Adventitious Shoot Regeneration and Agrobacterium tumefaciens-mediated Transient Transformation of Almond × Peach Hybrid Rootstock ‘Hansen 536’

in HortScience

‘Hansen 536’ (Prunus dulcis × Prunus persica) is an important commercial rootstock for peach and almond. However, susceptibility to wet soil and bacterial canker has limited its use primarily to areas with less annual rainfall. Genetic engineering techniques offer an attractive approach to improve effectively the current problems with this cultivar. To develop an efficient shoot regeneration system from leaf explants, 10 culture media containing Murashige and Skoog (MS) or woody plant medium (WPM) supplemented with different plant growth regulators were evaluated, and adventitious shoot regeneration occurred at frequencies ranging from 0% to 36.1%. Optimal regeneration with a frequency of 32.3% to 36.1% occurred with WPM medium containing 8.88 µm 6-benzylamino-purine (BAP) and 0.98 to 3.94 µm indole-3-butyric acid (IBA). The regenerated shoots had a high rooting ability, and 80% of the in vitro shoots tested rooted and survived after being transplanted to substrate directly. Transient transformation showed an efficient delivery of the β-glucuronidase (GUS) reporter gene (gusA) using all three Agrobacterium tumefaciens strains tested with a concentration of OD600 0.5 to 1.0 for 4 days of cocultivation. The protocols described provide a foundation for further studies to improve shoot regeneration and stable transformation of the important peach and almond rootstock ‘Hansen 536’.

Contributor Notes

This work was supported by Michigan State University under Grant of Project GREEEN (Generating Research and Extension to meet Economic and Environmental Needs), Michigan State University, AgBioResearch; and the National Natural Science Foundation of China (no. 31601732).

Corresponding author. E-mail: songg@msu.edu.

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    Adventitious shoot regeneration from leaf explants for peach rootstock ‘Hansen 536’. (A) Callus regenerated from the leaf explant media after 2 weeks of dark cultivation. (B) Adventitious shoots produced from leaf explants, which are ready to be transferred to the shoot proliferation medium. (C) Root formation on a regenerated shoot. (D) Rooted plants transferred to 4-inch pots.

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    Effects of Agrobacterium strains, concentration of cell suspensions, and duration of cocultivation on the transient expression of the gusA gene. Leaf explants were infected by soaking them in three Agrobacterium strain suspensions (EHA105, GV3101, and LBA4404), each with three concentrations (OD600 = 0.1, 0.5, 1.0) for 5 min at room temperature (25 °C) in the dark. Transient expression of the GusA gene was analyzed after 2 and 4 d of cocultivation separately.

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