Induction, preservation, propagation and cytological observation of proembryonic masses of grapevine

Abstract: 【Objective】Grapevine (Vitis vinifera L.) is one of the world's most important fruit crops because of its high yielding and value. However, it is susceptible to pathogens. Genetic engineering is a powerful tool for plant improvement and has the potential to allow the integration of desirable disease-resistance genes into existing genomes. Successful application of gene technology to grapevine requires an efficient regeneration system. Somatic embryogenesis is the most commonly adopted method in genetic engineering research in grapes. Flowering in grapes occurs only once a year, which limits the opportunity for induction and transformation. Therefore it is important to produce regenerative embryogenic tissues and maintain them over a long period. In previous reports, proembryonic masses (PEM) were used in genetic transformation and gene editing. Therefore, it is necessary to induce, preserve, propagate and cytologically understand proembryonic masses.【Methods】In this study, Vitis vinifera L.‘Thompson Seedless', ‘Semillon', ‘Riesling', ‘Shiraz'and Vitis yeshanensis J. X. Chen accession‘Yanshan-1'were used to induce somatic embryos (SE) . To obtain higher induction efficiency, the optimum sampling period was determined according to microscopic observation. The pistils, stamens and small buds from grapevine were used to induce somatic embryos on three embryogenic culture initiation mediums. A secondary embryogenesis system was utilized to obtain more proembryonic masses. Induced callus from different explants were observed under stereoscopic microscope. Embryonic callus (EC) and non-embryonic callus were also observed under stereoscopic microscope. Cells of embryonic callus and non-embryonic callus werestained with improved carbolfuchsin solution and observed under microscope. In secondary embryogenesis, primary proembryonic masses, primary somatic embryos, secondary embryonic callus and secondary proembryonic masses were observed, which were stained with improved carbolfuchsin solution and observed under microscope. The secondary proembryonic masses were cultured on X3 medium containing Murashige and Skoog's basal salts and 30 g·L^-1 sucrose. The stages of secondary somatic embryos were observed under stereoscopic microscope. Regeneration of the secondary proembryonic masses was studied.【Results】The floral organs were collected when the microspores were in uninucleate stage and cultured on three embryogenic culture initiation mediums. 20 days after stamens were inoculated in embryogenic culture initiation medium, calli were induced from filament tip or connective tissue. 10 days after pistils were inoculated in embryogenic culture initiation medium, calli were induced from pistil connective tissue or ovary wall. After small buds had been cultured in embryogenic culture initiation medium for 7 days, calli were induced from the flower buds. V. vinifera L.‘Shiraz'and V. yeshanensis J. X. Chen‘Yanshan-1'failed to produce somatic embryos from the stamens, pistils and small flower buds on any of the three embryogenic culture initiation mediums. Somatic embryos were successfully initiated from stamens of V. vinifera L.‘Thompson Seedless', ‘Riesling'and‘Semillon'.‘Pistils' and small flower buds of‘Thompson Seedless', ‘Riesling'and‘Semillon'failed to produce embryogenic callus. Highest frequency of embryogenic calli from the stamens of‘Thompson Seedless', ‘Riesling'and‘Semillon'was obtained on PIV medium containing Murashige and Skoog's basal salts, 60 g·L^-1 sucrose, 2.0 mg·L^-1 6-BA and 1.0mg·L^-1 2, 4-D. PIV media were supplemented with 1 g·L^-1 myo-inositol and 0.3 g·L^-1KNO3. The frequency of somatic embryos of‘Thompson Seedless', ‘Riesling', and‘Semillon'was 10.8%, 1.8% and2%, respectively. Induced calli had two types, the embryogenic and the non-embryogenic. Embryogenic calli were yellowish, hard and slow-growing and could produce pro-embryonic masses on X3 medium containing Murashige and Skoog's basal salts and 30 g·L^-1sucrose. Pro-embryonic masses readily developed into somatic embryos. Embryonic calli were composed of cells that were dense, deeply stained and with division activity. Non-embryogenic calli were loose, water-soaked and couldn't produce proembryonic masses or somatic embryos on X3 medium. They were composed of non-embryonic cells that were of different sizes, lightly stained and vacuolized. Cream-colored primary proembryogenic masses were composed of differentiated cells with large nuclei, small cell volume and strong ability of division. These cells were nearly elliptical with thick cytoplasm and deeply stained. The secondary embryogenic callus consisted of cells with sparse cytoplasm, irregular shape, large size and also strong ability of division. The secondary proembryonic masses could develop normally on X3 medium containing Murashige and Skoo's basal salts and 30 g·L^-1 sucrose. After the secondary proembryonic masses had been cultured on X3 medium for one month, they produced global embryo, heart-shaped embryo, torpedo-shaped embryo and cotyledonary embryo. Cotyledonary embryos developed into normal seedlings on GM medium containing Murashige and Skoo's basal salts and 15 g·L^-1 sucrose and on rooting medium containing Skoo's basal salts, 30 g·L^-1 sucrose and 1.0 mg·L^-1 IBA.【Conclusion】The induction of somatic embryos from grape floral parts is influenced by genotype and explants type. The circulatory secondary embryogenesis systems can be used for sustainable induction and long-term preservation and propagation of proembryonic masses. The secondary pro-embryonic masses have differentiation ability and can develop into normal seedlings.