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

doi:10.11983/CBB16257

Abstract

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

Guodong Wu, Yu Xiu, Huafang Wang. Breeding of MtDREB2A Transgenic Soybean by an Optimized Cotyledonary-Node Method[J]. Chinese Bulletin of Botany, 2018, 53(1): 59-71.

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URL: https://www.chinbullbotany.com/EN/10.11983/CBB16257

https://www.chinbullbotany.com/EN/Y2018/V53/I1/59

Figures/Tables 10

References 45

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

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

	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	-	-

	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	-	-

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