Optimizing Initiation and Maintenance of Vitis Embryogenic Cultures

Plant Cell Tiss Organ Cult

Gray

2010

Unopened leaves, petioles and fully opened leaves from micropropagation cultures of five Vitis rotundifolia Michx. varieties were cultured on induction medium to study their embryogenic response. Among the various explants tested, the maximum number of varieties produced embryogenic cultures from unopened leaves followed by fully opened leaves and petioles. Based on morphological differences, two types of embryogenic cultures were identified. Friable cultures typically arose as proembryonic masses (PEM) on induction medium, whereas somatic embryo production without an intervening PEM stage was observed in compact cultures. Of the five varieties tested, the highest frequency of embryogenic response was observed from fully opened leaves of 'Supreme' and unopened leaves and petioles of 'Delicious'. Attempts to initiate suspension cultures from varieties resulted in proliferation and maintenance of 'Alachua' and 'Carlos' cultures in liquid medium for 16 weeks. Embryogenic potential of varieties was studied on cultures growing on embryo development medium. The maximum number of cotyledonary stage somatic embryos from 0.2 g proembryonic masses were observed in 'Carlos' (379.3) followed by 'Alachua' (350.0) and 'Delicious' (305.0). Cotyledonary stage somatic embryos germinated when cultured on Murashige and Skoog medium containing 1 lM Benzyladenine (BA). Although high embryo germination rates (80-100%) were observed in the varieties tested, plant recovery from germinated somatic embryos ranged from 6-47%. Embryogenic cultures could be maintained on X6 medium and used in genetic engineering studies.

Section: Induction Of Embryogenic Culturessupporting

confidence: 80%

Section: Maintenance Of Embryogenic Culturesmentioning

confidence: 99%

Section: Maintenance Of Embryogenic Culturesmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 96%

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Factors influencing induction and maintenance of Vitis rotundifolia Michx. embryogenic cultures

Plant Cell Tiss Organ Cult

Gray

2010

“…The development of fungal disease-resistant transgenic grapevines that would greatly reduce or eliminate the need for fungicide sprays was envisioned at a time when genetic transformation and regeneration of this recalcitrant woody species was deemed difficult (Walker 1996). Recent advances in grapevine genetic engineering now make it possible to insert an array of genes for Vitis crop improvement (Dhekney et al , 2009a(Dhekney et al , b, 2010Li et al 2006Li et al , 2008.…”

Section: Introductionmentioning

confidence: 99%

Grapevines engineered to express cisgenic Vitis vinifera thaumatin-like protein exhibit fungal disease resistance

In Vitro Cell.Dev.Biol.-Plant

Gray

2011

Self Cite

Cisgenic engineering involves isolation and modification of genetic elements from the host genome, which are reinserted to develop plant varieties with improved characteristics. As a first step toward production of fungal-disease resistant cisgenic grapevines, the Vitis vinifera thaumatin-like protein (vvtl-1) gene was isolated from "Chardonnay" and reengineered for constitutive expression. Embryogenic cultures of "Thompson Seedless" were initiated from leaves and transformed with Agrobacterium to regenerate cisgenic VVTL-1 plants. Cisgene presence and copy number were confirmed by PCR and quantitative real-time PCR. Protein expression was measured using ELISA. Among the plant lines tested, two exhibited a 7-10 day delay in powdery mildew disease development during greenhouse screening and decreased severity of black rot disease in field tests. Berries exhibited a 42.5% reduction in sour-bunch rot disease incidence compared to non-transformed controls after 3 wk of storage at room temperature. Although plants recovered in this study contain viral promoters and reporter/ marker genes, this is the first report of a cisgenic approach to obtain broad-spectrum fungal-disease resistance in genetically engineered grapevine.

Somatic Embryogenesis and Genetic Modification of Vitis

In Vitro Embryogenesis in Higher Plants

Grant

et al. 2016

Grapevine embryogenic cultures are ideal target tissues for inserting desired traits of interest and improving existing cultivars via precision breeding (PB). PB is a new approach that, like conventional breeding, utilizes only DNA fragments obtained from sexually compatible grapevine plants. Embryogenic culture induction occurs by placing leaves or stamens and pistils on induction medium with a dark/light photoperiod cycle for 12-16 weeks. Resulting cultures produce sectors of embryogenic and non-embryogenic callus, which can be identified on the basis of callus morphology and color. Somatic embryo development occurs following transfer of embryogenic callus to development medium and cultures can be maintained for extended periods of time by transfer of the proliferating proembryonic masses to fresh medium at 4-6-week intervals. To demonstrate plant recovery via PB, somatic embryos at the mid-cotyledonary stage are cocultivated with Agrobacterium containing the desired gene of interest along with a, non-PB, enhanced green fluorescent protein/neomycin phosphotransferase II (egfp/nptII) fusion gene. Modified cultures are grown on proliferation and development medium to produce uniformly modified somatic embryos via secondary embryogenesis. Modified embryos identified on the basis of green fluorescence and kanamycin resistance are transferred to germination medium for plant development. The resulting plants are considered to prototype examples of the PB approach, since they contain egfp/nptII, a non-grapevine-derived fusion gene. Uniform green fluorescent protein (GFP) fluorescence can be observed in all tissues of regenerated plants.