伤组织,后进行体胚诱导和分化培养形成再生雪莲植株.结果表明,诱导愈伤组织的最适培养基为MS 2,4-D 0.5 mg/L BA 1.5 mg/L,诱导率可达到100%;愈伤组织转移至MS 2,4-D 0.5 mg/L BA 1.5 mg/L培养基进行继代培养,增殖后的愈伤组织转移到MS 2,4-D 0.2 mg/L的液体培养基后成功诱导出雪莲体胚,出胚率达40%;将体胚接至MS ABA 0.5 mg/L培养基后,结果分化生长出大量的再生雪莲幼苗.     

糖、GA3及ABA对印度娃儿藤（Tylophora indica (Burm. F.) Merrill）体细胞胚发生的影响

从印度娃儿藤节间外植体获取愈伤组织,分析了糖、赤霉素（GA₃）及脱落酸（ABA）对愈伤组织形成体细胞的影响。实验证明,含4 μmol/L 2, 4-二氯苯氧乙酸（2, 4-D）的MS培养基是获得具有成胚功能的愈伤组织的最佳培养基。在含有6 μmol/L激动素（Kn）的MS培养基上,高达69%的愈伤组织分化为体细胞胚,平均单位外植体（每克愈伤组织）产胚25个。在6 μmol/L Kn存在的条件下,分析了蔗糖、葡糖糖对胚产生的影响,不同的糖及不同糖浓度对体细胞胚的发生影响很大。6 μmol/L Kn 与200 mmol/L 蔗糖处理胚胎发生率最大（71%）,单位外植体生成49个胚。然而葡萄糖与Kn、或者葡糖糖、蔗糖与Kn三者加在一起则降低成胚率及产胚数。一定浓度的GA₃ 和 ABA能促进体细胞胚的产生。在含200 mmol/L蔗糖的培养基中加10 μmol/L GA₃胚的生成率为98%,单位外植体产胚51个。在含200 mmol/L蔗糖的培养基中加2 μmol/L ABA能显著增加体细胞胚的量,该培养基上每外植体平均生成44个胚,产率为95％。本研究显示,含200 mmol/L蔗糖的培养基中分别加入6 μmol/L Kn、10 μmol/L GA₃ 或者 2 μmol/L ABA能显著提高印度娃儿藤体细胞胚发生率,而单独的葡萄糖或葡糖糖和蔗糖则有抑制作用。得到的胚均能正常发育并分化为植株。     

不同培养基对水稻胚性愈伤组织诱导及分化的影响

以水稻成熟胚为材料诱导愈伤组织,统计在不同基本培养基上的愈伤诱导率以及绿苗分化率,分析不同基本培养基及外源激素的含量和比例对愈伤组织生长及分化的影响。结果表明,试验材料对基本培养基具有选择性,MS培养基对籼稻种胚愈伤的诱导培养效果较好,NB培养基则更适合粳稻种胚愈伤的诱导培养;诱导继代培养基中加入多种氨基酸组合可有效提高出愈率和分化率,特别是粳稻的愈伤组织的诱导和分化需要多种氨基酸的共同作用;不同基因型水稻材料对激素和氨基酸组合的需求不同。     

佳禾早占成熟种胚高效率组织培养方法的研究（简报）

In order to improve the embryogenic callus induction rate and the regeneration rate of JiaHe-ZaoZhan rice, the influence of different factors were investigated, media with different hormones were used. Induction medium was supplemented with 1.5 mg/L 2,4-D + 3 mg/L NAA + 0.1 mg/L KT + 1 mg/L phytic acid + 20 mg/L PAA. Embryogenic call were treated under the condition of 25 degrees C before transferring to regeneration medium, the regeneration medium contained 0.5 mg/L 6-BA + 3 mg/L NAA + 0.5 mg/L KT + 1 mg/L phytic acid. The experiment results indicated that the hormone treatments had certain effects on the callus induction. Under the optimal medium, culture condition and the hormone treatments, the embryogenic callus induction could reach over 95%, and dry treatment of embryogenic callus had been found to increase the frequency of plant regeneration, significantly the plant regeneration rate could reach over 80%. Transplanted into pots, the young plants grew well. Then a experimental system with stability and high regenerating efficiency has been established for the mature seeds of rice (JiaHe-ZaoZhan).     

Genetic variability of somatic embryogenesis in tissue cultures of sugar beet breeding lines

Genetic variability of callus initiation and plant regeneration has been investigated among three sugar beet genotypes. It was found that TDZ has a genotype-independent effect on callus initiation and is responsible for more than a two-fold increase in the friable callus induction rate and more than a three-fold increase in the shoot regeneration rate from this callus. Along with the genotype-independent organogenesis, regeneration from callus occasionally went through the process of somatic embryogenesis in a highly genotype-specific manner. Despite fast and uncontrollable conversion of embryos to normal plants, it was possible to select and maintain repetitive embryogenic culture without loosing regeneration and root formation capabilities. Extensive experimenting with medium composition and culture conditions resulted in an optimal medium for maintenance of repetitive embryos. Comparing with BAP, low concentrations of TDZ provide higher level of adventitious shoot formation and do not induce vitrification of tissues.     

Studies on Cell Suspension Culture and Plant Regeneration in Rice

The present paper reports the establishment of rice cell suspension culture system, including callus induction and proliferation, isolation of single cells and small aggregates, cell suspension culture and callus re-formation, as well as regeneration of plantlets. The results have been obtained as follows: 1. The compositions of the different media used for callus induction, callus proliferation, cell suspension and plant regeneration are summarized in Table 1.2. Two kinds of disifectants, mercuric chloride and sodium hypochlorite, were used for surface sterilization of brown rice. The percentage of callus formation and callus yields were much higher when sodium hypochlorite was used (Fig. 3). We suggest that the disinfactant is one of the important factors that affect callus formed at the initial stage has an influence upon subsequent isolation of cells and suspension culture and even plant regeneration. 3. Table 3 shows that addition of yeast extract to the medium improves callus yield greatly and the efficiency of callus formation to a lesser extent. 4. Both medium Ⅱ (modified B5 medium) and N6 medium were suitable for cell suspension culture, but medium II was more effective for cell growth and callus re-formation (Fig. 4 and Table 4). 5. Effect of 2, 4-D on cell growth was tested at the concentration range among 0, 10-6, 10-5, 10-4 to 10-3 M. The results indicated that 10-5 M of 2,4-D was most effective for induction of rice callus. It has also been found that absence of 2,4-D increased callus re-formation in suspension culture, but no plant regeneration was observed. 6. By using 7% sucrose in differentiation medium, for all the three varieties, the plant regeneration frequency was raised up to 3 or 4 times than those of the 3% ones (Table 6). Occurrence of albino plants is often reported as one of the problems in rice anther culture. It is, however, no problem in seed-derived rice cell culture.     

毛报春(Primula × pubescens)腋芽再生组织培养体系的建立

以毛报春(Primula × pubescens)无菌腋芽为外植体, 分析不同浓度激素配比对愈伤组织诱导和分化以及不定芽增殖和生根的影响, 筛选出不同阶段的最适培养基, 优化毛报春的组织培养再生体系。结果表明, 毛报春腋芽愈伤组织诱导及分化的最适培养基为MS+0.2 mg∙L^-1 NAA+1.0 mg∙L^-1 6-BA, 诱导率达84%, 出芽率达67%; 不定芽增殖最适培养基为MS+0.5 mg∙L^-1 NAA+0.2 mg∙L^-1 6-BA, 增殖率可达67%, 苗绿且健壮; MS+0.2 mg∙L^-1 NAA培养基最有利于组培苗的生根及伸长, 平均单株生根数为9条, 生根率高达70%。该研究建立了毛报春的组织培养再生体系, 可为报春属其它植物的遗传研究及种质创新提供参考。     

骨干玉米自交系丹598遗传再生体系的建立

目的:以玉米骨干自交系丹598的幼胚为外植体,诱导愈伤组织建立遗传再生体系。方法:探讨胚龄、培养基种类、2,4-D浓度对愈伤组织诱导的影响。结果:在授粉后16～18 d,2,4-D浓度为2.0 mg/L时诱导最佳;设置N6、NB、改良NB、MS、MB等5种培养基,筛选出改良NB培养基为最佳诱导培养基;分化培养基中添加1 mg/L激动素、0.5 mg/L 6-卞基嘌呤和0.5 mg/L萘乙酸能促进绿苗分化和根系生长。结论:建立了玉米自交系丹598的优良再生体系,为以后的基因转化工作打下了良好基础。     

一种快速、高效花生植株再生体系的建立

快速、高效、重复性好的植株再生体系是转基因育种的基础;本研究以14份不同花生品种的胚小叶为外植体,利用不同激素浓度、组合和不同花生基因型筛选最佳芽诱导培养基、伸长培养基和高效再生基因型。结果表明最佳丛生芽诱导培养基为MSB;＋0．2mg·L-1NAA＋6mg·L-1 6-BA,诱导率为89．50％;最佳伸长培养基为MSB5＋0．2mg.L-1 NAA＋3mg’L-1 6-BA和MSB;＋O．2mg·L～NAA＋4mg·L-1 6-BA＋2mg·L～GA,交替培养,每个丛生芽伸长数达到7．24,时间缩短至3-4周。不同品种再生率的变幅在25．51％～93．01％,大于80％的品种有‘麻油1-1’、‘弗落蔓生’、‘濮花23号’、‘海花1号’。利用‘弗落蔓生’在15周内得到了生根组培苗。     

Isolated microspore culture and plant regeneration in rye (Secale cereale L.)

 An isolated microspore culture and green plant regeneration method for rye (Secale cereale L.) was established. Rye isolated microspore androgenesis was genotype-dependent. PG-96M medium supplemented with 6% maltose gave the highest microspore survival rate after 48 h of culture and the highest embryo/callus yield (930 embryos/calli per 100 anthers from cv. Florida 401). Osmotic pressure in the induction medium played an important role. Pretreatment of the anthers with mannitol was beneficial for the microspore culture. Embryos/calli of a relatively younger age and smaller size had a higher regeneration ability, with the best green plant regeneration rate being 6%. Over 150 microspore-derived green plants have been obtained so far. About 90% of the regenerated plants were spontaneous doubled haploids. This is the first report of isolated microspore culture in true rye resulting in androgenic embryogenesis and plant regeneration. Received: 26 April 1999 / Accepted: 23 November 1999     

酿酒葡萄"梅尔诺"再生系统建立的研究

以酿酒葡萄“梅尔诺”离体胚珠、叶柄为材料．通过控制激素水平、光照和温度等,对建立再生体系的器官发生途径和体胚发生途径进行了研究。结果表明,体胚的诱导和不定芽的再生与基本培养基、叶柄的着生部位、生长调节物质种类和浓度等因素有关。由“梅尔诺”的胚珠愈伤组织再生出体细胞胚的最佳培养基配方为CPSE培养基(CP287 BA 0．2mg／L NOA 1．0mg／L),体细胞胚再生率可达47．50％。“梅尔诺”体细胞胚在CPSE培养基上100％萌发为芽状,将其切断置于培养基MS TDZ 4．0mg／L上可直接诱导出绿色不定芽,再生率为52．25％;同时在培养基MS TDZ2．0mg／L上获得了“梅尔诺”离体叶柄再生不定芽．再生率为62．42％．二者再生的不定芽的最他增殖培养基为MS BA0．5mg／L。“梅尔诺”体细胞胚的萌发芽在WPM培养基中能很好的生根及成苗,并建立了单芽茎段微繁体系。     

石蒜组织培养和植株再生的研究

以石蒜的双鳞片为外植体,MS为培养基,对石蒜组织培养适宜条件进行筛选,并对石蒜的植株再生进行了研究。结果表明：MS＋3mg/L6-BA＋0.3mg/L NAA是不定芽增殖的最佳激素组合,诱导率可达73.99%;石蒜生长旺盛期的11月是芽诱导率最高的时期,同时,MS＋0.5mg/L6-BA＋0.8mg/L NAA是较好的生根培养基,生根率可达95.00%;幼苗移栽至蛭石30%营养土中移栽成活率最高达91.67%。     

Effect of genotype and medium composition on linseed (<Emphasis Type="Italic">Linum usitatissimum</Emphasis>) ovary culture

Breeding linseed (Linum usitatissimum L.) using haploid techniques allows breeders to develop new cultivars in a shorter time period. Many research groups successfully created new linseed genotypes through anther culture; however ovary culture has been the subject of only a few earlier studies. In the present study, the effect of genotype and growth regulators combination on callus induction and shoots regeneration in ovary culture of nine commercially important linseed cultivars was investigated. Ovaries were cultured on modified MS medium supplemented with three different combinations of plant growth regulators. Variable callogenic responses were expressed by all of the genotypes tested on different induction media. The results suggested that specific combination of growth regulators for callus induction must be designed for each genotype. Shoot regeneration from ovary derived callus is a critical phase of the whole gynogenetic process. Differences in adventitious shoot formation frequency among genotypes were demonstrated and four responsive genotypes have been selected. Ovary derived callus from cultivar ‘Mikael’ manifested the highest adventitious shoot formation frequency with a high number of shoots per explant. The optimum ratio of growth regulators for shoot regeneration was shown to depend on the genotype. Cultivars ‘Linola’, ‘Mikael’ and ‘Szaphir’ showed the highest shoot regeneration frequency when callus had originated on induction medium supplemented with 2 mg L⁻¹ BAP and 2 mg L⁻¹ NAA, while combination of 1 mg L⁻¹ BAP and 2 mg L⁻¹ IAA promoted shoot formation in ovary-derived callus of ‘Barbara’. The highest rate of shoots per explant has been obtained in second subculture.