Crops ›› 2018, Vol. 34 ›› Issue (1): 35-40.doi: 10.16035/j.issn.1001-7283.2018.01.005

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Establishment of Sorghum Cell Suspensions with Young Leaves

Hao Yanfang1,2,Wang Liangqun1,2,Liu Yong1,Zhang Wei1,2,Yang Wei1,Bai Hongyan1,Wu Bo1   

  1. 1 Sorghum Institute, Shanxi Academy of Agricultural Sciences, Jinzhong 030600, Shanxi, China
    2 Shanxi Key Laboratory of Sorghum Genetic and Germplasm Innovation, Jinzhong 030600, Shanxi, China
  • Received:2017-07-28 Revised:2017-10-13 Online:2018-02-20 Published:2018-08-24

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Abstract:

In this study, the acquisition of granular embryogenic callus, the establishment of cell suspensions and the main factors affecting the growth of suspended cells were studied by using young leaves of sorghum sterile seedlings as experimental materials. The results showed that the callus were induced on MS+2mg/L 2,4-D medium, and the light yellow, granular embryogenic callus were obtained after 2-3 subcultures. These embryogenic callus were suspened in liquid medium at 25±1℃ under dark conditions for 45-60 days suspension shaking culture, a high quality cell suspension system was established. Cell growth curve showed a “S” curve,cell density was 6.45×10 5-5.08×10 6/mL above, living cells rate was 72.76%, suborbicular cell rate was 87.50%. The basic components, the type and level of the hormone of the medium had a great influence on the growth state of the cell suspension, compared to 1/2 MS medium, MS medium was suitable medium, 2,4-D is essential for maintaining stable proliferation of suspended cells with a suitable concentration of 1mg/L, the addition of 0.5mg/L KT or 6-BA in the culture medium resulted in browning suspension, which affected the growth of the suspended cells. The most suitable medium was L2 medium (MS+1mg/L 2,4-D, 30g/L sucrose, pH5.8), the rotation speed was 110-120r/min, the subculture period was 7d and the proportion of new and old culture medium was 2:1.

Key words: Sorghum bicolor L. Moench, Suspended cell lines, Living cells rate, Suborbicular cell rate, Cell density

Table 1

Liquid culture medium"

培养基
Medium		 培养基种类
Medium		 激素浓度(mg/L)
Hormone concentration
2,4-D KT 6-BA
L1 MS 0.5 0 0
L2 1.0 0 0
L3 2.0 0 0
L4 4.0 0 0
L5 1.0 0.5 0
L6 1/2MS 0.5 0 0
L7 1.0 0 0
L8 2.0 0 0
L9 1.0 0.5 0
L10 1.0 0 0.5

Table 2

Effects of hormone level and mediums type on suspended cell of sorghum"

培养基
Medium		 活细胞率(%)
Living cell rate		 近圆细胞率(%)
Suborbicular cell rate		 细胞分裂增殖速度
Cell division rate		 细胞系均一性、稳定性及分散度
Uniformity, stability and dispersion of cell lines		 悬浮液状态
State of suspension
L1 65.90±4.23abc 79.30±8.15ab 较快 较好,个别细胞结团 较多絮状物
L2 78.58±5.82a 89.18±1.68a 很快 好,个别细胞结团 极少絮状物
L3 70.87±7.84ab 84.24±4.58ab 很快 较好,少量细胞结团 少量絮状物
L4 54.16±6.07bcd 56.93±5.71cd 较快 很差,细胞结团严重 大量絮状物
L5 48.08±12.01cd 66.22±7.95bc 较快 较好,个别细胞结团 少量絮状物
L6 57.68±5.21bcd 67.82±6.79bc 较慢 很差,细胞结团严重 少量絮状物
L7 71.33±6.58ab 78.67±8.45ab 较快 较好,个别细胞结团 极少絮状物
L8 53.13±8.16bcd 74.03±6.29ab 很快 很差,细胞结团严重 极少絮状物
L9 24.79±7.69e 37.23±6.41e 慢 较差,细胞结团较多 较多絮状物
L10 40.39±10.74de 42.52±4.16de 慢 很差,细胞结团严重 大量絮状物

Fig.1

Effects of different primary cultural density on cell growth curve of R111"

Table 3

Effects of different shaking speeds on the growth state of R111 suspension cells"

震荡转速(r/min)
Shaking speed		 细胞分裂增殖速度
Cell division rate		 细胞破碎程度
Cell-breaking extent		 细胞系均一性和分散度
Uniformity and dispersion of cell lines		 悬浮液状态
State of suspension
100 较快 极少 较差,细胞结团较多 极少絮状物
110 很快 极少 较好,个别细胞结团 极少絮状物
120 很快 较少 较好,个别细胞结团 极少絮状物
130 较慢 较多 较好,少量细胞结团 少量絮状物
140 慢 很多 较好,少量细胞结团 较多絮状物
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