葡萄胚挽救技术优化及无核和玫瑰香味新种质创制

doi:10.3864/j.issn.0578-1752.2020.16.012

摘要/Abstract

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’×‘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。

关键词: 葡萄, 香味性状, 胚挽救, 分子标记辅助选择, 无核

Abstract:

【Objective】The aim of this study was to investigate the grape scent traits to provide important material basis for seedless aroma grape breeding. By exploring the parental genotype, embryo development morphology and growth regulator on embryo rescue efficiency, further optimizing the seedless × muscat scent combined embryo rescue system, and molecular marker technology was used to initially identify seedless traits of hybrid offspring.【Method】Headspace solid phase micro-extraction (HS-SPME) combined with gas chromatography-mass spectrometry technique was used to determine the content of aromatic substances in the grapes of 10 different muscat-scented and non-muscat scented varieties. Excellent aroma varieties were selected as parents, then combined with the previous research results of the group, 13 cross-combinations, including 6 female parents and 5 male parents, were used to obtain new grapevines with seedlessness and aroma. Under aseptic conditions, the ovules were cultured from young fruits in the dark for 8 weeks. The developed hybrid embryos were inoculated in a variety of embryo germination media, with different concentrations and different proportions of hormones to assess and improve the germination rate and germination seedling rate. After the embryo developed into plants, they were placed in the greenhouse for hardening, the survived F₁ generation was transplanted to the field. The molecular marker technology was used to select the seedless characters of F₁hybrid lines in the early stage. 【Result】Among 10 table grapes, it was the terpenes that determined the aroma of muscat, which could be detected in 8 muscat scent varieties, and the content of which ranged from 0.0246 to 1.3824. Based on the above evaluation, ‘Muscat of Alexandria’, ‘Shine-Muscat’ and ‘Muscat Hamburg’ were selected to create new seedless aroma germplasm because their high levels of terpenoids. The study showed 1 284 progeny were obtained by embryo rescue technique, and 697 of them survived after transplanting into field. Ruby Seedless and Flame Seedless with a higher seedling rate were suitable as female parent in embryo rescue. Muscat Hamburg and Shine-Muscat proved as the better male parents. Among them, the embryo development rate and seedling formation rate of Ruby seedless × Aishenmeigui were the highest, at 48.59% and 51.71%, respectively. The germination rate was the highest when embryos were inoculated into WPM as based medium, and the growth regulator (1.0 mg?L^-1 KT + 0.5 mg?L^-1 NAA + 1.0 mg?L^-1 ZT) was added, which was 11.33%, higher than the control which was 7.41%. Three molecular markers GLSP1-569, SCF27-2000 and SCC8-1018 were selected for detecting the cross parents and hybrid progenies. The results showed that 27 out of 101 hybrid strains had seedless genes. 【Conclusion】 Muscat of Alexandria and Sunshine-Muscat had a high scent content, and the embryo rescue efficiency was high after crossing with European grapes, which was a suitable scented parent material. Ruby Seedless and Flame Seedless was suitable as female parent. During embryo germination and culture, using WPM medium as the basic medium, adding appropriate concentrations of KT and ZT had a better effect of promoting root growth. The seedless traits of hybrid F₁ generation were detected by molecular markers, and the seedless rate was 26.73%. At present, few aroma markers have been developed, so the hybrid offspring obtained in this study could provide an important material basis for the further study of aroma gene markers.

Key words: grapevine, aromatic characteristic, embryo rescue, marker-assisted selection, seedlessness

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.

图/表 10

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
杂交组合（母本×父本） Cross combination (♀×♂)	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1

表2

表3

表4

图1

表5

图2

图3

图4

图5

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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

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
杂交组合 Cross	果粒数 Number of berries	胚珠数 Number of cultured ovule	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

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
杂交组合 Cross	处理组 Treatment	2,4-D	KT	ZT	NAA	胚珠数 Number of cultured ovules	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

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
杂交组合 Cross	胚类型 Embryo code	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