CHEN Xu-qing,LI Hong-chao,DU Yun-peng,et al.High Frequency Plant Regeneration from Mesophyll ?Protoplasts in Chicory ( Cichorium endivia ?L.)[J].HEILONGJIANG AGRICULTURAL SCIENCES,2017,(10):21-25.[doi:10.11942/j.issn1002-2767.2017.10.0021]
软化菊苣高效原生质体培养及植株再生
- Title:
- High Frequency Plant Regeneration from Mesophyll ?Protoplasts in Chicory ( Cichorium endivia ?L.)
- 文章编号:
- 1002-2767（2017）10-0021-05
- 分类号:
- S644.9
- 文献标志码:
- A
- 摘要:
- 为了进行体细胞杂交和基因工程改造的尝试，采用10 mL酶解液处理12 h，1 g鲜重叶片游离出4.5~5.9×106个原生质体，生活率达78%~85%。原生质体分别用3种培养基进行培养，以软化菊苣种子无菌苗的叶片为起始材料进行了叶肉原生质体分离、培养和植株分化的研究，并建立高频率的植株再生系统。结果表明：最适合培养基为改良KMP，原生质体的分裂率和植板率分别达到48.0% 和27.5%。同时对原生质体培养的时间长度与愈伤组织的胚胎发生能力及植株分化频率进行了试验比较，培养时间28~41 d为最佳时间，愈伤组织的胚性（81.2%）和分化率（70.9%）最高；培养时间42~55 d，胚性愈伤及植株分化的比例分别为55.0%和45.2%;培养时间56~69 d，愈伤组织的胚性（22.5%）和分化率（16.5%）较差。原生质体再生植株在不含激素的1/2MS培养基上，部分植株直接生根，未生根植株转至1/2MS+IBA 0.3 mg?L1+ 0.1%活性碳可以生根。
- Abstract:
- In order to make attempts on somatic hybridization and gene transformation,a procedure for high frequency plant regeneration from chicory (Cichorium endivia L.) mesophyll protoplasts was developed by using leaves of an in-vitro seedling culture as starting materials.Protoplasts with yields of 4.5~5.9×106 were routinely released from one gram fresh leaf which was digested in 10 mL of an enzyme solution for 12 h.After careful rinsing and purification,viability of protoplasts ranged from 78% to 85%.With the aim at selecting a suitable culture medium,three different media were employed to compare their efficiency in protoplast culture.The results showed that the medium “modified KMP” proved the best,leading to highest rates in cell division(48.0%) and plating efficiency(27.5%),respectively.It was found the length of culture period affected either embryogenesis or regeneration of protoplasts,optimal period was 28~41 d resulting in highest percentages of both embryogenic callus (81.2%) and regeneration（70.9%）,followed by 55.0% and 45.2% obtained from sub-optimal period 42~55 d,while poor rates (22.5% and 16.5%) occurred in prolonged period 56~69 d.A part of plantlets regenerated from protoplasts were able to root on hormone-free 1/2MS,whereas rests produced roots after transfer upon the rooting medium 1/2MS +IBA 0.3 mg?L-1 + 0.1% active charcoal.
参考文献/References:
备注/Memo
收稿日期：2017-08-19