In planta Agrobacterium-Mediated Transformation of Rice

doi:10.1016/j.rsci.2016.11.001

Abstract

Abstract:

The floral-dip transformation, the simplest technique, is no requirement of tissue culture procedure, and can directly transfer the interest gene into plant reproductive cells. It has been successfully applied to various plant species. In this study, the optimal conditions of a floral-dip method for production of transgenic rice variety RD41 were explored. The simple and effective inoculation medium was composed of Murashige and Skoog (MS) medium, 5% sucrose, 44 nmol/L benzylaminopurine, and 0.075% surfactant Tween-20 with pH 5.7. The transformation efficiencies of Agrobacterium tumefaciens strains AGL1 and EHA105 were compared with the Agrobacterium density at OD₆₀₀ = 0.8-1.0 and the co-cultivation at 25 ºC for 48 h. A. tumefaciens strain EHA105 gave slightly higher transformation efficiency than AGL1, with statistically non-significant difference. The floral-drop transformation using the optimal floral-dip conditions showed higher transformation efficiency than the floral-dip method, but the dropped flowers turned brown and died within 2 d. Production of transgenic rice variety RD41 by the floral-dip method was achieved using A. tumefaciens strain EHA105 with the optimal conditions. Screening for the gusA gene by PCR using the gusA specific primers in the T₀ lines, there were 4 transgenic lines from 286 T₀ lines (1.4% transformation efficiency). However, histochemical glucuronidase (GUS) assay demonstrated that only three of four transgenic lines exhibited gusA expression. These results indicated that floral-dip transformation is a potential tool for production of the transgenic rice, which can be used for molecular breeding via genetic engineering in the future.

Key words: Agrobacterium-mediated transformation, floral-dip, floral-drop, transformation efficiency, in planta, rice

Ratanasut Kumrop, Rod-In Weerawan, Sujipuli Kawee. In planta Agrobacterium-Mediated Transformation of Rice[J]. Rice Science, 2017, 24(3): 181-186.

Figures/Tables 4

References 27

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27	(Managing Editor: Wang Caihong)

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).

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).

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.