Regeneration system for grape and uses thereof

Abstract

The invention features methods of producing a plant or a mature somatic embryo from an embryogenic cell or embryogenic culture. The method includes the steps of: (a) providing a liquid culture that includes an embryogenic cell; (b) recovering embryogenic cell from the culture; (c) transferring the embryogenic cell to a second culture; and (d) growing a mature somatic grape embryo from the embryogenic cell.

We claim:  
     
         1 . A method of growing a somatic grape embryo, said method comprising the steps of: 
 (a) providing a liquid culture of grape explant comprising an embryogenic cell or an embyrogenic cell mass;    (b) visually identifying an embryogenic cell or embryogenic cell mass from the liquid culture;    (c) recovering said embryogenic cell or embryogenic cell mass from the liquid culture;    (d) transferring said embryogenic cell or embryogenic cell mass to a second culture; and    (e) growing a somatic grape embryo from said embryogenic cell or embryogenic cell mass.    
     
     
         2 . The method of  claim 1 , wherein said grape explant is obtained from anthers, ovaries, ovules, floral tissue, vegetative tissue, tendrils, leaves, roots, nucellar tissue, stems, seeds, protoplasts, pericycle, apical meristem tissue, embryogenic tissue, somatic embryos, or zygotic embryos.  
     
     
         3 . The method of  claim 1 , wherein said grape explant is selected from the group consisting of Alden, Almeria, Anab-E-Shahi, Autumn Black, Beauty Seedless, Black Corinth, Black Damascus, Black Malvoisie, Black Prince, Blackrose, Bronx Seedless, Burgrave, Calmeria, Campbell Early, Canner, Cardinal, Catawba, Christmas, Concord, Dattier, Delight, Diamond, Dizmar, Duchess, Early Muscat, Emerald Seedless, Emperor, Exotic, Ferdinand de Lesseps, Fiesta, Flame seedless, Flame Tokay, Gasconade, Gold, Himrod, Hunisa, Hussiene, Isabella, Italia, July Muscat, Khandahar, Katta, Kourgane, Kishmishi, Loose Perlette, Malaga, Monukka, Muscat of Alexandria, Muscat Flame, Muscat Hamburg, New York Muscat, Niabell, Niagara, Olivette blanche, Ontario, Pierce, Queen, Red Malaga, Ribier, Rish Baba, Romulus, Ruby Seedless, Schuyler, Seneca, Suavis (IP 365), Thompson seedless, Thomuscat, Aleatico, Alicante Bouschet, Aligote, Alvarelhao, Aramon, Baco blanc (22A), Burger, Cabernet franc, Cabernet Sauvignon, Calzin, Carignane, Charbono, Chardonnay CH 01, Chardonnay CH 02, Chardonnay CH Dijon, Chasselas dore, Chenin blanc, Clairette blanche, Early Burgundy, Emerald Riesling, Feher Szagos, Fernao Pires, Flora, French Colombard, Fresia, Furmint, Gamay, Gewurztraminer, Grand noir, Gray Riesling, Green Hungarian, Green Veltliner, Grenache, Grillo, Helena, Inzolia, Lagrein, Lambrusco de Salamino, Malbec, Malvasia bianca, Mataro, Melon, Merlot, Meunier, Mission, Montua de Pilas, Muscadelle du Bordelais, Muscat blanc, Muscat Ottonel, Muscat Saint-Vallier, Nebbiolo, Nebbiolo fino, Nebbiolo Lampia, Orange Muscat, Palomino, Pedro Ximenes, Petit Bouschet, Petite Sirah, Peverella, Pinot noir, Pinot Saint-George, Primitivo di Gioa, Red Veltliner, Refosco, Rkatsiteli, Royalty, Rubired, Ruby Cabernet, Saint-Emilion, Saint Macaire, Salvador, Sangiovese, Sauvignon blanc, Sauvignon gris, Sauvignon vert, Scarlet, Seibel 5279, Seibel 9110, Seibel 13053, Semillon, Servant, Shiraz, Souzao, Sultana Crimson, Sylvaner, Tannat, Teroldico, Tinta Madeira, Tinto cao, Touriga, Traminer, Trebbiano Toscano, Trousseau, Valdepenas, Viognier, Walschriesling, White Riesling, Zinfandel, Couderc 1202, Couderc 1613, Couderc 1616, Couderc 3309 (Vitis riparia X rupestris), Dog Ridge, Foex 33 EM, Freedom, Ganzin 1 (A x R #1), Harmony, Kober 5BB, LN33, Millardet & de Grasset 41B ( Vitis vinifera  X  berlandieri ), Millardet & de Grasset 420A, Millardet & de Grasset 101-14 ( Vitis riparia  X  rupestris ), Oppenheim 4 (S04), Paulsen 775, Paulsen 1045, Paulsen 1103, Richter 99, Richter 110, Riparia Gloire, Ruggeri 225, Saint-George, Salt Creek, Teleki 5A,  Vitis rupestris Constantia, Vitis california , and  Vitis girdiana, Vitis rotundifolia, Vitis rotundifolia  Carlos, Teleki 5C ( Vitis berlandieri  X  riparia ), 5BB Teleki (selection Kober,  Vitis berlandieri  X  riparia ), S04 ( Vitis berlandieri  X  rupestris ), and 039-16 ( Vitis vinifera  X  Muscadinia ).  
     
     
         4 . The method of  claim 1 , wherein said liquid culture comprises B-5 major salts and MS minor salts.  
     
     
         5 . The method of  claim 1 , wherein said embryogenic cell or embryogenic cell mass is white to pale yellow in color.  
     
     
         6 . The method of  claim 1 , wherein recovering said embryogenic cell or said embryogenic cell mass from said culture comprises filtration, sedimentation, or selection.  
     
     
         7 . The method of  claim 1 , wherein said liquid culture is sieved to synchronize the formation of differentiated somatic grape embryos.  
     
     
         8 . The method of  claim 1 , wherein said second culture comprises a liquid culture.  
     
     
         9 . The method of  claim 1 , wherein said second culture comprises a solid medium.  
     
     
         10 . The method of  claim 1 , wherein said second culture comprises a plant growth regulator.  
     
     
         11 . The method of  claim 10 , wherein said plant growth regulator is an auxin, NOA, dicamba, picloram, NAA, IAA, 2,4-D, a cytokinin, benzyladenine, zeatin, thidiazuron, abscisic acid, or gibberellic acid.  
     
     
         12 . The method of  claim 1 , further comprising growing a plantlet from the somatic grape embryo of step (e).  
     
     
         13 . The method of  claim 1 , wherein said embryogenic cell or embryogenic cell mass is transformed with DNA.  
     
     
         14 . The method of  claim 1 , wherein said somatic grape embryo of step (e) is transformed with DNA.  
     
     
         15 . A mature somatic grape embryo produced by subculturing a somatic grape embryo of  claim 1  on a solid culture medium.  
     
     
         16 . A plantlet germinated from the mature somatic grape embryo of  claim 15 .  
     
     
         17 . A method for long-term storage of a mature somatic grape embryo, said method comprising drying said embryo and storing said embryo at a temperature less than 8° C.  
     
     
         18 . A method for direct seeding of a somatic grape embryo, comprising placing said somatic grape embryo in potting medium comprising sand and potting mixture.  
     
     
         19 . A method of growing a mature somatic grape embryo, said method comprising the steps of: 
 (a) providing a liquid culture of grape explant comprising an embryogenic cell or an embryogenic cell mass;    (b) visually identifying said embryogenic cell or embryogenic cell mass from the liquid culture;    (c) recovering said embryogenic cell or embryogenic cell mass from the liquid culture;    (d) transferring said embryogenic cell or embryogenic cell mass recovered from step (c) to a second culture; and    (e) growing a mature somatic grape embryo from said embryogenic cell or embryogenic cell mass of step (d).    
     
     
         20 . A method of growing a somatic grape embryo, said method comprising the steps of: 
 (a) providing a liquid culture of grape explant comprising an embryogenic cell or embryogenic cell mass obtained from a perennial grape embryogenic culture;    (b) recovering said embryogenic cell or embryogenic cell mass from the culture;    (c) transferring said embryogenic cell or embryogenic cell mass recovered from step (b) to a second culture; and    (d) growing a somatic grape embryo from said embryogenic cell or embryogenic cell mass of step (c).    
     
     
         21 . A method of selecting for a somatic grape embryo having resistance to a plant pathogen, said method comprising the steps of: 
 (a) culturing a perennial grape embryogenic cell culture in a first liquid culture medium to produce a cellular suspension culture, said first liquid culture medium comprising a plant growth regulator and a toxin of a plant pathogen;    (b) recovering a grape cell or grape cell cluster from said cellular suspension culture;    (c) culturing said grape cell or grape cell cluster of step (b) in a second liquid culture medium; and    (d) recovering a somatic grape embryo from the culture of step (c) having resistance to said plant pathogen.    
     
     
         22 . The method of  claim 21 , wherein said toxin is a purified plant pathogen toxin.  
     
     
         23 . The method of  claim 21 , wherein said toxin is included in a plant pathogen filtrate.  
     
     
         24 . The method of  claim 23 , wherein said plant pathogen filtrate is prepared from a plant pathogen selected from the group consisting of a virus, nematode, insect, fungus, mycoplasma, or a bacterium.  
     
     
         25 . The method of  claim 24 , wherein said virus is selected from the group consisting of a grapevine fanleaf virus, a rupestris stem pitting virus, and a grapevine leafroll virus.  
     
     
         26 . The method of  claim 24 , wherein said nematode is selected from the group consisting of a root-knot nematode, a cyst nematode, a root-attacking nematode, and an above-ground nematode.  
     
     
         27 . The method of  claim 24 , wherein said insect is selected from the group consisting of the family Phylloxera, Coleoptera, Lepidoptera, Homoptera, Dermptera, and Diptera.  
     
     
         28 . The method of  claim 24 , wherein said fungus is selected from the group consisting of  Uncinula necator, Plasmopara viticola, Botrytis cinerea, Guignardia bidwellii, Phomophsis viticola, Elsinoë ampelina, Eutypa lata, Armillaria mellea , and  Verticillium dahlias.    
     
     
         29 . The method of  claim 24 , wherein said bacterium is selected from the group consisting of  Agrobacterium vitis, Agrobacterium tumefaciens, Xylellafastidosa , and  Xanthomonas ampelina.    
     
     
         30 . The method of  claim 21 , wherein said plant growth regulator of step (a) is an auxin.  
     
     
         31 . The method of  claim 21 , wherein said second liquid culture medium comprises a plant growth regulator.  
     
     
         32 . The method of  claim 21 , further comprising growing a grape plant from the somatic grape embryo of step (d).  
     
     
         33 . The method of  claim 21 , further comprising transferring said somatic grape embryo to a germination culture medium.  
     
     
         34 . A mature somatic grape embryo produced by subculturing a somatic grape embryo of  claim 21  on a solid culture medium.  
     
     
         35 . A plantlet germinated from the somatic grape embryo of  claim 21 .  
     
     
         36 . A method of selecting for a somatic grape embryo having resistance to a plant pathogen, said method comprising the steps of: 
 (a) culturing an embryogenic cell or an embryogenic cell mass in a first liquid culture medium to produce a cellular suspension culture, said first liquid culture medium comprising a plant growth regulator and a toxin of a plant pathogen;    (b) recovering a grape cell or grape cell cluster from said cellular suspension culture;    (c) culturing said grape cell or grape cell cluster in a second liquid culture medium to produce a grape somatic embryo; and    (d) recovering said somatic grape embryo having resistance to said plant pathogen.    
     
     
         37 . The method of  claim 36 , wherein said toxin is a purified plant pathogen toxin.  
     
     
         38 . The method of  claim 36 , wherein said toxin is included in a plant pathogen filtrate.  
     
     
         39 . The method of  claim 38 , wherein said plant pathogen filtrate is prepared from a plant pathogen selected from the group consisting of a virus, nematode, insect, fungus, mycoplasma, or bacterium.  
     
     
         40 . The method of  claim 39 , wherein said virus is selected from the group consisting of grapevine fanleaf virus, rupestris stem pitting virus, and grapevine leafroll virus.  
     
     
         41 . The method of  claim 39 , wherein said nematode is selected from the group consisting of a root-knot nematode, a cyst nematode, a root-attacking nematode, and an above-ground nematode.  
     
     
         42 . The method of  claim 39 , wherein said insect is selected from the group consisting of Phylloxera, Coleoptera, Lepidoptera, Homoptera, Dermptera, and Diptera.  
     
     
         43 . The method of  claim 39 , wherein said fungus is selected from the group consisting of  Uncinula necator, Plasmopara viticola, Botrytis cinerea, Guignardia bidwelli, Phomophsis viticola, Elsinoë ampelina, Eutypa lata, Armillaria mellea , and  Verticillium dahliae.    
     
     
         44 . The method of  claim 39 , wherein said bacterium is selected from the group consisting of  Agrobacterium vitis, Agrobacterium tumefaciens, Xylellafastidosa , and  Xanthomonas ampelina.    
     
     
         45 . The method of  claim 36 , wherein said plant growth regulator of step (a) is an auxin.  
     
     
         46 . The method of  claim 36 , wherein said second liquid culture medium comprises a plant growth regulator.  
     
     
         47 . The method of  claim 36 , further comprising growing a grape plant from the somatic grape embryo of step (d).  
     
     
         48 . The method of  claim 36 , further comprising transferring said somatic grape embryo of step (d) to a germination medium to grow a grape plantlet.  
     
     
         49 . A mature somatic grape embryo produced by subculturing a somatic grape embryo of  claim 36  on a solid culture medium.  
     
     
         50 . A plantlet germinated from the somatic grape embryo of  claim 36 .  
     
     
         51 . A grape plant regenerated from a cell or cell cluster selected in the presence of a toxin from a plant pathogen, wherein said plant has an increased level of resistance to said pathogen relative to a regenerated control grape plant not selected in the presence of said toxin.