植物学报 ›› 2016, Vol. 51 ›› Issue (6): 807-816.doi: 10.11983/CBB15222

• 技术方法 • 上一篇    下一篇

绒毛白蜡体胚诱导和植株再生

燕丽萍1,2,3, 李丽1,2, 刘翠兰1,2, 吴德军1,2,,A;*(), 王因花1, 任飞1, 赵梁军3   

  1. 1山东省林业科学研究院, 济南 250014
    2山东省林木遗传改良重点实验室, 济南 250014
    3中国农业大学观赏园艺与园林系, 北京 100193
  • 收稿日期:2015-12-23 接受日期:2016-05-11 出版日期:2016-11-01 发布日期:2016-12-02
  • 通讯作者: 吴德军 E-mail:sdlky412@163.com
  • 作者简介:

    # 共同第一作者

  • 基金资助:
    “十二五”科技支撑计划(No.2013BAD01B06-6)和山东省科技发展计划(No.2014GNC111006)

Somatic Embryo Induction and Plantlet Regeneration of Fraxinus velutina

Liping Yan1,2,3, Li Li1,2, Cuilan Liu1,2, Dejun Wu1,2*, Yinhua Wang1, Fei Ren1, Liangjun Zhao3   

  1. 1Shandong Provincial Academy of Forestry, Jinan 250014, China
    2Shandong Provincial Key Laboratory of Forest Tree Genetic Improvement, Jinan 250014, China
    3Department of Ornamental Horticulture and Landscape Architecture, China Agricultural University, Beijing 100193, China
  • Received:2015-12-23 Accepted:2016-05-11 Online:2016-11-01 Published:2016-12-02
  • Contact: Wu Dejun E-mail:sdlky412@163.com
  • About author:

    # Co-first authors

摘要:

该文探讨了基本培养基、外植体、培养条件以及植物生长调节剂配比对绒毛白蜡(Fraxinus velutina)体胚诱导的影响。结果表明, 胚根是诱导体胚发生的最佳外植体; 体胚诱导的最适培养基为改良MS+2 mg·L-1 6-BA+0.1 mg·L-1 NAA、30 g·L-1蔗糖、5.0 g·L-1琼脂; 暗培养20天后进行光照培养(14小时光照/10小时黑暗), 光密度为100-120 μmol·m-2·s-1, 昼温度(25±2)°C, 夜温度(18±2)°C; 成功诱导出体细胞胚并获得再生植株, 体胚诱导率可达59.8%, 体胚萌发率达81.2%。壮苗最适培养基为改良WPM+0.5 mg·L-1 6-BA+0.2 mg·L-1 ZT+0.01 mg·L-1 NAA。生根最适培养基为改良1/2MS+1.0 mg·L-1 IBA+0.05 mg·L-1 NAA+20 g·L-1蔗糖, 生根率高达97.3%, 试管苗移栽成活率达97.8%。

Abstract:

Using explants of Fraxinus velutina, we investigated the effects of basic medium, plant growth regulators, and concentrations of antioxidant medium, and culturing conditions on the induction of somatic embryogenesis in Fraxinus velutina. The best regeneration medium was Murashige and Skoog (MS) basal medium with Gamborg B5 vitamin supplementation, with 2.0 mg·L-1 6-benzyladenine (6-BA), 0.1 mg·L-1 naphthaleneacetic acid (NAA) at 20-day dark culture followed by light intensity of 100-120 μmol·m-2·s-1 irradiation for 14 h·d-1, and the culture temperature was (25±2)°C at daytime and (18±2)°C at night. More than 81.2% radicle segments produced adventitious shoots, with a mean number of adventitious shoots per explant of 9.3. The optimal growing medium was modified WPM medium supplemented with 0.5 mg·L-1 6-BA, 0.2 mg·L-1 ZT and 0.01 mg·L-1 NAA. More than 97.3% regeneration plantlets were able to root after transfer to modified half-strength MS medium supplemented with 1.0 mg·L-1 indole-3-butyric acid, 0.05 mg·L-1 NAA and 20 g·L-1 sucrose; 97.8% of the plantlets survived.

表1

基本培养基对绒毛白蜡胚根愈伤组织诱导、体胚诱导和体胚芽再生率的影响"

Basal medium 6-BA
(mg·L-1)
NAA
(mg·L-1)
Callus induction
rate (%)
Induction rate of
somatic embryo (%)
Somatic embryo shoot
regeneration rate (%)
MS 2.0 0.1 79.4±10.7 b 34.6±5.3 b 61.2±12.8 b
Modified MS 2.0 0.1 89.3±12.2 a 59.8±4.5 a 81.2±14.5 a
WPM 2.0 0.1 71.2±8.4 c 25.4±5.3 c 41.2±9.3 c
Modified WPM 2.0 0.1 75.5±9.8 c 25.9±4.7 c 45.7±11.4 c

表2

不同外植体类型对绒毛白蜡愈伤组织诱导、体胚诱导和体胚芽再生率的影响"

Explant type Callus induction rate (%) Induction rate of somatic embryo (%) Somatic embryo shoot regeneration rate (%)
Cotyledon 57.5±11.6 b 23.8±5.9 b 21.7±8.8 b
Epicotyl 29.4±10.7 c 10.8±5.5 bc 4.7±2.9 c
Hypocotyl 35.7±9.3 c 11.4±5.3 c 5.1±2.3 c
Radicle 85.5±11.8 a 65.9±12.7 a 75.9±10.5 a

表3

暗培养时间对绒毛白蜡体胚诱导和体胚芽再生率的影响"

Darkness
(days)
6-BA
(mg·L-1)
NAA
(mg·L-1)
Induction rate of somatic
embryo (%)
Somatic embryo shoot
regeneration rate (%)
0 2.0 0.1 10.8±3.7 c 0 c
10 2.0 0.1 30.8±5.5 b 41.2±9.3 b
20 2.0 0.1 59.7±7.1 a 79.2±14.5 a
30 2.0 0.1 25.9±4.8 a 45.7±11.4 c

表4

不同植物生长调节剂组合对绒毛白蜡胚根体胚诱导和体胚芽再生率的影响"

Plant growth regulator (mg·L-1) Induction rate of somatic embryo
(%)
Somatic embryo shoot regeneration
rate (%)
Plant growth regulator (mg·L-1) Induction rate of
somatic embryo
(%)
Somatic embryo shoot
regeneration rate
(%)
6-BA NAA TDZ NAA
1.0
0 0±0.0 d 0±0.0 d 1.0 0 0±0.0 d 0±0.0 d
0.1 50.3±11.7 bc 34.2±11.3 c 0.1 40.4±10.3 bc 24.8±8.7 c
0.25 40.7±9.5 c 44.3±13.0 bc 0.25 38.7±9.7 bc 31.5±12.3 bc
1.5
0 0±0.0 d 0±0.0 d 1.5 0 0±0.0 0±0.0 d
0.1 61.8±10.3 ab 46.4±21.9 bc 0.1 50.9±9.8 b 57.2±16.9 a
0.25 58.8±11.8 b 57.8±19.7 b 0.25 48.5±12.3 b 44.8±14.5 b
2.0
0 0±0.0 d 0±0.0 d 2.0 0 0±0.0 d 0±0.0 d
0.1 68.9±12.5 a 78.9±24.8 a 0.1 56.5±11.7 a 45.2±14.5 b
0.25 57.6±10.3 b 63.2±17.6 ab 0.25 59.4±12.4 a 43.9±20.0 b

表5

绒毛白蜡壮苗培养基配方及结果分析"

Treatment Plant growth regulator Average plantlet
(length·cm-1)
6-BA
(mg·L-1)
ZT
(mg·L-1)
NAA
(mg·L-1)
1 0.10 0.10 0.01 2.1
2 0.10 0.20 0.05 4.3
3 0.10 0.50 0.10 3.1
4 0.20 0.10 0.05 3.5
5 0.20 0.20 0.50 3.1
6 0.20 0.50 0.01 4.2
7 0.50 0.10 0.10 5.7
8 0.50 0.20 0.01 6.9
9 0.50 0.50 0.05 3.6
Average plantlet (length·
cm-1)
T1 3.6 4.0 4.9
T2 4.0 4.9 3.9
T3 5.2 3.8 4.0
R 1.6 1.1 1.0

表6

不同浓度IBA和NAA组合对绒毛白蜡体胚芽生根的影响同生长素组合对不定芽生根诱导的影响"

Treatment Plant growth regulator 1/2MS Modified 1/2MS
IBA
(mg·L-1)
NAA
(mg·L-1)
Root rate
(%)
The average number
of root
Root rate
(%)
The average number
of root
1 0.1 - 0.0±0.0 e 0.0±0.0 d 0.0±0.0 e 0.0±0.0 d
2 0.5 - 11.6±1.3 d 2.7±0.5 c 21.3±2.1 d 3.5±0.3 c
3 1.0 - 24.9±2.4 c 3.3±0.8 c 31.5±1.8 cd 4.1±0.5 b
4 1.5 - 17.5±3.1 cd 3.1±0.3 c 26.6±3.2 d 3.6±0.3 c
5 - 0.01 8.6±2.8 d 3.8±0.3 b 14.9±1.9 de 3.3±0.5 c
6 - 0.05 13.4±3.7 cd 3.4±0.6 c 18.8±1.7 d 4.1±0.6 b
7 - 0.1 9.6±1.9 d 4.1±0.8 b 12.3±2.2 de 4.2±0.8 b
8 - 0.2 3.9±1.1 de 4.4±0.7 b 9.4±1.9 de 3.9±0.8 b
9 0.5 0.05 31.8±4.8 bc 5.7±0.6 a 67.6±3.8 bc 4.9±0.7 a
10 1.0 0.05 69.4±3.9 a 5.0±0.9 a 97.3±5.1 a 5.1±0.9 a
11 0.5 0.1 53.7±5.2 ab 5.4±0.9 a 77.6±4.3 b 5.3±0.6 a
12 1.0 0.1 67.8±4.4 a 5.7±0.5 a 76.8±4.7 b 6.4±0.8 a
13 0.5 0.2 32.5±4.9 bc 5.0±0.7 a 44.9±3.3 c 6.3±1.1 a
14 1.0 0.2 46.9±5.3 b 5.3±0.8 a 56.4±4.4 bc 5.6±0.7 a

表7

不同配比基质对绒毛白蜡试管苗移栽成活率的影响"

Treatment Sand:soil:medium (v:v:v) Survival rate (%)
1 4:1:1 97.8 a
2 4:1:2 87.6 b
3 2:2:1 73.4 c

图1

绒毛白蜡体胚发生的形态观察和植株再生培养(A) 胚根; (B) 球形胚; (C) 心形胚; (D) 鱼雷形胚和子叶形胚; (E) 子叶期体胚(CE)见光后萌发; (F) 萌发正常的体胚苗; (G) 增殖培养; (H) 壮苗培养; (I) 体胚苗形成完整植株; (J) 移栽的植株。(B)-(D) Bar=500 μm"

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