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

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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

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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"

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