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

Abstract

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

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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References 41

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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

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

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培养基 Medium	时间Time (d)
培养基 Medium	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

培养基 Medium	时间Time (d)
培养基 Medium	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

培养基 Medium	时间Time (d)
培养基 Medium	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

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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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%