Systems and methods for cellular reprogramming of a plant cell

Abstract

Plant cell fate and development is altered by treating cells with cellular reprogramming factors. Embryogenesis inducing morphogenic developmental genes are used as cellular reprogramming factors, specifically comprising polypeptides or polynucleotides encoding gene products for generating doubled haploids or haploid plants from gametes. Maize microspores treated by contacting the isolated cells with an exogenous purified, recombinant embryogenesis inducing morphogenic developmental gene polypeptide results in embryogenesis. The gametes of a maize plant develop into embryoids when transformed with a genetic construct including regulatory elements and structural genes capable of acting in a cascading fashion to alter cellular fate of plant cells. Developmental morphogenic proteins expressed from a genetic construct are used for ex situ treatment methods and for in planta cellular reprogramming.

1 . A method of generating a haploid plant embryo comprising:
 (a) obtaining an embryogenic microspore by providing a plant microspore to modulate microspore embryogenesis in the plant microspore, an embryogenesis modulation factor selected from the group consisting of:
 (i) an embryogenesis inducing polypeptide; or 
 (ii) an embryogenesis inducing compound; or 
 (iii) a combination of (i) and (ii); and 
   (b) producing the haploid plant embryo from the embryogenic microspore.   
     
     
         2 . The method of  claim 1 , wherein the embryogenesis inducing polypeptide is not produced by a stably integrated recombinant DNA construct in the microspore. 
     
     
         3 . The method of  claim 1 , wherein the embryogenesis inducing compound is a kinase inhibitor selected from N-[(2R)-2,3-dihydroxypropoxy]-3,4-difluoro-2-(2-fluoro-4-iodoanilino)benzamide, anthra(1,9-cd)pyrazol-6(2H)-one:4-(4-Fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole, or N-benzyl-2-(pyrimidin-4-ylamino)-1,3-thiazole-4-carboxamide. 
     
     
         4 . The method of  claim 1 , wherein the embryogenesis inducing compound is hemin. 
     
     
         5 . The method of  claim 1 , wherein the embryogenesis inducing polypeptide is selected from the group consisting of:
 (i) a WUS/WOX homeobox polypeptide;   (ii) a Babyboom (BBM) polypeptide or an Ovule Development Protein 2 (ODP2) polypeptide;   (iii) a LEC1 polypeptide;   (iv) a combination of (i) and (ii); and   (v) a combination of (i) and (iii).   
     
     
         6 . The method of  claim 5 , wherein the embryogenesis inducing polypeptide further comprises a cell penetrating peptide (CPP). 
     
     
         7 . The method of  claim 1 , wherein the embryogenesis modulation factor is present in a tissue culture media. 
     
     
         8 . The method of  claim 1 , comprising co-culturing the microspore with an embryogenesis inducing suspension feeder cell culture, wherein the embryogenesis inducing suspension feeder cell culture expresses an embryogenesis inducing polypeptide or co-culturing the microspore with the embryogenesis modulation factor in the culture media. 
     
     
         9 . The method of  claim 8 , wherein the embryogenesis inducing polypeptide is selected from the group consisting of:
 (i) a WUS/WOX homeobox polypeptide;   (ii) a Babyboom (BBM) polypeptide or an Ovule Development Protein 2 (ODP2) polypeptide;   (iii) a LEC1 polypeptide;   (iv) a combination of (i) and (ii); and   (v) a combination of (i) and (iii).   
     
     
         10 . The method of  claim 1 , further comprising culturing the haploid plant embryo. 
     
     
         11 . The method of  claim 10 , comprising contacting the haploid plant embryo with a chromosome doubling agent for a period sufficient to generate a doubled haploid plant embryo. 
     
     
         12 . The method of  claim 1 , wherein the microspore is obtained from maize, rice,  sorghum, brassica , soybean, wheat, and cotton. 
     
     
         13 . The method of  claim 1 , wherein the embryogenesis modulation factor comprises a cell penetrating peptide. 
     
     
         14 . A method of generating a haploid plant embryo comprising:
 (a) providing a plant comprising an expression cassette, wherein the expression cassette comprises a tapetum cell preferred regulatory element operably linked to a polynucleotide encoding an embryogenesis inducing polypeptide;   (b) crossing the plant of (a) with a wild type inbred plant to provide an F 1  hybrid;   (c) recovering an embryogenic microspore from the F 1  hybrid of (b); and   (d) producing the haploid plant embryo from the embryogenic microspore.   
     
     
         15 . The method of  claim 14 , wherein the embryogenesis inducing polypeptide is a morphogenic developmental polypeptide. 
     
     
         16 . The method of  claim 15 , wherein the morphogenic developmental polypeptide is selected from the group consisting of:
 (i) a WUS/WOX homeobox polypeptide;   (ii) a Babyboom (BBM) polypeptide or an Ovule Development Protein 2 (ODP2) polypeptide;   (iii) a LEC1 polypeptide;   (iv) a combination of (i) and (ii); and   (v) a combination of (i) and (iii).   
     
     
         17 . The method of  claim 1  or  14 , further comprising modifying genomic DNA by a site-specific nuclease. 
     
     
         18 . The method of  claim 14 , wherein the expression cassette further comprises a polynucleotide encoding a site-specific nuclease. 
     
     
         19 . The method of  claim 17  or  18 , wherein the site-specific nuclease is selected from the group consisting of a zinc finger nuclease, a meganuclease, TALEN, and a CRISPR-Cas endonuclease. 
     
     
         20 . The method of  claim 19 , wherein the CRISPR-Cas nuclease is Cas9 or Cpfl nuclease. 
     
     
         21 . The method of  claim 17 , wherein the modification of genomic DNA is made by a Cas endonuclease during microspore embryogenesis. 
     
     
         22 . The method of  claim 21 , wherein the modification of DNA is an insertion, a deletion, or a substitution mutation. 
     
     
         23 . The method of  claim 21 , wherein the Cas endonuclease is expressed from the expression cassette, the Cas endonuclease further comprising a cell penetrating peptide. 
     
     
         24 . The method of  claim 23 , further comprising providing a guide RNA expressed from the expression cassette. 
     
     
         25 . The method of  claim 22 , wherein the modification of DNA is performed by providing a guide RNA and Cas endonuclease as a ribonucleoprotein complex exogenously to the embryogenic microspore. 
     
     
         26 . The method of  claim 14 , wherein the plant is homozygous for the expression cassette. 
     
     
         27 . The method of  claim 14 , wherein the expression cassette further comprises a signal peptide. 
     
     
         28 . The method of  claim 14 , wherein the expression cassette further comprises a cell penetrating peptide (CPP). 
     
     
         29 . The method of  claim 14 , further comprising contacting the haploid plant embryo with a chromosome doubling agent for a period sufficient to generate a doubled haploid plant embryo. 
     
     
         30 . The method of  claim 14 , wherein the plant is maize, rice,  sorghum, brassica , soybean, wheat, or cotton. 
     
     
         31 . The method of  claim 29 , further comprising regenerating a doubled haploid plant from the doubled haploid plant embryo. 
     
     
         32 . A method of generating a doubled haploid plant comprising:
 (a) providing a plant comprising an expression cassette, wherein the expression cassette comprises an endosperm cell preferred regulatory element operably linked to a polynucleotide encoding an embryogenesis inducing polypeptide;   (b) crossing the plant of (a) with a wild type F 1  hybrid;   (c) recovering a haploid embryo from the cross of (b);   (d) contacting the haploid embryo with a chromosome doubling agent for a period sufficient to generate a doubled haploid embryo; and   (e) regenerating the doubled haploid plant from the doubled haploid embryo of (d).   
     
     
         33 . The method of  claim 32 , wherein the embryogenesis inducing polypeptide is a morphogenic developmental polypeptide. 
     
     
         34 . The method of  claim 33 , wherein the morphogenic developmental polypeptide is selected from the group consisting of:
 (i) a WUS/WOX homeobox polypeptide;   (ii) a Babyboom (BBM) polypeptide or an Ovule Development Protein 2 (ODP2) polypeptide;   (iii) a LEC1 polypeptide;   (iv) a combination of (i) and (ii); and   (v) a combination of (i) and (iii).   
     
     
         35 . The method of  claim 32 , wherein the expression cassette further comprises a polynucleotide encoding a gene-editing nuclease. 
     
     
         36 . The method of  claim 32 , further comprising modifying genomic DNA by a site-specific nuclease. 
     
     
         37 . The method of  claim 32 , wherein the expression cassette further comprises a polynucleotide encoding a site-specific nuclease. 
     
     
         38 . The method of  claim 36  or  37 , wherein the site-specific nuclease is selected from the group consisting of a zinc finger nuclease, a meganuclease, TALEN, and a CRISPR-Cas endonuclease. 
     
     
         39 . The method of  claim 38 , wherein the CRISPR-Cas nuclease is Cas9 or Cpfl nuclease. 
     
     
         40 . The method of  claim 36 , wherein the modification of genomic DNA is made by a Cas endonuclease during haploid embryo embryogenesis. 
     
     
         41 . The method of  claim 40 , wherein the modification of DNA is an insertion, deletion, or a substitution mutation. 
     
     
         42 . The method of  claim 40 , wherein the Cas endonuclease is expressed from the expression cassette, the Cas endonuclease further comprising a cell penetrating peptide. 
     
     
         43 . The method of  claim 42 , further comprising providing a guide RNA expressed from the expression cassette. 
     
     
         44 . The method of  claim 41 , wherein the modification of DNA is performed by providing a guide RNA and Cas endonuclease as a ribonucleoprotein complex exogenously to the embryogenic haploid embryo. 
     
     
         45 . The method of  claim 32 , wherein the plant is homozygous for the expression cassette. 
     
     
         46 . The method of  claim 32 , wherein the expression cassette further comprises a signal peptide. 
     
     
         47 . The method of  claim 32 , wherein the expression cassette further comprises a cell penetrating peptide (CPP). 
     
     
         48 . The method of  claim 32 , wherein the expression cassette further comprises a polynucleotide encoding a color marker or a fluorescent marker operably linked to regulatory element. 
     
     
         49 . The method of  claim 48 , wherein recovering the haploid embryo comprises screening for the presence or the absence of the color marker, the fluorescent marker, or the regulatory element. 
     
     
         50 . The method of  claim 48 , wherein the screening occurs in a cell viability and cell sorting microfluidics device for automated fluorescence detection for identifying, sorting, and selecting a haploid embryo comprising the expression cassette from a haploid embryo not comprising the expression cassette. 
     
     
         51 . An embryogenic microspore comprising an increased amount of an embryogenesis inducing polypeptide compared to a control microspore, wherein the polypeptide is not produced in the microspore. 
     
     
         52 . An embryoid or embryogenic tissue produced from the embryogenic microspore of  claim 51 . 
     
     
         53 . An embryogenic microspore comprising a heterologous cellular reprogramming agent, wherein the heterologous cellular reprogramming agent is not produced in the microspore. 
     
     
         54 . The embryogenic microspore of  claim 53 , wherein the cellular reprogramming agent is selected from the group consisting of:
 (i) an embryogenesis inducing polypeptide; or   (ii) an embryogenesis inducing compound; or   (iii) a combination of (i) and (ii).   
     
     
         55 . The embryogenic microspore of  claim 54 , wherein the embryogenesis inducing polypeptide is selected from the group consisting of:
 (i) a WUS/WOX homeobox polypeptide;   (ii) a Babyboom (BBM) polypeptide or an Ovule Development Protein 2 (ODP2) polypeptide;   (iii) a LEC1 polypeptide;   (iv) a combination of (i) and (ii); and   (v) a combination of (i) and (iii).   
     
     
         56 . The embryogenic microspore of  claim 53 , wherein the embryogenesis inducing compound is hemin or a kinase inhibitor or a combination thereof. 
     
     
         57 . The embryogenic microspore of  claim 53 , capable of producing a haploid embryo. 
     
     
         58 . The embryogenic microspore of  claim 51  or  53  is a maize embryogenic microspore. 
     
     
         59 . The embryogenic microspore of  claim 51  or  53  is from rice,  sorghum, brassica , soybean, wheat, or cotton. 
     
     
         60 . A plant cell comprising an expression cassette, wherein the expression cassette comprises a tapetum cell preferred regulatory element operably linked to a polynucleotide encoding an embryogenesis inducing polypeptide, and wherein the embryogenesis inducing polypeptide is capable of being secreted or transported into a microspore. 
     
     
         61 . The plant cell of  claim 60 , wherein the embryogenesis inducing polypeptide comprises a cell penetrating peptide. 
     
     
         62 . The plant cell of  claim 60 , wherein the embryogenesis inducing polypeptide is a morphogenic developmental polypeptide selected from the group consisting of:
 (i) a WUS/WOX homeobox polypeptide;   (ii) a Babyboom (BBM) polypeptide or an Ovule Development Protein 2 (ODP2) polypeptide;   (iii) a LEC1 polypeptide;   (iv) a combination of (i) and (ii); and   (v) a combination of (i) and (iii).   
     
     
         63 . A plant cell comprising an expression cassette, wherein the expression cassette comprises an endosperm cell preferred regulatory element operably linked to a polynucleotide encoding an embryogenesis inducing polypeptide and wherein the embryogenesis inducing polypeptide is produced in an endosperm cell, the embryo surrounding region (ESR), the Basal Endosperm Transfer Layer (BETL) or a combination thereof and capable of being secreted or transported into an embryo cell. 
     
     
         64 . A population of plant cells comprising the plant cell of  claim 63  and the embryo cell, wherein the embryo cell comprises the secreted or transported embryogenesis inducing polypeptide. 
     
     
         65 . The plant cell of  claim 63 , wherein the embryogenesis inducing polypeptide is a morphogenic developmental polypeptide selected from the group consisting of:
 (i) a WUS/WOX homeobox polypeptide;   (ii) a Babyboom (BBM) polypeptide or an Ovule Development Protein 2 (ODP2) polypeptide;   (iii) a LEC1 polypeptide;   (iv) a combination of (i) and (ii); and   (v) a combination of (i) and (iii).