Biotechnology Bulletin ›› 2024, Vol. 40 ›› Issue (9): 33-41.doi: 10.13560/j.cnki.biotech.bull.1985.2024-0536
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SONG Qian-na1,2(), DUAN Yong-hong1, FENG Rui-yun1,2()
Received:
2024-06-07
Online:
2024-09-26
Published:
2024-10-12
Contact:
FENG Rui-yun
E-mail:songqianna1007@126.com;fengruiyun1970@163.com
SONG Qian-na, DUAN Yong-hong, FENG Rui-yun. Establishment of CRISPR/Cas9-mediated Highly Efficient Gene Editing System in Microtubers of Potatoes[J]. Biotechnology Bulletin, 2024, 40(9): 33-41.
蔗糖浓度 Sucrose concentration/% | 并薯6号 Bingshu No. 6 | 青薯9号 Qingshu No.9 | ||||
---|---|---|---|---|---|---|
单瓶平均薯数Number of microtubers per bottle/粒 | 单瓶平均薯重Weight of microtubers per bottle/g | 单瓶平均薯数Number of microtubers per bottle/粒 | 单瓶平均薯重Weight of microtubers per bottle/g | |||
6 | 0.17±0.40b | 0.02±0.04c | 0.20±0.45c | 0.02±0.03b | ||
8 | 2.83±1.17a | 0.29±0.19ab | 1.80±0.84ab | 0.19±0.06a | ||
10 | 3.50±1.64a | 0.48±0.20a | 2.60±1.34a | 0.28±0.13a | ||
12 | 0.67±0.82b | 0.09±0.10bc | 0.80±0.84bc | 0.05±0.05b | ||
15 | 0.67±1.03b | 0.06±0.11c | 0.40±0.55c | 0.02±0.03b |
Table 1 Micro tuberization under different sucrose concentrations for two potato varieties
蔗糖浓度 Sucrose concentration/% | 并薯6号 Bingshu No. 6 | 青薯9号 Qingshu No.9 | ||||
---|---|---|---|---|---|---|
单瓶平均薯数Number of microtubers per bottle/粒 | 单瓶平均薯重Weight of microtubers per bottle/g | 单瓶平均薯数Number of microtubers per bottle/粒 | 单瓶平均薯重Weight of microtubers per bottle/g | |||
6 | 0.17±0.40b | 0.02±0.04c | 0.20±0.45c | 0.02±0.03b | ||
8 | 2.83±1.17a | 0.29±0.19ab | 1.80±0.84ab | 0.19±0.06a | ||
10 | 3.50±1.64a | 0.48±0.20a | 2.60±1.34a | 0.28±0.13a | ||
12 | 0.67±0.82b | 0.09±0.10bc | 0.80±0.84bc | 0.05±0.05b | ||
15 | 0.67±1.03b | 0.06±0.11c | 0.40±0.55c | 0.02±0.03b |
不同激素配比 Different hormone combination | 并薯6号 Bingshu No. 6 | 青薯9号 Qingshu No.9 | ||||
---|---|---|---|---|---|---|
单瓶平均薯数/粒Number of microtubers per bottle | 单瓶平均薯重Weight of microtubers per bottle/g | 单瓶平均薯数Number of microtubers per bottle/粒 | 单瓶平均薯重Weight of microtubers per bottle/g | |||
0 | 3.50±1.64a | 0.48±0.20a | 2.60±1.34a | 0.28±0.13a | ||
4 mg/L 6-BA | 0.67±0.58b | 0.04±0.03b | 0.33±0.58b | 0.02±0.03b | ||
4 mg/L 6-BA + 3.5 mg/L NAA | 0.67±0.58b | 0.03±0.03b | 0.00±0.00b | 0.00±0.00b | ||
5 mg/L KT | 4.67±0.58a | 0.62±0.07a | 3.30±0.82a | 0.37±0.15a |
Table 2 Micro tuberization under different hormone combinations for two potato varieties
不同激素配比 Different hormone combination | 并薯6号 Bingshu No. 6 | 青薯9号 Qingshu No.9 | ||||
---|---|---|---|---|---|---|
单瓶平均薯数/粒Number of microtubers per bottle | 单瓶平均薯重Weight of microtubers per bottle/g | 单瓶平均薯数Number of microtubers per bottle/粒 | 单瓶平均薯重Weight of microtubers per bottle/g | |||
0 | 3.50±1.64a | 0.48±0.20a | 2.60±1.34a | 0.28±0.13a | ||
4 mg/L 6-BA | 0.67±0.58b | 0.04±0.03b | 0.33±0.58b | 0.02±0.03b | ||
4 mg/L 6-BA + 3.5 mg/L NAA | 0.67±0.58b | 0.03±0.03b | 0.00±0.00b | 0.00±0.00b | ||
5 mg/L KT | 4.67±0.58a | 0.62±0.07a | 3.30±0.82a | 0.37±0.15a |
品种 Variety | 处理 Treatment | 单瓶平均结薯数Number of microtubers per bottle/粒 | 单瓶平均薯重Weight of microtubers per bottle/g | 大薯重Weight of the large tuber/g | 大薯直径 Diameter of the large tuber/cm |
---|---|---|---|---|---|
并薯6号 | 顶部1个节段 | 2.00±2.00a | 0.13±0.15a | 0.08±0.08ab | 0.43±0.38a |
底部1个节段 | 0.67±0.58a | 0.04±0.04a | 0.04±0.04b | 0.38±0.34a | |
顶部2个节段 | 3.33±0.58a | 0.23±0.05a | 0.09±0.05ab | 0.65±0.09a | |
底部2个节段 | 2.67±1.53a | 0.45±0.37a | 0.22±0.13a | 0.80±0.18a | |
青薯9号 | 顶部1个节段 | 1.33±2.16a | 0.10±0.20b | 0.02±0.04b | 0.18±0.29b |
底部1个节段 | 2.83±1.72a | 0.20±0.17ab | 0.10±0.07ab | 0.44±0.06ab | |
顶部2个节段 | 3.17±1.60a | 0.21±0.13ab | 0.10±0.04ab | 0.55±0.07a | |
底部2个节段 | 3.33±0.82a | 0.37±0.15a | 0.15±0.05a | 0.59±0.06a |
Table 3 Induction of tuberization under different segment lengths for two potato varieties
品种 Variety | 处理 Treatment | 单瓶平均结薯数Number of microtubers per bottle/粒 | 单瓶平均薯重Weight of microtubers per bottle/g | 大薯重Weight of the large tuber/g | 大薯直径 Diameter of the large tuber/cm |
---|---|---|---|---|---|
并薯6号 | 顶部1个节段 | 2.00±2.00a | 0.13±0.15a | 0.08±0.08ab | 0.43±0.38a |
底部1个节段 | 0.67±0.58a | 0.04±0.04a | 0.04±0.04b | 0.38±0.34a | |
顶部2个节段 | 3.33±0.58a | 0.23±0.05a | 0.09±0.05ab | 0.65±0.09a | |
底部2个节段 | 2.67±1.53a | 0.45±0.37a | 0.22±0.13a | 0.80±0.18a | |
青薯9号 | 顶部1个节段 | 1.33±2.16a | 0.10±0.20b | 0.02±0.04b | 0.18±0.29b |
底部1个节段 | 2.83±1.72a | 0.20±0.17ab | 0.10±0.07ab | 0.44±0.06ab | |
顶部2个节段 | 3.17±1.60a | 0.21±0.13ab | 0.10±0.04ab | 0.55±0.07a | |
底部2个节段 | 3.33±0.82a | 0.37±0.15a | 0.15±0.05a | 0.59±0.06a |
Fig. 1 Construction of CRISPR/Cas9 deletion vector A: Schematic diagram of pKSE402 vector. B: The target site in gene StuPPO2. Red is PAM sequence; blue is restriction enzyme site. C: Correct target sequence is confirmed by sequencing
品种Variety | 培养基类型Media types | 接种薯片数Microtubers/个 | 芽分化薯片数Differentiation microtubers/个 | 分化效率Differentiation efficiency/% |
---|---|---|---|---|
青薯9号 | K1 | 106 | 8 | 7.5 |
K2 | 94 | 39 | 41.5 | |
并薯6号 | K1 | 58 | 26 | 44.8 |
K2 | 63 | 22 | 34.9 |
Table 4 Induction and differentiation of microtuber for two potato varieties
品种Variety | 培养基类型Media types | 接种薯片数Microtubers/个 | 芽分化薯片数Differentiation microtubers/个 | 分化效率Differentiation efficiency/% |
---|---|---|---|---|
青薯9号 | K1 | 106 | 8 | 7.5 |
K2 | 94 | 39 | 41.5 | |
并薯6号 | K1 | 58 | 26 | 44.8 |
K2 | 63 | 22 | 34.9 |
Fig. 2 Agrobacterium-mediated potato microtuber genetic transformation system A: Induction of microtuber. B: Infection of microtuber thin slices. C: Induction of microtuber thin slices. D: Differentiation of buds. E: Rooting of regenerated plants. F: Positive regenerated plants are selected by GFP
品种 Variety | 再生植株 Regenarated plants/株 | 荧光植株 Fluorescent plants/株 | 转化效率 Transformation frequency/% | 突变植株数 Mutant plants/株 | 突变效率 Mutation frequency/% |
---|---|---|---|---|---|
青薯9号 | 54 | 28 | 51.9 | 23 | 82.1 |
并薯6号 | 46 | 19 | 41.3 | 12 | 63.2 |
Table 5 Identification and analysis of StuPPO2 gene editing for fluorescent regenerated plants
品种 Variety | 再生植株 Regenarated plants/株 | 荧光植株 Fluorescent plants/株 | 转化效率 Transformation frequency/% | 突变植株数 Mutant plants/株 | 突变效率 Mutation frequency/% |
---|---|---|---|---|---|
青薯9号 | 54 | 28 | 51.9 | 23 | 82.1 |
并薯6号 | 46 | 19 | 41.3 | 12 | 63.2 |
Fig. 3 Detection of StuPPO2 knockout plants A: PCR/RE assay for mutants. WT/D indicates that wild is cut by the enzyme; WT/U indicates that wild can not be cut by the enzyme; T0-1-T0-4 are mutant plants. B: Mutation type analysis of stuppo2 mutants. The PAM sequence is highlighted in red
品种 Variety | 结薯效率 Potato yield/% | 最适再生培养基 Optimal medium | 芽分化效率 Differentiation efficiency/% | 遗传转化频率 Transformation frequency/% | 基因编辑效率 Mutation frequency/% |
---|---|---|---|---|---|
Desiree | (48/48)100 | K1 | (27/104)25.9 | (9/28)32.1 | (3/9)33.3 |
晋薯16号 | (15/48)31.3 | K2 | (30/51)58.8 | (10/25)40.0 | (1/10)10 |
并薯26号 | (31/48)64.6 | K2 | (28/154)18.2 | (7/28)25.0 | (0/7)0 |
Table 6 Testing of gene editing system for another three tetraploid potato varieties
品种 Variety | 结薯效率 Potato yield/% | 最适再生培养基 Optimal medium | 芽分化效率 Differentiation efficiency/% | 遗传转化频率 Transformation frequency/% | 基因编辑效率 Mutation frequency/% |
---|---|---|---|---|---|
Desiree | (48/48)100 | K1 | (27/104)25.9 | (9/28)32.1 | (3/9)33.3 |
晋薯16号 | (15/48)31.3 | K2 | (30/51)58.8 | (10/25)40.0 | (1/10)10 |
并薯26号 | (31/48)64.6 | K2 | (28/154)18.2 | (7/28)25.0 | (0/7)0 |
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