四倍体大白菜游离小孢子培养技术

doi:10.16420/j.issn.0513-353x.2023-0046

摘要/Abstract

摘要：

四倍体大白菜减数分裂过程中产生的配子具有多样性，开展四倍体大白菜游离小孢子培养，旨在获得更多变异程度较大的纯合二倍体新资源。从14份早熟四倍体大白菜中筛选出4份易生胚的材料，通过花蕾表型、热激温度、生根激素、倍性鉴定等方面开展研究，建立了四倍体大白菜游离小孢子培养技术体系。研究结果表明：14份不同基因型四倍体大白菜，盛花期花蕾的花瓣长度与花药长度的比值介于0 ~ 0.5时，其单核靠边期的小孢子占比高，此时的出胚率最高;热激温度为33.5 ℃时暗培养24 h有利于小孢子胚胎发生;在生根培养基MS中添加吲哚丁酸（IBA）0.3 mg · L^-1和萘乙酸（NAA）0.1 mg · L^-1，生根效果好。利用流式细胞仪、染色体镜检及杂交授粉3种方法检测倍性，39株再生植株只有二倍体和四倍体2种倍性，二倍体为37株，占94.87%，四倍体为2株，占5.13%。

关键词: 大白菜, 小孢子培养, 四倍体, 资源创制

Abstract:

The gametes produced during the meiosis process of tetraploid Chinese cabbage are diverse. The purpose of carrying out free microspore culture of tetraploid Chinese cabbage is to obtain more new homozygous diploid resources with a large degree of variation. In this study，four easy embryogenic materials were selected from 14 early-maturing tetraploid Chinese cabbages. The technical system of isolated microspore culture of tetraploid Chinese cabbage was established in the aspects of flower bud phenotype，heat shock temperature，rooting hormone and ploidy identification. The results were as follows：For different genotypes of tetraploid Chinese cabbage，when petal/anther of flower buds was 0-0.5，the microspore was in the stage of mononuclear sideline，and the embryo rate was the highest at this time. When the heat shock temperature was 33.5 ℃ and dark culture for 24 h，they were beneficial to microspore embryogenesis. For rooting medium，the combination of indole butyric acid（IBA）and naphthylacetic acid（NAA）was better than single hormone. The optimum concentration was IBA 0.3 mg · L^-1and NAA 0.1 mg · L^-1. The ploidy was detected by flow cytometry，chromosome microscopy and cross pollination. The results showed that the probability of diploid was 94.87% and the probability of tetraploid was 5.13% in 39 regenerated plants.

Key words: Chinese cabbage, microspore culture, tetraploid, creation

刘晓东, 吴芳, 孟川, 牟金贵, 王玉海, 王明秋. 四倍体大白菜游离小孢子培养技术[J]. 园艺学报, 2023, 50(12): 2641-2652.

LIU Xiaodong, WU Fang, MENG Chuan, MU Jingui, WANG Yuhai, WANG Mingqiu. The Culture Technique of Isolated Microspore in Tetraploid Chinese Cabbage[J]. Acta Horticulturae Sinica, 2023, 50(12): 2641-2652.

图/表 12

参考文献 35

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编号 No.	生育期/d Days	叶球类型 Leafy head	编号 No.	生育期/d Days	叶球类型 Leafy head
D20	55 ~ 60	花心 Spreading type	D522	55 ~ 65	叠抱 Overlapping type
D21	55 ~ 60	合抱 Outward-curling type	D523	55 ~ 65	叠抱 Overlapping type
D22	55 ~ 60	花心 Spreading type	D524	55 ~ 65	叠抱 Overlapping type
D23	55 ~ 60	花心 Spreading type	D525	55 ~ 65	合抱Outward-curling type
D24	55 ~ 60	叠抱 Overlapping type	D526	55 ~ 65	花心 Spreading type
D520	55 ~ 65	花心 Spreading type	D527	55 ~ 65	合抱Outward-curling type
D521	55 ~ 65	花心 Spreading type	D528	55 ~ 65	合抱Outward-Curling Type

编号 No.	生育期/d Days	叶球类型 Leafy head	编号 No.	生育期/d Days	叶球类型 Leafy head
D20	55 ~ 60	花心 Spreading type	D522	55 ~ 65	叠抱 Overlapping type
D21	55 ~ 60	合抱 Outward-curling type	D523	55 ~ 65	叠抱 Overlapping type
D22	55 ~ 60	花心 Spreading type	D524	55 ~ 65	叠抱 Overlapping type
D23	55 ~ 60	花心 Spreading type	D525	55 ~ 65	合抱Outward-curling type
D24	55 ~ 60	叠抱 Overlapping type	D526	55 ~ 65	花心 Spreading type
D520	55 ~ 65	花心 Spreading type	D527	55 ~ 65	合抱Outward-curling type
D521	55 ~ 65	花心 Spreading type	D528	55 ~ 65	合抱Outward-Curling Type

NAA/ （mg · L^-1）	IBA/ （mg · L^-1）	根长/cm Root length	须根数量 Fibrous root	生根率/% Rooting rate	移栽成活率/% Transplantation survival rate
0	0	—	—	—	—
	0.1	3.51 ± 0.83 de	少 Few	50.0 a	36.7 b
0.1	0	3.15 ± 0.07 e	较少 Fewer	33.3 a	10.0 a
	0.1	3.86 ± 0.06 de	多 More	90.0 ab	90.0 c
	0.2	4.59 ± 1.41 d	多 More	100.0 b	90.0 c
	0.3	8.26 ± 1.05 c	多 More	100.0 b	100.0 c
	0.4	10.19 ± 0.39 b	少 Few	100.0 b	100.0 c
	0.5	11.51 ± 1.42 a	少 Few	93.3 b	93.3 c

NAA/ （mg · L^-1）	IBA/ （mg · L^-1）	根长/cm Root length	须根数量 Fibrous root	生根率/% Rooting rate	移栽成活率/% Transplantation survival rate
0	0	—	—	—	—
	0.1	3.51 ± 0.83 de	少 Few	50.0 a	36.7 b
0.1	0	3.15 ± 0.07 e	较少 Fewer	33.3 a	10.0 a
	0.1	3.86 ± 0.06 de	多 More	90.0 ab	90.0 c
	0.2	4.59 ± 1.41 d	多 More	100.0 b	90.0 c
	0.3	8.26 ± 1.05 c	多 More	100.0 b	100.0 c
	0.4	10.19 ± 0.39 b	少 Few	100.0 b	100.0 c
	0.5	11.51 ± 1.42 a	少 Few	93.3 b	93.3 c

编号 No.	鉴定植株数 Number of plant	二倍体数 Number of diploid	四倍体数 Number of autotetraploid	二倍体率/% Diploid rate	四倍体率/% Autotetraploid rate
D24	8	8	0	100.00	0
D522	18	16	2	88.89	11.11
D524	7	7	0	100.00	0
D528	6	6	0	100.00	0
合计Sum	39	37	2	94.87	5.13