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作物学报 ›› 2024, Vol. 50 ›› Issue (9): 2408-2414.doi: 10.3724/SP.J.1006.2024.34204

• 研究简报 • 上一篇    下一篇

以甘蓝下胚轴为外植体的再生体系优化

赵美1,2(), 刘一玮1,2, 石翔天1,2, 李亚俐1,2, 申树林1,2, 尹能文1,2, 赵会彦1,2, 傅鹰3,*(), 曲存民1,2,*()   

  1. 1西南大学西部(重庆)科学城种质创制大科学中心, 重庆 400715
    2南方山地农业教育部工程研究中心 / 西南大学农学与生物科技学院, 重庆 400715
    3浙江省农业科学院作物与技术利用研究所, 浙江杭州 310021
  • 收稿日期:2023-12-05 接受日期:2024-05-21 出版日期:2024-09-12 网络出版日期:2024-06-11
  • 通讯作者: *傅鹰, E-mail: fy97@163.com; 曲存民, E-mail: drqucunmin@swu.edu.cn
  • 作者简介:E-mail: zhaomei123@email.swu.edu.cn
  • 基金资助:
    国家自然科学基金项目(32272150);重庆市自然科学基金项目(CSTB2022NSCQ-LZX0034);重庆市自然科学基金项目(CSTB2022TIAD-KPX0010)

Optimization of regeneration system from hypocotyls of Brassica oleracea L.

ZHAO Mei1,2(), LIU Yi-Wei1,2, SHI Xiang-Tian1,2, LI Ya-Li1,2, SHEN Shu-Lin1,2, YIN Neng-Wen1,2, ZHAO Hui-Yan1,2, FU Ying3,*(), QU Cun-Min1,2,*()   

  1. 1Integrative Science Center of Germplasm Creation in Western China (Chongqing) Science City, Southwest University, Chongqing 400715, China
    2Engineering Research Center of South Upland Agriculture, Ministry of Education / College of Agronomy and Biotechnology, Southwest University, Chongqing 400715, China
    3Institute of Crop and Nuclear Technology Utilization, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, Zhejiang, China
  • Received:2023-12-05 Accepted:2024-05-21 Published:2024-09-12 Published online:2024-06-11
  • Contact: *E-mail: fy97@163.com; E-mail: drqucunmin@swu.edu.cn
  • Supported by:
    National Natural Science Foundation of China(32272150);Natural Science Foundation of Chongqing(CSTB2022NSCQ-LZX0034);Natural Science Foundation of Chongqing(CSTB2022TIAD-KPX0010)

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摘要：

针对甘蓝组织培养过程中再生体系难度大, 培养效率低的问题, 本研究以白花芥蓝下胚轴为外植体, 对离体培养关键影响因素——无菌苗光/暗培养时间、NaClO消毒时间、激素配比对甘蓝种子、不定芽分化及再生的影响进行了比较分析。结果表明, 75%无水乙醇40 s+5% NaClO 15 min消毒后, 将种子暗培养3 d, 再光培养3 d, 无菌苗生长最好; 预培养时培养基最佳激素组合是0.05 mg L-1 NAA (α-萘乙酸)+4 mg L-1 6-BA (6-苄氨基嘌呤); 筛选培养基的最佳激素组合为0.05 mg L-1 NAA+4 mg L-1 6-BA+250 mg L-1 Cb (羧苄青霉素), 愈伤发生率91.1%, 不定芽分化率57.3%; 生根培养基MS+30 g L-1蔗糖+7 g L-1琼脂+0.1 mg L-1 NAA+0.1%活性炭, 不定根产生数量多, 获得植株根系发达、生长势强。本研究建立的甘蓝组织培养优化体系能快速获得再生植株, 有助于加速甘蓝良种的繁育与评价。

关键词: 白花芥蓝, 下胚轴, 离体再生, 激素

Abstract:

Addressing the challenges of a difficult regeneration system and low culture efficiency in the tissue culture process of Brassica oleracea L., this study focused on using the hypocotyl of Brassica albograbra Bailey as explants, which was identified as a key influencing factor in in vitro culture. A comparative analysis was conducted to assess the effects of light/dark culture time, NaClO disinfection time, and hormone ratio on the differentiation and regeneration of Brassica oleracea L. seeds and adventitious buds. The results showed that the best growth of sterile seedlings was achieved when the seeds were disinfected with 75% anhydrous ethanol for 40 s + 5% NaClO 15 minutes. The seeds were then cultured in the dark for 3 d and then, followed by 3 d in the light. The optimal hormone combination for the pre-culture medium was 0.05 mg L-1 NAA+4 mg L-1 6-BA. In the screening culture medium, the optimal hormone combination was 0.05 mg L-1 NAA + 4 mg L-1 6-BA + 250 mg L-1 Cb (Carbenicillin) incidence of 91.1% and a differentiation rate of adventitious buds of 57.3%. For the rooting medium MS+30 g L-1 sucrose+7 g L-1 agar+0.1 mg L-1 NAA+0.1% activated carbon yielded a large number of adventitious roots with well-developed root systems and strong growth potential. The optimized tissue culture system established in this study for Brassica oleracea L. allows for the rapid production of regenerated plants. This system is beneficial for accelerating the breeding and evaluation of improved varieties of Brassica oleracea L.

Key words: Brassica albograbra Bailey, hypocotyl, in vitro regeneration, hormone combination

表1

甘蓝遗传转化所用的培养基"

培养基
Medium		 配方
Formula		 pH 用途
Application
M0 1/2 MS+30 mg L-1 蔗糖+7 g L-1 琼脂
1/2 MS+30 mg L-1 sucrose+7 g L-1 agar		 5.84-5.88 无菌苗培养
Aseptic seedling cultivation
M1 MS+30 g L-1 蔗糖+7 g L-1 琼脂+4 mg L-1 6-BA+0.05 mg L-1 NAA
MS+30 g L-1 sucrose+7 g L-1 agar+4 mg L-1 6-BA+0.05 mg-1 NAA		 5.84-5.88 预培养/共培养
Pre-culture/co-culture
M2 MS+30 g L-1 蔗糖+7 g L-1 琼脂+4 mg L-1 6-BA+0.05 mg L-1 NAA+250 mg L-1 Cb (羧苄青霉素)
MS+30 g L-1 sucrose+7 g L-1 agar+4 mg L-1 6-BA+0.05 mg L-1 NAA+250 mg L-1 Cb (Carbenicillin)		 5.84-5.88 筛选培养
Screening culture
M3 MS+30 g L-1蔗糖+7 g L-1琼脂+0.1 mg L-1 NAA+0.1%活性炭
MS+30 g L-1 sucrose+7 g L-1 agar+0.1 mg L-1 NAA+0.1% activated carbon		 5.84-5.88 生根培养
Rooting culture

图1

白花芥蓝离体再生过程 A: 种子发芽; B: 无菌苗; C: 下胚轴长诱导愈伤组织; D: 愈伤组织分化不定芽; E: 不定芽生长及继代培养; F: 生根培养; G: 水培; H: 土培。"

表2

5% NaClO不同处理时间对种子发芽的影响"

5% NaClO消毒时间
5% NaClO elimination time (min)		 播种粒数
Seed number		 污染率
Contamination rate (%)		 种子发芽率
Germination rate (%)
12 750 31.2±0.50 a 95.4±0.03 a
13 750 20.4±1.25 b 96.5±0.02 a
14 750 9.6±0.61 c 95.8±0.03 a
15 750 0 98.0±0.02 a
16 750 0 71.0±0.04 b
17 750 0 54.1±0.02 c

表3

不同光培养条件对无菌苗生长及下胚轴分化的影响"

暗培养时间
Dark culture time
(d)		 光培养时间
Light culture time
(d)		 无菌苗长度
Growth length of aseptic seedlings (cm)		 接种外植体数
Number of inoculated explants		 愈伤发生率
Rate of callus
(%)		 不定芽分化率
Differentiation rate of
adventitious buds (%)
6 0 2.4 100 68.0±12.17 b 24.0±10.54 c
5 1 3.2 100 75.0±7.81 b 29.0±7.04 bc
4 2 6.1 200 79.0±4.77 ab 34.5±4.77 abc
3 3 8.3 300 89.3±1.19 a 46.0±3.61 a
2 4 5.7 200 77.5±3.04 ab 39.5±5.27 ab
1 5 4.6 200 73.0±4.82 b 35.5±3.77 abc
0 6 2.9 100 70.0±7.94 b 33.0±7.55 abc

表4

不同浓度6-BA和NAA对下胚轴不定芽分化的影响"

激素及其浓度配比Hormone and its concentration ratio (mg L-1) 愈伤发生率
Rate of callus (%)		 不定芽分化率
Differentiation rate of adventitious buds (%)
6-BA NAA
2.00 0.10 25.1±2.40 e 1.3±0.88 f
3.00 0.10 40.6±1.76 d 15.7±1.00 e
4.00 0.10 43.1±1.33 d 17.3±1.33 e
5.00 0.10 56.7±2.91 c 28.1±1.20 c
2.00 0.05 53.6±2.31 c 20.5±0.88 d
3.00 0.05 80.0±2.96 b 39.7±2.08 b
4.00 0.05 91.1±3.93 a 57.3±0.88 a
5.00 0.05 77.6±2.52 b 38.9±0.88 b
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