南丰蜜桔胚性愈伤组织的诱导

以南丰蜜桔"杨小—26"花药为外植体,培养诱导胚性愈伤组织。结果表明:在不同取材时期和培养基条件下,胚性愈伤组织发生率是不同的。南丰蜜桔花药培养的取材最佳时期是单核靠边期;培养基LP+TDZ3.0mg/L+A1.03.0mg/L+NAA1.0mg/L+10mg/LAgNO3较适于胚性愈伤组织再生,再生率高达20.3%;培养基配方中AgNO3对胚性愈伤组织再生无显著影响。本研究首次报道了南丰蜜桔花药离体培养,诱导出胚性愈伤组织。     

葡萄原胚团的诱导、保存、扩繁及其细胞学观察

【目的】通过诱导、保存和扩繁葡萄原胚团,为葡萄外源基因转化及基因编辑研究提供受体材料。【方法】以4个欧亚种葡萄品种和1个野生种葡萄的花器官为材料,比较不同外植体在3种培养基上胚性愈伤组织的诱导率,通过二次再生途径诱导、保存和扩繁次生原胚团并利用改良苯酚品红染色法对其进行细胞学观察。【结果】‘无核白’葡萄的雄蕊接种在PIV培养基上胚性愈伤组织诱导率最高(10.8%);初生原胚团细胞核大、染色深且具有旺盛分裂能力,二次再生途径诱导的次生原胚团具有同样的细胞学特征且可以正常成苗。【结论】葡萄体细胞胚诱导受基因型、外植体类型影响显著;二次胚再生途径可以长期诱导、保存和扩繁原胚团,能为葡萄遗传转化持续提供受体材料。     

番木瓜体胚发生及植株再生研究

研究了外植体类型及培养基的不同激素配比等因素对番木瓜愈伤组织诱导和体胚发生的影响。结果表明,番木瓜的子叶和幼胚两种不同的外植体均能诱导愈伤组织、体胚发生和实现植株再生,但在愈伤组织诱导上存在差异,在MS+2,4-D1.00mg/L+BA0.30mg/L+NAA0.05mg/L培养基上诱导的愈伤组织较好。添加一定浓度的硫酸腺嘌呤(ADS)、水解酪蛋白(CH)可以促进番木瓜体胚发生,在生根培养基上生根后,可移栽成活。     

黄瓜体细胞胚途径再生体系的建立及优化

以不同生态型的黄瓜（Cucumis sativus L.）为材料，利用不同基因型建立黄瓜体细胞胚再生体系，并在此基础上对 黄瓜体细胞胚再生体系进行了优化。结果表明：华南型黄瓜9101 和D4 能够诱导出正常的胚状体。以筛选出的9101 为材料， 进一步通过不同组织部位、激素配比、暗培养等对体细胞胚再生体系进行优化，认为子叶节是最佳的外植体，其胚性愈伤 组织诱导的最适培养基为MS + 2.00 mg·L-1 2，4-D，诱导率可达100.00%，出胚的最适培养基为MS+0.10 mg·L-1 2，4-D + 0.5 mg·L-1 KT，其出胚率可达33.33%。此外，在愈伤诱导培养前期给予暗培养条件能提高愈伤诱导率、出胚率及正常再生植株 的获得。对体细胞胚的形态学观察发现，黄瓜体细胞胚发源于子叶节表面的胚性愈伤组织，经历球形胚、心形胚、鱼雷形胚 和子叶胚等时期，最终发育成完整的黄瓜植株。     

长寿花胚性愈伤组织的诱导及胚状体再生

 研究了长寿花胚性愈伤组织的筛选，胚状体的诱导发生、发育过程及植株再生。经过对外观及细胞学观察，看出外观质地疏松、颗粒状的淡黄色愈伤组织细胞圆形且形状规则，是胚性愈伤组织。诱导胚性愈伤组织的最适培养基为：MS+2，4-D 2 mg·L^-1 +BA 0.2 mg·L^-1；胚状体的诱导培养基为MS+BA 2 mg·L^-1 +NAA 0.2 mg·L^-1 +活性炭4 g·L^-1+蔗糖30 g·L^-1，胚状体诱导率可达185个胚状体；胚状体的再生培养基为MS+蔗糖30 g·L^-1。利用石蜡切片对胚状体的发生过程进行了观察。     

辣椒花药培养影响因素的研究

以6种基因型辣椒的花蕾为材料,比较了不同基因型辣椒花蕾的消毒效果,辣椒基因型及花药接种量对花药培养的影响。研究结果表明,本试验的消毒方法获得了较好的消毒效果,3个基因型材料均未受污染,其他最高的污染率为23.08%;50%的基因型能诱导出胚状体,出胚率1.18%～1.38%,小果型的辣椒不能诱导出胚状体;所有基因型均能诱导出愈伤组织,出愈率17.65%～69.44%;花药接种量对花药愈伤组织诱导有影响,增大接种量,出愈率上升0.77%～13.67%,但对胚状体诱导影响不明显;在愈伤组织的继代培养中,接种量对其生长的影响因基因型不同而不同,所有基因型的愈伤组织在第一次继代培养中均不能分化出胚状体或不定芽。     

麦草畏、水杨酸对西洋参成熟种胚体细胞胚发生的影响

以西洋参成熟种胚为试材,以不加激素和添加0.5mg/L 2,4-D的MS培养基为对照,研究了不同浓度(0.05~2.00mg/L)麦草畏、水杨酸对西洋参体胚发生的影响。结果表明:未添加激素的空白培养基没有愈伤和体胚发生;在附加2,4-D的培养基中愈伤和体胚发生率为100%;5个浓度的麦草畏和水杨酸均能诱导出体胚,其中1.00mg/L麦草畏的效果最佳,体胚发生率为56.60%,水杨酸处理下体胚发生率最高为7.50%;在各种浓度的水杨酸中种胚生长量比对照小且褐化严重。说明麦草畏、水杨酸均可诱导西洋参成熟种胚体细胞胚发生,诱导能力2,4-D麦草畏水杨酸;水杨酸对种胚生长有抑制作用。     

黄瓜(Cucumis sativusL.)花药培养

以卡罗尔、HH1-8-57与津绿农家乐3个不同基因型黄瓜品种为试材,在Kumar等、Song等和詹艳等试验研究基础上,设计试验分别探讨低温预处理时间、培养基成分(胚性愈伤组织诱导培养基、胚状体诱导培养基)和基因型等因素对黄瓜花药培养的影响。结果表明:在4℃低温预处理2d时,胚性愈伤组织诱导率显著高于其他处理;在MS+1.0mg·L-12,4-D+0.5mg·L-16-BA+3%蔗糖+0.8%琼脂培养基上胚性愈伤组织诱导率最高,达到81.3%;在MS+0.1mg·L-1NAA+3.0mg·L-16-BA+3%蔗糖+0.8%琼脂培养基上胚状体诱导率最高,为40.0%;基因型间胚性愈伤组织诱导率及胚状体诱导率差异显著,津绿农家乐品种胚性愈伤组织诱导率最高,达到81.1%,HH1-8-57胚状体诱导率最高,为40.0%。本研究从2份试材中成功地诱导出胚状体并获得了黄瓜单倍体再生植株。     

杧果幼胚发育阶段对离体培养物诱导效果的影响

【目的】明确幼胚愈伤诱导和胚抢救最佳时期,为杧果高效体胚发生体系构建及胚抢救技术有效利用提供依据。【方法】以'热农1号'杧果为材料,探讨幼胚不同发育时期和植物生长调节剂对愈伤组织诱导的影响,同时比较了幼胚不同发育时期对幼胚萌发的影响。【结果】授粉后25 d幼胚处于球形胚期,30 d处于心形胚期,35 d处于鱼雷胚期,40 d之后处于子叶胚期。授粉后40 d早期子叶胚愈伤组织诱导率显著高于其他时期;早期子叶胚在含有3.0 mg·L~(~(-1))2,4-D、1.0 mg·L~(~(-1))KT(或0.5 mg·L~(~(-1))KT及0.5 mg·L~(~(-1))ZT)培养基(PM3和PM4)中愈伤组织诱导率最高,为54.4%～62.2%,显著高于其他培养基。授粉后35 d鱼雷胚的萌发率最高,为47.2%,显著高于其他时期。愈伤组织能经体胚发生途径正常成苗,幼胚萌发后也能正常发育成苗。【结论】'热农1号'杧果幼胚愈伤诱导最适宜的时期是早期子叶胚,胚抢救最适宜的时期是鱼雷胚阶段。     

山核桃间接体细胞胚发生和植株再生

 以山核桃（Carya cathayensis Sarg.）自然授粉后10周的幼胚为外植体,对影响胚性愈伤组织和体胚发生的主导因子（基本培养基、植物生长调节物质等）进行了比较分析;对影响体胚萌发的脱水处理时间进行了比较;并采用石蜡切片法对幼胚脱分化产生胚性愈伤组织及体胚发生发育过程进行了组织细胞学观察。结果表明,幼胚胚轴和子叶接种在基本培养基1/2 MS上,胚性愈伤组织诱导率显著高于其它处理,达45.3%。接种于基本培养基DW中的幼胚体细胞胚诱导率显著高于其它培养基处理,达16.7%。显微镜下可观察到球形胚、心形胚、鱼雷胚和子叶胚。培养基添加1.0 mg · L-1 6-BA和0.01 mg · L-1 picloram（氨氯吡啶酸）组合时,胚性愈伤组织诱导率最高,为48.6%;而1.0 mg · L-1 6-BA与0.001 mg · L-1 picloram组合,体胚诱导率最高,为23.6%。体细胞胚发生方式属于间接体胚发生。用饱和Ca(NO3)2 · 4H2O对体胚进行脱水处理,脱水处理3 d后萌发率为39.03%,显著高于对照及其余处理。将脱水处理后的体胚接种至WPM基本培养基中,光照条件下培养,10周后可长成具3 ~ 4片真叶的完整植株。     

仙客来体细胞胚发生和发育过程中淀粉粒的变化

 以仙客来（Cyclamen persicum Mill.）开花植株的新生叶片为外植体，诱导筛选胚性愈伤组织，并使其进一步发育成体细胞胚。组织切片观察结果表明：胚性愈伤组织由胚性和非胚性细胞组成，胚性细胞多以胚性细胞团的形式存在，胚性细胞团起源于诱导的胚性决定细胞。体细胞胚起源于单个胚性细胞，经多细胞原胚、球形胚、心形胚和鱼雷胚等时期发育成完整植株。在体细胞胚的发生发育过程中，淀粉粒出现4次积累高峰，分别为胚性细胞、球形胚、早期鱼雷胚和成熟胚发育成完整的小植株时期，淀粉代谢与体细胞胚发生、发育及小植株的形态建成密切相关。     

Effect of sucrose concentrations on somatic embryogenesis in carnation (Dianthus caryophyllus L.)

The effect of sucrose concentration on callus induction followed by differentiation of embryogenic callus derived from petal explants of four carnation cultivars (Nelson, Sagres, Spirit and Impulse) was investigated. Embryogenic calli were produced on Murashige and Skoog [Murashige, T., Skoog, F.A., 1962. Revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant 154, 73–479] basal medium (MS) culture medium containing six concentrations of sucrose (3, 6, 9, 12, 15 and 18%, w/v) all supplemented with 9 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.8 μM 6-benzyladenine (BA). Maximum frequency of embryogenic callus was obtained from the media containing 9 and 12% sucrose. Somatic embryos were induced on a hormone-free MS media containing the seven concentrations of sucrose. Development of somatic embryos was enhanced by increasing sucrose concentration from 1.5 to 12%, while it was reduced in higher concentrations of 15 and 18%. However, normal embryos were not developed in the media containing 1.5 and 3% sucrose. Ninety-five percent of somatic embryos were regenerated to form the entire plantlets when they transferred onto the half-strength hormone-free MS culture medium containing 3% sucrose. Plantlets were also continued to grow normally under greenhouse condition.     

河套蜜瓜子叶诱导形成体细胞胚的研究

 以河套蜜瓜成熟种子子叶为外植体, 研究体细胞胚途径再生培养的主要影响因子, 并对体胚发生过程进行了观察。结果表明; 体细胞胚发生对培养基糖源浓度要求较高, MS附加60 g/L的糖源效果最好; 最佳植物生长调节剂组合为2,4-D 2.0 mg/L + 6-BA 1.0 mg/L; 1日苗龄子叶为最佳外植体源; 在诱导培养基上经暗培养1周、光培养1周, 再转入胚发育培养基, 有利于成熟胚的产生; 体细胞胚可产生在愈伤组织内部或表面、外植体表面、不定根上及外植体维管束的束中分生组织等部位。     

Influence of high concentrations of cytokinins on the production of somatic embryos by germinating seeds of tomato,aubergine and pepper

Summary

Somatic embryos of tomato, aubergine and pepper were initiated from intact seedlings when seeds were cultured on medium containing 6-benzylaminopurine (BAP) or thidiazuron (TDZ). The percentage of explants producing somatic embryos was highest for aubergine on media containing low concentrations of BAP, i.e. 0–10 mg l^–1, for tomato at 15–20 mg l^–1 and for pepper at 40–80 mg l^–1. Aubergine and tomato produced fewer somatic embryos per responsive seedling when cultured on medium containing TDZ, and pepper did not produce any somatic embryos on media containing 0–20 mg l^–1 1 TDZ. Morphogenesis of the seedlings producing somatic embryos was similar for all the genotypes, i.e. the seedlings were dwarf, only the cotyledons expanded, development of the apical meristem and the root were suppressed and a ring-like crown of nodular tissue developed at the base of the hypocotyl from which somatic embryos were initiated. Co-cultivation of tomato and aubergine seeds with seeds of pepper in media containing 0, 5, 10, 15 and 20 mg l^–1 BAP inhibited somatic embryogenesis in tomato and aubergine instead of assisting somatic embryogenesis in pepper. This is discussed in relation to the recent findings for the induction of somatic embyrogenesis in peanut (Arachis hypogea L.) and the role of BAP and TDZ.     

Recurrent somatic embryogenesis and plant regeneration in Angelica glauca Edgew., a critically endangered medicinal plant of the Western Himalaya

Summary

Secondary somatic embryogenesis and plant regeneration from seedling explants of Angelica glauca, an endangered medicinal plant of the Himalaya, is reported for the first time. Callus was obtained from all the explants tested in the present study (i.e., epicotyls, hypocotyls, and cotyledonary nodes). The highest frequency of callus formation (95.8%) was observed using epicotyl explants on 4.0 µM 2,4-dichlorophenoxyacetic acid (2,4-D), whereas 70.8% of hypocotyl explants, and 58.3% of cotyledonary nodes produced callus. One-hundred percent embryogenic callus was induced from epicotyl explants in 2.0 µM 6-benzyladenine (BA) and 2.0 µM μnaphthaleneacetic acid (NAA), together with the maximum number of somatic embryos (34.2 embryos per explant). Cotyledonary nodes did not produce somatic embryos. Histological studies confirmed the induction of somatic embryogenesis. Somatic embryos germinated into plantlets upon transfer to half-strength Murashige and Skoog (MS) medium without added plant growth regulators. We observed 85% survival of these plantlets under field conditions. The development of secondary embryos was also observed when primary embryos were sub-cultured on full-strength MS medium containing 2.0 µM NAA plus 2.0 µM BA. This system of recurrent somatic embryogenesis provides a route for gene transfer and also for the large-scale production of this critically endangered medicinal plant.     

芸芥体细胞胚胎发生的组织细胞学研究

以芸芥子叶为外植体, 在MS + 2, 4-D 1.0 mg·L^-1培养基上培养2周后可诱导体细胞胚胎的发生。胚性愈伤组织在MS + 2, 4-D 0.2 mg·L^-1培养基上大量增殖, 体细胞胚胎在N₆培养基上成熟后, 可在附加0.1 mg·L^-1 IBA的1/2MS培养基上生长。切片观察表明: 芸芥体细胞胚胎为单细胞发生方式, 与合子胚形成过程相似。芸芥胚性细胞具有细胞小、胞质浓、核大、核仁明显, 排列紧密的特点, 首先由单个细胞分裂形成2 - 细胞原胚, 2 - 细胞原胚分裂成3 - 细胞原胚或4 - 细胞原胚, 3 - 细胞原胚或4 - 细胞原胚继续分裂形成多细胞原胚, 进一步发育成一个成熟的体细胞胚胎。     

百合类体细胞胚和体细胞胚的形态学与组织学研究

 以东方百合‘Siberia’的无菌苗叶柄为试材,诱导产生初始分生结节组织。其后对分生结节组织进行分化再生采用体式显微镜观察和石蜡切片分析的方法对诱导得到的类体细胞胚和体细胞胚再生结构进行形态学和组织学观察。百合类体细胞胚结构呈球形至心形结构或芽状,组织内部主要由薄壁细胞组成,与母体组织存在着明显的维管组织联系,具有多个维管组织中心,其外形与体细胞胚极为相似。百合体细胞胚发源于分生结节表面新生的胚性愈伤组织,经历球形、椭圆形和子叶形胚阶段发育形成完整的植株,在整个发育过程中与母体愈伤组织在整个发育过程中无维管组织联系,且它的根芽两极同时发生,相互联系。研究结果表明,两种再生结构虽然具有极相似的外观形态,但是在发育方式和内部组织结构上截然不同。     

Histological examination of callogenesis and adventitious embryogenesis in immature ovary culture of grapevine (Vitis vinifera L.)

Summary

Histological examinations of callogenesis and adventitious embryogenesis in immature ovary culture of grapevine (Vitis vinifera L. ‘Neo Mat’) were carried out during different phases of ontogenetic development. Adventitious embryos and/or embryogenic calli were obtained when immature ovary explants were cultured on a callus induction medium (C medium) for two months followed by transfer of callus-forming explants onto an embryogenesis induction medium (E medium). Microscopic observations of serial sections of the explants revealed that the calli formed on C medium were initiated preferentially from receptacle parenchyma cells. No cell division in the embryo sac was observed and most of them degenerated four weeks after the onset of culture. Two to four weeks after transfer of the explants onto E medium, calli characterized by dense cytoplasm, conspicuous nuclei and thick cell walls were newly formed in the initially-formed, receptacle-derived ones. Proembryos simultaneously developed in the newly formed calli, indicating that they were embryogenic calli. Cell division of embryo sacs was never observed even on E medium, and adventitious embryos and embryogenic calli were hence of somatic origin. Adventitious embryos developed asynchronously and passed through globular, heart, torpedo and cotyledonary stages. These adventitious embryos germinated and developed into plantlets following their transfer onto a plant growth regulator-free medium.     

Evaluation of the efficiency of somatic embryogenesis in guava (Psidium guajava L.)

Summary

An embryogenic protocol for plant regeneration of guava (Psidium guajava L.) was established using 10-week post-anthesis, zygotic embryo explants. Somatic embryogenesis was induced on Murashige and Skoog medium (MS) containing 3% (w/v) sucrose, 0.8% (w/v) agar and various concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) by continuous treatment of the zygotic embryo explants. Somatic embryos appeared as globular structures at the end of the third week from culture initiation, and heart-shaped, cotyledonary-stage, and torpedo-stage embryos appeared within the next few weeks. The development of somatic embryos was asynchronous and showed five-to-seven discernible stages. Depending upon the response of the somatic embryos during their maturation, germination, acclimatisation, and encapsulation, they were grouped into one of three categories. The preferred type of somatic embryos (≥ 1.5 mm) were called the “elongated torpedo” (ET) category. The slightly less-preferred type of stomatic embryos (from 1.0 – 1.5 mm) were termed the “short torpedo” (ST) category. The least preferred types of somatic embryos, at the cotyledonary, heart-shaped, and/or globular stages of development (< 1.0 mm), were grouped into a third category designated “CHG”. The suitability and efficacy of various growth regulators and other treatments were assessed based on six different embryogenic parameters: (i) the frequency of embryogenesis; (ii) the intensity of embryogenesis, defined as the average number of somatic embryos produced per culture (“ANEPC”); (iii) the frequency of ET somatic embryos; (iv) the frequency of ST somatic embryos; (v) the frequency of CHG somatic embryos; and (vi) the overall efficiency of embryogenesis, defined as the potential of a treament to produce somatic embryos at the ET or ST stages, or at both stages of development, that could be converted into plantlets. In the present report, we found that 0.01 mg l^–1 2,4-D gave the maximum frequency and intensity of embryogenesis. But the highest frequencies of ET and ST somatic embryos were produced on MS medium containing 3% (w/v) sucrose and 0.001 mg l^–1 2,4-D, while CHG embryos were produced at the highest frequency on the same medium, but containing 0.5 mg l^–1 2,4-D. It was difficult to calculate the most effective concentration of 2,4-D for somatic embryogenesis based on parameters (i) – (v) above. Hence, quantitative estimations of the efficiency of embryogenesis (sixth parameter) were imperative in order to analyse the potential of the different treatments. The highest efficiency of somatic embryogenesis was achieved by continuous treatment of 10-week post-anthesis, zygotic embryo explants with 0.01 mg l^–1 2,4-D on full-strength MS agar medium containing 3% (w/v) sucrose. These somatic embryos matured normally on the same medium, and germinated well both on half-strength solid and in half-strength liquid MS medium containing 3% (w/v) sucrose. They grew in full-strength liquid MS medium with 3% (w/v) sucrose and showed maximum survival upon transfer to soil and hardening. Evaluations of the efficiency of somatic embryogenesis in guava, based on the six parameters defined above, have also helped us to understand and evaluate processes for high efficiency micropropagation in other species.     

Somatic embryogenesis in a broad spectrum of grape genotypes

Somatic embryogenesis is the preferred method for cell-to-plant regeneration of grapevine. In this study, we tested the embryogenic capacity of anther-derived calli from 59 grape genotypes, representing a diverse group of Vitis vinifera and hybrid varieties, and hybrids and accessions of non-vinifera Vitis species. Most genotypes were tested on two types of media: MST1 medium, which contained plant growth regulators (PGRs) 2,4-dichlorophenoxyacetic acid (2,4-D) and thidiazuron (TDZ), and MSE medium, which contained 2,4-D and 6-benzylaminopurine (BAP). Twenty-four of the grape genotypes produced embryogenic callus on one or both of these media, eighteen of which have not been reported to form somatic embryos before. The results also suggested that the various PGR combinations are differentially effective at inducing somatic embryos in various classes of grape genotypes. For example, seven of the eight V. vinifera conv. occidentalis varieties brought forth somatic embryos on MSE medium, and three out of four American Vitis genotypes produced somatic embryos on MST1 medium. We could not observe any apparent association between frequency of callus formation and embryogenic capacity of the anthers.