Genetic Transformation Of Buckwheat (Fagopyrum Esculentum Moench) With AtNHX1 Gene And Regeneration Of Salt-tolerant Transgenic Plants

Buckwheat is a dicotyledonous crop of the Polygonaceae family. In general, only twospecies of buckwheat, common buckwheat (Fagopyrum esculentum) and tartarybuckwheat (Fagopyrum tartaricum) are used as food around the world. Flavones,flavonoids, sterols, fagopyrin, and thiamin-binding proteins in buckwheat seeds possesspotential effects in treating some chronic diseases. In buckwheat, the main component offlavonoids (80%) is rutin, a flavonol glycoside (quercetin 3-rhamnosylglucoside). Rutincan reduce high blood pressure, decrease the permeability of the blood vessels, reduce therisk of arteriosclerosis, antagonize the increase of capillary fragility associated withhaemorrhagic disease, and have an anti-oedema effect and antioxidant activity.Buckwheat is a salt-sensitive glycophyte cereal crop and the growth and grain yieldsof buckwheat are significantly affected by soil salinity. Therefore, cultivation ofbuckwheat with higher salt tolerance would produce remarkable economic andpharmaceutical benefits. With the development of genetic transformation technology ofmany plant species, a new engineering approach by introduction of a specific gene hasbeen employed to achieve the potentially radical improvement of plant salt tolerance.The purpose of our experiment was to optimize conditions for producing organogeniccalli and regenerating plants from hypocotyls and cotyledon segments, and to develop anefficient transformation protocol that can be used for genetic transformation and otherfundamental studies. A large number of adventitious shoots were induced indirectly fromcalli originated from hypocotyl and cotyledon explants on MS medium supplementedwith 2.0 mg/L 6-benzylaminopurine (BA), 1.0 mg/L kinetin (KT) and 0.1 mg/L indole-3-acetic acid (IAA). The effects of preculture, infection time, extent of cocultivation andaddition of acetosyringone were evaluated by the transformation frequency of explantsinfected with A. tumefaciens strain LBA4404 harboring the plasmid pHZX1. The resultsshowed that AtNHX1-positive transgenic plants could be regenerated effectively from hypocotyl and cotyledon calli cocultured for 1～2 days with Agrobacterium afterpreculture for 2 days and infection for 20～25 minutes. Another factor affectingtransformation efficiency, acetosyringone, did not evidently increase the transformationfrequency. Using optimized transformation system, the Arabidopsis thaliana tonoplastNa~+/H~+ antiporter gene, AtNHX1, was transferred into buckwheat by Agrobacterium-mediated method. Transgenic buckwheat plants were regenerated and selected on MSbasal medium supplemented with 2.0 mg/L 6-BA, 1.0 mg/L kinetin (KT) and 0.1 mg/Lindole-3-acetic acid (IAA), 50 mg/L kanamycin, and 500 mg/L carbenicillin. Thetransformants were confirmed by PCR, RT-PCR, Southern and Northern blotting analysis.After stress treatment for 6 weeks with 200mmol/L NaCl, transgenic plants survived,while wild-type plants did not. After 3 days of stress treatment through differentconcentrations of NaCl, the transgenic plants accumulated higher concentration of Na~+than those of the control plants. Moreover, the rutin content of roots, stems and leaves oftransgenic buckwheat increased in comparison with those of the control plants. Theseresults demonstrated the AtNHX1 gene had been successfully transferred into buckwheatby Agrobacterium-mediated method, and the salt-tolerance of buckwheat was improvedby overexpression of the AtNHX1 gene. At the same time, it also demonstrated thefeasibility of engineering salt-tolerance in plants.