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中国生物工程杂志

China Biotechnology
China Biotechnology
China Biotechnology  2024, Vol. 44 Issue (2/3): 85-93    DOI: 10.13523/j.cb.2307003
    
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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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     
Received: 04 July 2023      Published: 03 April 2024
ZTFLH:  Q813  
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Xuting ZHANG
Jiaxiu WANG
Bin LIU
Yanxia ZHANG
Xiaorui SHANG
Jinyuan SHAN
Guojing LI
Ruigang WANG
Cite this article:

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.

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

Fig.1 The effect of basic medium on callus induction
Fig.2 Callus induction at different concentrations of 2,4-D A-E: Callus tissue at 2,4-D concentrations of 0, 1 mg/L, 2 mg/L, 3 mg/L, and 4 mg/L, respectively F: The subcultured callus tissue
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
Table 1 Effects of different concentrations of 2,4-D on callus induction
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
Table 2 Effects of different hormone combinations on the induction of adventitious shoots
Fig.3 Adventitious shoot induction A:Adventitious buds induction, 2 weeks B:Adventitious buds induction, 4 weeks C:Adventitious buds induction, 6 weeks
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
Table 3 Effect of different hormone combinations on adventitious shoot elongation
Fig.4 Regeneration system of mature zygotic embryos in L. gmelinii A: After 3 days of growth on callus induction medium, mature zygotic embryos turn red and callus tissue begins to initiate B: Red callus tissue that is soft and fragile after growing on callus induction medium for 2-3 weeks C: Preliminary formation of adventitious buds on adventitious bud induction culture D: A large number of adventitious buds are formed on the proliferating callus tissue E: Inducing the initial elongation of adventitious buds on the culture medium F: After subculture with elongation of adventitious buds, the main stem is generated G-I: Rooting culture, with significantly elongated main roots J: acclimatization and transplant
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
Table 4 Effect of different concentrations of IBA on the rooting of adventitious shoots
[1]   王战. 中国落叶松林. 北京: 中国林业出版社,1992.
[1]   Wang Z. The Chinese larch forest. Beijing: China Forestry Press,1992.
[2]   彭倩倩. 落叶松种植技术研究. 中国农业信息, 2017(22): 73, 75.
[2]   Peng Q Q. Study on planting techniques of larch. China Agricultural Information, 2017(22): 73, 75.
[3]   马常耕, 孙晓梅. 我国落叶松遗传改良现状及发展方向. 世界林业研究, 2008, 21(3): 58-63.
[3]   Ma C G, Sun X M. Larch genetic improvement and its future development in China. World Forestry Research, 2008, 21(3): 58-63.
[4]   刘炳麟. 落叶松启动子元件挖掘及基因组编辑实现. 成都: 电子科技大学, 2020.
[4]   Liu B L. Mining promoter elements of Larix gmelinii and realization of genome editing. Chengdu: University of Electronic Science and Technology of China, 2020.
[5]   桂腾琴. 落叶松体细胞胚胎发生的研究进展. 黔西南民族师范高等专科学校学报, 2006(3): 91-94.
[5]   Gui T Q. Research progress on somatic embryogenesis of Larix gmelinii. Journal of Southwest Guizhou Teachers College for Nationalities, 2006(3): 91-94.
[6]   吕守芳, 张守攻, 齐力旺, 等. 落叶松体细胞胚胎发生研究进展. 林业科学研究, 2004, 17(3): 392-398.
[6]   Lu S F, Zhang S G, Qi L W, et al. The current status of Larix somatic embryogenesis research. Forest Research, 2004, 17(3): 392-398.
[7]   Von Aderkas P, Klimaszewska K, Bonga J M. Diploid and haploid embryogenesis in Larixleptolepis, L. decidua, and their reciprocal hybrids. Canadian Journal of Forest Research, 1990, 20(1): 9-14.
doi: 10.1139/x90-002
[8]   齐力旺. 华北落叶松体细胞胚胎发生与遗传转化系统建立的研究. 北京: 中国林业科学研究院, 2000.
[8]   Qi L W. Study on establishment of somatic embryogenesis and genetic transformation system of Larix principis-rupprechtii. Beijing: Chinese Academy of Forestry, 2000.
[9]   吕守芳, 张守攻, 齐力旺, 等. 日本落叶松体细胞胚胎发生的研究. 林业科学, 2005, 41(2): 48-52, 215.
[9]   Lü S F, Zhang S G, Qi L W, et al. Somatic embryogenesis from immature embryos of Larix kaempferi. Scientia Silvae Sinicae, 2005, 41(2): 48-52, 215.
[10]   赵晓敏. 兴安落叶松胚性愈伤组织诱导研究. 哈尔滨: 东北林业大学, 2007.
[10]   Zhao X M. Study on embryogenic callus induction of Larix gmelinii. Harbin: Northeast Forestry University, 2007.
[11]   宋跃. 长白落叶松体细胞胚胎发生及遗传转化研究. 哈尔滨: 东北林业大学, 2017.
[11]   Song Y. Study on somatic embryogenesis and genetic transformation of Larix olgensis. Harbin: Northeast Forestry University, 2017.
[12]   Thompson R G, von Aderkas P. Somatic embryogenesis and plant regeneration from immature embryos of western larch. Plant Cell Reports, 1992, 11(8): 379-385.
doi: 10.1007/BF00234365 pmid: 24201537
[13]   吴丽君. 湿地松、火炬松离体培养植株再生技术的研究. 南京: 南京林业大学, 2008.
[13]   Wu L J. In vitro culture and plantlet regeneration of slash pine and loblolly pine. Nanjing: Nanjing Forestry University, 2008.
[14]   黄健秋, 卫志明. 松属树种的组织培养和原生质体培养. 植物学通报, 1994, 11(1):34-42.
[14]   Huang J Q, Wei Z M. Tissue culture and protoplast culture of the pine species. Botanical Bulletin, 1994, 11 (1): 34-42.
[15]   Lin X F, Zhang W B, Takano H, et al. Efficient plant regeneration and micropropagation from callus derived from mature zygotic embryo of Larix gmelinii. Plant Biotechnology, 2004, 21(2): 159-163.
doi: 10.5511/plantbiotechnology.21.159
[16]   王伟达. 落叶松体细胞胚形成与不定芽诱导的研究. 哈尔滨: 东北林业大学, 2008.
[16]   Wang W D. Study on somatic embryogenesis and adventitious bud induction of Larix gmelinii. Harbin: Northeast Forestry University, 2008.
[17]   赵健, 李旦, 张金凤. 华北落叶松成熟合子胚不定芽诱导及伸长研究. 中国农学通报, 2014, 30(16): 12-17.
[17]   Zhao J, Li D, Zhang J F. Adventitious buds induction and elongation from mature zygotic embryo of Larix principis-rupprechtii Mayr. Chinese Agricultural Science Bulletin, 2014, 30(16): 12-17.
[18]   Brassard N, Brissette L, Lord D, et al. Elongation, rooting and acclimatization of micropropagated shoots from mature material of hybrid larch. Plant Cell, Tissue and Organ Culture, 1996, 44(1): 37-44.
doi: 10.1007/BF00045911
[19]   郑武林. 马尾松与落叶松高效离体再生体系建立. 南昌: 江西农业大学, 2014.
[19]   Zheng W L. Establishment of highly efficient in vitro regeneration system of masson pine and Larch. Nanchang: Jiangxi Agricultural University, 2014.
[20]   王可佳, 闫学彤, 郑珂媛, 等. 落叶松针叶直接不定芽发生体系的建立. 分子植物育种, 2021, 19(8): 2719-2724.
[20]   Wang K J, Yan X T, Zheng K Y, et al. Direct regeneration of adventitious bud from needles of Larix. Molecular Plant Breeding, 2021, 19(8): 2719-2724.
[21]   施翔. 杂交松组织培养研究. 南京: 南京林业大学, 2005.
[21]   Shi X. Study on tissue culture of hybrid pine. Nanjing: Nanjing Forestry University, 2005.
[22]   张莉. 落叶松高效离体再生体系建立及内源激素变化研究. 哈尔滨: 东北林业大学, 2016.
[22]   Zhang L. Establishment of efficient regeneration system in vitro of Larix gmelinii and study on changes of endogenous hormones. Harbin: Northeast Forestry University, 2016.
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