Acta Prataculturae Sinica ›› 2023, Vol. 32 ›› Issue (1): 165-177.DOI: 10.11686/cyxb2022189
Kai JIANG(), Xue-li WU(), Yi-jun LIU, Yue MA, Yang SONG, Wen-jie LU, Zeng-yu WANG
Received:
2022-04-27
Revised:
2022-06-16
Online:
2023-01-20
Published:
2022-11-07
Contact:
Xue-li WU
Kai JIANG, Xue-li WU, Yi-jun LIU, Yue MA, Yang SONG, Wen-jie LU, Zeng-yu WANG. Comparative study on transformation systems of seashore paspalum using hpt and bar genes as selection markers[J]. Acta Prataculturae Sinica, 2023, 32(1): 165-177.
培养基类型Medium type | 培养基配方Formula of medium | 其他Others |
---|---|---|
诱导和继代培养基Induction and subculture media | MS基本培养基+2.5 mg·L-1 2,4-二氯苯氧乙酸+0.6 mg·L-1 CuSO4 MS basic medium+2.5 mg·L-1 2,4-dichlorophenoxy acetic acid (2,4-D)+0.6 mg·L-1 CuSO4 | 添加30 g·L-1蔗糖,调pH值为5.8,加入8 g·L-1琼脂,121 ℃高温灭菌20 min。Added 30 g·L-1 sucrose, adjusted the pH value to 5.8, add 8 g·L-1 agar, and sterilized at 121 ℃ for 20 min. |
选择继代培养基Selective subculture medium | MS基本培养基+2.5 mg·L-1 2,4-D+0.6 mg·L-1 CuSO4+200 mg·L-1头孢噻肟+适宜浓度筛选剂MS basic medium+2.5 mg·L-1 2,4-D+0.6 mg·L-1 CuSO4+200 mg·L-1 cefotaxime+suitable concentration screening agent | |
再生培养基Regeneration medium | MS基本培养基+0.05 mg·L-1激动素+8.0 mg·L-1 6-苄氨基嘌呤+10 mg·L-1 CuSO4 MS basic medium+0.05 mg·L-1 kinetin (KT)+8.0 mg·L-1 6-benzylaminopurine (6-BA)+10 mg·L-1 CuSO4 | |
选择再生培养基Select regeneration medium | MS基本培养基+0.05 mg·L-1激动素+8.0 mg·L-1 6-苄氨基嘌呤+10 mg·L-1 CuSO4+200 mg·L-1头孢噻肟+适宜浓度筛选剂MS basic medium+0.05 mg·L-1 KT+8.0 mg·L-1 6-BA+10 mg·L-1 CuSO4+200 mg·L-1 cefotaxime+suitable concentration screening agent | |
壮苗培养基Strong seedling medium | 1/2 MS基本培养基1/2 MS basic medium | |
选择壮苗培养基Select seedling growth medium | 1/2 MS基本培养基+适宜浓度筛选剂1/2 MS basic medium+suitable concentration screening agent |
Table 1 Types and preparation methods of culture medium
培养基类型Medium type | 培养基配方Formula of medium | 其他Others |
---|---|---|
诱导和继代培养基Induction and subculture media | MS基本培养基+2.5 mg·L-1 2,4-二氯苯氧乙酸+0.6 mg·L-1 CuSO4 MS basic medium+2.5 mg·L-1 2,4-dichlorophenoxy acetic acid (2,4-D)+0.6 mg·L-1 CuSO4 | 添加30 g·L-1蔗糖,调pH值为5.8,加入8 g·L-1琼脂,121 ℃高温灭菌20 min。Added 30 g·L-1 sucrose, adjusted the pH value to 5.8, add 8 g·L-1 agar, and sterilized at 121 ℃ for 20 min. |
选择继代培养基Selective subculture medium | MS基本培养基+2.5 mg·L-1 2,4-D+0.6 mg·L-1 CuSO4+200 mg·L-1头孢噻肟+适宜浓度筛选剂MS basic medium+2.5 mg·L-1 2,4-D+0.6 mg·L-1 CuSO4+200 mg·L-1 cefotaxime+suitable concentration screening agent | |
再生培养基Regeneration medium | MS基本培养基+0.05 mg·L-1激动素+8.0 mg·L-1 6-苄氨基嘌呤+10 mg·L-1 CuSO4 MS basic medium+0.05 mg·L-1 kinetin (KT)+8.0 mg·L-1 6-benzylaminopurine (6-BA)+10 mg·L-1 CuSO4 | |
选择再生培养基Select regeneration medium | MS基本培养基+0.05 mg·L-1激动素+8.0 mg·L-1 6-苄氨基嘌呤+10 mg·L-1 CuSO4+200 mg·L-1头孢噻肟+适宜浓度筛选剂MS basic medium+0.05 mg·L-1 KT+8.0 mg·L-1 6-BA+10 mg·L-1 CuSO4+200 mg·L-1 cefotaxime+suitable concentration screening agent | |
壮苗培养基Strong seedling medium | 1/2 MS基本培养基1/2 MS basic medium | |
选择壮苗培养基Select seedling growth medium | 1/2 MS基本培养基+适宜浓度筛选剂1/2 MS basic medium+suitable concentration screening agent |
筛选标记 Selectable marker | 接种愈伤组织数No. of infected calli | 抗性愈伤组织数No. of resistant calli | 愈伤组织GUS染色数No. of GUS (+) calli | 抗性再生植株的克隆数No. of resistant plant clones | 再生植株的GUS染色数No. of GUS (+) plant lines | 转基因效率 Transformation efficiency (%) | 平均转基因效率 Average transformation efficiency (%, mean±SD) |
---|---|---|---|---|---|---|---|
hpt | 63 | 10 | 10 | 6 | 6 | 9.52 | 14.46±4.69 |
59 | 17 | 15 | 15 | 12 | 20.34 | ||
49 | 10 | 7 | 7 | 6 | 12.24 | ||
77 | 15 | 11 | 9 | 9 | 11.69 | ||
81 | 22 | 22 | 18 | 15 | 18.52 | ||
bar | 89 | 7 | 5 | 5 | 3 | 3.37 | 5.65±2.61 |
60 | 9 | 9 | 7 | 6 | 10.00 | ||
79 | 8 | 7 | 7 | 4 | 5.06 | ||
76 | 4 | 3 | 3 | 3 | 3.95 | ||
51 | 6 | 6 | 5 | 3 | 5.88 |
Table 2 Summary of transformation efficiencies of Agrobacterium transformation using embryogenic callus
筛选标记 Selectable marker | 接种愈伤组织数No. of infected calli | 抗性愈伤组织数No. of resistant calli | 愈伤组织GUS染色数No. of GUS (+) calli | 抗性再生植株的克隆数No. of resistant plant clones | 再生植株的GUS染色数No. of GUS (+) plant lines | 转基因效率 Transformation efficiency (%) | 平均转基因效率 Average transformation efficiency (%, mean±SD) |
---|---|---|---|---|---|---|---|
hpt | 63 | 10 | 10 | 6 | 6 | 9.52 | 14.46±4.69 |
59 | 17 | 15 | 15 | 12 | 20.34 | ||
49 | 10 | 7 | 7 | 6 | 12.24 | ||
77 | 15 | 11 | 9 | 9 | 11.69 | ||
81 | 22 | 22 | 18 | 15 | 18.52 | ||
bar | 89 | 7 | 5 | 5 | 3 | 3.37 | 5.65±2.61 |
60 | 9 | 9 | 7 | 6 | 10.00 | ||
79 | 8 | 7 | 7 | 4 | 5.06 | ||
76 | 4 | 3 | 3 | 3 | 3.95 | ||
51 | 6 | 6 | 5 | 3 | 5.88 |
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