Chin Bull Bot ›› 2016, Vol. 51 ›› Issue (4): 533-541.doi: 10.11983/CBB15087

• TECHNIQUES AND METHODS • Previous Articles     Next Articles

Introduction of Exogenous DNA of Populus alba into Sect. Aigeiros via Pollen-tube Pathway

Xinwen Zhao*   

  1. Research Institute of Poplar of Liaoning Province, Gaizhou 115200, China
  • Received:2015-05-17 Accepted:2015-09-04 Online:2016-08-05 Published:2016-07-01
  • Contact: Zhao Xinwen E-mail:zhaoxinwen2001@163.com
  • About author:

    # Co-first authors

Abstract:

Sect. Aigeiros is fast-growing and practical and has strong agamogenesis ability. Sect. Leuce has good quality and strong resistance. Many economic traits between them are complementary, so Sect. Aigeiros and Leuce hybrid advantages in polymerization can be obtained from a superior hybrid. However, there are natural incompatibility barriers between Sect. Aigeiros and Leuce. In this study, we introduced the genotype of P. alba into P. × Liaoninggensis sp. nov × P. deltoids cv. ‘N001’ by the pollen-tube pathway to overcome this difficulty. Four of the offspring were similar to P. alba by analysis of phenotype characteristics. From the molecular identification of AFLP, the 4 variant plants were determined to be positive transgenic plants from the genotype of P. alba. Meanwhile, the transformation system of the pollen-tube pathway for Sect. Aigeiros was optimized: the optimal treatment time was 30-72 h, optimal treatment method the uncut stigma method, and optimal treatment 200 μg·mL-1.

Table 1

Adaptors and selective primers used in AFLP analysis"

The adaptors and primers The nucleotide sequence of the adaptors and primers (5'→ 3')
PstI adaptors 1 CTCGTAGACTGCGTACATGCA
PstI adaptors 2 TGTACGCAGTCTAC
MseI adaptors 1 GACGATGAGTCCTGAG
MseI adaptors 2 TACTCAGGACTCAT
PstI GACTGCGTACATGCAG
MseI GATGAGTCCTGAGTAAC
PstI-1/MseI-2 GACTGCGTACATGCAGAA/GATGAGTCCTGAGTAACAC
PstI-1/MseI-7 GACTGCGTACATGCAGAA/GATGAGTCCTGAGTAACTG
PstI-2/MseI-1 GACTGCGTACATGCAGAC/GATGAGTCCTGAGTAACAA
PstI-2/MseI-2 GACTGCGTACATGCAGAC/GATGAGTCCTGAGTAACAC
PstI-2/MseI-3 GACTGCGTACATGCAGAC/GATGAGTCCTGAGTAACAG
PstI-3/MseI-2 GACTGCGTACATGCAGAG/GATGAGTCCTGAGTAACAC
PstI-3/MseI-3 GACTGCGTACATGCAGAG/GATGAGTCCTGAGTAACAG
PstI-3/MseI-7 GACTGCGTACATGCAGAG/GATGAGTCCTGAGTAACTG

Figure 1

Introduction of exogenous DNA of Populus alba into Sect. Aigeiros (A) Stigma uncut inflorescence after pollination; (B) Stigma cut inflorescence after pollination; (C) Introduction of exogenous DNA to the stigma uncut treatments; (D) Introduction of exogenous DNA to the stigma cut treatments"

Table 2

The effects of different treatment methods and different time on the introduction of exogenous DNA of Populus alba"

Code Treatment method Treatment time (h) The No. of seeds The No. of plants Survival rate (%) Variant plants
H1 Stigma uncut method 24 175 80 45.7 0
H2 Stigma uncut method 30 6 6 100 1
H3 Stigma uncut method 36 31 31 100 0
H4 Stigma uncut method 42 679 420 61.8 0
H5 Stigma uncut method 48 173 70 40.5 0
H6 Stigma uncut method 60 792 540 68.2 2
H7 Stigma uncut method 72 129 50 38.8 0
H8 Cut-stigma method 24 3 3 100 0
H9 Cut-stigma method 30 0 0 - 0
H10 Cut-stigma method 36 62 52 83.9 0
H11 Cut-stigma method 42 342 125 36.5 0
H12 Cut-stigma method 48 0 0 - 0
H13 Cut-stigma method 60 9 9 100 0
H14 Cut-stigma method 72 3 3 100 1
CK CK 839 180 21.5 0

Figure 2

The variant plants found in the D1 generation (A) CK (acceptor); (B)-(E) The variant plants found in H2 (B)、H6 (C)、H6 (D) and H14 (E) treatments, respectively"

Figure 3

AFLP electrophoretogram for parents, variant pl- ant and donor P1: Father; P2: Mother; CK: Plant CK (acceptor); No.1: Variant plant 1; No.2: Variant plant 2; No.3: Variant plant 3; No.4: Variant plant 4; M: Marker"

Figure 4

UPGMA dendrogram for variant plants, parents and donor based on AFLP analysis P1, P2, CK, No.1, No.2, No.3 and No.4 see Figure 3."

Table 3

The genetic similarity of variant plants, parents and donor"

P1 P2 Donor CK No.1 No.2 No.3 No.4
P1 1.00 - - - - - - -
P2 0.89 1.00 - - - - - -
Donor 0.76 0.77 1.00 - - - - -
CK 0.86 0.86 0.79 1.00 - - - -
No.1 0.76 0.76 0.84 0.78 1.00 - - -
No.2 0.75 0.75 0.82 0.78 0.83 1.00 - -
No.3 0.77 0.77 0.84 0.79 0.86 0.83 1.00 -
No.4 0.77 0.78 0.81 0.81 0.81 0.81 0.84 1.00
1 陈春洪, 王志坚, 孙玲, 操君喜, 赖来展 (1998). 用花粉管通道将外源DNA导入水稻的研究. 广东农业科学 4, 2-3.
2 陈洪伟 (2008). 白杨花粉管通道导入外源胡杨DNA技术研究. 博士论文. 北京: 北京林业大学. pp. 2-16.
3 邓德旺, 郭三堆, 杨志民 (1999). 棉花花粉管通道法转基因的分子细胞学机理研究. 云南大学学报(自然科学版) 21, 124-125.
4 郭三堆, 崔洪志, 夏兰芹, 武东亮, 倪万潮, 张震林, 张保龙, 徐英俊 (1999). 双价抗虫转基因棉花研究. 中国农业科学 32, 1-7.
5 侯立群, 李秀芬, 崔刚, 李际华, 乔玉玲, 王露琴 (2004). 几种遗传转化技术在核桃转基因育种中的应用. 山东林业科技 1, 8-9.
6 侯文胜, 郭三堆, 路明 (2003). 利用花粉管通道法获得转雪花莲凝集素基因(sgna)小麦. 植物学通报 20, 198-204.
7 李乃坚, 袁四清, 蒲汉丽, 周会光, 戴冕, 陈俊标, 罗战勇, 崔植琳, 黄自然, 李传瑛, 陈凤珍 (1998). 抗菌肽B基因转化烟草及转基因植株抗青枯病的鉴定. 农业生物技术学报 6, 178-184.
8 刘德璞, 廖林, 袁鹰, 刘玉芝 (1997). 导入外源DNA获得抗SMV大豆品系. 大豆科学 16, 277-282.
9 马庆国, 齐静, 裴东 (2010). 16个早实核桃良种遗传多样性的FISH-AFLP分析. 林业科学研究 23, 631-636.
10 庞金宣, 郑世锴, 刘国兴, 李际红, 张友朋 (2001). 窄冠型杨树新品种的选育. 林业科技通讯 4, 8-9.
11 王芙蓉, 张传云, 刘国栋, 刘任重, 刘勤红, 张军 (2006). 利用花粉管通道技术创造棉花变异种质及其SSR标记分析. 山东农业科学 1, 10-12.
12 王景雪, 孙毅, 崔贵梅, 胡晶晶 (2001). 花粉介导法获得玉米转基因植株. 植物学报 43, 275-279.
13 王永锋, 栾雨时, 高晓蓉 (2004). 花粉管通道法在植物转基因中的研究与应用. 东北农业大学学报 35, 764-768.
14 吴鸿锦, 刘志光, 韩克展, 许鹏 (1996). 新杂交种沙毛杨的选育. 北京林业大学学报 18, 48-54.
15 杨成超 (2005). 银白杨× 白榆亲子鉴定及子代利用研究. 博士论文. 北京: 中国林业科学研究院. pp. 28-41.
16 张金凤, 朱之悌, 张志毅, 杜宁霞, 张力刚 (2000). 中介亲本在黑白杨派间杂交中的应用. 北京林业大学学报 22, 35-38.
17 张文元 (2006). SOS1和SOS2基因在杨树上的遗传转化及其功能的初步验证. 博士论文. 北京: 中国林业科学研究院. pp. 20-29.
18 钟启宏, 孔繁瑞, 孙威, 李继耕, 熊耀国, 赵丹宁, 张维栋, 李峰 (1994). 通过花粉管途径建立泡桐转化体系的初探. 遗传 16, 16-19.
19 左开井, 张献龙, 聂以春, 刘金兰, 孙济中 (2002). 转基因抗虫棉转Bt基因插入区碱基组成分析. 遗传学报 29, 735-740.
20 Franco JA (1993). Populus. Flora Europaea. Cambridge: University Press. pp. 64-66.
21 Linaeus C (1753). Populus. Linn. Sp. Pl. London: Bernard Quaritch Ltd. pp. 1034-1035.
22 Ma QG, Zhang JP, Pei D (2011). Genetic analysis of walnut cultivarsin China using fluorescent amplified fragment length polymorphism.J Amer Soc Hort Sci 136, 422-428.
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