中国农业科学 ›› 2020, Vol. 53 ›› Issue (16): 3344-3355.doi: 10.3864/j.issn.0578-1752.2020.16.012
收稿日期:
2020-01-09
接受日期:
2020-06-05
出版日期:
2020-08-16
发布日期:
2020-08-27
通讯作者:
王跃进
作者简介:
贾姗姗,E-mail:基金资助:
JIA ShanShan1(),LUO QiangWei2,LI ShaSha1,WANG YueJin1(
)
Received:
2020-01-09
Accepted:
2020-06-05
Online:
2020-08-16
Published:
2020-08-27
Contact:
YueJin WANG
摘要:
【目的】研究葡萄玫瑰香味性状,为无核香味葡萄育种提供重要的材料基础,探究亲本基因型、胚发育形态以及生长调节剂对胚萌发的影响,进一步优化无核×玫瑰香味组合胚挽救体系,并结合分子标记技术初步对杂交后代进行无核性状鉴定。【方法】利用顶空固相微萃取结合气质联用方法,对10个玫瑰香味和无玫瑰香味葡萄品种的果实香味物质含量进行测定,从中筛选出浓香型品种作为亲本;结合课题组前期研究结果,以6个玫瑰香味品种为父本,5个欧洲无核品种为母本,配置13个杂交组合;无菌条件下,从幼果中剥离杂种胚珠,离体黑暗培养8周,随后将发育的杂种幼胚接种于添加不同激素浓度和比例的胚萌发培养基中,通过优化胚萌发培养基配方,提高幼胚萌发率和成苗率。幼苗经温室炼苗后,将成活的F1代移栽至大田。利用分子标记早期辅助选择具有无核性状的F1代杂种株系。【结果】萜烯类物质是玫瑰香味的主要呈香物质,8个玫瑰香味品种均能检测到萜烯类物质,其总含量为0.0246—1.3824。筛选出‘亚历山大’‘阳光玫瑰’‘玫瑰香’等玫瑰香味物质含量相对较高的品种,可作为杂交亲本,用于创制兼具无核和玫瑰香味新种质;利用胚挽救技术从13个杂交组合获得杂种株系1 284个,移栽成活697株;以‘红宝石无核’和‘火焰无核’作为母本的后代成苗率较高,以‘阳光玫瑰’‘爱神玫瑰’‘红亚历山大’为父本的胚挽救成苗较好,其中‘红宝石无核’ב爱神玫瑰’杂交后代胚发育率和成苗率相对较高,分别为48.59%和51.71%;幼胚萌发率以WPM为基础培养基,添加1.0 mg·L-1 KT+0.5 mg·L-1 NAA+1.0 mg·L-1 ZT的生长调节剂时较高,达11.33%,高于对照的7.41%;分别利用3种无核相关分子标记GLSP1-569、SCF27-2000和SCC8-1018,对101个株系进行无核性状检测,表明使用不同的标记在27个株系均检测到特异性条带,初步确定以上携带无核特异条带的株系为无核株系。【结论】'亚历山大'和'阳光玫瑰' 的香味物质含量高,并且与欧洲葡萄杂交后其胚挽救效率高,是合适的香味父本材料;而‘红宝石无核’‘火焰无核’适合作为母本。胚萌发培养时,以WPM培养基添加适当浓度的KT及ZT有较好的促生根效果。通过分子标记对杂交F1代无核性状检测率为26.73%。目前开发的玫瑰香味标记较少,本研究获得的杂种后代可为研究玫瑰香味基因标记提供重要的试材。
贾姗姗,骆强伟,李莎莎,王跃进. 葡萄胚挽救技术优化及无核和玫瑰香味新种质创制[J]. 中国农业科学, 2020, 53(16): 3344-3355.
JIA ShanShan,LUO QiangWei,LI ShaSha,WANG YueJin. Optimization of Embryo Rescue Technique and Production of Potential Seedless Grape Germplasm with Rosy Aroma[J]. Scientia Agricultura Sinica, 2020, 53(16): 3344-3355.
表1
杂交组合配置"
杂交组合(母本×父本) Cross combination (♀×♂) | 母本Female parent | 父本Male parent | ||
---|---|---|---|---|
种或杂种 Species or hybrid | 性状 Characteristic | 种或杂种 Species or hybrid | 性状 Characteristic | |
爱神玫瑰×玫瑰香 Aishenmeigui × Muscat Hamburg | 欧亚种 V. vinifera | 无核、玫瑰香味 Stenospermic, Muscat-scented | 欧亚种 V. vinifera | 有核、玫瑰香味 Seeded, Muscat-scented |
爱神玫瑰×阳光玫瑰 Aishenmeigui × Shine-Muscat | 欧亚种 V. vinifera | 无核、玫瑰香味 Stenospermic, Muscat-scented | 欧美杂种 V. labruscana ×V. vinifera | 有核、玫瑰香味 Seeded, Muscat-scented |
爱神玫瑰×昆香无核 Aishenmeigui × Kunxiang Seedless | 欧亚种 V. vinifera | 无核、玫瑰香味 Stenospermic, Muscat-scented | 欧亚种 V. vinifera | 无核、玫瑰香味 Stenospermic, Muscat-scented |
火焰无核×红亚历山大 Flame Seedless × Red Alexander | 欧亚种 V. vinifera | 无核 Stenospermic | 欧亚种 V. vinifera | 有核、玫瑰香味 Seeded, Muscat-scented |
火焰无核×阳光玫瑰 Flame Seedless × Shine-Muscat | 欧亚种 V. vinifera | 无核 Stenospermic | 欧美杂种 V. labruscana ×V. vinifera | 有核、玫瑰香味 Seeded, Muscat-scented |
火焰无核×新郁 Flame Seedless × Xinyu | 欧亚种 V. vinifera | 无核 Stenospermic | 欧亚种 V. vinifera | 有核、玫瑰香味 Seeded, Muscat-scented |
昆香无核×爱神玫瑰 Kunxiang Seedless × Aishenmeigui | 欧亚种 V. vinifera | 无核、玫瑰香味 Stenospermic, Muscat-scented | 欧亚种 V. vinifera | 无核、玫瑰香味 Stenospermic, Muscat-scented |
昆香无核×玫瑰香 Kunxiang Seedless × Muscat Hamburg | 欧亚种 V. vinifera | 无核、玫瑰香味 Stenospermic, Muscat-scented | 欧亚种 V. vinifera | 有核、玫瑰香味 Seeded, Muscat-scented |
克瑞森无核×玫瑰香 Crimson Seedless × Muscat Hamburg | 欧亚种 V. vinifera | 无核 Stenospermic | 欧亚种 V. vinifera | 有核、玫瑰香味 Seeded, Muscat-scented |
克瑞森无核×新郁 Crimson Seedless × Xinyu | 欧亚种 V. vinifera | 无核 Stenospermic | 欧亚种 V. vinifera | 有核、玫瑰香味 Seeded, Muscat-scented |
红宝石无核×玫瑰香 Ruby Seedless × Muscat Hamburg | 欧亚种 V. vinifera | 无核 Stenospermic | 欧亚种 V. vinifera | 有核、玫瑰香味 Seeded, Muscat-scented |
红宝石无核×红亚历山大 Ruby Seedless × Red Alexander | 欧亚种 V. vinifera | 无核 Stenospermic | 欧亚种 V. vinifera | 有核、玫瑰香味 Seeded, Muscat-scented |
红宝石无核×爱神玫瑰 Ruby Seedless × Aishenmeigui | 欧亚种 V. vinifera | 无核 Stenospermic | 欧亚种 V. vinifera | 无核、玫瑰香味 Stenospermic, Muscat-scented |
表2
玫瑰香味葡萄果实中所含化合物种类及含量"
化合物 Compound | 沙巴珍珠 Pearl of Csaba | 早玫瑰 Zao Meiguixiang | 玫瑰香 Muscat Hamburg | 亚历山大 Muscat of Alexandria | 巨玫瑰 Jumeigui | 金星无核 Venus Seedless | 意大利 Italia | 粉红玫瑰 Muscat Rose | 阳光玫瑰 Shine- Muscat | 无核白 Thompson Seedless | |
---|---|---|---|---|---|---|---|---|---|---|---|
醛类Aldehydes | 总醛 Total aldehydes | 8.9300 | 4.4924 | 9.2314 | 4.9050 | 15.8080 | 15.7184 | 8.0026 | 10.1754 | 2.1530 | 15.5756 |
醇类 Alcohols | 总醇 Total alcohols | 2.0900 | 0.0842 | 0.2296 | 1.4376 | 1.4816 | 0.3200 | 0.1754 | 0.5486 | 0.5210 | 0.1138 |
烯类 Olefins | 总烯 Total olefins | 0.3522 | 0.0504 | 0.0612 | 0.2074 | 0.1184 | 0.1266 | 0 | 0.0872 | 0.0816 | 0 |
烷类 Alkenes | 总烷 Total alkenes | 0.0362 | 0.0328 | 0.0098 | 0.0112 | 1.8162 | 0.2784 | 0.0154 | 0.0314 | 0.0588 | 0.3464 |
酮类 Ketones | 总酮 Total ketones | 0 | 0 | 0.0848 | 0.0076 | 0 | 0.0234 | 0.0382 | 0.0122 | 0 | 0 |
酯类 Esters | 总酯 Total esters | 0 | 0 | 0 | 0.1202 | 0.6918 | 0.0084 | 0 | 0.0122 | 0.0094 | 0 |
酸类 Acids | 总酸 Total acids | 0 | 0 | 0 | 0.0328 | 0 | 0.0116 | 0.0272 | 0 | 0 | 0 |
萜烯类 Terpenes | 香茅醇 Citronellol | 0.2912 | 0.0062 | 0 | 0 | 0.3490 | 0 | 0 | 0 | 0 | 0 |
香叶醇 Geraniol | 0.4432 | 0.0152 | 0.0088 | 0.1524 | 0.6448 | 0 | 0.0246 | 0.0446 | 0 | 0 | |
香叶醛 Citral | 0.0608 | 0.0024 | 0.0286 | 0.0244 | 0 | 0 | 0 | 0 | 0 | ||
里那醇 Linalool | 0.5532 | 0.0396 | 0.1656 | 1.0790 | 0.0980 | 0 | 0 | 0.1184 | 0.4812 | ||
松油醇 Terpineol | 0.0340 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
à-松油醇 à-Terpineolα- | 0 | 0 | 0 | 0.0102 | 0 | 0 | 0.0156 | 0 | 0 | ||
脱氢芳樟醇 1,5,7-Octatrien-3-ol, 3,7- dimethyl | 0 | 0 | 0 | 0.0035 | 0 | 0 | 0 | 0 | 0 | 0 | |
总萜烯Total terpenes | 1.3824 | 0.0634 | 0.1744 | 1.2636 | 1.1264 | 0 | 0.0246 | 0.1788 | 0.4812 | 0 | |
其他 Others | 0.0234 | 0.0274 | 0.0374 | 0.0454 | 0.2674 | 0.0516 | 0.0554 | 0.1038 | 0.0076 | 0.0194 | |
总挥发性物质含量 Total volatile matter content | 12.8142 | 4.7506 | 9.8286 | 8.0308 | 21.3098 | 16.5384 | 8.3388 | 11.1496 | 3.3126 | 16.0552 |
表3
无核香味葡萄胚挽救育种结果"
杂交组合 Cross | 果粒数 Number of berries | 胚珠数 Number of cultured ovule | 发育胚Developed embryos | 成苗Plantlets formation | ||
---|---|---|---|---|---|---|
数量 Number | 占比 Rate (%) | 数量 Number | 占比 Rate (%) | |||
爱神玫瑰×玫瑰香 Aishenmeigui × Muscat Hamburg | 285 | 508 | 75 | 15.69 | 14 | 18.67 |
爱神玫瑰×阳光玫瑰 Aishenmeigui × Shine-Muscat | 1504 | 2086 | 335 | 16.06 | 79 | 23.58 |
爱神玫瑰×昆香无核 Aishenmeigui × Kunxiang Seedless | 620 | 750 | 71 | 9.47 | 17 | 23.49 |
火焰无核×红亚历山大 Flame Seedless × Red Alexander | 925 | 1208 | 133 | 11.01 | 50 | 37.59 |
火焰无核×阳光玫瑰 Flame Seedless × Shine-Muscat | 638 | 922 | 98 | 10.80 | 20 | 20.41 |
火焰无核×新郁 Flame Seedless × Xinyu | 665 | 635 | 34 | 5.48 | 8 | 23.53 |
昆香无核×爱神玫瑰 Kunxiang Seedless × Aishenmeigui | 2050 | 2184 | 305 | 13.97 | 70 | 22.95 |
昆香无核×玫瑰香 Kunxiang Seedless × Muscat Hamburg | 385 | 405 | 102 | 25.19 | 0 | 0 |
克瑞森无核×玫瑰香 Crimson Seedless × Muscat Hamburg | 435 | 441 | 19 | 4.31 | 1 | 5.26 |
克瑞森无核×新郁 Crimson Seedless × Xinyu | 635 | 540 | 16 | 5.61 | 1 | 6.25 |
红宝石无核×玫瑰香 Ruby Seedless × Muscat Hamburg | 1160 | 1895 | 395 | 20.84 | 186 | 47.09 |
红宝石无核×红亚历山大 Ruby Seedless × Red Alexander | 1308 | 2956 | 763 | 25.81 | 249 | 32.63 |
红宝石无核×爱神玫瑰 Ruby Seedless × Aishenmeigui | 1243 | 2486 | 1139 | 48.59 | 589 | 51.71 |
合计 Total | 11853 | 17016 | 3485 | 20.48 | 1284 | 36.84 |
表4
不同生长调节剂对胚挽救成苗的影响"
杂交组合 Cross | 处理组 Treatment | 生长调节剂成分 Exogenous hormone (mg·L-1) | 胚珠数 Number of cultured ovules | 发育胚 Developed embryos | 萌发 Germination | |||||
---|---|---|---|---|---|---|---|---|---|---|
2,4-D | KT | ZT | NAA | 数量Number | 占比 Rate (%) | 数量Number | 占比 Rate (%) | |||
昆香无核×爱神玫瑰 Kunxiang Seedless × Aishenmeigui | T1 | 0 | 0 | 0 | 0 | 135 | 22 | 16.30b | 10 | 7.41a |
T2 | 0 | 1 | 1 | 0.5 | 150 | 33 | 22.00a | 17 | 11.33a | |
T3 | 0 | 2 | 2 | 1 | 150 | 26 | 17.33b | 14 | 9.33a | |
T4 | 1 | 0 | 1 | 0.5 | 150 | 7 | 4.67bc | 1 | 0.67b | |
T5 | 1 | 1 | 2 | 0 | 132 | 7 | 5.30b | 2 | 1.52b | |
T6 | 1 | 2 | 0 | 0.5 | 140 | 1 | 0.72c | 1 | 0.72b | |
T7 | 2 | 0 | 2 | 0.5 | 150 | 10 | 6.67b | 2 | 1.33b | |
T8 | 2 | 1 | 0 | 1 | 150 | 4 | 2.67c | 1 | 0.67b | |
T9 | 2 | 2 | 1 | 0 | 150 | 6 | 4.00bc | 1 | 0.67b |
表5
不同形态杂种胚的萌发成苗"
杂交组合 Cross | 胚类型 Embryo code | 接种胚数Cultured embryos | 发育胚Developed embryos | 成苗Plantlets formation | |||
---|---|---|---|---|---|---|---|
数量 Number | 占比 Rate (%) | 数量 Number | 占比 Rate (%) | 数量Number | 占比 Rate (%) | ||
昆香无核×爱神玫瑰 Kunxiang Seedless × Aishenmeigui | G | 55 | 47.41 | 23 | 41.82 | 12 | 21.82 |
H | 26 | 22.41 | 22 | 84.62 | 6 | 23.08 | |
F | 29 | 25.00 | 26 | 89.66 | 14 | 48.28 | |
C | 2 | 1.72 | 2 | 100.00 | 2 | 100.00 | |
A | 4 | 3.45 | 4 | 100.00 | 1 | 25.00 | |
红宝石无核×玫瑰香 Ruby Seedless × Muscat Hamburg | G | 77 | 39.49 | 32 | 41.56 | 17 | 22.08 |
H | 5 | 2.56 | 2 | 40.00 | 1 | 20.00 | |
F | 106 | 54.36 | 96 | 90.57 | 62 | 58.49 | |
C | 4 | 2.05 | 3 | 75.00 | 3 | 75.00 | |
A | 3 | 1.54 | 1 | 33.33 | 0 | 0 |
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