植物学报 ›› 2020, Vol. 55 ›› Issue (5): 605-612.DOI: 10.11983/CBB20030
刘建飞1, 刘炎1, 刘克俭2, 池阳3, 霍志发3, 霍永洪3, 由香玲1,*()
收稿日期:
2020-02-26
接受日期:
2020-06-05
出版日期:
2020-09-01
发布日期:
2020-09-03
通讯作者:
由香玲
作者简介:
E-mail: 185064633@qq.com基金资助:
Jianfei Liu1, Yan Liu1, Kejian Liu2, Yang Chi3, Zhifa Huo3, Yonghong Huo3, Xiangling You1,*()
Received:
2020-02-26
Accepted:
2020-06-05
Online:
2020-09-01
Published:
2020-09-03
Contact:
Xiangling You
摘要: 以长白落叶松(Larix olgensis)未成熟合子胚为外植体诱导胚性愈伤组织, 通过调节影响体胚发生的营养物质和植物生长调节剂配比, 进行愈伤组织的胚性恢复与保持以及体胚发生再生体系的优化。结果表明: 不同无性系之间胚性愈伤组织诱导率差异显著, 胚性愈伤组织在S+0.2 mg·L -1NAA+0.5 mg·L -1BA+0.5 mg·L -1KT+0.5 g·L -1谷氨酰胺+0.5 g·L -1水解酪蛋白+30 g·L -1蔗糖及3.0 g·L -1植物凝胶培养条件下, 可以恢复胚性并长久保持。在S+20 mg·L -1ABA+60 g·L -1PEG4000+60 g·L -1蔗糖及3.0 g·L -1植物凝胶条件下分化培养6周, 体胚发生率可达100%。将正常发育的体胚先在WPM+ 6 mg·L -1间苯三酚+1.0 g·L -1活性炭+3.0 mg·L -1VB1+20 g·L -1蔗糖及3.0 g·L -1植物凝胶条件下培养2周, 再转接至B5+ 0.4 mg·L -1NAA+1.0 mg·L -1IBA+0.5 mg·L -1GA3+2.0 mg·L -1VB1+1.0 g·L -1活性炭+20 g·L -1蔗糖及3.0 g·L -1植物凝胶条件下培养2周, 可见子叶舒展、下胚轴伸长且根系正常的体胚苗。该研究建立了长白落叶松胚性愈伤组织胚性恢复与保持方法, 并进一步优化了体胚发生的植株再生体系, 为林木资源快速繁育和遗传改良奠定了基础。
刘建飞, 刘炎, 刘克俭, 池阳, 霍志发, 霍永洪, 由香玲. 长白落叶松体胚发生再生体系优化. 植物学报, 2020, 55(5): 605-612.
Jianfei Liu, Yan Liu, Kejian Liu, Yang Chi, Zhifa Huo, Yonghong Huo, Xiangling You. Optimization of the Regeneration System from Somatic Embryogenesis in Larix olgensis. Chinese Bulletin of Botany, 2020, 55(5): 605-612.
图1 长白落叶松愈伤组织及镜检结构 (A) 愈伤组织; (B) 胚性愈伤组织; (C) 非胚性愈伤组织; (D) 胚性愈伤组织显微图像; (E) 非胚性愈伤组织显微图像。 (A)- (C) Bars=3 mm; (D) Bar=200 μm; (E) Bar=500 μm
Figure 1 Callus and its microscopic images of Larix olgensis (A) Callus; (B) Embryogenic callus; (C) Non-embryogenic callus; (D) The image of embryogenic callus; (E) The image of non-embryogenic callus. (A)-(C) Bars=3 mm; (D) Bar= 200 μm; (E) Bar=500 μm
Excellent trees | Callus induction rate (%) | Embryogenic callus formation rate (%) |
---|---|---|
061-1009 | 89.43±48.59 a | 0 |
091-1008 | 81.56±47.91 c | 0 |
058-842 | 91.25±53.17 a | 4.53±2.73 a |
024-922 | 85.91±37.29 b | 0 |
表1 不同长白落叶松株系愈伤组织与胚性愈伤组织诱导(平均值±标准差)
Table 1 Callus and embryogenic callus induction in different Larix olgensis lines (means±SD)
Excellent trees | Callus induction rate (%) | Embryogenic callus formation rate (%) |
---|---|---|
061-1009 | 89.43±48.59 a | 0 |
091-1008 | 81.56±47.91 c | 0 |
058-842 | 91.25±53.17 a | 4.53±2.73 a |
024-922 | 85.91±37.29 b | 0 |
The combination of plant growth regulators in the medium | Somatic embryogenesis (%) | Somatic embryo amount (numbers·g-1) |
---|---|---|
0.1 mg?L-1 2,4-D+0.04 mg·L-1 BA+0.02 mg·L-1 KT | 45.27±23.31 | 16.47±1.67 |
0.5 mg?L-1 NAA+0.5 mg·L-1 BA+0.5 mg·L-1 KT | 52.65±31.83 b | 25.31±9.85 c |
0.4 mg?L-1 NAA+0.5 mg·L-1 BA+0.5 mg·L-1 KT | 57.38±15.41 ab | 31.53±5.37 b |
0.3 mg?L-1 NAA+0.5 mg·L-1 BA+0.5 mg·L-1 KT | 64.41±11.79 a | 39.74±4.29 ab |
0.2 mg?L-1 NAA+0.5 mg·L-1 BA+0.5 mg·L-1 KT | 72.36±10.57 a | 48.56±3.77 a |
0.1 mg?L-1 NAA+0.5 mg·L-1 BA+0.5 mg·L-1 KT | 63.70±43.86 a | 41.92±7.22 a |
表2 不同浓度配比的植物生长调节剂对长白落叶松体胚发生率和体胚数量的影响(平均值±标准差)
Table 2 Effects of plant growth regulators at different concentration ratios on somatic embryogenesis and somatic embryo amount in Larix olgensis (means±SD)
The combination of plant growth regulators in the medium | Somatic embryogenesis (%) | Somatic embryo amount (numbers·g-1) |
---|---|---|
0.1 mg?L-1 2,4-D+0.04 mg·L-1 BA+0.02 mg·L-1 KT | 45.27±23.31 | 16.47±1.67 |
0.5 mg?L-1 NAA+0.5 mg·L-1 BA+0.5 mg·L-1 KT | 52.65±31.83 b | 25.31±9.85 c |
0.4 mg?L-1 NAA+0.5 mg·L-1 BA+0.5 mg·L-1 KT | 57.38±15.41 ab | 31.53±5.37 b |
0.3 mg?L-1 NAA+0.5 mg·L-1 BA+0.5 mg·L-1 KT | 64.41±11.79 a | 39.74±4.29 ab |
0.2 mg?L-1 NAA+0.5 mg·L-1 BA+0.5 mg·L-1 KT | 72.36±10.57 a | 48.56±3.77 a |
0.1 mg?L-1 NAA+0.5 mg·L-1 BA+0.5 mg·L-1 KT | 63.70±43.86 a | 41.92±7.22 a |
图2 长白落叶松体胚成熟过程 (A) 培养2周; (B) 培养4周; (C) 培养6周。Bars=2.5 mm
Figure 2 Maturation of somatic embryos of Larix olgensis (A) Cultured for 2 weeks; (B) Cultured for 4 weeks; (C) Cultured for 6 weeks. Bars=2.5 mm
图3 不同因素对长白落叶松体胚发生率的影响(平均值±标准误) (A) ABA; (B) PEG4000; (C) 蔗糖和麦芽糖; (D) 添加蔗糖后培养6周; (E) 添加麦芽糖后培养6周。 (D), (E) Bars=2.5 mm。不同小写字母表示差异显著(P<0.05)。* P<0.05; ** P<0.01
Figure 3 Effects of different factors on the rate of somatic embryogenesis in Larix olgensis (means±SE) (A) ABA; (B) PEG4000; (C) Sucrose and maltose; (D) Cultured with sucrose for 6 weeks; (E) Cultured with maltose for 6 weeks. (D), (E) Bars=2.5 mm. Different lowercase letters indicate significant differences (P<0.05). * P<0.05; ** P<0.01
图4 长白落叶松不同发育状态的体胚苗及其移栽 (A) 子叶胚; (B) 子叶胚萌发; (C) 对照体胚苗; (D) 优化体胚苗; (E) 生根体胚苗; (F) 移栽体胚苗。 (A) Bar=1.5 mm; (B) Bar=6.5 mm; (C), (D) Bars=1.0 cm; (E), (F) Bars=1.5 cm
Figure 4 Somatic embryo seedlings at different developmental states of Larix olgensis and their transplanting (A) Cotyledonary embryo; (B) Cotyledons germinate; (C) Control of somatic embryo seedling; (D) Optimization somatic embryo seedling; (E) Rooting somatic embryo seedlings; (F) Transplanting somatic embryo seedling. (A) Bar=1.5 mm; (B) Bar=6.5 mm; (C), (D) Bars=1.0 cm; (E), (F) Bars=1.5 cm
Growth regulator combination | Somatic embryo seedlings | |||
---|---|---|---|---|
NAA (mg·L-1) | IBA (mg·L-1) | Seedling rate (%) | Stem length (mm) | Main root length (mm) |
0.4 | 0.5 | 38.83±29.38 ab | 1.59±1.44 c | 16.38±12.74 b |
0.4 | 1.0 | 46.21±41.29 a | 1.75±0.77 ab | 19.17±14.52 a |
0.4 | 1.5 | 35.37±33.34 b | 1.67±1.75 b | 12.60±10.34 c |
0.8 | 0.5 | 22.57±25.43 c | 1.76±0.91 ab | 12.31±9.54 c |
0.8 | 1.0 | 22.65±15.83 c | 1.81±0.88 a | 15.18±13.37 bc |
0.8 | 1.5 | 21.43±19.68 c | 1.78±0.67 ab | 11.40±8.53 c |
表3 生长调节剂组合对长白落叶松体胚苗生长的影响(平均值±标准差)
Table 3 Effects of growth regulator combination on the growth of somatic embryo seedling in Larix olgensis (means±SD)
Growth regulator combination | Somatic embryo seedlings | |||
---|---|---|---|---|
NAA (mg·L-1) | IBA (mg·L-1) | Seedling rate (%) | Stem length (mm) | Main root length (mm) |
0.4 | 0.5 | 38.83±29.38 ab | 1.59±1.44 c | 16.38±12.74 b |
0.4 | 1.0 | 46.21±41.29 a | 1.75±0.77 ab | 19.17±14.52 a |
0.4 | 1.5 | 35.37±33.34 b | 1.67±1.75 b | 12.60±10.34 c |
0.8 | 0.5 | 22.57±25.43 c | 1.76±0.91 ab | 12.31±9.54 c |
0.8 | 1.0 | 22.65±15.83 c | 1.81±0.88 a | 15.18±13.37 bc |
0.8 | 1.5 | 21.43±19.68 c | 1.78±0.67 ab | 11.40±8.53 c |
Plant regeneration | GA3 concentration (mg·L-1) | ||
---|---|---|---|
0 | 0.5 | 1.0 | |
Seedling rate (%) | 45.61±52.27 b | 55.44±37.91 a | 45.91±35.81 b |
Stem length (mm) | 1.63±1.33 b | 2.65±2.61 a | 0.91±1.11 c |
Main root length (mm) | 17.98±14.22 a | 12.41±16.55 b | 8.65±5.49 c |
表4 不同浓度GA3对长白落叶松体胚苗生长的影响(平均值±标准差)
Table 4 Effects of different concentration of GA3 on the growth of somatic embryo seedling in Larix olgensis (means± SD)
Plant regeneration | GA3 concentration (mg·L-1) | ||
---|---|---|---|
0 | 0.5 | 1.0 | |
Seedling rate (%) | 45.61±52.27 b | 55.44±37.91 a | 45.91±35.81 b |
Stem length (mm) | 1.63±1.33 b | 2.65±2.61 a | 0.91±1.11 c |
Main root length (mm) | 17.98±14.22 a | 12.41±16.55 b | 8.65±5.49 c |
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