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中国生物工程杂志

China Biotechnology
China Biotechnology
China Biotechnology  2018, Vol. 38 Issue (1): 25-31    DOI: 10.13523/j.cb.20180103
Orginal Article     
Analysis of the Transformation Efficiency and Induced Differentiation of Lilium brownii Scales
Ting AN1,Jing JI1(),Yu-rong WANG2,3,Zhi-gang MA4,Gang WANG1(),Qian LI1,Dan YANG1,Song-hao ZHANG1
1 School of Environmental Science and Engineering,Tianjin University, Tianjin 300072, China
2 Tianda Tianfu Bio.Co.LTD, Tianjin 300072, China
3 UCSD, Biological Sciences, Molecular Biology, California 92093, USA
4 School of Chemical Engineering and Technology, Tianjin University ,Tianjin 300072,China
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Abstract  

Genetic engineering is an important means to improve the traits of lily. To establish an efficiency and stable transformation system is the basis of transgenic research. The different regeneration systems of lily were screened. The vector containing the target gene GR and selection gene NPTII was screened by Agrobacterium tumefaciens. Establish efficiency genetic transformation system for callus and scales through orthogonal test. The results showed that the optimum culture conditions as follow. The medium of lily scale inducing was MS+2mg/L 2,4-D + 0.1mg/L NAA + 90g/L + 0.2mg/L NAA + 30g/L sucrose. Indirect differentiation medium was MS+2.5mg/L 2,4-D+0.4mg/L 0.4mg/L TDZ+60g/L sucrose. The Kana selection pressure for scales was 100mg/L and for callus was 75mg/L. The direct genetic transformation of the scales was Agrobacterium OD600 = 0.6, pre-culture for 3 days, infiltrating for 40min, As 200μmol/L. The indirect callus transformation was: Agrobacterium OD600, pre-incubated for 5 days, infiltrating for 40min, As 200μmol/L. The direct and indirect positive plant transformation rates were 17.50% and 12.60%.

Key words： Lily      Genetic transformation      GR      Regeneration system      Direct differentiation     
Received: 14 July 2017      Published: 31 January 2018
ZTFLH:  Q813  
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Ting AN
Jing JI
Yu-rong WANG
Zhi-gang MA
Gang WANG
Qian LI
Dan YANG
Song-hao ZHANG
Cite this article:

Ting AN,Jing JI,Yu-rong WANG,Zhi-gang MA,Gang WANG,Qian LI,Dan YANG,Song-hao ZHANG. Analysis of the Transformation Efficiency and Induced Differentiation of Lilium brownii Scales. China Biotechnology, 2018, 38(1): 25-31.

URL:

https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20180103     OR     https://manu60.magtech.com.cn/biotech/Y2018/V38/I1/25

NPTII基因引物序列GR基因引物序列
引物名称引物序列引物名称引物序列
NPTII-F5'-GCTATGACTGGGCACAACAG-3'LcGR F5'-TCGTAATAGGTGCTGGAAGTGGT-3'
NPTII-R5'-AAGGAGCACGAAATGCCATA-3'LcGR R5'-ATGTGCTTCGCCGAATAGGTTA-3'
Table 1 Primers of gene sequences
Fig.1 The map of pCAMBIA2300-LcGR vector
编号2,4-D
(mg /L)		NAA
(mg /L)		蔗糖
(g /L)		增殖分
化率(%)
1103030 e
210.16040 de
310.29045 d
4206075 c
520.190100 a
620.23070 c
7306085 bc
830.13075 c
930.29090 ab
Table 2 Effects of different concentrations of 2,4-D, NAA and sucrose on the proliferation of scales
Fig.2 The regeneration process of lily(a) Proliferation and enlargement of lily scale (b) Buds inducement of scales (c) Callus inducement from scales (d) Rooting differentiation
编号6-BA
(mg /L)		NAA
(mg /L)		分化率(%)生长状态
10.50.270 bc浅绿,较弱
20.50.480 b浅绿,较弱
30.50.680 b浅绿,健康
410.295 a深绿,强壮
510.480 b深绿,健康
610.665 c深绿,较慢
71.50.265 c浅绿,健康
81.50.450 d细弱,缓慢
91.50.650 d细弱,缓慢
Table 3 Effects of 6-BA and NAA on adventitious bud formation in scale adventitious directly
编号2,4-D
(mg /L)		TDZ
(mg /L)		蔗糖
(g /L)		分化率(%)
120.13030 f
220.26070 e
320.39089 ab
42.50.46092 a
52.50.69070 e
62.50.23070 e
730.46085 bc
830.23075 de
930.69080 cd
Table 4 Effects of different concentrations of 2,4 - D, TDZ and sucrose on callus induction
Kan浓度(mg /L)鳞片褐化率(%)愈伤组织褐化率(%)
000
2522.426.1
5049.264.3
7568.892.3
10095.7100
125100100
Table 5 Sensitivity tests of antibiotics applied on tissues
序 号农杆菌
OD值		预培养
时间(d)		侵染时
间(min)		As浓度
(μmol/L)		分化率
(%)		最终转化
率(%)
10.41201009.64.69 f
20.433015010.686.52de
30.454020019.29.73 b
40.613015010.687.59cd
50.634020030.2012.9 a
60.652010020.309.21bc
70.813015012.907.67cd
80.832010010.405.84ef
90.854020013.407.91cd
Table 6 Optimization of scale transformation conditions
序号农杆菌
OD值		预培养
时间(d)		侵染时
间(min)		As浓度
(μmol/L)		分化率
(%)		最终转化
率(%)
10.412010012.802.10 d
20.433015031.007.05 bc
30.454020032.0011.6 a
40.613015012.404.58 cd
50.634020025.509.21 bc
60.652010020.208.20 bc
70.81301509.204.06 cd
80.832010011.27.83 cd
90.854020016.308.13 bc
Table 7 Optimization of callus transformation conditions
Fig.3 Trans formation(a) Direct differentiation in translation (b) Indirect differentiation in translation
Fig.4 Electrophoresis of PCR product of NPTII and LcGR genes in the transgenic lily lines
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