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。     

‘奥迪亚’葡萄杂交后代胚挽救及无核性状分子鉴定

【目的】为了研究影响杂交后代胚挽救萌发率的主要因素,并对其无核性状进行早期鉴定,【方法】以无核、抗病葡萄品种‘奥迪亚’为母本,‘玫瑰香’、‘摩尔多瓦’和‘红地球’3个主栽品种为父本进行杂交,通过L9(33)正交试验,建立简单高效的胚挽救体系,并用分子标记对杂交苗无核性状进行鉴定。【结果】结果表明,影响胚挽救萌发率的最关键因素为接种时期,其次为培养基种类和NAA浓度;以‘奥迪亚’为母本的杂交胚其适宜接种程序为授粉后49～56 d剥取胚珠,接种在B5或NN69培养基上充分发育70 d,再转入1/2 B5培养基上萌芽,‘奥迪亚’ב摩尔多瓦’杂交胚通过此程序萌发率达到了50.48%;经分子标记鉴定,各组合后代的无核株系均超过50%。【结论】利用胚挽救方法以‘奥迪亚’为杂交母本可高效创新葡萄无核种质。     

无核葡萄杂交胚珠的培养技术

将无核ｘ无核5个杂交组合的葡萄果德分别在授粉后35、40、45、50、55d采回，取出胚珠接种在幼胚培养基中进行培养。60～70d后转入胚萌发培养基中，待胚萌发10d左右，再转入成苗培养基中使其发育成正常幼苗。结果表明，较适宜胚发育的培养基是B。和Nitsch为基本培养基，附加GA30．2mg·l-1、IAA1．5mg·l-1、ZT1．0mg·l-1；5个接种时间以授粉后45d胚发育率最高；各组合中白乐ｘ红脸无核的出苗率最高，所获植株占发育胚的24．9％。     

抗病抗寒无核葡萄胚挽救研究

【目的】为了培育抗病抗寒无核葡萄新品种,【方法】以2个种子败育型无核品种‘波尔莱特’、‘红无籽露’作母本,以抗病抗寒的中国野生山葡萄株系‘黑龙江实生’、‘双优’及欧山杂种‘北醇’、‘00-1-10’(‘玫瑰香’ב黑龙江实生’)分别作父本杂交,授粉51 d后将胚珠分别接种于ER和MM4培养基上进行胚挽救,培养60 d后在WPM+BA 0.2 mg.L-1培养基上诱导成苗。【结果】结果表明,2个母本品种胚挽救的适宜培养基不同,‘波尔莱特’作母本适合于ER培养基,‘红无籽露’作母本适合于MM4培养基。2个母本基因型对胚珠的发育率和成苗率的影响差异不大,‘波尔莱特’略优于‘红无籽露’;但4个父本基因型对胚挽救效果影响较大,以欧山杂种‘00-1-10’和‘北醇’作父本的杂交组合胚株的发育率和成苗率明显高于山葡萄‘黑龙江实生’、‘双优’作父本的杂交组合。共获得无核葡萄胚挽救新种质50个株系。【结论】在胚挽救过程中,不同基因型的无核葡萄适宜于不同的基本培养基,欧山杂种比山葡萄更适宜于作杂交的父本。     

“新葡7号”葡萄胚挽救最佳接种时期研究

正近年来,随着生物技术的发展,胚挽救技术在葡萄育种上的研究应用,为培育优质无核葡萄品种提供了新思路和崭新的途径,丰富了无核葡萄有性杂交的组合方式,提高了无核葡萄品种的选育效果。无核葡萄胚的发育受基因型的严格控制。胚龄越小越难培养,而胚龄越大则存在胚萎缩退化的可能,所以掌握无核葡萄胚珠败育时期,确定胚珠接种的适宜时间,是胚珠培养能否成功的前提。郭修武等人研究了胚珠接种时间和培养基种类对无核葡萄胚挽救的影     

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。     

接种时期和培养基对无核葡萄胚挽救的影响

以4个组合康能无核×金星无核、雷明无核×金星无核、金星无核自交、雷蒙无核自交为试材, 采用L25 (55 ) 正交设计, 研究了接种时间和培养基种类对无核葡萄胚挽救的影响。结果表明, 影响无核葡萄胚挽救最重要的因素是接种时期, 其他因素影响程度因组合而不同, 最佳接种时期在花后45～60 d,最高出苗率13.64%～75.23%。对胚珠进行一定处理有助于促进胚珠的萌发出苗, 其中横切处理与低温处理可以显著提高胚珠萌发出苗率。     

红宝石无核葡萄胚珠培养成苗技术研究

以自然授粉的红宝石无核为试材,观察了胚珠生长状况,对胚珠离体培养与成苗技术进行了探索,结果表 明:红宝石无核的胚珠发育在花后35 d是其胚挽救的最佳时期。此时,取出胚珠接种在Nitsch+IAA 2.0 mg/L+GA 0.4 mg/L,附加水解酪蛋白100mg/L,活性炭2g/L的培养基中培养120 d,胚发育率为58％,后将胚珠切开处理并转接 到胚萌发培养基1/2MS+6-BA 0.5 mg/L中萌发,萌发率为16％,在1/2MS+6-IBA 0.2mg/L培养基上成苗,小苗生长 最好。通过前期保湿等措施,小苗移栽成活率达100％。     

无核品种中葡萄18号胚挽救技术体系的建立及其杂交后代早期选择

[目的]无核品种是葡萄的主要育种目标之一,通过建立无核品种中葡萄18号的胚挽救技术体系,为无核葡萄的胚挽救育种提供参考依据.[方法]通过研究不同胚珠取样时间(盛花后52、53、54、57、58、61和65 d,DAF),培养基类型(NN 和 ER)及相态(固-液双相和固相),氨基酸(0,2.5mmol·L-1 Ser、Cys、Gln),椰汁(0、100、200 mL·L-1),GA3/KT/NAA组合及4种细胞分裂素TDZ(0和0.2 mg·L-1)、6-BA(0和0.2 mg·L-1)、KT(0、0.2和0.4 mg·L-1)和ZT(0、0.2和0.4 mg·L-1)对中葡萄18号胚发育率、胚萌发率或成苗率的影响,并利用无核基因SSR标记对胚挽救杂种苗进行无核性状早期鉴定,初步建立该品种的胚挽救技术体系.[结果]中葡萄18号在DAF 61取样时胚发育率最高(34.33％);基本培养基NN(29.33％)较ER(25％)的胚发育率更高,且固-液双相培养基的效果较固相更佳;添加2.5 mmol·L-1 Ser(40.74％)、Cys(43.33％)或Gln(44.21％)的胚发育率均高于对照(29.33％);添加200mL·L-1椰汁的胚发育率(37.10％)显著高于对照(29.33％).在3种激素GA3/KT/NAA组合中,发现仅添加GA3和KT时,成苗率最高(60％),而添加NAA的组合均出现异常分化现象,且成苗率较低.四种细胞分裂素TDZ、6-BA、KT和ZT中,添加0.2mg·L-1 ZT更适合胚萌发及成苗,正常成苗的比率达76.36％.分别利用无核SSR标记P3_VvAGL11和5U_VviAGL11对中葡萄18号x玫瑰香的406个杂交株系进行无核性状检测,初步判定229个后代为无核株系,该组合的无核率为56.4％.[结论]中葡萄18号作为胚挽救母本的最佳取样时间为DAF 61前后,可利用固-液双相培养基NN+2.5 mmol·L-1 Cys/Gln+5.5 g·L-1琼脂+60 g·L-1蔗糖+2 g·L-1活性炭+0.5 g·L-1水解酪蛋白进行胚珠离体培养,暗培养9～10周后,接种裸胚于WPM+0.2 mg·L-1 ZT+20 g·L-1蔗糖+6 g·L-1琼脂+1 g·L-1活性炭进行胚萌发培养.鉴定出拥有无核基因SSR标记的胚挽救苗229株,是潜在的无核葡萄新种质.     

花前施用6-BA对抗寒无核葡萄胚挽救效果的影响

在花前14 d,分别对4个抗寒无核葡萄杂交组合‘火焰无核’×00-1-5、‘秦红10号’×00-1-5、‘秦红2号’ב木星’和‘红宝石无核’×00-1-5的母本花序施用不同浓度6-BA,以MM3固液双相培养基作为胚发育培养基,WPM固体培养基作为胚萌发培养基进行胚挽救,研究花前施用6-BA对胚挽救效果的影响。结果表明:花前施用6-BA对胚的发育起促进作用,但不同浓度6-BA对不同杂交组合的胚挽救效果不同,其中‘秦红10号’×00-1-5、‘秦红2号’ב木星’和‘红宝石无核’×00-1-5在6-BA浓度为50mg/L时,胚发育率、萌发率和成苗率最高;而‘火焰无核’×00-1-5在6-BA浓度为30 mg/L时,胚发育率、萌发率和成苗率最高。无核品种‘秦红2号’‘红宝石无核’最适宜作母本,‘火焰无核’胚挽救效果较差。     

Effects of exogenous application of plant growth regulators on the development of ovule and subsequent embryo rescue of stenospermic grape (Vitis vinifera L.)

Seedless grapevine cultivars (Vitis vinifera L.) are widely grown in Europe, America and Asia. Abortion of zygotic embryos in seedless grapes largely limits the efficiency of breeding of seedless cultivars through genetic crossing. The present study was designed to investigate effects of exogenously applied plant growth regulators (PGRs) to the grapevines in field condition on ovule and subsequent embryo rescue of seedless grapes of small seed traces. First experiment was performed by measuring ovules weight, proportion of each category ovules in maturity and embryo development in vitro of seedless grape cv. Centennial Seedless, Thompson Seedless and Crimson Seedless sprayed by chlormequat (CCC), benzyladenine (BA), ethephon (CEPA) and putrescine (Put). The effects of different application concentration and date of CCC were further evaluated in Centennial Seedless in later experiment. The results showed that exogenous application of all PGRs did not affect the total number of ovules per berries in maturity. CCC increased the ovules weight and proportion of ovules >4 mm in length of three varieties in maturity. The effects of two application times of PGRs on weight of berries and ovules and proportion of each category ovules in maturity were not significantly different. In the proceeding of embryo rescue, CCC at 100 and 1000 mg l⁻¹, BA at 100 mg l⁻¹ and Put at 20 mg l⁻¹ increased the percentage of developed embryos of Centennial Seedless and Thompson Seedless. The results showed that the size of ovules excised for embryo rescue significantly affected embryo formation and plant regeneration. The percentage of embryos formation in ovules >4 mm in length was significantly more than in ovules 2–4 mm in length, no embryo was found in ovules <2 mm in length. Exogenous application of CCC at 100–500 mg l⁻¹ significantly increased percentage of ovules >2 mm in length by 80.0–82.7% in Centennial Seedless, therefore improving embryo formation. The statistical correlation was found between the proportion of ovules >2 mm and embryo formation (r = 0.92) in Centennial Seedless. Among the different spraying time in Centennial Seedless, CCC applied 14 days before bloom produced significantly more ovules >2 mm in length and embryos formation.     

Investigating the most effective factors in the embryo rescue technique for use with ‘Flame Seedless’ grapevine (Vitis vinifera)

Embryo rescue is among the most important techniques used in seedless grapevine (Vitis vinifera L.) breeding and is applied to the female parent. A variety of factors that affect embryo development and germination, as well as plantlet production, in the seedless grapevine cultivar ‘Flame Seedless’ were investigated. The results showed that using Nitsch and Nitsch (NN) medium rather than Emershad and Ramming (ER) medium for double-phase rather than solid phase media produced better developed embryos (DE; 47.49% compared to 24.85%, and 40.69% compared to 31.66%, respectively) and germinated embryos (GE; 28.46% compared to 19.16%, and 29.71% compared to 17.91%, respectively). Preceding embryo rescue, pre-treatment of grapevine clusters with 0.34 mM putrescine (Put) or 0.82 mM cycocel (CCC) increased the percentages of DE and GE. The results indicated that 1.0 mM spermine (Spm), 0.5 mM spermidine (Spd), or 1.0 mM Put in NN culture medium increased the percentages of DE and GE. Using Woody Plant Medium (WPM) as the secondary medium gave higher percentages of GE and developed plantlets (DP) than using Gamborg (B5) medium or Murashige and Skoog (MS) medium.     

High-frequency somatic embryo production from unfertilized ovules of grapes

Somatic embryos were produced in large numbers from cultured, unfertilized ovules of Vitis vinifera, cultivars ‘Cabernet Sauvignon’ and ‘Grenache’, and of the hybrid grape ‘Gloryvine’ (V. vinifera × V. rupestris). Callus of nucellar origin was produced by culturing the ovule explants initially in Nitsch medium containing 5 μM 2,4-dichlorophenoxy acetic acid (2,4-D) or 5 μM β-naphthoxyacetic acid (NOA) plus 1 μM benzyladenine (BA), and then in a medium supplemented with NOA (10 μM) plus BA (1 μM). Embryos were produced when callus was transferred to basal medium containing no auxin or cytokinin. Secondary and tertiary embryos were formed by budding of primary somatic embryos. Prolonged culture of ovular callus in NOA (10 μM) plus BA (1 μM) gave rise to cell aggregates which developed into free-floating roots when transferred to basal medium.     

Breeding of disease-resistant seedless grapes using Chinese wild Vitis spp.: I. In vitro embryo rescue and plant development

Seedless grape cultivars (Vitis vinifera L.) are widely grown in Europe, America and Asia. Fungal diseases are a great threat of them. Several wild Chinese Vitis species showed high resistance to many fungal diseases. Therefore, an investigation was conducted to assess the potential to incorporate these species in a breeding project for development of the disease-resistant seedless cultivars. Hybridization was conducted using V. vinifera as female parents and the wild Chinese Vitis spp. as male parents. In-ovulo embryo rescue was used to develop hybrid plants from the seedless females. An efficient protocol is reported here for the in vitro embryo rescue and plant development from the cross Emerald Seedless × Beichun. Ovules were excised from immature fruits 7 weeks after pollination (WAP) and cultured in the double-phase ER medium supplemented with 6.0% sucrose and 0.3% activated charcoal (AC). Following 8–12 weeks of culture, embryos were removed from the ovules and transferred onto WPM supplemented with 1.0 μM 6-benzyladenine (6-BA), 2.0% sucrose and 0.2% AC and solidified with 0.6% agar. After 8 weeks of culture, the embryos germinated and subsequently grew into whole plantlets. With the optimized parameters developed in the present study, about 34.0%, 91.2% and 77.4% of embryo formation, embryo germination and plant development were obtained, respectively. When this protocol was applied to 11 other cross combinations, genotype was found to significantly influence embryo formation, embryo germination and plant development, with different frequencies of hybrid plants successfully obtained in all crosses.     

无核葡萄品种资源性状的聚类分析

对国家果树种质郑州葡萄圃内的37个无核葡萄品种的16个描述性状和11个测量性状进行了调查,借助SPSS10.0统计软件对结果聚类分析,根据其性状相近程度,将供试的无核葡萄品种划分为3个品种群和5个亚群。同一品种群或亚群内的品种在形态特征上较为相似,遗传背景较为近似,利用不同品种群间的品种进行杂交有望提高无核葡萄育种效率。聚类分析结果可为无核葡萄育种中无核品种×无核品种杂交组合的亲本选择提供参考依据。     

三倍体无核葡萄育种研究进展

综合有关文献综述了三倍体无核葡萄育种研究的最新进展。探讨了三倍体葡萄育种的主要途径如利用二倍体和四倍体杂交、胚乳培养、利用2n配子和芽变选择等。概述了三倍体葡萄的无核机理、三倍体鉴定方法、胚挽救技术应用。简要介绍了国内外育成的7个三倍体无核葡萄新品种尾玲、戴拉王、蜜无核、美嶺、夏黑、无核早红和马格拉契的特征特性,并讨论了三倍体无核葡萄育种存在问题和未来的研究重点如杂交亲本的选择、三倍体杂合胚的发育特点和无核性状的遗传规律等。     

无核葡萄与中国野生葡萄杂种胚败育的某些生理生化变化

 通过对无核葡萄、有核葡萄以及无核葡萄与中国野生葡萄杂种的胚珠进行离体培养, 并结合生理生化指标测定发现: 不同的无核葡萄及其杂种胚败育的时间不同, 胚败育时, 保护酶活性下降, 反应膜脂过氧化水平的MDA 含量上升, 可溶性糖与可溶性蛋白质含量降低, 可溶性淀粉含量变化不大。进行胚挽救时, 应根据不同的杂交组合, 在最佳时间, 即胚发育程度最高、MDA 含量较低时取胚进行培养。