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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2024, Vol. 44 Issue (2/3): 85-93    DOI: 10.13523/j.cb.2307003
技术与方法     
兴安落叶松成熟合子胚高效离体再生体系的建立*
张旭婷,王佳秀,刘槟,张燕霞,尚晓蕊,单金元,李国婧,王瑞刚***()
内蒙古农业大学旱寒区植物逆境适应与遗传修饰改良自治区重点实验室 呼和浩特 010018
Establishment of an Efficient in Vitro Regeneration System for Mature Zygotic Embryos in Larix gmelinii
ZHANG Xuting,WANG Jiaxiu,LIU Bin,ZHANG Yanxia,SHANG Xiaorui,SHAN Jinyuan,LI Guojing,WANG Ruigang***()
Key Laboratory of Plants Adversity Adaptation and Genetic Improvement in Cold and Arid Regions of Inner Mongolia, Inner Mongolia Agricultural University, Hohhot 010018, China
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摘要：

以兴安落叶松成熟合子胚为外植体,开展落叶松离体器官发生的探究。实验选用多种培养基,以6-BA和2,4-D等生长调节剂进行愈伤组织和不定芽诱导,进行了不定芽伸长和生根诱导。结果表明:愈伤组织在1/2 MS+2 mg/L 2,4-D培养基中诱导率最高,愈伤组织诱导率为91.11%;不定芽的诱导培养基为1/2 MS+1 mg/L 6-BA+0.07 mg/L 2,4-D,不定芽诱导率为87.78%;不定芽伸长培养基为MS+0.20 mg/L 6-BA+0.01 mg/L 2,4-D+1 mg/L活性炭,不定芽伸长率达到48.33%;不定根生根培养基为1/2 MS+1 mg/L IBA,不定根诱导率达到36.67%;落叶松的再生苗移植成活率已达到了100%。上述研究成果为落叶松组培器官再生技术和进一步稳定遗传改良工作提供了良好的理论基础和实验依据。
关键词： 兴安落叶松;  成熟合子胚;  再生体系    
Abstract:

The mature zygotic embryos of Larix gmelinii were used as explants to study the isolated organogenesis of larch. The experimental design used different medium, including 6-BA and 2,4-D to induce healing and adventitious shoots, elongation and rooting. The results showed that the best callus induction medium was 1/2 MS + 2 mg/L 2, 4-D, and callus induction rate was 91.11%. Induction medium for adventitious budding was 1/2 MS+1 mg/L 6-BA+0.07 mg/L 2, 4-D, and the adventitious shoot induction rate was 87.78%. The indefinite bud elongation medium was MS+0.20 mg/L 6-BA+0.01 mg/L 2, 4-D + 1 mg/L activated carbon, and the elongation rate of adventitious buds reached 48.33%. Adventitious root rooting medium was 1/2 MS + 1 mg/L IBA, and the induction rate of indefinite root reached 36.67%. The survival rate of transplanted seedlings was 100%. The above studies provide a good theoretical and practical basis for larch tissue culture organogenesis and further research on stable genetic transformation.
Key words: Larix gmelinii    Mature zygotic embryo    Regeneration system
收稿日期: 2023-07-04 出版日期: 2024-04-03
ZTFLH:  Q813  
基金资助: *国家转基因生物新品种培育专项(2018ZX08020-003);呼和浩特市重大科技专项(2022-社-重-1-3);内蒙古自治区高校创新团队(NMGIRT2222)
通讯作者: ***电子信箱:ruigangwang@126.com   
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张旭婷
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尚晓蕊
单金元
李国婧
王瑞刚

引用本文:

张旭婷, 王佳秀, 刘槟, 张燕霞, 尚晓蕊, 单金元, 李国婧, 王瑞刚. 兴安落叶松成熟合子胚高效离体再生体系的建立*[J]. 中国生物工程杂志, 2024, 44(2/3): 85-93.

ZHANG Xuting, WANG Jiaxiu, LIU Bin, ZHANG Yanxia, SHANG Xiaorui, SHAN Jinyuan, LI Guojing, WANG Ruigang. Establishment of an Efficient in Vitro Regeneration System for Mature Zygotic Embryos in Larix gmelinii. China Biotechnology, 2024, 44(2/3): 85-93.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2307003    或    https://manu60.magtech.com.cn/biotech/CN/Y2024/V44/I2/3/85

图1  不同基本培养基对愈伤组织诱导的影响
图2  不同2,4-D浓度下诱导的愈伤组织状态 A~E: 0、1 mg/L、2 mg/L、3 mg/L和4 mg/L 2,4-D下诱导的愈伤组织 F:继代培养的愈伤组织
2,4-D浓度/(mg/L) 接种成熟合子胚个数 愈伤组织诱导率/% 均值
重复1 重复2 重复3
0 30 46.67 63.33 53.33 54.44±4.84 cd
1 30 86.67 73.33 80.00 80.00±3.84 ab
2 30 90.00 96.67 86.67 91.11±5.09 a
3 30 76.67 66.67 60.00 67.78±4.48 bc
4 30 53.33 36.67 50.00 46.00±5.09 d
表1  不同浓度2,4-D对愈伤组织诱导的影响
6-BA浓度
/(mg/L)		 2,4-D浓度
/(mg/L)		 接种愈伤组织
块个数		 不定芽的诱导率/% 均值
重复1 重复2 重复3
0.5 0.05 30 30.00 36.67 26.67 31.11±2.94fghi
0.5 0.07 30 30.00 23.33 30.00 27.78±2.22ghi
0.5 0.1 30 30.00 20.00 30.00 26.67±3.33hi
0.5 0.2 30 10.00 30.00 26.67 22.22±6.19hi
0.5 0.3 30 23.33 20.00 20.00 21.11±1.11i
0.7 0.05 30 20.00 30.00 26.67 25.56±2.94hi
0.7 0.07 30 50.00 60.00 43.33 51.11±4.84cde
0.7 0.1 30 46.67 30.00 53.33 43.33±6.94defg
0.7 0.2 30 66.67 50.00 23.33 46.67±6.61de
0.7 0.3 30 36.67 30.00 46.67 37.78±4.84efgh
1.0 0.05 30 70.00 63.33 80.00 71.11±4.84b
1.0 0.07 30 90.00 86.67 86.67 87.78±1.11a
1.0 0.1 30 76.67 63.33 66.67 68.89±4.00b
1.0 0.2 30 53.33 50.00 50.00 51.11±1.11cde
1.0 0.3 30 40.00 36.67 50.00 42.22±3.22efg
2.0 0.05 30 43.33 50.00 40.00 44.44±2.94def
2.0 0.07 30 53.33 50.00 43.33 48.89±2.94de
2.0 0.1 30 63.33 56.67 56.67 58.89±2.22bcd
2.0 0.2 30 73.33 60.00 60.00 64.44±4.44bc
2.0 0.3 30 53.33 53.33 40.00 48.89±4.41de
3.0 0.05 30 46.67 36.67 60.00 47.78±3.75de
3.0 0.07 30 53.33 40.00 36.67 43.33±5.09defg
3.0 0.1 30 30.00 36.67 26.67 31.11±2.94fghi
3.0 0.2 30 30.00 36.67 23.33 30.00±3.85fghi
3.0 0.3 30 23.33 33.33 30.00 28.89±2.94fghi
表2  不同激素组合对不定芽诱导的影响
图3  不定芽诱导 A:诱导2周的不定芽 B:诱导4周的不定芽 C:诱导6周的不定芽
6-BA浓度
/(mg/L)		 2,4-D浓度
/(mg/L)		 接种不定芽丛数 不定芽的伸长率/% 均值
重复1 重复2 重复3
0.1 0.01 20 30 20 30 26.67±3.33bcd
0.1 0.02 20 15 20 30 21.67±4.41cd
0.1 0.04 20 15 15 20 16.67±1.676d
0.2 0.01 20 55 45 45 48.33±2.89a
0.2 0.02 20 40 35 35 36.67±1.67b
0.2 0.04 20 35 25 40 33.33±4.41b
0.4 0.01 20 25 30 35 30.00±2.89bc
0.4 0.02 20 20 25 15 20.00±2.89cd
0.4 0.04 20 20 20 10 16.67±3.33d
表3  不同激素组合对不定芽伸长的影响
图4  兴安落叶松成熟合子胚的再生体系 A:在愈伤组织诱导培养基上生长3天,成熟合子胚变红,愈伤组织开始启动 B:在愈伤组织诱导培养基上生长2~3周后松软易碎的红色愈伤组织 C:在不定芽诱导培养上初步形成不定芽 D:增殖的愈伤组织上形成大量不定芽 E:不定芽伸长诱导培养基上不定芽的初步伸长 F:不定芽伸长继代培养后,主茎生成 G~I:生根培养,主根明显伸长 J:落叶松组培苗的炼苗移栽
IBA浓度/(mg/L) 接种伸长芽苗个数 不定根诱导率/% 均值
重复1 重复2 重复3
0 10 0 0 0 0c
0.5 10 30 10 20 20.00±5.77b
1.0 10 40 40 30 36.67±3.33a
1.5 10 10 20 30 16.67±6.67b
2.0 10 10 0 10 10.00±5.77bc
表4  不同浓度IBA对不定芽生根的影响
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