无核葡萄离体胚珠发育影响因子及其生理变化

doi:10.3864/j.issn.0578-1752.2020.22.011

摘要/Abstract

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.

Key words: seedless grape, embryo rescue, ovule development, in vitro culture, physiological indexes

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.

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.

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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

培养基 Medium	时间Time (d)
培养基 Medium	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

培养基 Medium	时间Time (d)
培养基 Medium	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

培养基 Medium	时间Time (d)
培养基 Medium	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

培养基 Medium	时间Time (d)
培养基 Medium	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