Rice Science ›› 2017, Vol. 24 ›› Issue (3): 181-186.DOI: 10.1016/j.rsci.2016.11.001
• Orginal Article • Previous Articles
Ratanasut Kumrop(), Rod-In Weerawan, Sujipuli Kawee
Online:
2017-05-28
Published:
2017-03-03
Ratanasut Kumrop, Rod-In Weerawan, Sujipuli Kawee. In planta Agrobacterium-Mediated Transformation of Rice[J]. Rice Science, 2017, 24(3): 181-186.
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Transformation method | Floral tissue | Anther |
---|---|---|
Floral-dip | 11.12 ± 1.76 b (94 GUS-stained/837 dipped flowers) | 77.79 ± 7.71 a (84 GUS-stained anthers/94 GUS-stained flowers) |
Floral-drop | 14.92 ± 0.81 a (117 GUS-stained/784 dropped flowers) | 81.94 ± 5.47 a (96 GUS-stained anthers/117 GUS-stained flowers) |
GUS-stained flowers indicate flowers with transformed floral tissues. GUS-stained anthers indicate flowers with transformed anthers. | ||
Values are expressed as Mean ± SE (n = 9). The different lowercase letters after the numbers in the same column indicate significant differences between treatments by the Duncan’s multiple range test (P < 0.05). |
Table 1 Transient transformation efficiency of the floral-dip and floral-drop methods in floral tissues and anthers. %
Transformation method | Floral tissue | Anther |
---|---|---|
Floral-dip | 11.12 ± 1.76 b (94 GUS-stained/837 dipped flowers) | 77.79 ± 7.71 a (84 GUS-stained anthers/94 GUS-stained flowers) |
Floral-drop | 14.92 ± 0.81 a (117 GUS-stained/784 dropped flowers) | 81.94 ± 5.47 a (96 GUS-stained anthers/117 GUS-stained flowers) |
GUS-stained flowers indicate flowers with transformed floral tissues. GUS-stained anthers indicate flowers with transformed anthers. | ||
Values are expressed as Mean ± SE (n = 9). The different lowercase letters after the numbers in the same column indicate significant differences between treatments by the Duncan’s multiple range test (P < 0.05). |
Inoculation media | Floral tissue | Anther |
---|---|---|
Standard | 11.91 ± 1.76 a (50 GUS-stained/413 dipped flowers) | 85.53 ± 6.77 a (42 GUS-stained anthers/50 GUS-stained flowers) |
No pH adjustment | 9.68 ± 0.06 b (34 GUS-stained/351 dipped flowers) | 82.61 ± 7.35 a (28 GUS-stained anthers/34 GUS-stained flowers) |
No Murashige and Skoog | 8.14 ± 1.52 b (34 GUS-stained/419 dipped flowers) | 74.93 ± 12.16 ab (26 GUS-stained anthers/34 GUS-stained flowers) |
No benzylaminopurine | 5.51 ± 1.20 c (26 GUS-stained/477 dipped flowers) | 59.84 ± 14.80 bc (16 GUS-stained anthers/26 GUS-stained flowers) |
No Tween-20 | 2.45 ± 1.22 d (10 GUS-stained/414 dipped flowers) | 58.89 ± 8.39 bc (7 GUS-stained anthers/10 GUS-stained flowers) |
No sucrose | 3.81 ± 0.93 d (13 GUS-stained/352 dipped flowers) | 55.00 ± 18.03 c (8 GUS-stained anthers/13 GUS-stained flowers) |
No sucrose, 5% glucose | 9.42 ± 0.91 b (34 GUS-stained/359 dipped flowers) | 80.27 ± 7.53 a (27 GUS-stained anthers/34 GUS-stained flowers) |
No sucrose, 10% glucose | 4.39 ± 1.90 cd (18 GUS-stained/390 dipped flowers) | 75.56 ± 7.70 ab (14 GUS-stained anthers/18 GUS-stained flowers) |
Add 200 µmol/L AS | 10.19 ± 0.40 ab (36 GUS-stained/353 dipped flowers) | 83.77 ± 6.40 a (30 GUS-stained anthers/36 GUS-stained flowers) |
GUS-stained flowers indicate flowers with transformed floral tissues. GUS-stained anthers indicate flowers with transformed anthers. | ||
AS, Acetosyringone. | ||
Values are expressed as Mean ± SE (n = 9). The different lowercase letters after the number in the same column indicate significant differences among treatments by the Duncan’s multiple range test (P < 0.05). |
Table 2 Transient transformation efficiency in floral tissues and anthers at different composition in inoculation medium. %
Inoculation media | Floral tissue | Anther |
---|---|---|
Standard | 11.91 ± 1.76 a (50 GUS-stained/413 dipped flowers) | 85.53 ± 6.77 a (42 GUS-stained anthers/50 GUS-stained flowers) |
No pH adjustment | 9.68 ± 0.06 b (34 GUS-stained/351 dipped flowers) | 82.61 ± 7.35 a (28 GUS-stained anthers/34 GUS-stained flowers) |
No Murashige and Skoog | 8.14 ± 1.52 b (34 GUS-stained/419 dipped flowers) | 74.93 ± 12.16 ab (26 GUS-stained anthers/34 GUS-stained flowers) |
No benzylaminopurine | 5.51 ± 1.20 c (26 GUS-stained/477 dipped flowers) | 59.84 ± 14.80 bc (16 GUS-stained anthers/26 GUS-stained flowers) |
No Tween-20 | 2.45 ± 1.22 d (10 GUS-stained/414 dipped flowers) | 58.89 ± 8.39 bc (7 GUS-stained anthers/10 GUS-stained flowers) |
No sucrose | 3.81 ± 0.93 d (13 GUS-stained/352 dipped flowers) | 55.00 ± 18.03 c (8 GUS-stained anthers/13 GUS-stained flowers) |
No sucrose, 5% glucose | 9.42 ± 0.91 b (34 GUS-stained/359 dipped flowers) | 80.27 ± 7.53 a (27 GUS-stained anthers/34 GUS-stained flowers) |
No sucrose, 10% glucose | 4.39 ± 1.90 cd (18 GUS-stained/390 dipped flowers) | 75.56 ± 7.70 ab (14 GUS-stained anthers/18 GUS-stained flowers) |
Add 200 µmol/L AS | 10.19 ± 0.40 ab (36 GUS-stained/353 dipped flowers) | 83.77 ± 6.40 a (30 GUS-stained anthers/36 GUS-stained flowers) |
GUS-stained flowers indicate flowers with transformed floral tissues. GUS-stained anthers indicate flowers with transformed anthers. | ||
AS, Acetosyringone. | ||
Values are expressed as Mean ± SE (n = 9). The different lowercase letters after the number in the same column indicate significant differences among treatments by the Duncan’s multiple range test (P < 0.05). |
A. tumefaciens strain | Floral tissue | Anther |
AGL1 | 12.26 ± 0.65 a (106 GUS-stained flowers/866 dipped flowers) | 77.11 ± 4.73 a (82 GUS-stained anthers/106 GUS-stained flowers) |
EHA105 | 14.34 ± 3.25 a (132 GUS-stained flowers/904 dipped flowers) | 80.74 ± 1.43 a (107 GUS-stained anthers/132 GUS-stained flowers) |
GUS-stained flowers indicate flowers with transformed floral tissues. GUS-stained anthers indicate flowers with transformed anthers. | ||
Values are expressed as Mean ± SE (n = 9). The same lowercase letter after the numbers in the same column indicates non-significant differences between treatments by the independent sample t-test. |
Table 3 Transient transformation efficiency in floral tissue and anther of the rice spikelets dipped in two different A. tumefaciens strains, AGL1 and EHA105, harbouring pCAMBIA1304 determined by GUS assay. %
A. tumefaciens strain | Floral tissue | Anther |
AGL1 | 12.26 ± 0.65 a (106 GUS-stained flowers/866 dipped flowers) | 77.11 ± 4.73 a (82 GUS-stained anthers/106 GUS-stained flowers) |
EHA105 | 14.34 ± 3.25 a (132 GUS-stained flowers/904 dipped flowers) | 80.74 ± 1.43 a (107 GUS-stained anthers/132 GUS-stained flowers) |
GUS-stained flowers indicate flowers with transformed floral tissues. GUS-stained anthers indicate flowers with transformed anthers. | ||
Values are expressed as Mean ± SE (n = 9). The same lowercase letter after the numbers in the same column indicates non-significant differences between treatments by the independent sample t-test. |
Fig. 1. Analysis of gusA transgenes and expression in T0 bulk tests Nos. 2, 4, 7 and 13. (A, PCR amplification of a 989 bp expected product for positive transformed plants using a pair of primers gusA-F1 and gusA-R1; B, PCR amplification of a 361 bp expected product for positive transformed plants using a pair of primers gusA-F2 and gusA-R2; C, gusA expression analysis of 20 individual T0 plants from the PCR-positive bulk tests Nos. 2, 4, 7 and 13.M, 1 kb Plus DNA Ladder (Invitrogen, USA).)
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