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。     

不同因素对离体培养条件下莲幼胚萌发的影响

为了探索不同因素对离体培养条件下莲幼胚萌发的影响,以正常授粉的莲幼胚为外植体,分别进行了不同胚龄、不同蔗糖浓度和不同激素浓度配比下莲幼胚的萌发试验。结果表明,在含有1.0mg/L NAA和0.5mg/L ZT的诱导培养基上,以3d和4d胚龄幼胚的萌发效果较好,萌发率分别达到31%和36%;以4d胚龄幼胚为材料,当培养基中蔗糖浓度为3%和5%时,幼胚萌发较好,萌发率分别为38%和32%;以4d胚龄幼胚为材料,在含有3.0mg/L NAA和0.5mg/L ZT的诱导培养基上,幼胚萌发率显著提高,萌发效果最好,萌发率达71%。     

假单性结实型无核葡萄胚挽救影响因子研究

【目的】探究无核葡萄胚发育和胚萌发的影响因子,有利于优化无核葡萄胚挽救技术体系及提高其育种效率。【方法】以早黑宝盛花后100 d种胚为试材,采用剥种取胚进行胚离体培养,研究外源激素和营养添加物质对胚萌发成苗的影响,基于熵权TOPSIS(优劣解距离法)分析法筛选出最佳胚萌发培养基;以无核翠宝(盛花后28～38 d)、丽红宝(盛花后28～32 d)和晶红宝(盛花后28～32 d)未成熟幼果为试材,研究不同取样时间和不同培养时间对胚珠发育率的影响,基于熵权TOPSIS分析法综合评价不同形态胚的萌发成苗效果。【结果】在胚萌发培养基中添加0.1 mg·L-1IAA+0.2 mg·L-16-BA+0.5 g·L-1葡萄汁时,胚的萌发率和成苗率显著高于其他。取样时间接近(或达到)胚败育始期时,胚发育率最高。无核翠宝和丽红宝胚珠离体培养10周后的胚萌发率最高(≥50.00%)。不同形态胚的萌发成苗效果不同,子叶形胚的萌发成苗效果最好,其他排序为:鱼雷形胚﹥球形胚﹥心形胚﹥畸形胚。【结论】胚萌发培养基中添加外源物质有利于胚萌发成苗;离体培养败育时期的胚珠能获得更高的胚发育率和多胚率;子叶形胚和鱼雷形胚更易萌发成苗。     

枣的胚败育及幼胚培养研究

 对30 个枣优良品种坐果后30 d 的胚败育情况进行了调查, 结果表明, 败育率高于75 %的高度败育品种占53.4 % , 败育率介于25 %至75 %的中度败育品种占3.3 % , 败育率低于25 %的轻度败育品种占43.3 %。以优良品种‘冬枣’、‘金丝丰’等为试材, 对坐果后10～70 d 幼胚进行培养的结果表明, 枣离体胚接种后主要有3 种生长方式, 即形成愈伤组织、子叶间小叶增生和胚直接萌发成苗。本文还提出了提高枣胚培养及胚培养苗移栽成功的技术要点。     

紫香无核胚培养技术研究

以紫香无核为母本,黎明无核、克瑞森无核、京早晶分别为父本杂交,对杂交胚珠进行培养挽救。结果表明:在授粉后52d接种,3个杂交组合的胚珠均达到最大发育率和最大萌发率;胚珠发育阶段采用ER培养基培养的发育率比用Nitsch培养基的高,但在Nitsch接种的萌发率却比ER的都高;低温(7℃)处理的胚萌发率高于常温(24℃)培养的萌发率。     

磨盘柿败育杂种胚胎发育的细胞学特征

为揭示磨盘柿杂种败育的细胞学机制,采用常规石蜡切片法观察了磨盘柿正常杂种及早期和晚期败育型杂种的胚胎发育过程。结果表明,正常杂种的胚在授粉后35、40、45、55d依次发育至球形期、心形期、鱼雷期和子叶期;与正常杂种相比,早期败育型杂种的初生胚乳核分裂出现异常,多数胚最终只能发育至球形早期;晚期败育型杂种的胚乳没有明显的异常现象,但胚发育滞后,且着生位置偏离珠孔,呈横生或斜生状态;2种败育杂种在种形异常之前胚基本正常,可望借助胚挽救手段分别于授粉后30～40d及60～70d进行离体培养获得F1杂种。     

苦瓜辐照花粉授粉离体胚挽救的研究

为提高辐照花粉授粉不同发育阶段胚的萌发,以剂量为150 Gy的60Co-γ射线进行辐照花粉授粉,利用胚挽救技术进行苦瓜离体胚培养,从培养基、接种方式、光暗处理等方面进行优化研究。结果表明,在不同类型苦瓜中,整粒种子经暗处理10 d后,切掉3/4并保留含胚切块,在MS基本培养基中转光下培养20 d后,可使胚挽救时期提前到授粉后5 d,使授粉后9 d的胚萌发出苗率达9.6%以上,并免去了剥离种皮取胚的步骤,简化了苦瓜离体胚挽救技术。     

冬枣极早期幼胚培养成苗技术研究

以胚龄15 ~ 20 d的球形期后期‘冬枣’幼胚为试材,对培养基种类、种皮保留与否、接种方式、生长调节剂种类、水解乳蛋白（LH）和活性炭（AC）用量、蔗糖浓度等影响极早期幼胚培养的诸因素进行了系统研究。结果表明,采用MS + 3.0 mg ? L-1 IAA + 0.5 mg ? L-1 6- BA + 1 g ? L-1 AC + 0.8 g ? L-1 LH + 30 g ? L-1蔗糖的培养基,去除种皮后将带有胚乳的幼胚合点端接入培养基,在21 ℃下进行培养,胚萌发率和正常胚比率最高可分别达到93%和84%。萌发后的幼胚在MS + IBA 0.4 mg ? L-1的培养基中继续培养,生根率可达40%。其中,去除种皮带胚乳培养和幼胚合点端朝下接种对极早期幼胚的培养至关重要。     

香樟子叶胚培养的研究

以香樟(Cinnamomum caphora L.)子叶胚为外植体,以MS为基本培养基,研究不同浓度植物激素组合对香樟子叶胚培养的影响。结果表明:在该研究所设浓度范围内,MS+BA2.0mg/L+IBA 1.0mg/L子叶胚萌发率为41.7%,对香樟子叶胚萌发的诱导效果相对较好。     

Identification and analysis of differentially expressed proteins during cotyledon embryo stage in longan

The cotyledon stage is a crucial developmental stage during longan embryo development. Two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption ionization-time of flight-tandem mass spectrometry (MALDI-TOF/TOF-MS) were conducted to separate and identify proteins expressed in the longan cotyledon embryos at different development stages. A total of 28 proteins that exhibited regulated expression were successfully identified with a protein identification success rate of 72.2%. The 28 proteins were assigned to six functional classes based on their putative biological functions: energy and metabolism (21%), secondary metabolism (18%), protein metabolism (21%), cell division (11%), antioxidation (4%), nucleic acid metabolism (4%), hormonal regulation (4%) and unknown proteins (18%). Interestingly, three enzymes involved in flavonoid biosynthesis, whose expression in embryos had not been observed previously were clearly up-regulated during the cotyledon stage of embryo development in longan. Identification and analysis of the 28 proteins would shed new lights on further understanding of the biochemical and physiological processes of the embryo development at cotyledon stage in longan.     

Temperatures affecting embryo development and seed germination of Christmas rose (Helleborus niger) after sowing

Embryo development followed by seed germination was investigated in seeds of Christmas rose (Helleborus niger) held under different temperature conditions. The seeds had a heart-shaped embryo when dispersed from parent plants. That embryo developed rapidly to the torpedo stage at 25 °C, after which no further development occurred. When seeds with a torpedo-stage embryo were held at 15 °C, the embryos developed to the cotyledon stage, but no further germination occurred. Similar phenomena in the course of embryo development were observed in seeds held in a non-heated polyethylene house, in which the embryos developed to the torpedo stage at 20 °C or more, while further development to the cotyledon stage was attained only at temperatures of 15 °C or less. Seeds with a cotyledon-stage embryo rarely germinated until they were treated at 4 °C for more than 8 weeks. These results suggest that seeds of H. niger have a deep, simple morphophysiological dormancy caused by the combination of rudimentary embryos and a physiological dormancy that can be broken by cycles of warming and chilling.     

用于无核葡萄选育的胚挽救技术研究

通过4a的连续试验,观察了无核葡萄果粒及胚珠发育变化,对胚挽救的多个影响因素包括品种、接种时间、培养基和培养方式进行了研究。结果表明,红脸无核、底来特、黎明无核作母本的胚珠萌发率较高;各品种适宜的接种时间为无核白花后26d,红脸无核花后35d,爱莫无核×火焰无核花后48d,底来特×红宝石无核花后56d;用Nitsch作基本培养基优于B5;添加BA0.2mg/L获得的胚珠萌发率最高;常温黑暗、固体培养有利于胚珠的发育。已定植结果的无核葡萄杂交胚挽救后代的无核比率平均为58.6%。将胚挽救技术用于无核葡萄育种是十分有效的。     

陇东马牙枣幼胚培养

以陇东马牙枣盛花后50～60d的幼胚为试材,采用L9(3)4正交设计,筛选适宜的幼胚萌发培养基。结果表明,基本培养基、激素配比、蔗糖的含量对幼胚萌发都有显著的影响;其中MS-H培养基的萌发率最高为47.62%;ZT最适宜含量为0.5mg/L,IAA最适宜含量为0.6mg/L;较高的蔗糖含量(50g/L)有利于胚的萌发。幼胚萌发的适宜培养基为MS-H+ZT0.5mg/L+IAA0.6mg/L+LH0.5g/L+蔗糖50g/L+琼脂4.0g/L,剥去种皮,幼胚萌发效果较好。当胚萌发后,将胚从胚乳中或愈伤组织中剥离继续诱导愈伤组织,再经诱导不定芽,生根获得正常植株。     

Embryogenesis and plant regeneration from anther culture in loquat (Eriobotrya japonica L.)

The object of this study was to induce embryogenesis and establish plant regeneration system for anther culture in loquat (Eriobotrya japonica L.). Cold pretreatment was a key factor, and supplement of 2,4-D in the media was absolutely necessary for induction of calluses from cultured loquat anthers. The best response of anthers to in vitro culture was obtained when a 48-h cold pretreatment was employed to flower buds at 4 °C in darkness. Genotype was a decisive factor for embryo differentiation. When anther-derived calluses of three loquat cultivars, i.e., cv. ‘Longquan1’, ‘Dawuxing’ and ‘Zaozhong6’, were transferred to embryo differentiation medium, embryos were induced only for cv. ‘Dawuxing’ on MS medium containing 3% sucrose, 0.23 μM ZT in combination with 0.05 μM NAA + 0.05 μM IBA or 0.11 μM NAA + 0.10 μM IBA, and the differentiation rates were 3.33% and 10.00%, respectively. The results of histological studies showed that embryos developed through typical globular, heart, torpedo and cotyledon stages after 4 weeks of culture. The treatment designed to mature the embryos on medium containing 3% of sucrose at 4 °C under darkness for 4 weeks was effective for subsequent embryo germination and plant conversion, which gave rise to 72.5% plant recovery. Cytological studies showed that 26 plantlets were haploids (n = 17) and the remaining 4 plantlets were diploids for the 30 regenerants tested.     

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。     

六月鲜枣愈伤组织诱导及胚状体发生

采用5因素4水平的正交试验设计,对六月鲜枣的幼叶、嫩茎、冷藏5d后的花药、花后48～50d的胚乳,利用3因素3水平的正交试验设计,对花后58～60d的幼胚及子叶,进行愈伤组织诱导再分化试验。探讨了不同激素、碳源和基本培养基对产生愈伤组织的影响。结果表明:碳源对胚乳、花药器官,基本培养基对嫩茎、幼叶、花药愈伤组织的诱导有显著影响,而且生长素种类和浓度对不同外植体的愈伤组织诱导作用不同;嫩茎、子叶及幼胚较易诱导产生愈伤组织,但只有子叶及幼胚产生的愈伤组织具较强的再生能力,并可诱导出不定芽,不定芽分化率分别为51.1%和9.5%,ZT较BA易诱导愈伤组织产生不定芽,不定芽转移生根成苗获得了完整植株。另外,用子叶及幼胚培养获得了大量胚状体,且胚状体最终可发育成苗。     

‘乌叶’荔枝胚胎发育过程特异蛋白的变化

 应用改进的IEF2SDS2PAGE 技术比较分析了荔枝胚胎分化发育过程中蛋白质组分 的变化。结果表明, 大多数蛋白质组分在各发育时期的图谱相似, 但不同发育时期存在变化。 试验共发现了前期鱼雷胚的43. 7 kD、pI 3. 5 和55 kD、pI 9. 4 , 后期鱼雷胚的23. 4 kD、pI 6. 6和25. 1 kD、pI 6. 8 以及前期子叶胚的25. 1 kD、pI 9. 3 和64. 6 kD、pI 5. 9 等6 个新出现的特异蛋白。后期心形胚的图谱上蛋白质点数最多, 可分辨蛋白质斑点数约300 个, 这与蛋白质含量的测定结果一致。以上特异蛋白在多次重复中均显示出良好的重现性。因此认为, 这些特异蛋白在胚胎分化发育中可能起重要作用。