中国农业科学 ›› 2020, Vol. 53 ›› Issue (22): 4646-4657.doi: 10.3864/j.issn.0578-1752.2020.22.011
李桂荣1,2,全冉1,程珊珊1,侯小进1,樊秀彩3,扈惠灵1()
收稿日期:
2020-03-25
接受日期:
2020-07-13
出版日期:
2020-11-16
发布日期:
2020-11-28
通讯作者:
扈惠灵
作者简介:
李桂荣,E-mail: 基金资助:
LI GuiRong1,2,QUAN Ran1,CHENG ShanShan1,HOU XiaoJin1,FAN XiuCai3,HU HuiLing1()
Received:
2020-03-25
Accepted:
2020-07-13
Online:
2020-11-16
Published:
2020-11-28
Contact:
HuiLing HU
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。
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.
表1
不同培养基处理"
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 |
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"
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 |
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"
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 |
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"
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 |
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"
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 |
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"
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 |
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"
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 |
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"
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 |
表9
不同生理指标与胚珠发育率的相关系数"
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** |
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