农杆菌介导的杜仲叶片愈伤组织遗传转化体系

doi:10.11707/j.1001-7488.20200209

Abstract

Abstract:

Objective: To optimize the callus regeneration system for Eucommia ulmoides leaf explants, to assess the sensitivity of callus to Km and Cef, and to determine the optimum factor levels for Agrobacterium tumefaciens-mediated transformation, in order to allow for genetic transformation of adult plant of E. ulmoides. Method: Callus was induced from leaves of E. ulmoides adult plants. It was inoculated in different concentrations of MS medium, NAA and 6-BA to optimize culture conditions for adventitious bud induction and regeneration. In addition, different concentrations of Km and Cef were added to the culture medium to study callus sensitivity. Based on the obtained callus receptor system for leaves, an L₁₆ (4⁵) orthogonal array experiment was conducted to explore the effect of different conversion factors on Agrobacterium-mediated transformation efficiency. Transformation with an optimum combination of factors was used to obtain Km-resistant buds, which were assessed as transformants by GUS histochemical staining and PCR. Result: MS medium at 3/4 strength promoted the induction and growth of adventitious buds in E. ulmoides. The optimum medium for induction of adventitious buds in callus was 3/4MS + 0.27 μmol·L^-1 NAA + 4.4 μmol·L^-1 6-BA, with induction rate of 83%±10.0%; The optimal medium for adventitious bud rejuvenation was 3/4MS + 0.054 μmol·L^-1 NAA + 4.4 μmol·L^-1 6-BA, with an average growth length of (2.47 ±1.33) cm. Sensitivity testing of antibiotic and bacteriostatic agents showed that in the selected medium for genetic transformation, the optimal concentrations of Cef and Km for screening were 200 mg·L^-1 and 70 mg·L^-1, respectively. The orthogonal test showed that the optimum combination of factors for Agrobacterium-mediated transformation was 5 days of pre-culture, 10 min of infection time and 3 days of co-cultivation. Genetic transformation of about 200 calli was carried out using the optimum instantaneous conversion system, and three Km-resistant buds were obtained. GUS histochemical staining showed that the GUS gene was expressed in these Km-resistant buds, and PCR analysis demonstrated the presence of the NPTⅡ gene. Conclusion: For E. ulmoides callus, this study defined an adventitious bud induction medium, 3/4MS+0.27 μmol·L^-1 NAA + 4.4 μmol·L^-1 6-BA, and an adventitious bud rejuvenation medium:3/4MS + 0.054 μmol·L^-1 NAA + 4.4 μmol·L^-1 6-BA. Agrobacterium-mediated transformation of callus involved 5 days of pre-culture, 10 min of infection time and 3 days of co-cultivation, and the selection medium, 3/4MS + 0.054 μmol·L^-1 NAA + 4.4 μmol·L^-1 6-BA + 200 mg·L^-1 Cef + 70 mg·L^-1 Km. Three resistant buds were obtained using this system. PCR analysis and GUS histochemical staining indicated that T-DNA had been integrated into the genome of these Km-resistant buds. This report demonstrated that genetic transformation could be studied in a mature E. ulmoides tree, laying a foundation for studying gene functions and for objective-specific improvement of E. ulmoides.

Key words: Eucommia ulmoides, Agrobacterium-mediated transformation, regeneration process, GUS transient expression

CLC Number:

S718.46

Minhao Liu,Junlei Xu,Jing Ye,Zhouqi Li,Ruishen Fan,Long Li. Agrobacterium tumefaciens-Mediated Transformation of Leaf Callus in Eucommia ulmoides[J]. Scientia Silvae Sinicae, 2020, 56(2): 79-88.

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培养基 Medium	6-BA浓度 Concentration of 6-BA/ (μmol·L^-1)	NAA浓度 Concentration of NAA/(μmol·L^-1)	不定芽诱导率 Percentage of shoot induction (%) (mean± SD)	不定芽伸长长度 Shoot length/cm (mean ± SD)
MS	0	0.27	0±0 f	—
MS	0	0.54	0±0 f	—
MS	4.4	0.054	—	0.51±0.48 b
MS	4.4	0.27	53±7.0 abcd	0.86±0.91 ab
MS	4.4	0.54	20±8.7 ef	-
MS	6.7	0.27	48±15.6 bcde	-
MS	8.8	0	24±9.1 def	-
MS	8.8	0.054	—	0.34±0.41 b
MS	8.8	0.27	19±4.5 ef	0.48±0.27 b
MS	8.8	0.54	55±14.4 abc	-
3/4MS	0	0.27	0±0 f	-
3/4MS	0	0.54	0±0 f	-
3/4MS	4.4	0.054	—	2.47±1.33 a
3/4MS	4.4	0.27	83±10.0 a	1.39±1.01 ab
3/4MS	4.4	0.54	43±10.7 bcde	-
3/4MS	6.7	0.27	60±16.8 ab	-
3/4MS	8.8	0	47±21.9 bcde	-
3/4MS	8.8	0.054	-	1.21±0.65 ab
3/4MS	8.8	0.27	26±6.0 cdef	1.03±0.82 ab
3/4MS	8.8	0.54	66±11.0 ab	-

编号 No.	Km浓度 Concentration of Km/(mg·L^-1)	Cef浓度 Concentration of Cef/(mg·L^-1)	不定芽诱导数 Number of shoot induced	愈伤组织状况 Growing states of callus
1	0	0	21	黄绿色,致密Yellowish green, compact
2	20	0	15	黄绿色,致密Yellowish green, compact
3	50	0	0	黄褐色,致密Yellowish brown, compact
4	70	0	0	褐色,死亡Brown, dead
5	100	0	0	褐色,死亡Brown, dead
6	0	0	18	黄褐色,致密Yellowish brown, compact
7	0	100	24	黄褐色,致密Yellowish brown, compact
8	0	200	20	黄褐色,致密Yellowish brown, compact
9	0	300	5	黄褐色,致密Yellowish brown, compact
10	0	400	0	褐色,死亡Brown, dead

方差来源 Sources of variance	平方和 Sum of squares	自由度 Degree of freedom	均方 Mean square	F	P
A:预培养时间Pre-culture duration	0.082	3	0.027	7.746	0.063
B:AS浓度AS concentration	0.003	3	0.001	0.262	0.850
C:侵染时间Infection duration	0.102	3	0.034	9.658	0.047^*
D:共培养时间Co-culture duration	0.281	3	0.094	26.575	0.012^*
误差Error	0.011	3	0.004
总计Total	1.618	16

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Agrobacterium tumefaciens-Mediated Transformation of Leaf Callus in Eucommia ulmoides

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编号 No.	预培养时间Pre-culture duration/d	AS浓度AS concentration/(μmol·L^-1)	侵染时间Infection duration/min	共培养时间Co-culture duration/d
1	0	0	6	1
2	0	127	8	2
3	0	255	10	3
4	0	510	15	4
5	2	0	8	3
6	2	127	6	4
7	2	255	15	1
8	2	510	10	2
9	5	0	10	4
10	5	127	15	3
11	5	255	6	2
12	5	510	8	1
13	10	0	15	2
14	10	127	10	1
15	10	255	8	4
16	10	510	6	3

编号No.	染色率Dyeing rate(%)			GUS瞬时表达率Transient GUS expression frequency(%)
编号No.	+	++	+++	GUS瞬时表达率Transient GUS expression frequency(%)
1	2	0	0	2
2	6	0	0	6
3	2	26	20	48
4	0	28	12	40
5	4	12	0	16
6	2	26	4	32
7	6	4	0	10
8	8	4	0	12
9	2	26	34	62
10	0	28	28	56
11	4	16	0	20
12	10	2	0	12
13	4	16	8	28
14	16	2	0	18
15	4	18	8	30
16	2	22	10	34