An Agrobacterium-mediated transformation via organogenesis regeneration of a facultative CAM plant, the common ice plant Mesembryanthemum crystallinum L

Sakae Agarie,Makiko Umemoto,Haruki Sunagawa,Toyoaki Anai,John C Cushman

doi:10.1080/1343943x.2020.1730700

Sakae Agarie, Makiko Umemoto + Show 3 more

Open Access

https://doi.org/10.1080/1343943x.2020.1730700

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Abstract

ABSTRACT The common ice plant, Mesembryanthemum crystallinum L. provides a useful model for the study of environmentally induced photosynthetic conversion and abiotic stresses tolerance. However, a procedure for the production of transgenic ice plant, which is essential for functional genomics, has not been fully established. Here we tested the factors on the transformation of cotyledonary nodes excised from the ice plant seedlings such as thidiazuron (TDZ), NaCl and phytosulfokine (PSK), a peptidyl plant growth factor using Agrobacterium tumefaciens strains EHA101 and EHA105 harboring binary vector plasmids pBI7EGFP and pCAMBIA1302, respectively. The established procedure is as follows: the explants (cotyledonary nodes) were co-cultivated with Agrobacterium for 3 days, and the explants were cultured in the medium with 0.5 mg l−1 kinetin and 100 mg l−1 carbenicillin for 72 h, and they were cultured in the medium with 0.5 mg l−1 kinetin and 100 nM PSKfor 4 weeks. Thidiazuron and NaCl enhanced the production of multiple adventitious shoot formation during regeneration but reduced the transformation efficiency due to the vitrification of adventitious shoots. PSK was effective in the production of healthy adventitious shoots. The transformation frequency at the stage of whole plants was 0.6% and 4.6% per inoculated cotyledonary nodes using the Agrobacterium strain EHA101 (pBI7EGFP) and EHA105 (pCAMBIA1302), respectively.

Highlights

The common ice plant, Mesembryanthemum crystallinum L. (Aizoaceae, Caryophyllales), a facultative halophyte, provides a useful model for the study of environmentally inducible switch from C3 photosynthesis to Crassulacean acid metabolism (CAM) and abiotic stresses tolerance (Bohnert & Cushman, 2001)
Similar phenomena were observed in the previous report (Sunagawa et al, 2007) during the regeneration of cotyledonary nodes which had been infected with Agrobacterium
To examine combination effects of plant growth regulators on the induction of healthy shoot formation of transgenic ice plant, the explants of cotyledonary node infected with Agrobacterium strain EHA101 were cultured on MS medium supplement with 1 mg l−1 TDZ, 0.25 mg L−1 1-naphthaleneacetic acid (NAA) (Sunagawa et al, 2007), and 150 mg L−1 carbenicillin for 1 week and transferred onto the media supplement with TDZ, 6-benzylaminopurine (BAP), NAA and gibberellic acid (GA3) without antibiotics (Table 1)

Summary

Introduction

The common ice plant, Mesembryanthemum crystallinum L. (Aizoaceae, Caryophyllales), a facultative halophyte, provides a useful model for the study of environmentally inducible switch from C3 photosynthesis to Crassulacean acid metabolism (CAM) and abiotic stresses tolerance (Bohnert & Cushman, 2001). We have developed a transformation procedure using Agrobacterium-mediated transformation of cotyledonary node explants coupled with the regeneration of transformed plants via direct organogenesis from cotyledonary node (Sunagawa, Agarie, Umemoto, Makishi & Nose, 2007). The transformation efficiency obtained in the present study was higher than that of reports published previously on the production of transgenic ice plant (whole plant) (Sunagawa et al, 2007). It has been achieved by infection with Agrobacterium strain EHA105 harboring a binary vector pCAMBIA. The PSK was effective in shortening the period of adventitious shoot formation and enhanced the production of healthy adventitious shoots

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