Methods for Producing Fertile Crosses between Wild and Domestic Soybean Species

Abstract

Methods for producing hybrids between domestic and wild soybean that are fertile and can be further bred with other soybean plants are provided, thus allowing transfer of desirable traits and genes from the wild soybean into the domestic soybean. This invention also provides novel media for producing callus and multiple somatic embryos, as well as novel media for producing multiple shoots from the embryos. The hybrid plants are made fertile by colchicine treatment to double their chromosome number so that they can be backcrossed into domestic soybean. These methods and media allow the production of elite soybean lines containing traits or genes from wild soybean as well as a minimum amount of additional wild soybean DNA. Backcrosses containing only one wild soybean chromosome can be produced, as well as sets of such backcrossed lines that each contain one chromosome from the wild ancestor, but collectively all the wild chromosomes from the hybrid ancestor. Plants and plant progeny and plant tissue (tissue including seeds) of plants produced by the foregoing methods are also provided. The methods do not require genetic modification, and thus this invention allows production of domestic soybean plants that are not genetically-modified organisms (non-GMO) but that express desirable traits derived from wild soybean.

1 . An amphidiploid plant produced by a method comprising crossing a first parent plant of a wild perennial  Glycine  species with a domestic annual  Glycine  cultivar, wherein said amphidiploid plant carries a desirable agronomic trait inherited from the wild perennial  Glycine  species, or progeny, or plant tissue thereof carrying said trait. 
     
     
         2 . A progeny plant of  claim 1 , which is a BC 1  plant carrying said desirable agronomic trait inherited from the wild perennial  Glycine  species, and which is produced by a method comprising crossing a plurality of said amphidiploid plants with domestic soybean plants and selecting those having 40 paired chromosomes and half the number of unpaired chromosomes as were present in the original wild perennial  Glycine  species, or progeny or plant tissue thereof carrying said trait. 
     
     
         3 . A progeny plant of  claim 2 , which is a BC 2  plant carrying said desirable agronomic trait inherited from the wild perennial  Glycine  species, and which is produced by a method comprising crossing said BC 1  plant with a domestic soybean plant and selecting BC 2  plants carrying said trait, or progeny or plant tissue thereof carrying said trait. 
     
     
         4 . A progeny plant of  claim 3 , which is a BC 3  plant carrying said desirable agronomic trait inherited from the wild perennial  Glycine  species, and which is produced by a method comprising crossing said BC 2  plant with a domestic soybean plant and selecting BC 3  plants carrying said trait, or progeny or plant tissue thereof carrying said trait. 
     
     
         5 . A seed of a plant of  claim 4 . 
     
     
         6 . A plant of  claim 4  comprising only one chromosome inherited from its wild perennial  Glycine  ancestor, or progeny or plant tissue thereof carrying said one chromosome. 
     
     
         7 . A plant, progeny or tissue, which is produced by a method comprising crossing a wild perennial  Glycine  species with a domestic  Glycine  species, and which contains only one wild perennial chromosome of the wild perennial  Glycine  species. 
     
     
         8 . A seed of a plant of  claim 7 . 
     
     
         9 . A plurality of plants, progeny or tissues of  claim 7  collectively containing all the wild perennial chromosomes of the wild perennial  Glycine  species. 
     
     
         10 . A seed maturation medium comprising sufficient growth hormones to produce morphogenic callus, wherein said medium prevents seed germination, said medium comprising:
 (a) major salts;   (b) minor salts;   (c) iron;   (d) vitamins including L-glutamine;   (e) growth hormones; and   (f) sugar.   
     
     
         11 . The medium of  claim 10  wherein said growth hormones comprise a growth hormone selected from the group consisting of indole acetic acid (IAA), 1-naphthalene acetic acid (NAA), benzylaminopurine (BAP), kinetin and combinations of any of the foregoing. 
     
     
         12 . The medium of  claim 10  wherein said sugar comprises D-mannitol. 
     
     
         13 . The medium of  claim 10  wherein said sugar is present in an amount approximately double the amount present in Murashige and Skoog medium. 
     
     
         14 . The medium of  claim 10  wherein said iron is present in one-half the amount used in Murashige and Skoog medium. 
     
     
         15 . The medium of  claim 10  wherein said major salts comprise those of Murashige and Skoog and Gamborg B5 media. 
     
     
         16 . The medium of  claim 10  wherein said minor salts comprise those of Gamborg B5 medium. 
     
     
         17 . The medium of  claim 10  wherein said vitamins comprise those of Murashige and Skoog medium and in addition comprise myo-inositol, ascorbic acid, L-glutamine and casein hydrolysate. 
     
     
         18 . Singh Seed Maturation medium, comprising the following components:
 (a) 10×, 100 mL/L major salts, consisting essentially of: 30 g/L KNO 3 , 1.34 g/L (NH 4 ) 2 SO 4 , 16.5 g/L NH 4 NO 3 , 1.5 g/L NaH 2 PO 4 . H 2 O, 1.7 g/L KH 2 PO 4 , 3.7 g/L MgSO 4 .7H 2 O, and 4.4 g/L CaCl 2  2H 2 O;   (b) 100×, 10 mL/L first minor salts, consisting essentially of: 0.3 g/L H 3 BO 3 , 1.0 g/L MnSO 4 .4H 2 O, and 0.46 g/L ZnSO 4 .7H 2 O;   (c) 100×, 10 mL/L second minor salts, consisting essentially of: 0.042 g/L KI, 0.0013 g/L CuSO 4 .5H 2 O, 0.013 g/L Na 2 MoO 4 .2H 2 O, 0.0015 g/L and CoCl 2 .6H 2 O;   (d) 10×, 100 mL/L iron components, consisting essentially of: 0.185 g/L Na 2 .EDTA, and 0.139 g/L FeSO 4 .7H 2 O;   (e) vitamins, consisting essentially of: 2 mL/L Nicotinic acid (500 mg/100 mL), 2 mL/L Pyridoxine HCl (100 mg/100 mL), 1 mL/L Thiamine (100 mg/100 mL), 2 mL/L  Glycine  (100 mg/100 mL), 0.1 g/L Myo-inositol, 0.1 g/L Ascorbic acid, 292 mg/L L-Glutamine, and 0.5 g/L Casein hydrolysate;   (f) growth hormones consisting essentially of: 2 mL/L IAA (10 mg/50 mL) EtOH, 2 mL/L NAA (100 mg/50 mL) EtOH, 1.30 mL/L Kinetin (50 mg/50 mL) NaOH, 400 μg/L BAP (100 mg/100 mL); and   (g) sugars consisting essentially of: 40 g/L Sucrose, 18 g/L D-mannitol; and 3 g/L Gelrite; and   (h) having a pH of 5.8.   
     
     
         19 . A multiple shoot regeneration medium comprising:
 (a) major and minor salts, including less than about 2 g/L NH 4+ ;   (b) iron;   (c) vitamins;   (d) BAP; and   (e) sugar.   
     
     
         20 . The medium of  claim 19  which is Singh C5 medium, comprising:
 (a) 10×, 100 mL/L major salts, consisting essentially of: 20 g/L KNO 3 , 10 g/L NH 4 NO 3 , 1 g/L NaH 2 PO 4 . H 2 O, 3 g/L KH 2 PO 4 , 3.75 g/L MgSO 4 .7H 2 O, and 6 g/L CaCl 2  2H 2 O;   (b) 100×, 10 mL/L first minor salts, consisting essentially of: 0.3 g/L H 3 BO 3 , 1.0 g/L MnSO 4 . H 2 O, and 0.46 g/L ZnSO 4 .7H 2 O;   (c) 100×, 10 mL/L second minor salts, consisting essentially of: 0.042 g/L KI,   
       0.0013 g/L CuSO 4 .5H 2 O, 0.013 g/L Na 2 MoO 4 .2H 2 O, and 0.0015 g/L CoCl 2 .6H 2 O;
 (d) 10×, 100 mL/L iron components, consisting essentially of: 0.261 g/L Na 2 .EDTA, 0.249 g/L FeSO 4 .7H 2 O, and 0.1503 g/L KOH; 
 (e) vitamins, consisting essentially of: 250 μl/L Nicotinic acid (500 mg/100 mL), 1 mL/L Pyridoxine HCl (100 mg/100 mL), 2.25 mL/L Thiamine (100 mg/100 mL), 2 mL/L  Glycine  (100 mg/100 mL), and 0.250 g/L Myo-inositol; 
 (f) a growth hormone, which is 1 mL/L BAP (100 mg/100 mL); and 
 (g) sugars consisting essentially of: 25 g/L Sucrose, and 3 g/L Gelrite; and 
 (h) having a pH of 5.6.