Establishment of Agrobacterium rhizogenes-mediated Genetic Transformation System of Eucalyptus urophylla × E. grandis

doi:10.7525/j.issn.1673-5102.2022.03.021

Abstract

Abstract:

Agrobacterium rhizogenes-mediated genetic transformation systemplays an important role in the identification of functional genes. To establish Agrobacterium rhizogenes-mediated genetic transformation system in Eucalyptus， the different strains infected the leaves and stems of Eucalyptus urophylla × E. grandis were used to select the appropriate A. rhizogenes strains and explant types， and the effects of Agrobacterium concentration and infection time on hairy root induction were explored. The results showed that MSU440 was the optimal A. rhizogenes strain and leaves was the preferred explants for hairy root induction. The highest hairy root induction rate reached 81.0% with the average root length of hairy roots was 3.23 cm. The leaves were infected by MSU440 which grown to a bacterial density of 0.3（OD₆₀₀） for 30 min， then they were co-cultured for 48h and transferred the selection medium supplemented with 20 mg·L^-1 Kanamycin. The exogenous genes were stably integrated in the genome of Eucalyptus hair roots which confirmed by PCR analyses and GUS staining， and the transformation efficiency reached 20.2%. In our study， an A. rhizogenes-mediated genetic transformation system of Eucalyptus was preliminarily established， which laid a foundation for gene function identification and further transgenic breeding of Eucalyptus.

Key words: Eucalyptus, Agrobacterium rhizogenes, hairy root, transgene, transformation

CLC Number:

S184

Ping LUO, Haonan ZHANG, Jianmin XU, Bing HU, Xiaoping WANG, Guangyou LI, Chunjie FAN. Establishment of Agrobacterium rhizogenes-mediated Genetic Transformation System of Eucalyptus urophylla × E. grandis[J]. Bulletin of Botanical Research, 2022, 42(3): 512-520.

Figures/Tables 9

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菌株 Agrobacterium strain	冠瘿碱类型 Crown gallin type	抗性 Resistance
ArA4	农杆碱型 Agropine	卡那霉素 Kanamycin
MSU440	农杆碱型 Agropine	链霉素 Streptomycin
Ar1193	农杆碱型 Agropine	链霉素 Streptomycin
ArQual	农杆碱型 Agropine	链霉素 Streptomycin
K599	黄瓜碱型 Cucumopine	链霉素 Streptomycin

引物名称 Primer name	引物序列 Primer sequence（5′—3′）	产物长度 Product length /bp
rola-F	AGCGTTCGGACAGCCACCA	519
rola-R	GGCGTGGAAATGAATCGAAGAC
rolb-F	GCTCTTGCAGTGCTAGATTT	423
rolb-R	GAAGGTGCAAGCTACCTCTC
GUS-F	ACTCAGCAAGCGCACTTACAGG	496
GUS-R	TCCATACCTGTTCACCGACGAC
NPTⅡ-F	ATTGAACAAGATGGATTGCACGCA	742
NPTⅡ-R	GAGCGGCGATACCGTAAAGCA

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