Method for producing direct in vitro flowering and viable seed from cotyledon, radicle, and leaf explants, and plants produced therefrom

Abstract

The present invention relates to a method of reprogramming plant development that allows flower buds and seeds to arise de novo, directly from a cotyledon or radicle explants or from shoots produced on a cotyledon or radicle. The present invention also provides for an improved culturing media that provide for in vitro flowering.

1 . A method for eliciting direct flower bud production in vitro on a cotyledon, the method comprising:
 1) germinating at least one seed on a MSB 5  medium and splitting the two cotyledons,   2) transferring the cotyledon to a cotyledon-flowering medium comprising at least one substituted phenylurea cytokinin analog, and   3) allowing the cotyledon to develop at least one flower bud.   
   
   
       2 . The method of  claim 1  wherein the at least at least one substituted phenylurea cytokinin analog comprises TDZ. 
   
   
       3 . The method of  claim 2  wherein TDZ is present at a concentration of 1.5 mg/L to 2.5 mg/L. 
   
   
       4 . The method of  claim 3  wherein TDZ is present at a concentration of 2.0 mg/L. 
   
   
       5 . The method of  claim 2  wherein the cotyledon-flowering medium further comprises at least one cytokinin. 
   
   
       6 . The method of  claim 5  wherein the cytokinin is BAP. 
   
   
       7 . The method of  claim 6  wherein the BAP is present at a concentration from 0.75 mg/L to 1.5 mg/L. 
   
   
       8 . A method of  claim 7  wherein BAP is present at a concentration of 1 mg/L. 
   
   
       9 . The method of  claim 1  wherein the cotyledon-flowering medium comprises TDZ and BAP and wherein the concentration of TDZ in the cotyledon-flowering medium is 2.0 mg/L and the concentration of BAP in the cotyledon-flowering medium is 1.0 mg/L. 
   
   
       10 . The method of  claim 1 , further comprising allowing the flower bud to develop seed. 
   
   
       11 . A method for producing at least one transgenic seed in vitro, the method comprising eliciting direct flower bud production on a cotyledon, the method comprising:
 germinating at least one seed on a MSB5 medium and splitting the two cotyledons,   transforming the cotyledon with a gene of interest; transferring a cotyledon to a cotyledon-flowering medium comprising at least one substituted phenylurea cytokinin analog,   allowing the transformed cotyledon to develop at least one flower bud, and   allowing the at least one flower bud to develop at least one transgenic seed.   
   
   
       12 . The method of  claim 11  wherein the transforming is selected from the group consisting of  agrobacterium  mediated transformation, biolistic mediated transformation or whisker mediated transformation. 
   
   
       13 . The method of  claim 1 , wherein the seed is soybean. 
   
   
       14 . Plants or plant parts derived from the transgenic seed produced by  claim 10 . 
   
   
       15 . A method for producing transgenic seeds from an in vitro developed flower bud on a cotyledon, the method comprising:
 germinating at least one seed on a MSB5 medium and splitting the two cotyledons,   transforming the cotyledon with a gene of interest,   transferring the cotyledon to a cotyledon-flowering medium comprising MSB5 medium augmented with TDZ at a concentration of 2.0 mg/L and BAP at a concentration of 1.0 mg/L,   allowing the cotyledon to develop at least one flower bud,   transferring said cotyledon with the at least one flower bud to MSB5 medium, and   allowing said flower bud to produce at least one seed.   
   
   
       16 . The method of  claim 15  wherein the seed is a soybean seed. 
   
   
       17 . The method of  claim 15  wherein the transformation is chloroplast transformation. 
   
   
       18 . Plants or plant parts derived from the transgenic seed produced by  claim 15 . 
   
   
       19 . A cotyledon-flowering medium comprising a substituted phenylurea cytokinin analog and a cytokinin wherein a cotyledon placed onto said medium produces at least one flower bud. 
   
   
       20 . The cotyledon-flowering medium of  claim 18  wherein the substituted phenylurea cytokinin analog comprises TDZ and the cytokinin comprises BAP. 
   
   
       21 . The cotyledon-flowering medium of  claim 19  wherein the TDZ is present at a concentration of 1.5 to 2.5 mg/L and the BAP is present at a concentration of 0.75 mg/L to 1.5 mg/L. 
   
   
       22 . The cotyledon-flowering medium of  claim 20  wherein the TDZ is present at a concentration of 2.0 mg/L and the BAP is present at a concentration of 1.0 mg/L. 
   
   
       23 . A method for eliciting direct flower bud production in vitro on a shoot developed on a cotyledon, the method comprising:
 germinating at least one seed on a MSB5 medium and splitting the two cotyledons,   transferring the cotyledon to a cotyledon-shoot flowering medium comprising at least one substituted phenylurea cytokinin analog and a cytokinin, and   allowing the cotyledon to develop at least one shoot, and   allowing the at least one shoot to develop at least one flower bud.   
   
   
       24 . The method of  claim 23  wherein the at least at least one substituted phenylurea cytokinin analog comprises TDZ and the cytokinin comprises BAP. 
   
   
       25 . The method of  claim 24  wherein the TDZ is present at a concentration of 0.75 mg/L to 2.5 mg/L. 
   
   
       26 . The method of  claim 25  wherein the TDZ is present at a concentration of 1.0 mg/L to 2.0 mg/L. 
   
   
       27 . The method of  claim 24  wherein the BAP is present at a concentration of 2.5 mg/L to 3.5 mg/L. 
   
   
       28 . The method of  claim 27  wherein the BAP is present at a concentration of 3.0 mg/L. 
   
   
       29 . The method of  claim 24  wherein the TDZ is present at a concentration of 1.0 mg/L to 2.0 mg/L and the BAP is present at a concentration of 3.0 mg/L. 
   
   
       30 . The method of  claim 23 , further comprising allowing the flower bud to develop seed. 
   
   
       31 . A method for producing at least one transgenic seed in vitro, the method comprising eliciting direct flower bud production on a shoot developed on a cotyledon, the method comprising:
 germinating at least one seed on a MSB5 medium and splitting the two cotyledons,   transforming the cotyledon with a gene of interest,   transferring the cotyledon to a cotyledon-shoot flowering medium comprising at least one substituted phenylurea cytokinin analog and a cytokinin,   allowing the transformed cotyledon to develop at least one shoot,   allowing the at least one shoot to develop at least one flower bud, and   allowing the at least one flower bud to develop at least one transgenic seed.   
   
   
       32 . The method of  claim 31 , wherein the transforming of said cotyledon is chloroplast transformation. 
   
   
       33 . The method of  claim 23  or  31 , wherein the seed is soybean. 
   
   
       34 . Plants or plant parts derived from the transgenic seed produced by  claim 31 . 
   
   
       35 . A method for producing at least one transgenic seed in vitro, the method comprising eliciting direct flower bud production on a shoot developed on a cotyledon, the method comprising:
 germinating at least one seed on a MSB5 medium and splitting the two cotyledons,   transforming the cotyledon with a gene of interest,   transferring the cotyledon to a cotyledon-shoot-flowering medium comprising TDZ at a concentration of 1.0 to 2.0 mg/L and BAP at a concentration of 3.0 mg/L,   allowing the transformed cotyledon to develop at least one shoot,   allowing the at least one shoot to develop at least one flower bud, and   allowing the at least one flower bud to develop at least one transgenic seed.   
   
   
       36 . The method of  claim 35  wherein the seed is a soybean seed. 
   
   
       37 . The method of  claim 36  wherein the transforming comprises chloroplast transformation. 
   
   
       38 . A cotyledon-shoot-flowering medium comprising a substituted phenylurea cytokinin analog and a cytokinin wherein a cotyledon placed onto said medium produces at least one flower bud. 
   
   
       39 . The cotyledon-shoot-flowering medium of  claim 38  wherein the substituted phenylurea cytokinin analog comprises TDZ and the cytokinin comprises BAP. 
   
   
       40 . The cotyledon-shoot-flowering medium of  claim 39  wherein the TDZ is present at a concentration of 0.75 mg/L to 2.5 mg/L and the BAP is present at a concentration of 2.5 mg/L to 3.5 mg/L. 
   
   
       41 . The cotyledon-shoot-flowering medium of  claim 40  wherein the TDZ is present at a concentration of 1.0 mg/L to 2.0 mg/L and the BAP is present at a concentration of 3.0 mg/L. 
   
   
       42 . A method for eliciting direct flower bud production in vitro on a radicle, the method comprising:
 germinating at least one seed on a MSB5 medium and removing a radicle from the seed,   transferring the radicle to a radicle-flowering medium comprising at least one substituted phenylurea cytokinin analog, and   allowing the radicle to develop at least one flower bud.   
   
   
       43 . The method of  claim 42  wherein the at least one substituted phenylurea cytokinin analog comprises TDZ. 
   
   
       44 . The method of  claim 43  wherein TDZ is present at a concentration of 1.5 mg/L to 2.5 mg/L. 
   
   
       45 . The method of  claim 44  wherein TDZ is present at a concentration of 2.0 mg/L. 
   
   
       46 . The method of  claim 43  wherein the radicle-flowering medium further comprises at least one cytokinin. 
   
   
       47 . The method of  claim 46  wherein the cytokinin is BAP. 
   
   
       48 . The method of  claim 47  wherein the BAP is present at a concentration of 0.75 mg/L to 1.5 mg/L. 
   
   
       49 . The method of  claim 42  wherein the radicle-flowering medium comprises TDZ and BAP and wherein the concentration of TDZ in the radicle-flowering medium is 2.0 mg/L and the concentration of BAP in the radicle-flowering medium is 1.0 mg/L. 
   
   
       50 . The method of  claim 42 , further comprising allowing the flower bud to develop seed. 
   
   
       51 . A method for producing at least one transgenic seed in vitro, the method comprising eliciting direct flower bud production on a radicle, the method comprising:
 germinating at least one seed on a MSB5 medium and removing a radicle from the seed,   transforming the radicle with a gene of interest, transferring the radicle to a radicle-flowering medium comprising at least one substituted phenylurea cytokinin analog,   allowing the transformed radicle to develop at least one flower bud, and   allowing the at least one flower bud to develop at least one transgenic seed.   
   
   
       52 . The method of  claim 51 , wherein the transforming of said radicle is chloroplast transformation. 
   
   
       53 . The method of  claim 42  or  51 , wherein the seed is soybean. 
   
   
       54 . Plants or plant parts derived from the transgenic seed produced by  claim 51 . 
   
   
       55 . A method for producing at least one transgenic seed from an in vitro developed flower bud on a radicle, the method comprising:
 germinating at least one seed on a MSB5 medium and removing a radicle,   transforming the radicle with a gene of interest,   transferring the radicle to a radicle-flowering medium comprising MSB5 medium augmented with TDZ at a concentration of 2.0 mg/L and BAP at a concentration of 1.0 mg/L,   allowing the radicle to develop at least one flower bud,   transferring said radicle with the at least one flower bud to MSB5 medium, and   allowing said flower bud to produce at least one seed.   
   
   
       56 . The method of  claim 55  wherein the seed is a soybean seed. 
   
   
       57 . The method of  claim 55  wherein the transforming comprises chloroplast transformation. 
   
   
       58 . A radicle-flowering medium comprising a substituted phenylurea cytokinin analog and a cytokinin wherein a radicle placed onto said medium produces at least one flower bud. 
   
   
       59 . The radicle-flowering medium of  claim 58  wherein the substituted phenylurea cytokinin analog comprises TDZ and the cytokinin comprises BAP. 
   
   
       60 . The radicle-flowering medium of  claim 59  wherein the TDZ is present at a concentration of 1.5 mg/L to 2.5 mg/L and the BAP is present at a concentration of 0.75 mg/L to 1.5 mg/L. 
   
   
       61 . The radicle-flowering medium of  claim 60  wherein the TDZ is present at a concentration of 2.0 mg/L and the BAP is present at a concentration of 1.0 mg/L. 
   
   
       62 . A method for eliciting direct flower bud production in vitro on a shoot developed on a radicle, the method comprising:
 germinating at least one seed on a MSB5 medium and removing a radicle,   transferring a radicle to a radicle-shoot flowering medium comprising at least one substituted phenylurea cytokinin analog and a cytokinin, and   allowing the radicle to develop at least one shoot, and   allowing the at least one shoot to develop at least one flower bud.   
   
   
       63 . The method of  claim 1  wherein the at least one substituted phenylurea cytokinin analog comprises TDZ and the cytokinin comprises BAP. 
   
   
       64 . The method of  claim 2  wherein the TDZ is present at a concentration of 0.75 mg/L to 2.5 mg/L. 
   
   
       65 . The method of  claim 3  wherein the TDZ is present at a concentration of 1.0 mg/L to 2.0 mg/L. 
   
   
       66 . The method of  claim 2  wherein the BAP is present at a concentration of 2.5 mg/L to 3.5 mg/L. 
   
   
       67 . The method of  claim 5  wherein the BAP is present at a concentration of 3.0 mg/L. 
   
   
       68 . The method of  claim 2  wherein the TDZ is present at a concentration of 1.0 mg/L to 2.0 mg/L and the BAP is present at a concentration of 3.0 mg/L. 
   
   
       69 . The method of  claim 1 , further comprising allowing the flower bud to develop seed. 
   
   
       70 . A method for producing at least one transgenic seed in vitro, the method comprising eliciting direct flower bud production on a shoot developed on a radicle, the method comprising:
 germinating at least one seed on a MSB5 medium and removing a radicle,   transforming the radicle with a gene of interest,   transferring a radicle to a radicle-shoot flowering medium comprising at least one substituted phenylurea cytokinin analog and a cytokinin,   allowing the transformed radicle to develop at least one shoot,   allowing the at least one shoot to develop at least one flower bud, and   allowing the at least one flower bud to develop at least one transgenic seed.   
   
   
       71 . The method of  claim 70 , wherein the transforming of said radicle is chloroplast transformation. 
   
   
       72 . The method of  claim 62 , wherein the seed is soybean. 
   
   
       73 . Plants or plant parts derived from the transgenic seed produced by  claim 70 . 
   
   
       74 . A method for producing at least one transgenic seed in vitro, the method comprising eliciting direct flower bud production on a shoot developed on a radicle, the method comprising:
 germinating at least one seed on a MSB5 medium and removing a radicle, transforming the radicle with a gene of interest,   transferring the radicle to a radicle-shoot-flowering medium comprising TDZ at a concentration of 1.0 to 2.0 mg/L and BAP at a concentration of 3.0 mg/L,   allowing the transformed radicle to develop at least one shoot,   allowing the at least one shoot to develop at least one flower bud, and   allowing the at least one flower bud to develop at least one transgenic seed.   
   
   
       75 . The method of  claim 74  wherein the seed is a soybean seed. 
   
   
       76 . The method of  claim 75  wherein the transforming comprises chloroplast transformation. 
   
   
       77 . A radicle-shoot-flowering medium comprising a substituted phenylurea cytokinin analog and a cytokinin wherein a radicle placed onto said medium produces at least one shoot. 
   
   
       78 . The radicle-shoot-flowering medium of  claim 77  wherein the substituted phenylurea cytokinin analog comprises TDZ and the cytokinin comprises BAP. 
   
   
       79 . The radicle-shoot-flowering medium of  claim 78  wherein the TDZ is present at a concentration of 0.75 mg/L to 2.5 mg/L and the BAP is present at a concentration of 2.5 mg/L to 3.5 mg/L. 
   
   
       80 . The radicle-shoot-flowering medium of  claim 79  wherein the TDZ is present at a concentration of 1.0 mg/L to 2.0 mg/L and the BAP is present at a concentration of 3.0 mg/L. 
   
   
       81 . A method for eliciting flower bud production in vitro developed shoot from a leaf explant, the method comprising:
 culturing a leaf explant on an in vitro shoot multiplication medium comprising MS medium supplemented with an auxin and a cytokinin, allowing at least one shoot to develop on the leaf explant,   transferring at least one shoot to an in vitro shoot elongation medium comprising a gibberellic acid,   allowing the at least one shoot to elongate, transferring the at least one elongated shoot to a MS medium, and   allowing the at least one elongated shoot to develop at least one flower bud.   
   
   
       82 . The method of  claim 81  wherein auxin in the in vitro shoot multiplication medium comprises IAA and the cytokinin in the in vitro shoot multiplication medium comprises BAP. 
   
   
       83 . The method of  claim 83  wherein the IAA is present at a concentration of 0.05 mg/L to 0.25 mg/L and the BAP is present at a concentration of 1.75 mg/L to 2.25 mg/L. 
   
   
       84 . The method of  claim 83  wherein the IAA is present at a concentration of 0.1 mg/L and the BAP is present at a concentration of 2.0 mg/L. 
   
   
       85 . The method of  claim 81 , further comprising allowing the flower bud to develop seed. 
   
   
       86 . A method for producing at least one transgenic seeds in vitro, the method comprising eliciting direct flower bud production on a shoot developed on a leaf explant, the method comprising:
 transforming a leaf explant with a gene of interest,   culturing a leaf explant on an in vitro shoot multiplication medium comprising MS medium supplemented with an auxin and a cytokinin,   allowing at least one shoot to develop on the leaf explant,   transferring at least one shoot to an in vitro shoot elongation medium comprising a gibberellic acid, allowing the at least one shoot to elongate,   transferring the at least one elongated shoot to a MS medium,   allowing the at least one elongated shoot to develop at least one flower bud, and   allowing the at least one flower bud to develop at least one seed.   
   
   
       87 . Plants or plant parts derived from the transgenic seed produced by  claim 86 . 
   
   
       88 . A method for producing at least one transgenic seed in vitro, the method comprising eliciting flower bud production on in vitro developed shoots from leaf explant, the method comprising:
 transforming a leaf explant with a gene of interest,   culturing a leaf explant on an in vitro shoot multiplication medium comprising MS medium supplemented with IAA at a concentration of 0.05 mg/L to 0.25 mg/L of and BAP at a concentration of 1.75 mg/L to 2.25 mg/L,   allowing at least one shoot to develop on the leaf explant,   transferring at least one shoot to an in vitro shoot elongation medium comprising a gibberellic acid,   allowing the at least one shoot to elongate,   transferring the at least one elongated shoot to a MS medium,   allowing the at least one elongated shoot to develop at least one flower bud, and   allowing the at least one flower bud to develop at least one seed.