Chin Bull Bot ›› 2018, Vol. 53 ›› Issue (1): 59-71.doi: 10.11983/CBB16257

• EXPERIMENTAL COMMUNICATIONS • Previous Articles     Next Articles

Breeding of MtDREB2A Transgenic Soybean by an Optimized Cotyledonary-Node Method

Guodong Wu, Yu Xiu, Huafang Wang*()   

  1. Key Laboratory of Genetics and Breeding in Forest Trees and Ornamental Plants of Ministry of Education, National Engineering Laboratory for Tree Breeding, College of Biological Sciences and Biotechnology, Beijing Forestry University, Beijing 100083, China
  • Received:2016-12-23 Accepted:2017-05-04 Online:2018-08-10 Published:2018-01-01
  • Contact: Huafang Wang E-mail:hfwang@bjfu.edu.cn

Abstract:

Orthogonal factorial experiments and histochemical GUS staining were combined to optimize the genetic transformation system of Glycine max cv. ‘Dongnong 50’ and transfer the key gene MtDREB2A for drought resistance into the soybean. Sterile of soybean seeds used as explants with NaClO solution and Cl2 gas fumigation methods reached 98.67% and 93.33% germination, respectively. Histochemical staining rate of the tissues transformed with GUS by the cotyledon node method was 68.33%, significantly higher than that by the hypocotyl (14.00%) and embryo tip (0.67%) methods (P<0.05). The cotyledon node-transformed GUS gene was up to 72.00% in germinated sterile seeds for 5 days, mediated by Agrobacterium tumefaciens cultured at 25°C, OD600 0.9, and co-cultured for 5 days. The shoots were induced and differentiated with cotyledon node-transformed GUS up to 3.33% by optimal recovery culture for 5 days and were screened on culture medium containing phosphinothricin (3 mg·L-1), cefotaxime sodium (200 mg·L-1) and carbenicillin (300 mg·L-1). The transgenic efficiency was 1.11% with the optimized soybean genetic transformation system. The MtDREB2A transgenic plant roots of soybean ‘Dongnong 50’ were more dense and both taproot length and lateral root number were significantly longer and greater than those of the control (P<0.05). The study verified that the MtDREB2A gene plays a role in promoting soybean root growth, which lays a solid foundation and provides a theoretical basis for the gene using in drought resistance breeding of soybean.

Key words: soybean, optimization of gene transformation system, GUS, MtDREB2A, root enhancement

Table 1

The primers used in construction of plant express vector"

Primer name Primer sequence (5′-3′) Tm (°C) Product length (bp)
rd29A-F1 GGCTTTACACTTTATGCTTCC 49.2 859
rd29A-F2 TTGTTAGGCTCCCTCATTTC
rd29A-T1 CAGTTTGAAAGAAAAGGGAA 46.7 71
rd29A-T2 GCTTTTTGGAACTCATGTCG
MtDREB2A-F1 CATGCCATGGTGGAAATTGAAAGATGGGTGCT 53.5 971
MtDREB2A-F2 GGGTGACCGGATTATTATCTAGTTGCCCAAACG
MtDREB2A-T1 ACTTTTCCGACGGCTCAA 44.0 472
MtDREB2A-T2 GTCATTACACACACCCTCTC

Table 2

List of components used in media preparation for transformation method"

Germination medium Re-suspension medium Co-cultivation medium Shoot induction medium Shoot elongation medium Rooting
medium
MS salts 1/2 × 1/2 × - - - -
MS iron stock 1/2 × 1/2 × 1 × 1 × 1 × 1 ×
MS vitamins 1/2 × 1/2 × - - - -
B5 salts - - 1 × 1 × 1 × 1/2 ×
B5 vitamins - - 1 × 1 × 1 × -
Sucrose (g·L-1) 15 - 30 30 30 30
Glucose (g·L-1) - 10 - - - -
Agar (g·L-1) 8 - 5 8 8 8
pH 5.8 5.8 5.5 5.5 5.5 5.6
6-BA (mg·L-1) - - 1.7 1.7 1.7 -
GA (mg·L-1) - - - - 1.0 -
IBA (mg·L-1) - - - - - 1.0
MES (g·L-1) - - 0.6 0.6 0.6 0.6
L-cys (mg·L-1) - - 182.5 182.5 182.5 -
Na2S2O3 (mg·L-1) - - 250 250 250 -
DTT (mg·L-1) - - 154.3 - - -
AS (mg·L-1) - 39.2 39.2 - - -
Cef (mg·L-1) - - - 100-400 100-400 -
Cb (mg·L-1) - - - 100-400 100-400 -
PPT (mg·L-1) - - - 0, 2-5 - -

Figure 1

Construction of plant express vector pCAMBIA3301-rd29A-MtDREB2A(A) PCR confirmation of rd29A promoter (1: Marker; 2: pCAMBIA3301-35S-GUS; 3-6: PCR product of rd29A); (B) PCR confirmation of MtDREB2A genes (1: Marker; 2: pCAMBIA3301-35S-GUS; 3-6: PCR product of MtDREB2A); (C) Structure of pCAMBIA3301-rd29A-MtDREB2A"

Table 3

Effect of NaClO method on seed sterilization of Glycine max cv. ‘Dongnong 50’ (means±SD)"

Treatment NaClO concentration (%) Time (min) Sterilization rate (%) Contamination rate (%) Death rate (%)
1 0.10 3 81.33±3.06 e 16.00±2.00 b 2.67±1.16 bcd
2 0.10 5 72.67±1.16 f 24.00±2.00 a 3.33±1.16 bc
3 0.10 10 88.67±1.16 bc 10.67±1.16 cd 0.67±1.16 d
4 0.25 3 86.67±1.16 cd 10.00±2.00 cde 3.33±1.16 bc
5 0.25 5 70.67±2.31 f 23.33±4.16 a 6.00±2.00 a
6 0.25 10 90.67±1.16 b 8.00±2.00 de 1.33±1.16 cd
7 0.50 3 83.33±3.06 de 13.33±3.06 bc 3.33±1.16 bc
8 0.50 5 89.33±2.31 bc 6.00±2.00 ef 4.67±1.16 ab
9 0.50 10 98.67±1.16 a 0.67±1.16 g 0.67±1.16 d

Table 4

Effect of Cl2 method on seed sterilization of Glycine max cv. ‘Dongnong 50’"

Treatment Time (h) Sterilization rate (%) Contamination rate (%) Death rate (%)
1 1 82.00±5.29 b 13.33±5.78 a 4.66±1.16 b
2 2 82.67±6.11 b 12.00±6.00 a 5.33±3.06 b
3 4 92.00±3.46 a 2.67±2.31 b 5.33±3.06 b
4 6 93.33±1.16 a 2.67±2.31 b 4.00±2.00 b
5 8 88.00±3.46 ab 1.33±2.31 b 10.67±1.16 a

Figure 2

GUS gene histochemical staining(A) The effect of transgenic methods on histochemical GUS staining rate, different lowercase letters indicate significant differences at 0.05 level according to Duncan’s test; (B), (C) The GUS gene histochemical staining of cotyledonary node after co-cultivation ((B) Negative control; (C) Positive result of histochemical stain)"

Table 5

The results of orthogonal experiment for Agrobacterium-mediated transformation of Glycine max cv. ‘Dongnong 50’"

Treatment Factor Histochemical staining rate
of cotyledonary-node
(%)
Germination time
(d) (A)
Culture temperature
(°C) (B)
OD600
value (C)
Co-cultivation time
(d) (D)
1 3 25 0.3 2 8.00±2.00 g
2 3 26 0.5 3 31.05±10.46 def
3 3 27 0.7 4 38.27±5.46 cde
4 3 28 0.9 5 46.30±17.65 bcd
5 4 25 0.5 4 39.70±19.34 cde
6 4 26 0.3 5 59.33±3.06 ab
7 4 27 0.9 2 4.00±2.00 g
8 4 28 0.7 3 21.33±6.43 efg
9 5 25 0.7 5 68.33±12.42 a
10 5 26 0.9 4 51.06±7.65 abc
11 5 27 0.3 3 30.56±16.17 def
12 5 28 0.5 2 4.00±5.29 g
13 6 25 0.9 3 40.95±11.61 bcd
14 6 26 0.7 2 8.52±1.70 g
15 6 27 0.5 5 20.36±9.30 efg
16 6 28 0.3 4 12.67±10.26 fg
K1 30.91 39.25 27.64 6.13
K2 31.09 37.49 23.78 30.97
K3 38.49 23.30 34.12 35.43
K4 20.62 21.07 35.58 48.58
R 17.87 18.18 11.80 42.45

Figure 3

Transformation system of Glycine max cv. ‘Dongnong 50’ using optimized cotyledonary-node method(A) Co-cultivation after transformation; (B) Shoot induction; (C) Shoot elongation; (D) Resistant bud transplant; (E) PCR confirmation of GUS gene (1: Marker; 2: pCAMBIA3301-35S-GUS; 3: Control soybean; 4-13: Resistant plant); (F) RT-PCR confirmation of GUS gene (1: Marker; 2: Control soybean; 3-9: Resistant plant); (G) Histochemical staining of leaves from control (left) and resistant plant (middle and right)"

Figure 4

Confirmation and root system analyses of MtDREB2A transgenic soybean Dongnong 50(A) PCR result of MtDREB2A transgenic soybean (1: Marker; 2: pCAMBIA3301-rd29A-MtDREB2A; 3: Control soybean; 4-6: MtDREB2A transgenic soybean); (B) The root of control soybean (Bar=1 cm); (C) The length of taproot of control and transgenic soybeans; (D) The pod of MtDREB2A transgenic soybean in greenhouse; (E) The root of MtDREB2A transgenic soybean (Bar=1 cm); (F) The number of lateral root of control and transgenic soybeans. Different lowercase letters in Figures (C) and (F) indicate significant differences at 0.05 level according to Duncan’s test."

Table 6

The adventitious bud induction rate and histochemical staining rate of orthogonal experiment"

Treatment Factor Adventitious bud induction rate
(%)
Histochemical staining rate of adventitious bud (%)
Recovery
time (d) (A)
PPT concentration (mg·L-1) (B) Cef concentration (mg·L-1) (C) Cb concentration (mg·L-1) (D)
1 0 2 100 100 1.11±1.92 f 0.00±0.00
2 0 3 200 200 1.11±1.92 f 1.11±1.92
3 0 4 300 300 3.33±5.77 f 2.22±1.92
4 0 5 400 400 1.11±1.92 f 1.11±1.92
5 5 2 200 300 22.22±9.62 cd 2.22±1.92
6 5 3 100 400 7.78±3.85 ef 3.33±0.00
7 5 4 400 100 4.45±3.85 f 1.11±1.92
8 5 5 300 200 2.22±3.85 f 1.11±1.92
9 10 2 300 400 31.11±5.09 bcd 0.00±0.00
10 10 3 400 300 18.89±3.85 de 0.00±0.00
11 10 4 100 200 27.78±10.18 cd 0.00±0.00
12 10 5 200 100 21.11±8.39 cd 1.11±1.92
13 15 2 400 200 55.56±5.09 a 0.00±0.00
14 15 3 300 100 42.22±12.62 b 0.00±0.00
15 15 4 200 400 33.33±13.33 bc 0.00±0.00
16 15 5 100 300 23.33±8.82 cd 0.00±0.00
Adventitious bud induction rate K1 1.67 27.50 15.00 17.22
K2 9.17 17.50 19.44 21.67
K3 24.72 17.22 19.72 16.94
K4 38.61 11.94 20.00 18.33
R 36.94 15.56 5.00 4.72
Histochemical staining rate K1 1.11 0.56 0.83 0.56
K2 1.94 1.11 1.11 0.56
K3 0.28 0.83 0.83 1.11
K4 0.00 0.83 0.56 1.11
R 1.94 0.56 0.56 0.56
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