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
[1] 刘庆昌, 吴国良 . 植物细胞组织培养. 北京: 中国农业大学出版社, 2003: 139-162.
[2] 杜晓映 . 葡萄细胞的悬浮培养及诱导子对悬浮细胞多酚产量影响的研究. 杨凌:西北农林科技大学, 2008.
[3] 葛台明, 章荣德, 秦发兰 , 等. 冬小麦原生质体培养的胚状体直接发生. 生物工程学报, 2000,16(5):609-613.
[4] 蔺忠龙, 白现广, 吕广磊 , 等. 疣粒野生稻胚性悬浮细胞系的建立及其原生质体的培养和植株再生. 植物生理学通讯, 2008,44(6):1181-1184.
[5] Xiang F N, Xia G M, Chen H M . Asymmetric somatic hybridization between wheat (Triticum aestivum) and Avena sativa L. Science in China Series C-Life Sciences, 2003,46(3):243-252.
doi: 10.1360/03yc9026 pmid: 18763139
[6] Xiao W, Huang X, Gong Q , et al. Somatic hybrids obtained by asymmetric protoplast fusion between Musa Silk cv. Guoshanxiang (AAB) and Musa acuminata cv. Mas (AA). Plant Cell, Tissue and Organ Culture, 2009,97:313-321.
doi: 10.1007/s11240-009-9530-1
[7] Wagiran A, Ismail I, Zain C R , et al. Agrobacterium tumefaciens-mediated transformation of the isopentenyltransferase gene in japonica rice suspension cell culture. Australian Journal of Crop Science, 2010,4(6):421-429.
[8] Masteller V J, Holden D J . The growth of and organ formation from callus tissue of sorghum. Plant Physiology, 1970,45:362-364.
doi: 10.1104/pp.45.3.362 pmid: 16657320
[9] Wernicke W, Brettel R I S . Morphogenesis from cultured leaf tissue of Sorghum bicolor-culture initiation. Protoplasma, 1982,111:19-27.
doi: 10.1007/BF01287643
[10] Kumaravadivel N, Rangasamy S R S . Plant regeneration from sorghum anther cultures and field evaluation of progeny. Plant Cell Reports, 1994,13:286-290.
doi: 10.1007/BF00233321 pmid: 24193766
[11] Jogeswar G, Ranadheer D, Anjaiah V , et al. High frequency somatic embryogenesis and regeneration in different genotypes of Sorghum bicolor (L. ) Moench from immature inflorescence explants. In Vitro Cellular and Developmental Biology-Plant, 2007,43:159-166.
[12] Oldach C H, Morgenstern A, Rother S , et al. Efficient in vitro plant regeneration from immature zygotic embryos of pearl millet [Pennisetum glaucum (L.) R. Br. ] and Sorghum bicolor (L.) Moench. Plant Cell Reports, 2001,20:416-421.
doi: 10.1007/s002990100335
[13] Kishore N S, Visarada K B R S,Lakshmi Y A , et al. In vitro culture methods in sorghum with shoot tip as the explant material. Plant Cell Reports, 2006,25:174-182.
doi: 10.1007/s00299-005-0044-y pmid: 16402251
[14] Mythili P K, Seetharama N, Reddy V D . Plant regeneration from embryogenic cell suspension cultures of wild sorghum (Sorghum dimidiatum Stapf. ). Plant Cell Reports, 1999,18:424-428.
doi: 10.1007/s002990050597
[15] 王良群, 刘勇, 郝艳芳 , 等. 高粱茎尖和幼穗细胞悬浮系的建立. 农学学报, 2013,3(6):1-3.
doi: 10.3969/j.issn.1007-7774.2013.06.001
[16] 韩福光, 张颖 . 高粱不同外植体愈伤组织诱导的研究. 辽宁农业科学, 1993(1):45-48.
[17] 王良群, 白志良, 王呈祥 , 等. 高粱不同外植体再生苗分化培养的研究. 杂粮作物, 2004,24(5):262-263.
doi: 10.3969/j.issn.2095-0896.2004.05.005
[18] Baker C J, Mock N M . An improved method for monitoring cell death in cell suspension and leaf disc assays using evans blue. Plant Cell, Tissue and Organ Culture, 1994,39:7-12.
doi: 10.1007/BF00037585
[19] 赖钟雄, 陈振光 . 龙眼胚性细胞悬浮培养再生植株. 应用与环境生物学报, 2002,8(5):485-491.
doi: 10.3321/j.issn:1006-687X.2002.05.008
[20] 袁文娅, 关淑艳, 马红丹 , 等. 玉米自交系H99与齐319幼胚愈伤组织诱导条件的优化. 作物杂志, 2013(4):48-51.
[21] 蔡汉权, 赖钟雄, 林珊珊, 等 . 罗勒 ( Ocimum basilicum)悬浮细胞系的建立与保持. 热带作物学报, 2006,27(1):44-48.
doi: 10.3969/j.issn.1000-2561.2006.01.009
[22] 欧巧明, 厚毅清, 包梅年 , 等. 中药半夏单细胞悬浮培养、胚胎发生及植株再生. 中国生物工程杂志, 2012,32(10):39-49.
[23] 戴雪梅, 华玉伟, 李哲 , 等. 植物悬浮细胞培养的关键技术及存在问题. 热带生物学报, 2013,4(4):381-385.
doi: 10.3969/j.issn.1009-1823.2013.04.016
[24] 芦笛, 杨清, 陆巍 . 影响禾谷类作物胚性细胞悬浮系建立的一些因素. 植物生理学通讯, 2007,43(3):399-406.
[25] 陈琰 . 不同抗叶锈小麦品种悬浮细胞系的建立. 保定:河北农业大学, 2005.
doi: 10.7666/d.y740357
[26] 乔妹 . 小麦“5389”悬浮细胞系的建立和植株再生. 保定:河北农业大学, 2011.
doi: 10.7666/d.y1897526
[27] 梁军, 魏刚, 吕全 , 等. 印楝细胞悬浮培养系的建立及悬浮培养. 林业科学研究, 2003,16(5):568-574.
doi: 10.3321/j.issn:1001-1498.2003.05.008
[28] 朱至清 . 植物细胞工程. 北京: 化学工业出版社, 2003: 25-26.
[29] 方文娟, 韩烈保, 曾会明 . 植物细胞悬浮培养影响因子研究. 生物技术通报, 2005(5):11-15.
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