李桂荣^1,2, 全冉¹, 程珊珊¹, 侯小进¹, 樊秀彩³, 扈惠灵^,11 河南科技学院园艺园林学院,河南新乡 453003
2 河南省园艺植物资源利用与种质创新工程研究中心,河南新乡 453003
3 中国农业科学院郑州果树研究所,郑州 450009

The Influencing Factors of in-vitro Ovule Development in Seedless Grape and Its Physiological Changes

LI GuiRong^1,2, QUAN Ran¹, CHENG ShanShan¹, HOU XiaoJin¹, FAN XiuCai³, HU HuiLing^,1 1 School of Horticulture and Landscape Architecture, Henan Institute of Science and Technology, Xinxiang 453003, Henan
2 Henan Province Engineering Research Center of Horticultural Plant Resource Utilization and Germplasm Enhancement, Xinxiang 453003, Henan
3 Zhengzhou Fruit Research Institute, Chinese Academy of Agricultural Sciences Zhengzhou 453003

通讯作者: 扈惠灵,Tel：13523220523;E-mail：hu-huiling@163.com

责任编辑: 赵伶俐
收稿日期:2020-03-25接受日期:2020-07-13网络出版日期:2020-11-16

基金资助:

河南省自然科学基金.182300410045

Received:2020-03-25Accepted:2020-07-13Online:2020-11-16
作者简介 About authors
李桂荣,E-mail： liguirong10@163.com。








摘要
【目的】 研究无核葡萄离体胚珠发育影响因素,为提高无核葡萄胚挽救育种效率提供技术参考。【方法】 选择‘红宝石’无核葡萄为材料,在自然授粉后65 d摘取葡萄幼果,消毒处理后,剥取胚珠接种到不同培养基上。研究不同培养基处理（4种不同的基本培养基、附加4种不同蔗糖含量,共计16个处理）和不同培养时间对胚珠发育率和生理指标的影响,并对胚珠发育率和不同生理指标进行相关分析。【结果】 在胚珠离体培养阶段,不同处理对胚珠发育率影响不同。采用固液双层基本培养基（改良MM3固体培养基+8 mg·L ^-1 ER液体培养基）、附加45 g·L ^-1蔗糖、培养49 d时,胚珠发育率最高,达到（42.23±6.93）%。不同处理对离体培养胚珠各生理指标影响不同。当培养时间相同、基本培养基为固液双层培养基时,胚珠淀粉含量变化差异不显著,而可溶性蛋白含量、总酚含量和超氧化物歧化酶（SOD）活性均增加,过氧化物酶（POD）和多酚氧化酶（PPO）活性均降低;随着附加蔗糖含量的增加,胚珠淀粉含量变化差异不显著,而可溶性蛋白、总酚含量和SOD酶活性均出现先增加后下降的趋势,POD酶和PPO酶活性均出现逐渐升高的趋势。当培养基处理相同时,随着离体培养时间的延长,胚珠淀粉含量出现逐渐下降的趋势,可溶性蛋白含量、总酚含量和SOD酶活性均出现先增加后下降的趋势,POD酶和PPO酶活性均出现逐渐升高的趋势。相关分析发现,胚珠发育率与胚珠可溶性蛋白含量、总酚含量和SOD酶活性呈极显著正相关,与POD酶活性和PPO酶活性呈极显著负相关,与淀粉含量相关性不显著。 【结论】 采用固液双层基本培养基（改良MM3固体培养基+8 mg·L ^-1 ER液体培养基）,附加45 g·L ^-1蔗糖,培养时间49 d时,胚珠发育率最高,离体培养的胚珠生理活性也较强。胚珠发育率与离体培养胚珠的可溶性蛋白含量、总酚含量和SOD酶活性呈极显著正相关,与POD酶活性和PPO酶活性呈极显著负相关。
关键词： 无核葡萄;胚挽救;离体培养;胚珠发育;生理指标

Abstract
【Objective】The influencing factors of in-vitro ovule development in seedless grape were studied to improve the efficiency of seedless grape embryo rescue breeding.【Method】 ‘Ruby’ seedless grape was used as the material. Young fruits were at harvested 65 days after natural pollination. Then, these fruits were disinfected, and ovules were stripped and inoculated on different medium. The effects of different medium treatments (4 different basic medium with 4 different sucrose quality added, thus 16 treatments in total) and different culture time on ovule development rate and physiological indexes were studied, and the correlation between ovule development rate and different physiological indexes was also analyzed. 【Result】Different treatments had different effects on development rate of ovule cultured in vitro. When solid-liquid double-layer medium (modified MM3 solid medium + 8 g·L ^-1 ER liquid medium), 45 g·L ^-1 sucrose was added and the culture time was 49 d, the ovule development rate was the highest of (42.23 ± 6.93) %. Different treatments had different effects on the physiological indexes of ovule cultured in vitro. When the basic medium was solid-liquid double-layer medium, the change of starch content was not significant, and soluble protein content, total phenol content and SOD enzyme activity increased, while POD enzyme and PPO enzyme activity showed a decreasing trend. With the increase of sucrose quality in the basic medium, the difference in starch content was not significant, while soluble protein, total phenol content and SOD enzyme activity showed a trend of first increasing and then decreasing, while POD and PPO enzyme activities showed a trend of gradually increasing. When the medium treatment was the same, with the extension of the in vitro culture time, starch content gradually decreased, while soluble protein content, total phenol content and SOD enzyme activity increased first and then decreased, and POD enzyme and PPO enzyme activities increased gradually. Correlation analysis showed that ovule development rate had a significant positive correlation with soluble protein content, total phenol content and SOD enzyme activity, and a significant negative correlation with POD enzyme activity and PPO enzyme activity, but no significant correlation with starch content were found. 【Conclusion】When using the solid-liquid double-layer medium (modified MM3 solid medium + 8 g·L ^-1 ER liquid medium) with 45 g·L ^-1 sucrose added and the culture time of 49 d, the physiological activity of in-vitro ovule was strong. Ovule development rate had a significant positive correlation with the content of soluble protein, total phenol content and SOD enzyme activity, and a significant negative correlation with POD enzyme activity and PPO enzyme activity.
Keywords：seedless grape;embryo rescue;ovule development;in vitro culture;physiological indexes


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本文引用格式
李桂荣, 全冉, 程珊珊, 侯小进, 樊秀彩, 扈惠灵. 无核葡萄离体胚珠发育影响因子及其生理变化[J]. 中国农业科学, 2020, 53(22): 4646-4657 doi:10.3864/j.issn.0578-1752.2020.22.011
LI GuiRong, QUAN Ran, CHENG ShanShan, HOU XiaoJin, FAN XiuCai, HU HuiLing. The Influencing Factors of in-vitro Ovule Development in Seedless Grape and Its Physiological Changes[J]. Scientia Agricultura Sinica, 2020, 53(22): 4646-4657 doi:10.3864/j.issn.0578-1752.2020.22.011


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0 引言

【研究意义】种子败育型无核葡萄获得种胚后代的最有效方式是采用离体胚挽救技术^[1]。无核葡萄胚挽救技术包括胚珠离体培养、胚萌发和成苗3个阶段^{[2,3,4,5,6,7,8]},其中在第一个阶段获得大量的发育胚珠是基础,但前期的研究实践发现胚珠离体培养阶段的发育率低,一般都低于30%^{[9,10,11,12,13,14,15]},这是目前无核葡萄胚挽救育种过程中的一个瓶颈问题。因此,摸索无核葡萄离体胚珠发育的最佳培养条件,探究培养条件与离体胚珠发育生理变化的关系,减少胚败育,有助于提高胚珠发育率,对于今后无核葡萄胚挽救育种效率的提高至关重要。【前人研究进展】1982年,RAMMING等^[9]首次报道利用改良的White培养基培养无核葡萄胚珠,获得两株实生苗。此后,很多研究者继续深入研究亲本基因型、取样时期、培养基及其相互作用等对无核葡萄胚挽救育种的影响^{[16,17,18,19,20,21]}。亲本基因型的筛选与胚挽救技术是提高育种效率的重要因素^[1]。‘红宝石’无核葡萄（Ruby seedless grape）属无核晚熟红色葡萄品种。选用‘红宝石’无核葡萄作为无核葡萄胚挽救杂交亲本易获得杂交后代^[22,23,24],目前以‘红宝石’无核葡萄为亲本,通过胚挽救技术已获得了‘紫丰’^[25]和‘秦红1号’^[26]无核葡萄新品种。因此,‘红宝石’无核葡萄是适宜开展无核葡萄胚挽救育种的主要品种之一。而以‘红宝石’无核葡萄作母本进行胚珠离体培养时,胚珠发育率也很低,仅为2%—28.3%^{[4,15,18,21,27-29]}。前人在无核葡萄胚珠离体培养阶段,多数是从抑制幼胚早期萌发、防止幼胚夭亡或者畸形胚萌发的角度出发,采用各种培养方式抑制离体胚珠内胚的早期萌发,如采用低温处理胚珠、培养基附加60 g·L^-1蔗糖等手段离体培养胚珠,而且培养时间较长,最少8周,有的甚至达到9—10周^{[16,17,18,19,20,21]}。这个培养方式已经采用了很多年,但这是否为无核葡萄胚挽救育种过程中胚珠发育率低的限制因素,目前尚未进行深入研究。【本研究切入点】无核葡萄早期胚珠离体培养的研究报道中优选或采用的基本培养基有多种类型,其中MM3、改良MM3以及固液双层培养基（改良MM3固体培养基+少量ER液体培养基）都表现出较好的使用效果^[4,12,18],附加60 g·L^-1的蔗糖含量最为常见,没有更多的比较研究^[1],而这个阶段离体培养时间最少都是8周,胚珠发育率一直都低于30%^{[9,10,11,12,13,14,15]}。据此,本研究以无核葡萄胚挽救育种中常用母本品种‘红宝石’无核为试材,比较研究基本培养基、蔗糖浓度、培养时间等关键因子在不同处理组合下对早期离体培养胚珠发育的影响,并探究培养条件与胚珠发育生理状态间的关系。【拟解决的关键问题】确定提高胚珠发育率的离体培养条件;明确不同培养条件与离体胚珠生理活性间的相关程度,为进一步优化培养条件,减少葡萄胚败育发生奠定基础。

1 材料与方法

试验于2018—2019年在河南科技学院和中国农业科学院郑州果树研究所进行。

1.1 试验材料

选择种子败育型、品质优良的欧亚种（Vitis Vinifera L.）无核葡萄品种‘红宝石’无核（Ruby seedless grape）为试验材料,于自然授粉65 d后摘取葡萄幼果（图1-A）,4℃冷藏备用。

1.2 试验方法

1.2.1 胚珠离体培养 在超净工作台上,将消过毒的无菌幼果粒剖开,选取大小一致的胚珠（图1-B）接种在附加不同蔗糖含量的不同培养基上培养,固体培养基添加7.0 g·L^-1琼脂和1.5 g·L^-1活性炭,pH为6.0（表1）。每个培养基处理接种30瓶,每瓶10个胚珠。培养的环境条件为：温度（25±2）℃,相对湿度50%—60%,暗培养。

Table 1
表1
表1不同培养基处理
Table 1Different medium treatments

编号 NO.	基本培养基 Basic medium	蔗糖 Sugar (g·L-1)
M1	MS固体培养基MS solid medium	30
M2	MM3固体培养基MM3 solid medium	30
M3	改良MM3固体培养基Modified MM3 solid medium	30
M4	固液双层培养基（改良MM3固体培养基+8 mg·L-1 ER液体培养基） Solid-liquid double-layer medium (modified MM3 solid medium + 8mg·L-1 ER liquid medium)	30
M5	MS固体培养基MS solid medium	45
M6	MM3固体培养基MM3 solid medium	45
M7	改良MM3固体培养基Modified MM3 solid medium	45
M8	固液双层培养基（改良MM3固体培养基+8 mg·L-1 ER液体培养基） Solid-liquid double-layer medium (modified MM3 solid medium + 8mg·L-1 ER liquid medium)	45
M9	MS固体培养基MS solid medium	60
M10	MM3固体培养基MM3 solid medium	60
M11	改良MM3固体培养基Modified MM3 solid medium	60
M12	固液双层培养基（改良MM3固体培养基+8nmg·L-1 ER液体培养基） Solid-liquid double-layer medium (modified MM3 solid medium + 8 mg·L-1 ER liquid medium)	60
M13	MS固体培养基 MS solid medium	75
M14	MM3固体培养基 MM3 solid medium	75
M15	改良MM3固体培养基Modified MM3 solid medium	75
M16	固液双层培养基（改良MM3固体培养基+8 mg·L-1 ER液体培养基） Solid-liquid double-layer medium (modified MM3 solid medium + 8 mg·L-1 ER liquid medium)	75

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1.2.2 不同离体培养时间处理 不同培养基处理下接种胚珠后,分别培养42、49、56和63 d,研究不同离体培养时间对胚珠发育的影响。

1.2.3 胚珠发育率统计 统计不同处理下的发育胚珠数,胚珠里没有胚视为未发育胚珠,含有1个胚视为发育胚珠（图1-C）。每3瓶共计30个离体胚珠算1次重复,共3次重复。胚珠发育率（%）=发育胚珠个数/接种胚珠总数×100。

1.2.4 生理指标测定 淀粉含量、可溶性蛋白含量、总酚含量、超氧化物歧化酶（SOD）活性、过氧化物酶（POD）活性和多酚氧化酶（PPO）活性的测定均采用北京索莱宝公司试剂盒进行测定,型号分别为BC0700、PR1400、BC1340、BC0170、BC0090、BC0195。每个指标类型重复测定3次。

1.2.5 胚萌发率统计 胚珠离体培养42、49、56和63 d后,分别将发育胚珠内的胚（分别标记为E1、E2、E3和E4）剥出,统计总胚数,接种到胚萌发培养基上,离体培养30 d后,统计胚萌发总数（胚的子叶、胚根有伸长的视为萌发胚）。胚萌发率（%）=萌发胚个数/接种胚总数×100。胚萌发培养基为WPM+IAA 1.0 mg·L^-1+6-BA 0.5 mg·L^-1+GA₃ 2.0 mg·L^-1+琼脂7.0 g·L^-1+蔗糖20 g·L^-1+活性炭 1.5 g·L^-1,添加Put 2 mmol·L^-1+ Spm 2 mmol·L^-1+ Spd 2 mmol·L^-1,pH为6.0。培养环境条件：温度（25±2）℃,相对湿度50%—60%,光照强度40 μmol·m^-2·s^-1,光周期为16 h/8 h^[18]。

1.2.6 成苗及驯化移栽 待胚萌发长成正常完整苗（苗高6—8 cm,6—8片真叶）时,剪成带一个芽的茎段接种到培养基上继代生根成苗,培养基为：1/2 MS+IBA 0.1 mg·L^-1+琼脂7.0 g·L^-1+蔗糖20 g·L^-1+活性炭1.5 g·L^-1,pH 6.0。培养室条件：温度（25±2）℃,相对湿度50%—60%,光照强度40 μmol·m^-2·s^-1,光周期为16 h/8 h。成苗后的试管苗在炼苗室炼苗7 d后,移栽到营养钵中,继续培养30 d;然后将营养钵转移到温室内驯化30 d,移植至大棚圃地,按照田间管理的程序管理。翌年3月定植到大田^[18]。

1.2.7 数据分析 采用SPSS 22.0软件进行方差分析及相关分析,相关系数为简单相关系数。

2 结果

2.1 对离体培养胚珠发育率的影响

由表2可以看出,当M8培养基处理（基本培养基是固液双层培养基、附加45 g·L^-1的蔗糖）、培养时间为49 d时,胚珠发育率最高,达到（42.23±6.93）%;其次是M12培养基处理（基本培养基是固液双层培养基、附加60 g·L^-1的蔗糖）、培养时间为49 d时,胚珠发育率较高,达到（40.00±3.30）%,发育胚珠即为离体胚珠培养后获得至少1个活性胚的胚珠,见图1-B—D。

Table 2
表2
表2不同培养基和不同离体培养时间对离体培养胚珠发育率的影响
Table 2Effects of different medium and culture time on ovule development rate in vitro (%)

培养基 Medium	时间Time (d)
	42	49	56	63
M1	0.00±0.00f	1.10±1.91j	2.20±1.91d	1.10±1.91b
M2	1.10±1.91ef	5.57±1.96ij	4.43±1.96cd	4.47±3.87ab
M3	2.20±1.91ef	10.00±3.30hi	6.70±0.00cd	5.57±5.10ab
M4	3.33±3.35def	14.43±1.96gh	15.57±1.96b	8.87±5.10a
M5	4.47±3.87cdef	16.67±3.35f	24.43±1.96a	5.57±1.96ab
M6	5.57±5.10cdef	25.57±1.96ef	26.67±3.35a	5.57±5.10ab
M7	8.87±5.10abcdef	32.20±1.91cd	28.90±3.81a	6.70±0.00ab
M8	17.77±13.85a	42.23±6.93a	30.00±3.30a	8.90±1.91a
M9	7.77±5.08bcde	23.33±3.35e	25.57±1.96a	7.77±5.08ab
M10	12.23±5.08abcd	30.00±3.30de	26.67±3.35a	7.77±5.08ab
M11	13.33±3.35abc	36.67±3.35bc	28.90±3.81a	8.90±3.81a
M12	16.67±3.35bc	40.00±3.30ab	28.90±3.81a	8.90±3.81a
M13	3.33±3.35def	7.80±1.91i	4.47±3.87cd	3.33±3.35ab
M14	4.47±3.87cdef	8.90±1.91hi	5.57±5.10cd	4.47±3.87ab
M15	7.80±1.91bcde	8.90±1.91hi	8.87±5.10c	4.47±3.87ab
M16	10.00±0.00abcde	10.00±3.30hi	10.00±0.00c	6.67±3.35ab

同一列不同小写字母表示差异显著（P＜0.05）。下同 Means in the same column with the same letter are not significantly different at P＜0.05

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2.2 对离体培养胚珠生理指标的影响

2.2.1 对胚珠淀粉含量的影响 由表3可以看出,当培养时间相同、培养基不同时,胚珠淀粉含量变化差异不显著（P＞0.05）。当培养基处理相同时,随着离体培养时间的延长,胚珠淀粉含量出现逐渐下降的趋势,其中,离体胚珠在处理M12上培养42 d时,淀粉含量最高,达到（10.21±0.02）%;而当培养时间为63 d时,胚珠淀粉含量仅为（5.11±0.02）%。其他培养基处理变化趋势与M12处理一致,均为逐渐下降趋势。

Table 3
表3
表3不同培养基和不同离体培养时间对离体培养胚珠淀粉含量的影响
Table 3Effects of different medium and culture time on starch content of ovule in vitro (%)

培养基 Medium	时间Time (d)
	42	49	56	63
M1	10.06±0.09c	7.95±0.20b	7.15±0.05a	5.05±0.03b
M2	10.09±0.10bc	8.06±0.08a	7.16±0.03a	5.06±0.03ab
M3	10.13±0.07abc	8.09±0.03a	7.17±0.02a	5.09±0.05ab
M4	10.19±0.03a	8.11±0.05a	7.18±0.03a	5.10±0.03ab
M5	10.18±0.02ab	8.11±0.02a	7.17±0.06a	5.04±0.02b
M6	10.16±0.06ab	8.10±0.03a	7.18±0.03a	5.07±0.02ab
M7	10.15±0.04ab	8.12±0.03a	7.19±0.02a	5.08±0.06ab
M8	10.18±0.03ab	8.13±0.04a	7.20±0.02a	5.09±0.03ab
M9	10.16±0.02ab	8.10±0.03a	7.16±0.03a	5.09±0.02ab
M10	10.18±0.02ab	8.11±0.03a	7.18±0.04a	5.09±0.05ab
M11	10.19±0.03a	8.12±0.11a	7.19±0.03a	5.09±0.04ab
M12	10.21±0.02a	8.10±0.03a	7.20±0.05a	5.11±0.02a
M13	10.14±0.01abc	8.11±0.03a	7.16±0.05a	5.04±0.01b
M14	10.15±0.01ab	8.19±0.01a	7.16±0.01a	5.04±0.02b
M15	10.15±0.02ab	8.07±0.05a	7.19±0.03a	5.05±0.02b
M16	10.17±0.04ab	8.09±0.02a	7.19±0.03a	5.07±0.02ab

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2.2.2 对可溶性蛋白含量的影响 由表4可以看出,当培养时间相同、基本培养基为固液双层培养基时,可溶性蛋白含量均增加,与其他基本培养基相比,可溶性蛋白含量差异显著（P＜0.05）,其中M8培养效果最好,可溶性蛋白含量最高,达到（0.49±0.03）mg·g^-1。随着附加蔗糖含量的增加,可溶性蛋白含量出现先增加后下降的趋势,如当基本培养基为固液双层培养基,培养42 d时,附加蔗糖含量为30 g·L^-1,可溶性蛋白含量为（0.36±0.01）mg·g^-1;附加蔗糖含量为45 g·L^-1时,可溶性蛋白含量增加,达到（0.46±0.03）mg·g^-1;而当附加蔗糖含量为75 g·L^-1时,可溶性蛋白含量降低,仅为（0.16±0.03）mg·g^-1。当培养基处理相同时,随着离体培养时间的延长,可溶性蛋白含量出现先增加后下降的趋势。说明胚珠离体培养阶段,附加蔗糖含量不宜过高,培养时间也不宜过长,以防止可溶性蛋白含量的降低。

Table 4
表4
表4不同培养基和离体培养时间对离体培养胚珠可溶性蛋白含量的影响
Table 4Effects of different medium and culture time on soluble protein content of ovule in vitro (mg·g^-1)

培养基 Medium	时间Time (d)
	42	49	56	63
M1	0.28±0.04cde	0.32±0.03def	0.31±0.03cde	0.18±0.04bcde
M2	0.30±0.01cd	0.34±0.04de	0.32±0.03bcd	0.20±0.02abc
M3	0.33±0.04bc	0.37±0.04bcd	0.33±0.04bc	0.22±0.04abc
M4	0.36±0.01b	0.40±0.04bc	0.36±0.03b	0.22±0.03ab
M5	0.23±0.02fg	0.31±0.05def	0.3±0.01cde	0.20±0.05abc
M6	0.26±0.02def	0.36±0.02cd	0.36±0.01b	0.23±0.04ab
M7	0.35±0.03b	0.44±0.03b	0.43±0.03a	0.26±0.04a
M8	0.46±0.03a	0.49±0.03a	0.47±0.05a	0.26±0.05a
M9	0.19±0.02hi	0.21±0.04h	0.21±0.01gh	0.18±0.04bcde
M10	0.21±0.02gh	0.25±0.01gh	0.24±0.01fg	0.19±0.04abcd
M11	0.25±0.04efg	0.27±0.03fg	0.27±0.01ef	0.21±0.04abc
M12	0.26±0.05defg	0.28±0.05ef	0.27±0.01def	0.25±0.03ab
M13	0.11±0.02k	0.13±0.02j	0.13±0.03j	0.09±0.04f
M14	0.12±0.02jk	0.14±0.01ij	0.14±0.04ij	0.11±0.04ef
M15	0.14±0.02jk	0.18±0.02hij	0.17±0.04hi	0.12±0.03def
M16	0.16±0.03jk	0.19±0.03hi	0.18±0.04hi	0.14±0.05cdef

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2.2.3 对总酚含量的影响 由表5可以看出,当培养时间相同、基本培养基为固液双层培养基时,总酚含量均增加,与其他基本培养基相比,总酚含量差异显著（P＜0.05）,其中M12培养效果较好,总酚含量最高,达到（3.37±0.04）mg·g^-1;随着附加蔗糖含量的增加,总酚含量出现先增加后下降的趋势,如当基本培养基为固液双层培养基,培养42 d时,附加蔗糖质量为30 g·L^-1,总酚含量为（1.36±0.03）mg·g^-1,附加蔗糖含量为60 g·L^-1时,总酚含量升高,达到（3.37±0.04） mg·g^-1,而当附加蔗糖含量为75 g·L^-1时,总酚含量降低,为（2.19±0.03）mg·g^-1。当培养基处理相同时,随着离体培养时间的延长,总酚含量出现先增加后下降的趋势。

Table 5
表5
表5不同培养基和不同离体培养时间对离体培养胚珠总酚含量的影响
Table 5Effects of different medium and culture time on phenolics content of ovule in vitro (mg·g^-1)

培养基 Medium	时间Time (d)
	42	49	56	63
M1	1.10±0.04l	2.32±0.03i	2.30±0.03gh	0.71±0.03h
M2	1.16±0.03k	2.33±0.03i	2.32±0.03gh	0.82±0.06g
M3	1.26±0.03j	2.37±0.04hi	2.35±0.02g	0.83±0.06g
M4	1.36±0.03i	2.41±0.04h	2.41±0.03f	0.90±0.03f
M5	1.78±0.03h	3.29±0.03g	3.30±0.01e	0.97±0.04e
M6	1.96±0.03g	3.37±0.02f	3.36±0.01d	1.00±0.03e
M7	2.08±0.04f	3.42±0.03e	3.43±0.03c	1.02±0.01de
M8	2.19±0.04e	3.48±0.04d	3.47±0.05c	1.07±0.01d
M9	2.74±0.07d	4.21±0.04c	4.21±0.01b	1.32±0.03c
M10	3.00±0.03c	4.22±0.02bc	4.22±0.03ab	1.40±0.03b
M11	3.20±0.03b	4.27±0.03ab	4.27±0.02a	1.42±0.03b
M12	3.37±0.04a	4.28±0.05a	4.27±0.01a	1.54±0.04a
M13	2.09±0.02f	2.13±0.02k	2.13±0.03j	0.60±0.03i
M14	2.11±0.03f	2.14±0.01k	2.14±0.06j	0.70±0.04h
M15	2.17±0.02e	2.25±0.03j	2.24±0.04i	0.75±0.03h
M16	2.19±0.03e	2.31±0.03i	2.28±0.06hi	0.81±0.03g

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2.2.4 对离体培养胚珠SOD酶活性的影响 由表6可以看出,当培养时间相同、基本培养基为固液双层培养基时,SOD酶活性均增加,与其他基本培养基相比,SOD酶活性差异显著（P＜0.05）,其中M12培养效果较好,SOD酶活性最高,达到（32.4±0.35）U·g^-1 FW;随着附加蔗糖含量的增加,SOD酶活性出现先升高后下降的趋势,如当基本培养基为固液双层培养基,培养42 d时,附加蔗糖含量为30 g·L^-1,SOD酶活性为（21.27±0.32）U·g^-1 FW,附加蔗糖含量为60 g·L^-1时,SOD酶活性升高,达到（32.4±0.35）U·g^-1 FW,而当附加蔗糖含量为75 g·L^-1时,SOD酶活性降低,为（22.9 ± 0.3）U·g^-1 FW。当培养基处理相同时,随着离体培养时间的延长,SOD酶活性出现先升高后下降的趋势,说明胚珠离体培养阶段,附加蔗糖含量不宜过高,培养时间也不宜过长,有利于SOD酶清除活性氧,防止胚败育。

Table 6
表6
表6不同培养基和不同离体培养时间对离体培养胚珠SOD酶活性的影响
Table 6The effect of different medium and culture time on SOD activity (U·g^-1 FW) of ovule in vitro

培养基 Medium	时间Time (d)
	42	49	56	63
M1	17.57±0.31l	28.93±0.45g	28.63±0.42i	12.33±0.15i
M2	18.90±0.30k	29.90±1.13g	28.90±0.30i	13.90±0.30h
M3	19.30±0.46jk	31.90±0.20f	31.00±0.26f	15.00±0.28g
M4	21.27±0.32h	33.13±0.68e	32.9±0.26de	16.43±1.08f
M5	24.00±0.46e	29.47±0.51g	29.30±0.56hi	17.57±0.31e
M6	26.60±0.26d	31.07±0.47f	30.43±0.50fg	19.50±0.20c
M7	29.20±0.30c	33.03±0.71e	32.37±0.45e	20.60±0.42b
M8	32.23±0.31a	36.20±0.56c	35.53±0.40c	21.55±0.70a
M9	29.20±0.30c	29.93±0.55g	29.83±0.65gh	15.57±0.301
M10	30.60±0.30b	34.27±0.51d	33.53±0.64d	18.50±0.20d
M11	31.07±0.47b	38.07±0.38b	37.17±0.61b	19.53±0.35c
M12	32.40±0.35a	40.17±1.00a	39.20±0.52a	20.60±0.30b
M13	19.50±0.20j	19.57±0.25k	18.90±0.40m	10.17±0.42j
M14	20.60±0.30i	22.23±0.35j	20.87±0.31l	11.60±0.30i
M15	21.87±0.35g	24.00±0.46i	25.90±0.46k	13.87±0.35h
M16	22.90±0.30f	26.60±0.26h	27.53±0.29j	13.90±0.30h

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2.2.5 对POD酶活性的影响 由表7可以看出,当培养时间相同、基本培养基为固液双层培养基时,POD酶活性均较低;随着附加蔗糖含量的增加,POD酶活性出现逐渐升高的趋势,当附加蔗糖含量为75 g·L^-1时,POD酶活性均升高,其中M13处理下,POD酶活性最高,达到（22.17±10.00）U·g^-1 FW。当培养基处理相同时,随着离体培养时间的延长,POD酶活性出现逐渐升高的趋势,说明胚珠离体培养阶段,适宜的蔗糖含量和培养时间控制POD酶活性的升高,有助于防止离体培养胚珠的老化。

Table 7
表7
表7不同培养基和不同离体培养时间对离体培养胚珠POD酶活性的影响
Table 7Effect of different medium and culture time on POD activity of ovule in vitro (U·g^-1 FW)

培养基 Medium	时间Time (d)
	42	49	56	63
M1	9.43±0.32bcd	9.37±0.15bcd	13.27±0.29ef	15.13±0.68e
M2	8.83±0.12def	8.87±0.21def	11.57±0.21h	13.93±0.25fg
M3	7.53±0.12g	7.63±0.25g	10.37±0.21j	11.75±0.64h
M4	7.23±0.15g	7.23±0.15g	9.57±0.12k	11.00±0.39h
M5	9.67±0.57bc	9.97±0.38ab	12.83±0.32fg	16.23±0.74d
M6	9.13±0.25cde	9.27±0.47cd	11.07±0.15i	14.87±0.57ef
M7	8.87±0.31def	8.97±0.12de	9.03±0.23lm	13.25±0.49g
M8	8.43±0.25f	8.47±0.21ef	8.63±0.23m	11.95±0.68h
M9	10.03±0.21ab	9.97±0.38ab	13.63±0.23e	18.17±0.55c
M10	9.77±0.76bc	9.67±0.71abc	12.57±0.32g	15.03±0.71e
M11	8.23±0.35f	8.33±0.51f	10.97±0.40i	13.07±0.47g
M12	7.47±0.40g	7.53±0.15g	9.37±0.06kl	11.47±0.51h
M13	10.57±0.25a	10.17±0.32a	18.87±0.35a	22.17±10.00a
M14	9.97±0.31ab	10.17±0.32a	17.33±0.25b	20.07±0.38b
M15	9.77±0.40bc	9.87±0.21abc	15.73±0.59c	19.53±0.15b
M16	8.57±0.31ef	8.87±0.25def	14.87±0.35d	18.50±0.10c

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2.2.6 对离体培养胚珠PPO酶活性的影响 由表8可以看出,当培养时间相同、基本培养基为固液双层培养基时,PPO酶活性均较低;随着附加蔗糖含量的增加,PPO酶活性出现逐渐升高的趋势,当附加蔗糖质量为75 g·L^-1时,PPO酶活性均升高,其中M13处理下,PPO酶活性最高,达到（8.67±0.25）U·g^-1 FW。结合2.2.3,当蔗糖含量为75 g·L^-1时,总酚含量较低,可能是由于此时离体胚珠内PPO酶活性偏高,造成离体培养胚珠总酚物质的减少,醌类化合物质的积累,胚珠组织出现褐变,造成离体胚珠褐化,加剧了离体胚珠内胚的败育,从而影响了离体胚珠的发育率。当培养基处理相同时,随着离体培养时间的延长,PPO酶活性出现逐渐升高的趋势,说明胚珠离体培养阶段控制PPO酶活性的升高,有助于防止离体培养胚珠的褐化。

Table 8
表8
表8不同培养基和不同离体培养时间对离体培养胚珠PPO酶活性的影响
Table 8Effect of different medium and culture time on PPO activity of ovule in vitro (U·g^-1 FW)

培养基 Medium	时间Time (d)
	42	49	56	63
M1	2.67±0.40efg	3.67±0.25bc	3.77±0.47ef	5.77±0.31fg
M2	2.57±0.35fg	3.37±0.31bcde	3.33±0.15h	5.57±0.35gh
M3	2.47±0.35fg	3.23±0.12e	3.17±0.12j	5.10±0.01hi
M4	2.47±0.40fg	3.07±0.23e	3.03±0.31k	4.98±0.22i
M5	2.57±0.31fg	3.77±0.47b	3.83±0.31fg	7.03±0.32cd
M6	2.47±0.40fg	3.33±0.15bcde	3.77±0.23i	6.87±0.06cd
M7	2.37±0.46g	3.17±0.12de	3.67±0.25lm	6.55±0.21de
M8	2.37±0.38g	3.03±0.31e	3.53±0.40m	6.25±0.33ef
M9	3.67±0.25abc	3.57±0.21bcd	3.97±0.21e	7.63±0.25b
M10	3.33±0.21bcd	3.57±0.15bcd	3.87±0.06g	6.33±0.38e
M11	3.23±0.12cde	3.53±0.25bcd	3.47±0.25i	6.17±0.32ef
M12	3.07±0.23def	3.17±0.06de	3.37±0.23kl	5.77±0.31fg
M13	4.10±0.17a	4.87±0.25a	5.77±0.31a	8.67±0.25a
M14	3.87±0.40ab	4.77±0.23a	5.57±0.21b	8.33±0.38a
M15	3.33±0.21bcd	4.63±0.31a	5.53±0.29c	7.63±0.21b
M16	3.23±0.12cde	4.43±0.15a	5.53±0.31d	7.27±0.15bc

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2.3 不同生理指标与胚珠发育率的相关性

从各个生理指标与胚珠发育率的相关性分析可以看出,胚珠发育率与离体培养胚珠的可溶性蛋白含量、总酚含量和SOD酶活性呈极显著正相关,与POD酶活性和PPO酶活性呈极显著负相关,与淀粉含量相关性不显著（表9）。

Table 9
表9
表9不同生理指标与胚珠发育率的相关系数
Table 9Correlation coefficient of different physiological indexes and ovule development rate

生理指标 Physiological index	相关系数 Correlation coefficient
淀粉含量 Starch content (%)	0.062
可溶性蛋白含量 Soluble protein content (mg·g-1)	0.430**
总酚含量 Phenolics content (mg·g-1)	0.800**
SOD酶活性 SOD activity (U·g-1 FW)	0.733**
POD酶活性 POD activity (U·g-1 FW)	-0.331**
PPO酶活性 PPO activity (U·g-1 FW)	-0.325**

**表示0.01的显著水平 ** represents statistical significance at the 0.01 level

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2.4 胚萌发成苗及移栽驯化

由表10可以看出,E2胚萌发率最高,达到88.2%,即在胚珠离体培养49 d时,总胚数、萌发胚总数、胚萌发率均达到最高;而胚珠离体培养63 d,总胚数、萌发胚总数、胚萌发率均为最低。成苗移栽驯化后获得一批胚挽救苗（图1-E—H）。

图1

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图1自然授粉的‘红宝石’无核葡萄胚挽救过程中离体胚珠发育及胚萌发成苗

A：自然授粉的‘红宝石’无核葡萄果实;B：离体胚珠培养;C—D：发育胚珠;E—F：胚萌发;G-H：移栽驯化
Fig. 1In vitro ovule development and embryo germination during embryo rescue of selfing

‘Ruby seedless’ grape A: Selfing ‘Ruby seedless grape’ fruit; B: In vitro ovule cultured; C-D: Developmental ovule; E-F: Embryo germination; G-H: Transplanting domestication


Table 1
表1
表10 胚萌发率
Table 10 Embryo germination rate

材料 Material	胚珠发育总数（个） No. of ovule development	胚萌发总数（个） No. of embryo germination	胚萌发率 Embryo germination rate (%)
E1	107	87	81.3%
E2	272	240	88.2%
E3	250	202	80.8%
E4	89	68	76.4%

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3 讨论

影响无核葡萄离体胚珠发育的主要因素包括亲本基因型、基本培养基、附加成分、培养时期等^[1]。在胚珠离体培养阶段,为胚珠发育提供营养物质最主要的成分是培养基。本试验选择基本培养基为固液双层基本培养基时,胚珠发育率最高,说明在胚珠离体培养阶段,固液双层基本培养基有利于胚珠发育率的提高,且胚珠发育率均高于前人研究结果,唐冬梅等^[28]采用MM3基本培养基时,‘红宝石’无核葡萄×新郁杂交胚珠发育率为28.3%;LI等^[7]采用改良MM3基本培养基时,‘红宝石’无核葡萄×北醇杂交胚珠发育率为28.3%。在基本培养基中添加一定量的蔗糖,既为胚珠发育提供了能源,也起到调节培养基渗透压的作用。本试验发现在基本培养基中添加不同含量的蔗糖时,随着蔗糖含量的升高,胚珠发育率出现先升高后下降的趋势。与前人^[7,22,28,30]研究结果相比,减少基本培养基中的蔗糖含量,有助于胚珠发育率的提高,因此胚珠离体培养阶段,蔗糖含量不能过高,建议在前人一直沿用的60 g·L^-1蔗糖的基础上,进行下调。

胚珠离体培养时间的长短也会影响胚珠发育率。本试验发现随着离体培养时间的延长,胚珠发育率呈先增高后下降的趋势,离体培养49 d（7周）胚珠发育率较高,最高达到（42.23 ± 6.93）%,离体培养63 d（9周）胚珠发育率均降低。前人以‘红宝石’无核葡萄作母本,获得杂种胚珠进行离体培养时,离体培养胚珠时间不同,胚珠发育率不同,王飞等^[31]离体培养杂种胚珠40 d时,胚珠发育率为56.5%;LI等^[7]离体培养杂种胚珠56 d时,胚珠发育率为28.3%;唐冬梅等^[28]离体培养杂种胚珠60 d时,胚珠发育率为27.8%;罗尧幸^[29]离体培养杂种胚珠9周,即63 d时,胚珠发育率分别为13.7%和13.8%;李志瑛等^[15]离体培养杂种胚珠10周,即70 d时,胚珠发育率为13.81%,前人研究结果均表明随着离体培养时间的延长,从40 d至70 d,胚珠发育率越来越低,和本研究结果一致。因此胚珠离体培养阶段,缩短培养时间,有利于胚珠的发育;及时取出幼胚,进行第二阶段胚萌发的培养,有助于提高无核葡萄育种效率。

无核葡萄胚败育是一种细胞程序性死亡现象,种子败育型无核葡萄胚珠可能由于胚乳提前降解,导致胚营养缺乏而败育。在胚胎发育过程中,可溶性糖、可溶性蛋白、淀粉、多酚物质、酶活性及内源激素等的变化与细胞的生物功能和生理活动密切相关^{[32,33,34,35]}。本研究关于无核葡萄胚挽救过程中胚珠离体培养阶段,发育胚珠生理代谢物质的变化目前尚未有人开展研究。前人^{[36,37,38,39,40,41]}对相关生理指标测定研究主要是从不同品种、幼果不同取材时期等方面开展,本研究结果是对前人研究的一个补充。研究离体胚珠内生理指标的变化在一定程度上可以反映幼胚败育进程,说明在胚珠离体培养阶段,当胚珠生理活性较高时,应该及时取出幼胚,可以防止胚败育,提高胚珠发育率,进而提高胚萌发率和成苗率。如何进一步提高该阶段发育胚珠的生理活性,需要不断改良胚珠离体培养方法;提高胚珠发育率,是今后开展无核葡萄胚挽救育种工作需要考虑的一个重要研究内容。

4 结论

本研究在胚珠离体培养阶段,培养自然授粉65 d的‘红宝石’无核葡萄胚珠,采用固液双层基本培养基（改良MM3固体培养基+8 mg·L^-1 ER液体培养基）,附加45 g·L^-1蔗糖,培养时间49 d时,胚珠发育率最高,达到（42.23±6.93）%,胚珠生理活性也较强。通过相关性分析发现,胚珠发育率与离体培养胚珠的可溶性蛋白含量、总酚含量和SOD酶活性呈极显著正相关,与POD酶活性和PPO酶活性呈极显著负相关,与淀粉含量相关性不显著。胚珠发育率的提高是无核葡萄胚挽救育种效率提高的前提。

参考文献 View Option 原文顺序
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[7] LI G R, JI W, WANG G, ZHANG J X, WANG Y J . An improved embryo-rescue protocol for hybrid progeny from seedless Vitis vinifera grapes?×?wild Chines. Vitis species
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A highly efficient technique of embryo rescue is critical when using stenospermocarpic Vitis vinifera cultivars (female parents) to breed novel, disease-resistant, seedless grape cultivars by hybridizing with wild Chinese Vitis species (male parents) having many disease-resistance alleles. The effects of various factors on the improvement of embryo formation, germination, and plantlet development for seven hybrid combinations were studied. The results indicated that Beichun and Shuangyou were the best male parents. The best sampling time for ovule inoculation differed among the female parents. When hybrid ovules were cultured on a double-phase medium with five different solid medium types, percent embryo formation was highest (11.3-28.3%) on a modified MM3 medium. Percentages of embryo germination (15.4-55.4%) and plantlet development (11.15-44.6%) were all highest when embryos were cultured on Woody Plant Medium + 5.7 muM indole-3-acetic acid + 4.4 muM 6-benzylaminopurine + 1.4 muM gibberellic acid + 2% sucrose + 0.05% casein hydrolysate + 0.3% activated charcoal + 0.7% agar. In the absence of other amino acids, the addition of proline significantly increased embryo formation (36.1%), embryo germination (64.6%), and plantlet development (90.5%). A highly efficient protocol has been developed for hybrid embryo rescue from seedless V. vinifera grapes x wild Chinese Vitis species that results in a significant improvement in breeding efficiency for new disease-resistant seedless grapes.

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[13] 王爱玲, 王跃进, 唐冬梅, 张剑侠, 张朝红 . 提高无核葡萄胚挽救中幼胚成苗率的研究
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【Objective】The objective of the study was to improve the breeding efficiency of seedless grape through embryo rescue in vitro and investigate the effects of various factors on seedlings in vitro. 【Method】 Hybrid ovules of 7 cross combinations were used to investigate the effect of different genotypes, different ovule culture media, supplementation of different aminophenols, low-temperature treatment on the proportion of seedlings of harvested fruits in vitro. 【Result】 The results indicated that among the investigated embryoes of seedless grape, the female parent and male parent had influence on seedlings. In cross combinations with diploid seedless grapes as female parent, diploid seedless grapes as male parent was suitable for seedlings than tetraploid seeded grapes. Among the three different media, MM4 medium was most suitable for embryo development and seedlings in vitro culture of seedless grapes. ER medium supplemented with 4 mmol?L-1 proline was most suitable for embryo development in vitro culture of seedless grapes. The effect of low-temperature treatment of Ruby Seedless×Centennial Seedless and Ruby Seedless×Black Olympia for 3 days was the best in seedling growth. 【Conclusion】 ER medium supplemented with 4 mmol?L-1 proline or MM4 medium are suitable for embryo rescue in vitro culture of seedless grapes. Low-temperature treatment of fruits for 3 days, has a positive effect on improvement of embryo rescue efficiency of diploid seedless grape×diploid seedless grape.


WANG A L, WANG Y J, TANG D M, ZHANG J X, ZHANG C H . Research on improvement of seedling rate in embryo rescue of seedless grapes
Scientia Agricultura Sinica, 2010,43(20):4238-4245. (in Chinese)
URL [本文引用: 2]
【Objective】The objective of the study was to improve the breeding efficiency of seedless grape through embryo rescue in vitro and investigate the effects of various factors on seedlings in vitro. 【Method】 Hybrid ovules of 7 cross combinations were used to investigate the effect of different genotypes, different ovule culture media, supplementation of different aminophenols, low-temperature treatment on the proportion of seedlings of harvested fruits in vitro. 【Result】 The results indicated that among the investigated embryoes of seedless grape, the female parent and male parent had influence on seedlings. In cross combinations with diploid seedless grapes as female parent, diploid seedless grapes as male parent was suitable for seedlings than tetraploid seeded grapes. Among the three different media, MM4 medium was most suitable for embryo development and seedlings in vitro culture of seedless grapes. ER medium supplemented with 4 mmol?L-1 proline was most suitable for embryo development in vitro culture of seedless grapes. The effect of low-temperature treatment of Ruby Seedless×Centennial Seedless and Ruby Seedless×Black Olympia for 3 days was the best in seedling growth. 【Conclusion】 ER medium supplemented with 4 mmol?L-1 proline or MM4 medium are suitable for embryo rescue in vitro culture of seedless grapes. Low-temperature treatment of fruits for 3 days, has a positive effect on improvement of embryo rescue efficiency of diploid seedless grape×diploid seedless grape.


[14] 赵雅楠, 骆强伟, 王跃进 . 利用胚挽救技术创制无核抗寒葡萄新种质
中国农业科学, 2018,51(21):4119-4130.
DOI:10.3864/j.issn.0578-1752.2018.21.010URL [本文引用: 2]
【Objective】 The objective of this study is to obtain new cold-resistant seedless grape germplasm and improve the breeding efficiency through embryo rescue in vitro. 【Method】 The different genotypes, basal culture media (MM3 and ER), amino acids (2.5 mmol·L ^-1cysteine, asparagine, glutamine and serine were added in MM3 medium) were used to investigate the effect on the embryo development rates and seedling rates from ovules of 7 cross combinations. From the parents, the molecular markers GSLP1-569 and SCC8-1018 for seedlessness and the marker S241-717 for cold-resistance were used to identify the strains with specific bands, and then the three markers were used to screen the hybrids by recognizing the specific bands. 【Result】 Of the 7 combinations, 1 168 ovules cultured in MM3 were obtained 331 embryos and 97 hybrid strains. Among these crosses, ‘Ruby Seedless’ × ‘Beichun’ got the highest embryo development rate and seedling rate, which were 46.00% and 17.33%, respectively. Compared with female parents, for ‘Shuangyou’ as male parent and ‘Blush Seedless’ ‘Thompson Seedless’ ‘Kunxiang Seedless’ as female parents, ‘Kunxiang Seedless’ × ‘Shuangyou’ had the highest embryo development rate and seedling rate, which were 45.39% and 16.31%, respectively. For ‘Beibinghong’ as male parent and ‘Blush Seedless’ ‘Beauty Seedless’ ‘Thompson Seedless’ as female parents, ‘Blush Seedless’ × ‘Beibinghong’ had the highest embryo development rate and seedling rate, which were 33.52% and 6.59%, respectively. Different male parents also influenced seedlings formation. The embryo development rates of ‘Blush Seedless’ × ‘Beibinghong’ and ‘Blush Seedless’ × ‘Shuangyou’ were 33.52% and 39.00%, and the seedling rates of them were 6.59% and 9.50%, respectively. The embryo development rates of ‘Thompson Seedless’ × ‘Beibinghong’ and ‘Thompson Seedless’ × ‘Shuangyou’ were 7.64% and 19.26%, and the seedling rates of them were 1.91% and 8.15%, respectively. The rate of embryo development and seedling of ovule cultured in MM3 medium was higher than that of ER medium. For ‘Kunxiang Seedless’ × ‘Shuangyou’, the ovules cultured in MM3 medium with 2.5 mmol·L ^-1 glutamine showed the highest embryo development rate, which was 59.69%. The seedling rate of ovules cultured in basal media with asparagine and glutamine was 21.43% and 20.93%, respectively. The effect of promoting seedlings development was significant. For ‘Ruby Seedless’ × ‘Beichun’, the ovules in media with 2.5 mmol·L ^-1 asparagine showed the highest embryo development rate, which was 55.71%. The seedling rate of ovules cultured in basal media with asparagine and glutamine was 21.43% and 22.22%, respectively. The effect of promoting seedlings development was significant. By using the molecular markers GSLP1-569 and SCC8-1018 for seedlessness and the marker S241-717 for cold-resistance, 83 hybrid strains from 6 cross combinations were detected, and there were 49 strains with specific bands of the molecular markers linked to the seedlessness, 55 strains with specific bands of the markers S241-717 for cold-resistance. Importantly, 36 strains with both seedlessness and cold-resistance were identified in this study. 【Conclusion】 ‘Kunxiang Seedless’ and ‘Blush Seedless’ are suitable as the female parents for embryo rescue in vitro of seedless grapes. The embryo rescue efficiency of ‘Shuangyou’ as the male parent is higher than that of ‘Beibinghong’. MM3 is suitable for embryo rescue in vitro, and the addition of amide acids to MM3 culture medium is helpful to the seedling formation. Among the 83 hybrid strains detected by molecular markers, 36 strains may have the seedless and cold-resistant characteristics.
ZHAO Y N, LUO Q W, WANG Y J . Breeding for grape germplasm involved in seedlessness with cold-resistant using embryo rescue
Scientia Agricultura Sinica, 2018,51(21):4119-4130. (in Chinese)
DOI:10.3864/j.issn.0578-1752.2018.21.010URL [本文引用: 2]
【Objective】 The objective of this study is to obtain new cold-resistant seedless grape germplasm and improve the breeding efficiency through embryo rescue in vitro. 【Method】 The different genotypes, basal culture media (MM3 and ER), amino acids (2.5 mmol·L ^-1cysteine, asparagine, glutamine and serine were added in MM3 medium) were used to investigate the effect on the embryo development rates and seedling rates from ovules of 7 cross combinations. From the parents, the molecular markers GSLP1-569 and SCC8-1018 for seedlessness and the marker S241-717 for cold-resistance were used to identify the strains with specific bands, and then the three markers were used to screen the hybrids by recognizing the specific bands. 【Result】 Of the 7 combinations, 1 168 ovules cultured in MM3 were obtained 331 embryos and 97 hybrid strains. Among these crosses, ‘Ruby Seedless’ × ‘Beichun’ got the highest embryo development rate and seedling rate, which were 46.00% and 17.33%, respectively. Compared with female parents, for ‘Shuangyou’ as male parent and ‘Blush Seedless’ ‘Thompson Seedless’ ‘Kunxiang Seedless’ as female parents, ‘Kunxiang Seedless’ × ‘Shuangyou’ had the highest embryo development rate and seedling rate, which were 45.39% and 16.31%, respectively. For ‘Beibinghong’ as male parent and ‘Blush Seedless’ ‘Beauty Seedless’ ‘Thompson Seedless’ as female parents, ‘Blush Seedless’ × ‘Beibinghong’ had the highest embryo development rate and seedling rate, which were 33.52% and 6.59%, respectively. Different male parents also influenced seedlings formation. The embryo development rates of ‘Blush Seedless’ × ‘Beibinghong’ and ‘Blush Seedless’ × ‘Shuangyou’ were 33.52% and 39.00%, and the seedling rates of them were 6.59% and 9.50%, respectively. The embryo development rates of ‘Thompson Seedless’ × ‘Beibinghong’ and ‘Thompson Seedless’ × ‘Shuangyou’ were 7.64% and 19.26%, and the seedling rates of them were 1.91% and 8.15%, respectively. The rate of embryo development and seedling of ovule cultured in MM3 medium was higher than that of ER medium. For ‘Kunxiang Seedless’ × ‘Shuangyou’, the ovules cultured in MM3 medium with 2.5 mmol·L ^-1 glutamine showed the highest embryo development rate, which was 59.69%. The seedling rate of ovules cultured in basal media with asparagine and glutamine was 21.43% and 20.93%, respectively. The effect of promoting seedlings development was significant. For ‘Ruby Seedless’ × ‘Beichun’, the ovules in media with 2.5 mmol·L ^-1 asparagine showed the highest embryo development rate, which was 55.71%. The seedling rate of ovules cultured in basal media with asparagine and glutamine was 21.43% and 22.22%, respectively. The effect of promoting seedlings development was significant. By using the molecular markers GSLP1-569 and SCC8-1018 for seedlessness and the marker S241-717 for cold-resistance, 83 hybrid strains from 6 cross combinations were detected, and there were 49 strains with specific bands of the molecular markers linked to the seedlessness, 55 strains with specific bands of the markers S241-717 for cold-resistance. Importantly, 36 strains with both seedlessness and cold-resistance were identified in this study. 【Conclusion】 ‘Kunxiang Seedless’ and ‘Blush Seedless’ are suitable as the female parents for embryo rescue in vitro of seedless grapes. The embryo rescue efficiency of ‘Shuangyou’ as the male parent is higher than that of ‘Beibinghong’. MM3 is suitable for embryo rescue in vitro, and the addition of amide acids to MM3 culture medium is helpful to the seedling formation. Among the 83 hybrid strains detected by molecular markers, 36 strains may have the seedless and cold-resistant characteristics.

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Sino-Overseas Grapevine & Wine, 2018(4):69-72. (in Chinese)
[本文引用: 1]

[25] 郝燕, 杨瑞, 王玉安, 张坤, 李红旭 . 无核葡萄新品种‘紫丰’的选育
果树学报, 2019,36(4):533-536.
[本文引用: 1]

HAO Y, YANG R, WANG Y A, ZHANG K, LI H X . A new seedless grape cultivar 'Zifeng'
Journal of Fruit Science, 2019,36(4):533-536. (in Chinese)
[本文引用: 1]

[26] 张剑侠, 唐冬梅, 王跃进 . 无核葡萄新品种‘秦红1号’
园艺学报, 2015,42(S2):2831-2832.
DOI:10.16420/j.issn.0513-353x.2015-0654URL [本文引用: 1]
‘Qinhong 1’was selected from sexual cross between seedless grape cultivars‘Delight’（Vitis vinifera L.）and‘Ruby Seedless’（V. vinifera L.）through the embryo rescue techniques. Its fruit characteristics are as following：Fruit color is red to pale purple，cluster is branch conical，single cluster weight is more than 800 g，the biggest one reaches 1 130 g，the shape of berry is nearly round or oval，the average berry weight is 3.4 g. The fruit has a good quality，contains 18% soluble solids，and 0.9% titratable acid. Its flesh is delicate and crisp. It has high yield which can reaches 49 395 kg · hm-2 during full bearing period. The cultivar is suitable for open field cultivation in the arid areas of north China and facility cultivation in the north and south China.
ZHANG J X, TANG D M, WANG Y J . A new seedless grape cultivar 'Qinhong 1'
Acta Horticulturae Sinica, 2015,42(S2):2831-2832. (in Chinese)
DOI:10.16420/j.issn.0513-353x.2015-0654URL [本文引用: 1]
‘Qinhong 1’was selected from sexual cross between seedless grape cultivars‘Delight’（Vitis vinifera L.）and‘Ruby Seedless’（V. vinifera L.）through the embryo rescue techniques. Its fruit characteristics are as following：Fruit color is red to pale purple，cluster is branch conical，single cluster weight is more than 800 g，the biggest one reaches 1 130 g，the shape of berry is nearly round or oval，the average berry weight is 3.4 g. The fruit has a good quality，contains 18% soluble solids，and 0.9% titratable acid. Its flesh is delicate and crisp. It has high yield which can reaches 49 395 kg · hm-2 during full bearing period. The cultivar is suitable for open field cultivation in the arid areas of north China and facility cultivation in the north and south China.

[27] 田莉莉 . 抗病无核葡萄胚挽救育种及种质创新
[D]. 杨凌: 西北农林科技大学, 2007.
[本文引用: 1]

TIAN L L . Breeding for the disease-resistant seedless grape novel varieties and innovating of new germplasms using embryo rescue
[D]. Yangling: Northwest A & F University, 2007. (in Chinese)
[本文引用: 1]

[28] 唐冬梅, 王跃进, 赵荣华, 潘学军, 蔡军社, 张剑侠, 张朝红, 骆强伟 . 无核葡萄胚挽救中影响胚发育的因子
中国农业科学, 2009,42(7):2449-2457.
URL [本文引用: 3]
【Objective】 The objective of this study was to improve breeding efficiency of seedless grape through embryo rescue in vitro, to investigate the effects of various correlative factors on embryo development in vitro. 【Method】 The open pollination ovules from 12 seedless grape varieties (lines), including Thompson Seedless, Black Monukka, Centennial Seedless, Delight, Flame Seedless, Superior Seedless, Autumn Royal, Fantasy Seedless, Dawn Seedless, Otilia Seedless, 00-3-1, Blush Seedless and hybrid ovules of 6 cross combinations Ruby Seedless×Guifeimeigui, Ruby Seedless×Xinyu, Delight×Kyoho, Xinyu×Kyoho, Emerald Seedless×Taishan-2, Delight ×Jiangxi-2 were used to investigate the effect of different ovule development media, different medium phases, low temperature, and light/dark condition on the proportion of embryo development in vitro, and evaluate the optimal sampling dates after bloom for picking the young berries, culturing the excised ovules in vitro in different cultivars and crosses. 【Result】 The results indicated that among the 8 media, Nitsch medium supplemented with 0.5 mg?L-1 GA3 and 1.5 mg?L-1 IAA, MM3 medium were found to be most suitable for embryo development in vitro of seedless grapes, and the next was ER medium. Among the solid, liquid and solid-liquid double phase media, solid-liquid double phase medium had the most positive effect on embryo development of middling ovules of seedless grape cv. Delight, yet three phase media showed no significant different effect on embryo development of bigger ovules of grape line 00-3-1. The results also showed that low temperature at the stage of ovule culture in vitro had a negative effect on embryos development. Dark culture had a positive effect on embryo development in ovule cultures in vitro. The optimal sampling dates after bloom of culturing ovules in vitro dissected from berries varied among different cultivars and crosses, the optimal sampling dates was 35 day after bloom (DAB) in Thompson Seedless, 40 DAB in Centennial Seedless, 40 DAB in Flame Seedless, 60 DAB in Delight, 55 DAB in Dawn Seedless, 70 DAB in Black Monukka, 50 DAB in Superior Seedless, 70 DAB in Autumn Royal, 60 DAB in Fantasy Seedless, 40 DAB in Otilia Seedless, 70 DAB in Ruby Seedless×Guifeimeigui, respectively. 【Conclusion】 The results indicated that Nitsch medium supplemented with 0.5 mg?L-1 GA3 and 1.5 mg?L-1 IAA, MM3 and ER media were suitable for embryo rescue in vitro of seedless grapes. The culture environment of room temperature (25±2)℃, solid-liquid double phase medium and dark culture had a positive effect on improvement of embryo rescue efficiency.


TANG D M, WANG Y J, ZHAO R H, PAN X J, CAI J S, ZHANG J X, ZHANG C H, LUO Q W . Factors influencing embryo development in embryo rescue of seedless grapes
Scientia Agricultura Sinica, 2009,42(7):2449-2457. (in Chinese)
URL [本文引用: 3]
【Objective】 The objective of this study was to improve breeding efficiency of seedless grape through embryo rescue in vitro, to investigate the effects of various correlative factors on embryo development in vitro. 【Method】 The open pollination ovules from 12 seedless grape varieties (lines), including Thompson Seedless, Black Monukka, Centennial Seedless, Delight, Flame Seedless, Superior Seedless, Autumn Royal, Fantasy Seedless, Dawn Seedless, Otilia Seedless, 00-3-1, Blush Seedless and hybrid ovules of 6 cross combinations Ruby Seedless×Guifeimeigui, Ruby Seedless×Xinyu, Delight×Kyoho, Xinyu×Kyoho, Emerald Seedless×Taishan-2, Delight ×Jiangxi-2 were used to investigate the effect of different ovule development media, different medium phases, low temperature, and light/dark condition on the proportion of embryo development in vitro, and evaluate the optimal sampling dates after bloom for picking the young berries, culturing the excised ovules in vitro in different cultivars and crosses. 【Result】 The results indicated that among the 8 media, Nitsch medium supplemented with 0.5 mg?L-1 GA3 and 1.5 mg?L-1 IAA, MM3 medium were found to be most suitable for embryo development in vitro of seedless grapes, and the next was ER medium. Among the solid, liquid and solid-liquid double phase media, solid-liquid double phase medium had the most positive effect on embryo development of middling ovules of seedless grape cv. Delight, yet three phase media showed no significant different effect on embryo development of bigger ovules of grape line 00-3-1. The results also showed that low temperature at the stage of ovule culture in vitro had a negative effect on embryos development. Dark culture had a positive effect on embryo development in ovule cultures in vitro. The optimal sampling dates after bloom of culturing ovules in vitro dissected from berries varied among different cultivars and crosses, the optimal sampling dates was 35 day after bloom (DAB) in Thompson Seedless, 40 DAB in Centennial Seedless, 40 DAB in Flame Seedless, 60 DAB in Delight, 55 DAB in Dawn Seedless, 70 DAB in Black Monukka, 50 DAB in Superior Seedless, 70 DAB in Autumn Royal, 60 DAB in Fantasy Seedless, 40 DAB in Otilia Seedless, 70 DAB in Ruby Seedless×Guifeimeigui, respectively. 【Conclusion】 The results indicated that Nitsch medium supplemented with 0.5 mg?L-1 GA3 and 1.5 mg?L-1 IAA, MM3 and ER media were suitable for embryo rescue in vitro of seedless grapes. The culture environment of room temperature (25±2)℃, solid-liquid double phase medium and dark culture had a positive effect on improvement of embryo rescue efficiency.


[29] 罗尧幸 . 耐寒无核葡萄胚挽救种质创新研究
[D]. 山西: 山西农业大学, 2018.
[本文引用: 2]

LUO Y X . Study on germplasm innovation of cold-tolerant and seedless grapes by embryo rescue
[D]. Shanxi: Shanxi Agricultural University, 2018. (in Chinese)
[本文引用: 2]

[30] GRAY D J, MORTENSEN J A, BENTON C M, DURHAM R E, MOORE G A . Ovule culture to obtain progeny from hybrid seedless bunch grapes
Journal of the American Society for Horticultural Science, 1990,115(6):1019-1024.
DOI:10.21273/JASHS.115.6.1019URL [本文引用: 1]

[31] 王飞, 王跃进, 周建锡 . 无核葡萄与中国野生葡萄杂种的胚挽救技术研究
园艺学报, 2006,33(5):1079-1082.
URL [本文引用: 1]
Through crossing between seedless grapes and Chinese wild grapes we got four group hybrid grape seedlings. The best time to harvest the hybrid grapes ovule for embryo rescued were as follows: 45 d after pollination are (1) Flame seedless with Vitis amurensis, (2) Ruby seedless with Olmo seedless, (3)Ruby seedless with Beichun, (4) Olmo seedless with Vitis amurensis; 30 d after pollination is (5) Changsui saltanina seedless with Vitis amarensis; 35 d after pollination is (6) Self-pollinated saltanina. Nistschs medium with additional 0.5 mg·L ^{- 1} 6-BA + 0.5 mg·L^-1 GA₃ + 2.5 mg·L^-1 IBA + 0.1 mg·L^-1 ZT was suitable for cross (1) , (3) and (5) , but Nistschs medium with additional 0.5 mg·L^-1 6-BA + 0.5 mg·L^-1GA₃ + 2.0 mg·L^-1 IBA + 0.1 mg·L^-1 ZT was suitable for cross (2) , (4) and (6). For embryo regeneration Nistschs medium with additional 2.0 mg·L^-1 IBA + 1.0 mg·L^-1 6-BA + GA₃ 0.2 mg·L^-1 was suitable for cross (1) , (2) , (3) , (5) and (6) , but for cross (4) medium with 1.5 mg·L^-1 IBA + 0.2 mg·L ^{- 1} GA₃ + 1.0 mg·L ^{- 1} 6-BA was more suitable. 1/2MS medium adding 0.15 mg·L ^{- 1} IBA + 0.02 mg·L ^{- 1} 6-BA were suitable for cross (1) and (5) to acquire embryo rescue plants, but 1 /2MS medium with addition 0.1 mg·L ^{- 1} IBA + 0.02 mg·L ^{- 1} 6-BA was suitable for cross (2) and (4).
WANG F, WANG Y J, ZHOU J X . Breeding technology of embryo rescue in seedless grape with Chinese wild grape
Acta Horticulturae Sinica, 2006,33(5):1079-1082. (in Chinese)
URL [本文引用: 1]
Through crossing between seedless grapes and Chinese wild grapes we got four group hybrid grape seedlings. The best time to harvest the hybrid grapes ovule for embryo rescued were as follows: 45 d after pollination are (1) Flame seedless with Vitis amurensis, (2) Ruby seedless with Olmo seedless, (3)Ruby seedless with Beichun, (4) Olmo seedless with Vitis amurensis; 30 d after pollination is (5) Changsui saltanina seedless with Vitis amarensis; 35 d after pollination is (6) Self-pollinated saltanina. Nistschs medium with additional 0.5 mg·L ^{- 1} 6-BA + 0.5 mg·L^-1 GA₃ + 2.5 mg·L^-1 IBA + 0.1 mg·L^-1 ZT was suitable for cross (1) , (3) and (5) , but Nistschs medium with additional 0.5 mg·L^-1 6-BA + 0.5 mg·L^-1GA₃ + 2.0 mg·L^-1 IBA + 0.1 mg·L^-1 ZT was suitable for cross (2) , (4) and (6). For embryo regeneration Nistschs medium with additional 2.0 mg·L^-1 IBA + 1.0 mg·L^-1 6-BA + GA₃ 0.2 mg·L^-1 was suitable for cross (1) , (2) , (3) , (5) and (6) , but for cross (4) medium with 1.5 mg·L^-1 IBA + 0.2 mg·L ^{- 1} GA₃ + 1.0 mg·L ^{- 1} 6-BA was more suitable. 1/2MS medium adding 0.15 mg·L ^{- 1} IBA + 0.02 mg·L ^{- 1} 6-BA were suitable for cross (1) and (5) to acquire embryo rescue plants, but 1 /2MS medium with addition 0.1 mg·L ^{- 1} IBA + 0.02 mg·L ^{- 1} 6-BA was suitable for cross (2) and (4).

[32] 李顺雨 . 种子败育型葡萄胚败育的形态学观察及生理基础
[D]. 贵阳: 贵州大学, 2009.
[本文引用: 1]

LI S Y . Morphological observation and physiological basis of embryo abortion in seed-aborting grape
[D]. Guiyang: Guizhou University, 2009. (in Chinese)
[本文引用: 1]

[33] 朱林, 李佩芬, 卢炳芝, 于向荣 . 无核葡萄品种的胚珠培养和胚分化(简报)
植物生理学通讯, 1992,28(4):273-274.
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ZHU L, LI P F, LU B Z, YU X R . Study on ovule culture and embryo differentiation of seedless grape varieties
Plant Physiology Communications, 1992,28(4):273-274. (in Chinese)
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[34] LAFON-PLACETTE C, K?HLER C. Embryo and endosperm, partners in seed development
Current Opinion in Plant Biology, 2014,17:64-69.
DOI:10.1016/j.pbi.2013.11.008URL [本文引用: 1]
Angiosperm seeds are the major source of human calories, generating a pressing need to understand the underlying processes governing seed growth and development. They are composed of the two fertilization products, embryo and endosperm surrounded by the maternally derived seed coat. The successful interaction of all three seed components is a requirement for seeds to complete their development and to produce viable embryos that are competent to establish a new sporophytic generation. Here, we review recent reports investigating signal exchange between embryo and endosperm, focusing in particular on the transport of metabolites and small RNAs between both fertilization products.

[35] 寇艳茹 . 板栗胚胎发育生理机制及调控技术研究
[D]. 北京: 北京林业大学, 2015.
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KOU Y R . Studies on physiological mechanism of embryonic development and its regulation in Chinese chesnut
[D]. Beijing: Beijing Forestry University, 2015. (in Chinese)
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[36] 江淑平 . 葡萄无核品种胚败育因子及其机理的研究
[D]. 杨凌: 西北农林科技大学, 2004.
[本文引用: 1]

JIANG S P . Factors and mechanism of embryonic abortion in seedless grapes
[D]. Yangling: Northwest A & F University, 2004. (in Chinese)
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[37] 董姝娟, 郭修武 . 几个葡萄品种胚胎发育过程中保护酶活性、丙二醛及内含营养物质含量的变化
中外葡萄与葡萄酒, 2008(2):12-16.
[本文引用: 1]

DONG S J, GUO X W . Change of protective enzyme activity, contents of MDA and inner nutriments during the embryonic development of several grape cultivars
Sino-Overseas Grapevine & Wine, 2008(2):12-16. (in Chinese)
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[38] 王静波 . 无核葡萄胚胎发育形态学与生理特性研究
[D]. 太谷: 山西农业大学, 2018.
[本文引用: 1]

WANG J B . Research on morphological and physiological characteristics of embryo development in seedless grapes
[D]. Taigu: Shanxi Agricultural University, 2018. (in Chinese)
[本文引用: 1]

[39] 张朝红, 王跃进, 王西平, 杨亚州 . ‘杨格尔’与‘京秀’葡萄胚珠发育过程中的活性氧代谢比较
园艺学报, 2007,34(6):1361-1366.
URL [本文引用: 1]
The O₂^- production rate and H₂O₂ content and the activities of the antioxidant enzymes in the ovule of seedless grape ‘Youngle’and seeded grape ‘Jingxiu’were studied in the different periods after bloom. The results indicated that the concentrations of O₂^- rose all the time, and the increasing rate in the ovule of seedless cultivar ‘Youngle’was greater than that in the ovule of seeded cultivar ‘Jingxiu’during middle and later periods of development; H₂O₂ concentration increased initially and decreased before it increased again. The subsequent increase in the seedless cultivar ‘Youngle’was significantly greater than seeded cultivar‘Jingxiu’. The activities of SOD, CAT and AsA-POD increased initially and dropped drastically after ovule abortion in the seedless cultivar‘Youngle’. But it maintained high level in seeded cultivar‘Jingxiu’. The activity of POD in the ovule of the seedless cultivar ‘Youngle’displayed a similar trend to the other enzymes, but the activity in seeded cultivar‘Jingxiu’ increased constantly, but the increasing speed was lower in later stages. The changes of reactive oxygen content and the activity of antioxidant enzymes in ovules have some relevance to ovule abortion in the seedless cultivar 'Youngle'.
ZHANG C H, WANG Y J, WANG X P, WANG Y Z . A comparative study on metabolism of reactive oxygen species during ovule development in seeded and seedless grapes
Acta Horticulturae Sinica, 2007,34(6):1361-1366. (in Chinese)
URL [本文引用: 1]
The O₂^- production rate and H₂O₂ content and the activities of the antioxidant enzymes in the ovule of seedless grape ‘Youngle’and seeded grape ‘Jingxiu’were studied in the different periods after bloom. The results indicated that the concentrations of O₂^- rose all the time, and the increasing rate in the ovule of seedless cultivar ‘Youngle’was greater than that in the ovule of seeded cultivar ‘Jingxiu’during middle and later periods of development; H₂O₂ concentration increased initially and decreased before it increased again. The subsequent increase in the seedless cultivar ‘Youngle’was significantly greater than seeded cultivar‘Jingxiu’. The activities of SOD, CAT and AsA-POD increased initially and dropped drastically after ovule abortion in the seedless cultivar‘Youngle’. But it maintained high level in seeded cultivar‘Jingxiu’. The activity of POD in the ovule of the seedless cultivar ‘Youngle’displayed a similar trend to the other enzymes, but the activity in seeded cultivar‘Jingxiu’ increased constantly, but the increasing speed was lower in later stages. The changes of reactive oxygen content and the activity of antioxidant enzymes in ovules have some relevance to ovule abortion in the seedless cultivar 'Youngle'.

[40] 潘学军, 李顺雨, 张文娥, 刘崇怀 . 种子败育型葡萄胚珠败育前后抗氧化物质及丙二醛含量的变化
园艺学报, 2011,38(增刊):2482.
[本文引用: 1]

PAN X J, LI S Y, ZHANG W E, LIU C H . Changes of antioxidant and malondialdehyde contents in ovules of seed abortive grapes before and after abortion
Acta Horticulturae Sinica, 2011,38(Suppl.):2482. (in Chinese)
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[41] 纪薇, 郭荣荣, 王静波, 焦晓博, 闫钊, 昌秦湘, 董志刚, 王跃进 . 无核葡萄胚败育生理生化因子灰色关联分析
园艺学报, 2019,46(8):1473-1485.
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JI W, GUO R R, WANG J B, JIAO X B, YAN Z, CHANG Q X, DONG Z G, WANG Y J . Grey correlation analysis of physiological and biochemical factors in embryo abortion of seedless grape
Acta Horticulturae Sinica, 2019,46(8):1473-1485. (in Chinese)
[本文引用: 1]