农杆菌介导的草莓转化法

Jan G. Schaart

doi:10.21769/BioProtoc.1022

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Peer-reviewed

Agrobacterium-mediated Transformation of Strawberry

农杆菌介导的草莓转化法

JS Jan G. Schaart email

发布: 2014年01月05日第4卷第1期 DOI: 10.21769/BioProtoc.1022 浏览次数: 14201

评审: Tie Liu

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Cover of New Phytologist, featuring study using the protocol.

Jan 2013

Special Issue on Protocols for Organoid Research

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Abstract

Traditional breeding for improvement of strawberry (Fragaria x ananassa) is difficult because strawberry is an octoploid, hybrid species. Genetic modification of strawberry would though be a promising alternative for obtaining the desired improvements in existing elite strawberry cultivars (Schaart et al., 2011). The availability of suitable genes for trait improvements in strawberry has however been a rate-limiting step until recently, but with the completion of the sequencing of the genome of woodland strawberry (F. vesca) (Shualev et al., 2011), we now have access to a treasure chest with valuable candidate genes. For strawberry, methods for genetic transformation have originally been described by Nehra et al. (1990) and James et al. (1990) and success of transformation was shown to be highly cultivar dependent. The latest progress in strawberry transformation is reviewed by Husaini et al. (2011). In our lab transformation of strawberry is based on the method for shoot regeneration described by Passey et al. (2003) and the use of the supervirulent Agrobacterium strain AGL0 (Lazo et al., 1991). We mainly make use of the strawberry transformation as a tool for functional analysis of candidate genes. For this the cultivar Calypso is a very suitable genotype because of its high transformation efficiencies (up to 100%) and ever-bearing fruiting characteristic, which provides a continuous supply of strawberry fruits once the plants start flowering.

Keywords: Strawberry (草莓)

Agrobacterium (农杆菌)

Transformation (转化)

Genetic modification (遗传修饰)

Materials and Reagents

Sucrose (Duchefa Biochemie BV, catalog number: S0809 )
Daishin agar (Duchefa Biochemie BV, catalog number: D1004 )
6-Benzylaminopurine (BAP) (Duchefa Biochemie BV, catalog number: B0904 ) Indole-3-butyric acid (IBA) (Duchefa Biochemie BV, catalog number: I0902 )
Murashige and Skoog salts including vitamins (MS) (Duchefa Biochemie BV, catalog number: M0221 )
Gelrite (Duchefa Biochemie BV, catalog number: G1101 )
3’,5’Dimethoxy-4’-hydroxy-acetophenone (Acetosyringone) (Sigma-Aldrich, catalog number: D134406 )
LB broth (LB) (Sigma-Aldrich, catalog number: L3022 )
Rifampicin (Duchefa Biochemie BV, catalog number: R0146 )
Dimethyl sulfoxide (DMSO) (Merck KGaA, catalog number: 802912 )
MN615 filter paper 82 mm (MACHEREY-NAGEL GmbH & Co. KG, catalog number: MN615)
Thidiazuron (TDZ) (Duchefa Biochemie BV, catalog number: T0916 )
1-Naphthalene acetic acid (NAA) (Duchefa Biochemie BV, catalog number: N0903 )
Cefotaxime sodium salt (cefotaxime) (Duchefa Biochemie BV, catalog number: C0111 )
Kanamycin monosulphate monohydrate (kanamycin) (Duchefa Biochemie BV, catalog number: K0126 )
D-glucose monohydrate (glucose) (Duchefa Biochemie BV, catalog number: G0802 )
Shoot multiplication medium (SMM) (see Recipes)
Shoot propagation medium (SPM) (see Recipes)
Shoot regeneration medium (SRM) (gluc.) (see Recipes)
SRM + AS (see Recipes)
SRM SEL (see Recipes)
MS-liquid + AS (see Recipes)
1 mg/ml NAA (see Recipes)
0.22 mg/ml TDZ (see Recipes)
1 mg/ml IBA (see Recipes)
1 mg/ml BAP (see Recipes)
100 mM AS (see Recipes)
125 mM Cefotaxime (see Recipes)
50 mg/ml Kanamycin (see Recipes)
50 mg/ml Rifampicin (see Recipes)
30 g/100 ml Glucose (see Recipes)