Methods of Transforming Plants and Identifying Parental Origin of a Chromosome In Those Plants

Abstract

Methods for plant transformation, for improving transformation efficiency, and for producing transgenic plants are provided. The methods comprise crossing a recipient plant from a genetic line of a plant species of interest with a donor plant selected from a transformation competent genetic line of the same plant species or of another closely related plant species to obtain a hybrid plant. Tissues obtained from the hybrid plant are transformation competent. These tissues can then be transformed with one or more nucleotide sequences of interest and selected for transgenic events having the nucleotide sequence of interest integrated within a chromosome derived from the recipient plant. Transformed cells can be selected and transgenic hybrid plants regenerated. The nucleotide sequence of interest can be introgressed into the genetic line from which the original recipient parent was derived, or into other genetic lines. Transformed plants and seeds are additionally provided.

1 . A method of identifying the parental origin of a chromosome containing transferred DNA comprising:
 a) crossing a recipient parent with a donor parent to produce a F1 hybrid plant;   b) transforming at least one cell or tissue of the F1 hybrid plant with transferred DNA;   c) finding at least one transformed F1 hybrid plant cell containing the transferred DNA; and   d) using flanking DNA of at least one transformed F1 hybrid plant cell to identify the parental origin of the chromosome containing the transferred DNA.   
     
     
         2 . The method of  claim 1 , further comprising regenerating a transgenic plant from the transformed F1 hybrid plant cell identified as containing the transferred DNA in a chromosome of the recipient parent. 
     
     
         3 . The method of  claim 1  wherein the transferred DNA comprises at least one or combinations of a target site, a targeting cassette, a functional expression unit, a transposon or a transgene. 
     
     
         4 . The method of  claim 1  wherein the tissue of the F1 hybrid plant comprises embryos, cells, cell suspension cultures, callus, meristems, axillary meristems, leaf discs, or pollen. 
     
     
         5 . The method of  claim 1 , wherein at least one nucleotide of the flanking DNA of the F1 hybrid, and of at least one nucleotide of the corresponding region in the recipient parent and/or in the donor parent, is used one or more times in plasmid rescue, inverse polymerase chain reaction (PCR), Southern hybridization, sequencing, Restriction Fragment Length Polymorphism (RFLP), Single Nucleotide Polymorphism (SNP) or cloning to identify the parental origin of the chromosome containing the transferred DNA. 
     
     
         6 . The method of  claim 1  wherein transforming comprises  Agrobacterium -mediated or particle gun transformation. 
     
     
         7 . The method of  claim 1  wherein the recipient parent is more recalcitrant to transformation than the donor parent. 
     
     
         8 . The method of  claim 1  wherein the recipient parent comprises one or more desired characteristics in disease resistance, insect resistance, yield, stalk strength, standability, green snap, oil, phytate, silage, herbicide resistance, starch, carbohydrate, sugar, sterility, fertility, transgenicly produced protein, amino acid content, or height than the donor parent. 
     
     
         9 . The method of  claim 1  wherein the recipient parent and the donor parent are dicots. 
     
     
         10 . The method of  claim 9  wherein the dicot is soybean, sunflower, canola, cotton, alfalfa, potato, sugar beet or safflower. 
     
     
         11 . The method of  claim 1  wherein the recipient parent and the donor parent are monocots. 
     
     
         12 . The method of  claim 11  wherein the monocot is maize, wheat, sorghum, millet rice, barley, oats or rye. 
     
     
         13 . The method of  claim 12  wherein the maize is inbred maize. 
     
     
         14 . The method of  claim 11  wherein the recipient parent is PHTE4, PHAA0, PHP18, PH05F, PH09B, PHP02, PHJ90, PH24E, PHN46, PHT05, ASKC27 or PH21T. 
     
     
         15 . The method of  claim 11  wherein the donor parent is Hi-II, A188, H99, DAB01, DAB02, or DAB012. 
     
     
         16 . A method of identifying the parental origin of a chromosome containing transferred DNA comprising:
 a) crossing a recipient parent with a donor parent to produce at least one F1 hybrid plant;   b) transforming at least one transformed F1 hybrid plant cell or tissue of the F1 hybrid plant with a first transferred DNA, wherein the first transferred DNA comprises a target site;   c) finding at least one cell containing the first transferred DNA;   d) using flanking DNA of the transferred DNA to identify the parental origin of the chromosome containing the first transferred DNA.   e) regenerating a transgenic plant from a F1 hybrid plant cell identified as comprising the first transferred DNA in a chromosome of the recipient parent;   f) introgressing, through at least one backcrossing and selection, the transferred DNA to produce a second recipient parent containing the transferred DNA;   g) crossing the second recipient parent with a donor parent to produce at least one F2 hybrid plant;   h) transforming at least one cell or tissue of the F2 hybrid plant with a second transferred DNA;   i) introducing a recombinase that recognizes and implements recombination; and   j) finding at least one transformed F2 hybrid plant cell comprising the first and the second transferred DNA.   
     
     
         17 . The method of  claim 16 , further comprising using the flanking DNA of at least one transformed F2 hybrid plant cell to identify the parental origin of the chromosome containing the transferred DNA. 
     
     
         18 . The method of  claim 16 , further comprising regenerating a first transgenic plant from a transformed F2 hybrid plant cell comprising the first and second transferred DNA in a chromosome of the recipient parent. 
     
     
         19 . The method of  claim 16  wherein the first transferred DNA comprises a targeting cassette. 
     
     
         20 . The method of  claim 19  wherein the targeting cassette comprises a nucleotide sequence flanked by at least one non-identical recombination site and wherein the second transferred DNA is flanked by non-identical recombination sites corresponding to said recombination sites contained in the target site of said chromosome. 
     
     
         21 . A method of identifying the parental origin of a chromosome containing a transgene comprising:
 a) crossing a recipient parent with a donor parent to produce at least one F1 hybrid plant;   b) transforming at least one cell or tissue of the F1 hybrid plant with a transferred DNA;   c) finding at least one transformed F1 hybrid plant cell containing the transferred DNA;   d) digesting flanking DNA with restriction enzymes;   e) ligating adapters to the digested flanking DNA;   f) amplifying digested flanking DNA of the transformed F1 hybrid plant cell, and comparable DNA from the donor parent and/or recipient parent at least once with at least one vector-specific DNA primer and/or at least one nested primer to produce a PCR product; and   g) sequencing the PCR product or digesting PCR product with at least one restriction enzyme to identify parental origin of chromosome.   
     
     
         22 . The method of  claim 21  wherein the adapters are bubble adapters.