Crops ›› 2023, Vol. 39 ›› Issue (4): 98-103.doi: 10.16035/j.issn.1001-7283.2023.04.015

Previous Articles     Next Articles

Study on Embryogenic Callus Induction and Embryoid Differentiation of Alfalfa

Wang Xiaochun1(), Gao Ting1(), Yang Weidi1, Wang Chuan1, Chen Caijin2   

  1. 1Institute of Animal Sciences, Ningxia Academy of Agriculture and Forestry Sciences, Yinchuan 750000, Ningxia, China
    2Guyuan Branch of Ningxia Academy of Agriculture and Forestry Sciences,Guyuan 756000, Ningxia, China
  • Received:2021-12-28 Revised:2022-03-28 Online:2023-08-15 Published:2023-08-15

RichHTML

5

PDF (PC)

180

Abstract:

The induction and differentiation of embryogenic callus were studied using hypocotyls, stem segments and leaves of Guyuan alfalfaas explants. The results showed that the callus induction rate was 100% when the concentrations of 2,4-D was 2 and 4mg/L. When the concentration of 2,4-D added to 6or 8mg/L, the induction rate gradually decreased, indicating that high concentration of 2,4-D could inhibit the callus induction. The induction rates were 100% when the concentrations of 2,4-D (1.0, 2.0, 3.0, 4.0mg/L) combined with 0.5mg/L6-BA or KT using three kinds of explants cultured on MS medium.The callus could dedifferentiate completely within 24days, and there was no difference in texture and morphology.Embryoids could be differentiated from all the formulations in the differentiation medium of 1/2MS, but the highest differentiation rate was only 12.6% when the concentration of 2,4-D was 2.0mg/L combined with 0.5mg/LKT. The differentiation rate was increased to 42.7% by supplementing 0.4mg/L KT in differentiation medium. Using the leaves and petioles of the regenerated plantlets as explants, the embryoids could be easy to induced and developed into strong plants in the medium supplemented with 2,4-D (1.0-8.0mg/L) and 0.5mg/L 6-BA or KT because the inducible ability of somatic embryoid was highly inherited. In conclusion, the high frequency regeneration system of alfalfa could be established by regenerating plants.

Key words: Alfalfa, Callus, Somatic embryo, Regeneration plants

Table 1

2,4-D combined with different kinds of cytokininsmg/L"

激素Hormone I0 (CK) I1 I2 I3 I4 I5 I6 I7 I8 I9 I10
2,4-D 0 1.0 2.0 3.0 4.0 5.0 1.0 2.0 3.0 4.0 5.0
6-BA 0 0.5 0.5 0.5 0.5 0.5 － － － － －
KT 0 － － － － － 0.5 0.5 0.5 0.5 0.5

Table 2

Induction effects of different concentrations of 2,4-D on alfalfa callus"

2,4-D浓度
2,4-Dconcentration(mg/L)		 诱导率
Induction rate (%)		 愈伤组织生长状态及大小
Growth status and size of callus
0 0.0d 外植体死亡
2 100.0a 白色，愈伤块体积比原外植体增殖约8倍，继代约200d后逐渐发黏褐化
4 100.0a 白色，愈伤块体积比原外植体增殖约10倍，继代约20d后逐渐发黏褐化
6 84.7b 白色，愈伤块体积比原外植体增殖约4倍，继代约10d后逐渐发黏褐化
8 76.6c 白色，愈伤块体积比原外植体增殖约3倍，继代约10d后逐渐发黏褐化

Fig.1

Different explants ofGuyuan alfalfa and corresponding callus status"

Table 3

Diff erentiation of callus in different induction media"

指标Index I1 I2 I3 I4 I5 I6 I7 I8 I9 I10
接种数Number of explants 120 120 120 120 120 120 120 120 120 120
诱导率Rate of callus induction (%) 100 100 100 100 100 100 100 100 100 100
分化率Rate of callus embryogenesis (%) 7.2b 10.2a 8.4b 6.3b 9.8ab 8.6b 12.6a 8.6b 7.4b 9.8ab

Fig.2

Callus formed by combinations of differentcytokinins (6-BA, KT) with2,4-D"

Fig.3

Two different types of calluses"

Fig.4Comp

arison of differentiation rates of embryo/seedlings in different differentiation media The different lowercase indicate significant difference at 0.05 level"

[1] 李阳, 亓雯雯, 李绍阳, 等. 苜蓿种子萌发和幼苗生长对温度、光照和埋深的响应. 生态学杂志, 2021, 40(2):332-339.
[2] 金太成, 王悦, 吴艳菊, 等. 苜蓿次生胚遗传转化系统优化. 分子植物育种, 2019, 17(20):6685-6690.
[3] Von Arnold S, Sabala I, Bozhkov P, et al. Developmental pathways ofsomatic embryogenesis. Plant Cell, Tissue and Organ Culture, 2002, 69(3):233-249.
doi: 10.1023/A:1015673200621
[4] 李玉珠, 李巧璐, 范艳红, 等. 苜蓿下胚轴愈伤组织诱导和体细胞胚的发生与观察. 中国草地学报, 2014, 36(6):97-102.
[5] 温之雨, 董福双, 张华宁, 等. 紫花苜蓿高频体胚发生及萌发成苗技术与转基因应用. 西北农业学报, 2017, 26(6):882-889.
[6] 王晓春, 师尚礼, 梁慧敏, 等. 2,4-D和6-BA组合配比对金达苜蓿愈伤组织诱导与分化的影响. 草地学报, 2010, 18(2):219-222.
doi: 10.11733/j.issn.1007-0435.2010.02.014
[7] 张凌云, 师尚礼, 尹成学, 等. 俄罗斯杂花苜蓿再生体系的建立. 草业科学, 2013, 30(12):1995-2000.
[8] 王成龙, 周美亮, 董雪妮, 等. 紫花苜蓿两种再生体系的优化及比较. 中国农业科技导报, 2015, 17(4):53-61.
[9] 李娟, 张万军, 王涛. 黄花苜蓿高频植株再生体系建立的研究. 草地学报, 2014, 22(4):834-838.
doi: 10.11733/j.issn.1007-0435.2014.04.024
[10] 史毅, 马晖玲. 三个苜蓿品种植株高频再生体系的研究. 甘肃农业大学学报, 2014, 49(2):125-132.
[11] 王静, 麻冬梅, 许兴, 等. 苜蓿高频再生体系的建立. 北方园艺, 2012(2):109-112.
[12] 靳慧卿. 豆科牧草体细胞胚再生体系构建及其差异性比较研究. 呼和浩特:内蒙古农业大学, 2015.
[13] 王英哲, 任伟, 徐博, 等. 根蘖型苜蓿高频再生体系的建立. 黑龙江畜牧兽医, 2017(7):174- 176,297-298.
[14] 段亚楠, 卢福荣, 茹艺, 等. 紫花苜蓿组织培养高频再生体系的建立. 北方园艺, 2013(15):127-129.
[15] 许来俊, 王成章, 严学兵, 等. 苜蓿植株再生体系研究进展. 草业科学, 2012, 29(8):1294-1301.
[16] 赵金梅, 李芳, 周禾, 等. 紫花苜蓿组织培养体系的建立. 核农学报, 2010, 24(3):507-512.
doi: 10.11869/hnxb.2010.03.0507
[17] Saunders J W, Bingham E T. Production of alfalfa plants from callus tissue. Crop Science, 1972, 12:804-808.
doi: 10.2135/cropsci1972.0011183X001200060026x
[18] 李云玲, 苗锦山. 紫花苜蓿的组织培养与快繁技术研究. 安徽农学通报, 2013, 19(7):51-52.
[19] 徐春波, 王勇, 赵海霞, 等. 紫花苜蓿高频组培再生体系影响因素研究. 草原与草坪, 2010, 30(4):55-63.
[20] Sairam R V, Franklin G, Hassel R, et al. A study on the effect of genotypes, plant growth regulators and sugars in promoting plant regeneration via organogenesis from soybean cotyledonary nodal callus. Plant Cell, Tissue and Organ Culture, 2003, 75(1):79-85.
doi: 10.1023/A:1024649122748
[1] Wang Xiaochun, Zhu Dexin, Yang Tianhui, Wang Chuan, Yang Weidi, Gao Ting, Liang Xiaojun. Correlation Analysis of Main Agronomic Characteristics of Different Alfalfa Varieties and Comparison of Hay Yield in Yellow River Irrigation Area of Ningxia [J]. Crops, 2022, 38(4): 32-36.
[2] Yang Wanjun, Pan Xiangyu, Wang Xiuhua, Wang Lu, Zhao Yan. Genetic Diversity Analysis of Yield and Agronomic Traits of 119 Alfalfa Varieties (Lines) [J]. Crops, 2020, 36(6): 17-22.
[3] Li Yanfang,Du Yanwei,Zhang Zheng,Wang Gaohong,Zhao Genyou,Zhao Jinfeng,Yu Aili. Establishment and Optimization of Agrobacterium Mediated Transformation System for Mature Embryo of Foxtail Millet [J]. Crops, 2019, 35(3): 73-79.
[4] Liu Shuxia,Wei Guojiang,Jing Ruiyong,Wang Liyan,Guan Xiangjun,Sun Yufeng,Wang Guangda,Han Ruyue,Shi Jie,Guo Yongxia. Effects of Sinorhizobium SD101 Inoculation and Shading on Nitrogen Fixation and Photosynthesis of Medicago sativa L. [J]. Crops, 2018, 34(5): 156-161.
[5] Wei Zhang,Liangqun Wang,Yong Liu,Yanfang Hao,Wei Yang,Hongyan Bai,Bo Wu. Optimization of the Factors Related to the Efficiency of Agrobacterium-Mediated Transformation of Sorghum [J]. Crops, 2018, 34(1): 56-61.
[6] Jingdong Wang,Hongwen Ma,Yunfang Fan,Xiaojun Chen. The Study on Antibacterial Effects of Different Antibiotics and Concentrations in a Genetic Transformation System of Yandao8 [J]. Crops, 2016, 32(5): 61-66.
[7] Lijuan Zhang,Jianying Lu,Chang Wang,Gengmei Min,Xiaoming Yang. The Study on the Influence Factors of Callus and Adventitious Bud Induction in Pea [J]. Crops, 2016, 32(3): 33-36.
[8] Hao Yang,Yanming Zhu. Isolation of a Alkali Stress Responsive Gene GsCHX19 and Transformation into Medicago sativa L． [J]. Crops, 2016, 32(3): 37-44.
[9] Zhongyong Cen,Jiang Su,Xichao Deng,Yanjun Xie. Study on Total Phenolic Content and Correlation between PPO Activity and Browning of Corydalis saxicola Bunting Callus [J]. Crops, 2016, 32(1): 149-153.
[10] . [J]. Crops, 2013, 29(3): 45-48.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!