Chinese Bulletin of Botany ›› 2023, Vol. 58 ›› Issue (5): 760-769.DOI: 10.11983/CBB22141
• TECHNIQUES AND METHODS • Previous Articles Next Articles
Yu Xiaomin1, Wang Yaqin1, Liu Yuhan1, Yi Qingping2, Cheng Wenhan2, Zhu Yu1, Duan Feng1, Zhang Lixue1, He Yanhong1,2,*(
)
HomeReceived:2022-07-02
Accepted:2022-12-02
Online:2023-09-01
Published:2023-09-21
Contact:
*E-mail: hyh2010@mail.hzau.edu.cn
Yu Xiaomin, Wang Yaqin, Liu Yuhan, Yi Qingping, Cheng Wenhan, Zhu Yu, Duan Feng, Zhang Lixue, He Yanhong. Establishment of Agrobacterium tumefaciens-mediated Genetic Transformation System of Marigold (Tagetes erecta)[J]. Chinese Bulletin of Botany, 2023, 58(5): 760-769.
| Culture medium types | Formula |
|---|---|
| MS | 4.405 g·L-1 MS+30 g·L-1 sucrose+8 g·L-1 agar |
| Regeneration medium | 4.405 g·L-1 MS+40 g·L-1 sucrose+8 g·L-1 agar+0.2 mg·L-1 TDZ+0.5 mg·L-1 IBA |
| Co-culture medium | 4.405 g·L-1 MS+40 g·L-1 sucrose+8 g·L-1 agar+0.2 mg·L-1 TDZ+0.5 mg·L-1 IBA+0.5 g·L-1 MES+ 100 μmol·L-1 AS |
| Screening medium | 4.405 g·L-1 MS+40 g·L-1 sucrose+8 g·L-1 agar+0.2 mg·L-1 TDZ+0.5 mg·L-1 IBA+0.5 g·L-1 MES+10 mg·L-1 Kan+100 mg·L-1 Cef |
| Elongation medium/ rooting medium | 4.405 g·L-1 MS+30 g·L-1 sucrose+8 g·L-1 agar+0.5 g·L-1 MES+10 mg·L-1 Kan+100 mg·L-1 Cef |
Table 1 Medium used for genetic transformation of marigold Milestone Yellow
| Culture medium types | Formula |
|---|---|
| MS | 4.405 g·L-1 MS+30 g·L-1 sucrose+8 g·L-1 agar |
| Regeneration medium | 4.405 g·L-1 MS+40 g·L-1 sucrose+8 g·L-1 agar+0.2 mg·L-1 TDZ+0.5 mg·L-1 IBA |
| Co-culture medium | 4.405 g·L-1 MS+40 g·L-1 sucrose+8 g·L-1 agar+0.2 mg·L-1 TDZ+0.5 mg·L-1 IBA+0.5 g·L-1 MES+ 100 μmol·L-1 AS |
| Screening medium | 4.405 g·L-1 MS+40 g·L-1 sucrose+8 g·L-1 agar+0.2 mg·L-1 TDZ+0.5 mg·L-1 IBA+0.5 g·L-1 MES+10 mg·L-1 Kan+100 mg·L-1 Cef |
| Elongation medium/ rooting medium | 4.405 g·L-1 MS+30 g·L-1 sucrose+8 g·L-1 agar+0.5 g·L-1 MES+10 mg·L-1 Kan+100 mg·L-1 Cef |
| Kan concentration (mg·L-1) | Callus induction rate (%) | Regeneration rate (%) |
|---|---|---|
| 0 | 100.00±0.00 a | 72.50±5.00 a |
| 2 | 100.00±0.00 a | 67.50±12.58 a |
| 4 | 100.00±0.00 a | 50.00±14.14 b |
| 6 | 97.50±5.00 a | 15.00±10.00 c |
| 8 | 95.00±5.77 ab | 7.50±5.00 d |
| 10 | 92.50±5.00 b | 0.00±0.00 e |
| 15 | 65.00±5.77 c | 0.00±0.00 e |
| 20 | 37.50±12.58 d | 0.00±0.00 e |
| 30 | 27.50±5.00 d | 0.00±0.00 e |
| 40 | 15.00±5.77 e | 0.00±0.00 e |
| 50 | 0.00±0.00 f | 0.00±0.00 e |
Table 2 Effect of Kan concentration on leaflet regeneration of marigold
| Kan concentration (mg·L-1) | Callus induction rate (%) | Regeneration rate (%) |
|---|---|---|
| 0 | 100.00±0.00 a | 72.50±5.00 a |
| 2 | 100.00±0.00 a | 67.50±12.58 a |
| 4 | 100.00±0.00 a | 50.00±14.14 b |
| 6 | 97.50±5.00 a | 15.00±10.00 c |
| 8 | 95.00±5.77 ab | 7.50±5.00 d |
| 10 | 92.50±5.00 b | 0.00±0.00 e |
| 15 | 65.00±5.77 c | 0.00±0.00 e |
| 20 | 37.50±12.58 d | 0.00±0.00 e |
| 30 | 27.50±5.00 d | 0.00±0.00 e |
| 40 | 15.00±5.77 e | 0.00±0.00 e |
| 50 | 0.00±0.00 f | 0.00±0.00 e |
| Cef concentration (mg·L-1) | Callus induction rate (%) | Regeneration rate (%) | Growth of bacteria |
|---|---|---|---|
| 0 | 100.00±0.00 a | 60.00±8.16 a | Grow well |
| 100 | 97.50±5.00 a | 47.50±12.58 ab | Completely inhibited |
| 200 | 97.50±5.00 a | 40.00±8.16 abc | Completely inhibited |
| 300 | 65.00±5.77 b | 35.00±12.91 bc | Completely inhibited |
| 400 | 37.50±12.58 c | 37.50±17.08 bc | Completely inhibited |
| 500 | 27.50±5.00 c | 27.50±12.58 c | Completely inhibited |
Table 3 Effect of Cef concentration on leaflet regeneration of marigold and bacteria growth
| Cef concentration (mg·L-1) | Callus induction rate (%) | Regeneration rate (%) | Growth of bacteria |
|---|---|---|---|
| 0 | 100.00±0.00 a | 60.00±8.16 a | Grow well |
| 100 | 97.50±5.00 a | 47.50±12.58 ab | Completely inhibited |
| 200 | 97.50±5.00 a | 40.00±8.16 abc | Completely inhibited |
| 300 | 65.00±5.77 b | 35.00±12.91 bc | Completely inhibited |
| 400 | 37.50±12.58 c | 37.50±17.08 bc | Completely inhibited |
| 500 | 27.50±5.00 c | 27.50±12.58 c | Completely inhibited |
Figure 1 Effects of strain types on genetic transformation efficiency of marigold (A) Schematic diagram of transient expression of GUS gene and resistant callus in three strains (bars=1 cm); (B) Effect of strain types on transient expression efficiency of GUS gene and induction rate of resistant calli (different lowercase letters on the same color histogram indicate significant differences at 0.05 level)
| No. | OD600 | Infection time (d) | Co-culture time (d) | Transient expression rate of GUS gene (%) | Callus induction rate (%) | Resistance bud rate (%) | PCR positive rate (%) |
|---|---|---|---|---|---|---|---|
| 1 | 0.1 | 5 | 1 | 100 | 90.67±2.31 abcd | 45.33 | 4.00 |
| 2 | 0.1 | 10 | 2 | 100 | 91.85±7.83 abcd | 27.55 | 0.00 |
| 3 | 0.1 | 20 | 3 | 100 | 77.11±6.99 e | 20.25 | 0.00 |
| 4 | 0.1 | 30 | 4 | 100 | 86.20±11.95 cde | 0.00 | 0.00 |
| 5 | 0.5 | 5 | 2 | 100 | 96.67±2.88 abc | 28.33 | 0.00 |
| 6 | 0.5 | 10 | 1 | 100 | 86.67±0.00 bcde | 22.22 | 3.33 |
| 7 | 0.5 | 20 | 4 | 100 | 93.62±6.45 abc | 3.16 | 0.00 |
| 8 | 0.5 | 30 | 3 | 100 | 96.30±3.21 abc | 0.93 | 0.00 |
| 9 | 1 | 5 | 3 | 100 | 97.10±2.51 ab | 23.53 | 0.00 |
| 10 | 1 | 10 | 4 | 100 | 91.61±4.93 abcd | 13.51 | 0.00 |
| 11 | 1 | 20 | 1 | 100 | 81.25±6.25 de | 28.57 | 0.00 |
| 12 | 1 | 30 | 2 | 100 | 86.03±1.27 cde | 9.00 | 0.00 |
| 13 | 2 | 5 | 4 | 100 | 95.45±7.87 abc | 17.65 | 1.96 |
| 14 | 2 | 10 | 3 | 100 | 100.00±0.00 a | 5.71 | 0.00 |
| 15 | 2 | 20 | 2 | 100 | 86.18±3.66 cde | 12.64 | 0.00 |
| 16 | 2 | 30 | 1 | 100 | 86.11±4.34 cde | 0.00 | 0.00 |
Table 4 Effects of different infection conditions on transformation efficiency of marigold
| No. | OD600 | Infection time (d) | Co-culture time (d) | Transient expression rate of GUS gene (%) | Callus induction rate (%) | Resistance bud rate (%) | PCR positive rate (%) |
|---|---|---|---|---|---|---|---|
| 1 | 0.1 | 5 | 1 | 100 | 90.67±2.31 abcd | 45.33 | 4.00 |
| 2 | 0.1 | 10 | 2 | 100 | 91.85±7.83 abcd | 27.55 | 0.00 |
| 3 | 0.1 | 20 | 3 | 100 | 77.11±6.99 e | 20.25 | 0.00 |
| 4 | 0.1 | 30 | 4 | 100 | 86.20±11.95 cde | 0.00 | 0.00 |
| 5 | 0.5 | 5 | 2 | 100 | 96.67±2.88 abc | 28.33 | 0.00 |
| 6 | 0.5 | 10 | 1 | 100 | 86.67±0.00 bcde | 22.22 | 3.33 |
| 7 | 0.5 | 20 | 4 | 100 | 93.62±6.45 abc | 3.16 | 0.00 |
| 8 | 0.5 | 30 | 3 | 100 | 96.30±3.21 abc | 0.93 | 0.00 |
| 9 | 1 | 5 | 3 | 100 | 97.10±2.51 ab | 23.53 | 0.00 |
| 10 | 1 | 10 | 4 | 100 | 91.61±4.93 abcd | 13.51 | 0.00 |
| 11 | 1 | 20 | 1 | 100 | 81.25±6.25 de | 28.57 | 0.00 |
| 12 | 1 | 30 | 2 | 100 | 86.03±1.27 cde | 9.00 | 0.00 |
| 13 | 2 | 5 | 4 | 100 | 95.45±7.87 abc | 17.65 | 1.96 |
| 14 | 2 | 10 | 3 | 100 | 100.00±0.00 a | 5.71 | 0.00 |
| 15 | 2 | 20 | 2 | 100 | 86.18±3.66 cde | 12.64 | 0.00 |
| 16 | 2 | 30 | 1 | 100 | 86.11±4.34 cde | 0.00 | 0.00 |
| Factor | OD600 | Infection time (d) | Co-culture time (d) |
|---|---|---|---|
| K1 | 4.00 | 5.96 | 7.33 |
| K2 | 3.33 | 3.33 | 0.00 |
| K3 | 0.00 | 0.00 | 0.00 |
| K4 | 1.96 | 0.00 | 1.96 |
| K1 | 1.00 | 1.49 | 1.83 |
| K2 | 0.83 | 0.83 | 0.00 |
| K3 | 0.00 | 0.00 | 0.00 |
| K4 | 0.49 | 0.00 | 0.49 |
| R | 1.00 | 1.49 | 1.83 |
Table 5 Range analysis of PCR positive rate under different infection conditions
| Factor | OD600 | Infection time (d) | Co-culture time (d) |
|---|---|---|---|
| K1 | 4.00 | 5.96 | 7.33 |
| K2 | 3.33 | 3.33 | 0.00 |
| K3 | 0.00 | 0.00 | 0.00 |
| K4 | 1.96 | 0.00 | 1.96 |
| K1 | 1.00 | 1.49 | 1.83 |
| K2 | 0.83 | 0.83 | 0.00 |
| K3 | 0.00 | 0.00 | 0.00 |
| K4 | 0.49 | 0.00 | 0.49 |
| R | 1.00 | 1.49 | 1.83 |
Figure 2 Effect of acetosyringone (AS) on transformation efficiency of marigold Different lowercase letters on the same color histogram indicate significant differences at 0.05 level.
| Anti-browning agent and concentration | No. of resistant buds | Resistance buds rate (%) | No. of positive seedlings | PCR positive rate (%) | Description of budding state |
|---|---|---|---|---|---|
| CK | 4 | 4.00 | 0 | 0.00 | Grow well |
| 0.2 g·L-1 CA | 6 | 6.45 | 0 | 0.00 | Hard texture, more clustered buds, basically no main stem |
| 0.4 g·L-1 CA | 9 | 9.00 | 0 | 0.00 | Hard texture, more clustered buds, basically no main stem |
| 0.6 g·L-1 CA | 12 | 12.00 | 0 | 0.00 | Hard texture, more clustered buds, basically no main stem |
| 0.2 g·L-1 PVP | 12 | 12.00 | 4 | 4.00 | Grow well |
| 0.4 g·L-1 PVP | 7 | 7.00 | 0 | 0.00 | Grow well |
| 0.6 g·L-1 PVP | 7 | 7.37 | 1 | 1.11 | Grow well |
Table 6 Effects of citric acid (CA) and polyvinyl pyrrolidone (PVP) on transformation efficiency of marigold
| Anti-browning agent and concentration | No. of resistant buds | Resistance buds rate (%) | No. of positive seedlings | PCR positive rate (%) | Description of budding state |
|---|---|---|---|---|---|
| CK | 4 | 4.00 | 0 | 0.00 | Grow well |
| 0.2 g·L-1 CA | 6 | 6.45 | 0 | 0.00 | Hard texture, more clustered buds, basically no main stem |
| 0.4 g·L-1 CA | 9 | 9.00 | 0 | 0.00 | Hard texture, more clustered buds, basically no main stem |
| 0.6 g·L-1 CA | 12 | 12.00 | 0 | 0.00 | Hard texture, more clustered buds, basically no main stem |
| 0.2 g·L-1 PVP | 12 | 12.00 | 4 | 4.00 | Grow well |
| 0.4 g·L-1 PVP | 7 | 7.00 | 0 | 0.00 | Grow well |
| 0.6 g·L-1 PVP | 7 | 7.37 | 1 | 1.11 | Grow well |
Figure 3 Genetic transformation process diagram of marigold (A) Marigold compound leaves; (B) Leaflet culture; (C) Leaflet-induced calli; (D) GUS staining of transiently transformed leaves; (E) Resistance buds; (F) Resistant seedlings; (G) Rooting culture of resistant seedlings; (H) Positive seedlings; (I) Transplantation of positive seedlings. (A)-(D), (G)-(I) Bars=1 cm; (E), (F) Bars=0.5 cm
Figure 4 GUS staining effect on different organs of marigold positive plants Bars=2 mm
Figure 5 PCR amplification detection and Southern blot results of marigold resistant seedlings (A) PCR amplification detection of resistant seedlings (M: 2000 DNA marker; 1: ddH2O; 2: Plasmid; 3, 4: Negative control, DNA of uninfected leaf; 5-15: DNA of resistant seedlings); (B) Southern blot results of positive seedlings (the NPT-II probe was digested with HindIII. M: DNA marker; 1: Negative control, DNA of uninfected leaf; 2: Positive control, plasmid; 3-5: DNA of positive seedlings).
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