以甘蓝下胚轴为外植体的再生体系优化

doi:10.3724/SP.J.1006.2024.34204

Abstract

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

ZHAO Mei, LIU Yi-Wei, SHI Xiang-Tian, LI Ya-Li, SHEN Shu-Lin, YIN Neng-Wen, ZHAO Hui-Yan, FU Ying, QU Cun-Min. Optimization of regeneration system from hypocotyls of Brassica oleracea L.[J].Acta Agronomica Sinica, 2024, 50(9): 2408-2414.

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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

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

暗培养时间 Dark culture time (d)	光培养时间 Light culture time (d)
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

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	愈伤发生率 Rate of callus (%)	不定芽分化率 Differentiation rate of adventitious buds (%)
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

Optimization of regeneration system from hypocotyls of Brassica oleracea L.

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[2]	ZHANG Qiao-Yu, ZHANG Rui, LIU Zhao-Hui,TIAN Xiao-Li. Effect of Root-shoot Interaction on Cotton Hypocotyl Anatomy by Using Grafting Technique under Premature Senescence Induced by Potassium Deficiency [J]. Acta Agron Sin, 2017, 43(10): 1527-1535.
[3]	Amarasinghe Arachchige Yasarathna AMARASINGHE;YANG Yue-Sheng. High Efficient Organogenic Regeneration from Hypocotyl Explants of Soybean (Glycine max L.) on BA-free Medium [J]. Acta Agron Sin, 2006, 32(08): 1204-1208.
[4]	Zhou Hanyang; She Jianming; Lu Weizhong; Wu Heming. Study on Plantlet Regeneration from Protoplasts of Brassicanapus [J]. Acta Agron Sin, 1991, 17(05): 340-345.