Methods for Reproducing Plants Asexually and Compositions Thereof

Abstract

Disclosed are methods of obtaining clonal seeds, methods of plant cloning, methods of screening for maternal plants that produce clonal seeds asexually and methods of increasing yield of clonal seeds. Also disclosed are constructs comprising a nucleic acid that may silence the activity of a RNA-dependent DNA methylation pathway gene. Further disclosed are maternal plants comprising a construct wherein the construct comprises an exogenous nucleic acid sequence, wherein the construct renders the maternal plant defective for RNA-dependent DNA methylation.

What is claimed is: 
     
         1 . A method of obtaining seeds comprising clonal embryos comprising
 (a) collecting one or more seeds produced by a maternal plant, wherein the maternal plant is defective for RdDM activity for:
 (i) a nucleic acid having at least 90% identity to the full length of a nucleic acid of any of the nucleotide sequences set forth in SEQ ID NOs: 1-64, 100-230, 300-365, 399-541, 616-708, 757-820, 853-911, or 942-980, or a fragment, complement or combination thereof; or 
 (ii) a polypeptide having at least 90% identity to the full length of a polypeptide of any of the amino acid sequences set forth in SEQ ID NOs: 65-99, 231-299, 366-398, 542-615, 709-756, 821-852, 912-941, or 981-1005, or a fragment, complement or combination thereof; and 
   (b) sorting the seeds to separate the seeds comprising clonal embryos from the seeds comprising non-clonal embryos;   
       wherein the maternal plant is pollinated prior to collecting the seeds, and wherein the one or more seeds produced by the maternal plant comprise an embryo that is a clone of the maternal plant. 
     
     
         2 . The method of  claim 1 , wherein the plant defective for RdDM activity has an insertion, deletion, substitution or mutation of one or more nucleotides that makes RdDM activity defective. 
     
     
         3 . The method of  claim 1 , wherein the plant defective for RdDM activity is male-sterile. 
     
     
         4 . The method of  claim 1 , wherein the maternal plant is unable to self-pollinate. 
     
     
         5 . The method of  claim 1 , the method further comprising sorting seeds comprising parthenogenically-derived clonal embryos from seeds not comprising parthenogenically-derived clonal embryos. 
     
     
         6 . The method of  claim 1 , the method further comprising sorting the seeds comprising parthenogenically-derived clonal embryos based on the absence of a paternal-inherited trait or marker in the seeds. 
     
     
         7 . The method of  claim 1 , wherein the plant defective for RdDM activity is maize, rice, sorghum, sugarcane, barley, oat, wheat, turfgrass, soybean, canola, cotton, tobacco, sunflower, safflower, or alfalfa. 
     
     
         8 . A method of screening for maternal plants that produce seeds comprising parthenogenically-derived clonal embryos comprising
 a) obtaining a maternal plant unable to pass on paternally-derived chromosomes to embryos, wherein the plant is silenced for RdDM activity for:
 a. a nucleic acid having at least 90% identity to the full length of a nucleic acid of any of the nucleotide sequences set forth in SEQ ID NOs: 1-64, 100-230, 300-365, 399-541, 616-708, 757-820, 853-911, or 942-980, or a fragment, complement or combination thereof; or 
 b. a polypeptide having at least 90% identity to the full length of a polypeptide of any of the amino acid sequences set forth in SEQ ID NOs: 65-99, 231-299, 366-398, 542-615, 709-756, 821-852, 912-941, or 981-1005, or a fragment, complement or combination thereof; and 
   b) harvesting the seeds; and   c) determining whether the seeds comprise clonal embryos, wherein the presence of seeds comprising clonal embryos indicates the maternal plant can produce parthenogenically-derived clonal embryos.   
     
     
         9 . The method of  claim 8 , wherein the plant defective for RdDM activity has an insertion, deletion, substitution or mutation of one or more nucleotides that makes RdDM activity defective. 
     
     
         10 . The method of  claim 8 , wherein the plant defective for RdDM activity is male-sterile. 
     
     
         11 . The method of  claim 8 , wherein the maternal plant is unable to self-pollinate. 
     
     
         12 . The method of  claim 8 , the method further comprising sorting seeds comprising parthenogenically-derived clonal embryos from seeds not comprising parthenogenically-derived clonal embryos. 
     
     
         13 . The method of  claim 8 , the method further comprising sorting the seeds comprising parthenogenically-derived clonal embryos based on the absence of a paternal-inherited trait or marker in the seeds. 
     
     
         14 . The method of  claim 8 , wherein the plant defective for RdDM activity is maize, rice, sorghum, sugarcane, barley, oat, wheat, turfgrass, soybean, canola, cotton, tobacco, sunflower, safflower, or alfalfa. 
     
     
         15 . A method for the enriching for asexual embryos and endosperm in the seed population of a plant comprising providing a plant comprising an asexual female gametophyte defective for RdDM activity, wherein the plant is disrupted for an activity that regulates endosperm development and wherein the seeds formed comprise parthenogenically-derived clonal embryos. 
     
     
         16 . The method of  claim 15 , wherein an activity that regulates endosperm development in the PRC2 complex is disrupted. 
     
     
         17 . The method of  claim 15 , wherein the asexual female gametophyte is disrupted for activity in for CYCLIN DEPENDENT KINASE A, cdka, or MULTIPLE SUPPRESSOR OF IRA1 (MSI1) CUL4-DDB1, Fertilization Independent Embryo (FIE), Fertilization Independent Seed-1 (FIS1), Fertilization Independent Seed-1 (FIS2), or Fertilization Independent Seed-3 (FIS3). 
     
     
         18 . The method of  claim 15 , wherein the plant is defective for RdDM activity is defective for:
 (a) a nucleic acid having at least 90% identity to the full length of a nucleic acid of any of the nucleotide sequences set forth in SEQ ID NOs: 1-64, 100-230, 300-365, 399-541, 616-708, 757-820, 853-911, or 942-980, or a fragment, complement or combination thereof; or   (b) a polypeptide having at least 90% identity to the full length of a polypeptide of any of the amino acid sequences set forth in SEQ ID NOs: 65-99, 231-299, 366-398, 542-615, 709-756, 821-852, 912-941, or 981-1005, or a fragment, complement or combination thereof.   
     
     
         19 . The method of  claim 15 , wherein the plant defective for RdDM activity is defective for AGO4 (ARGONAUTE 4), AGOG (ARGONAUTE 6), AGO8 (ARGONAUTE 8), AGO9 (ARGONAUTE 9), CMT3 (CHROMOMETHYLASE 3), DCL3 (DICER-LIKE 3), DRM2 (DOMAINS REARRANGED METHYLASE 2), EXS1 (EXTRA SPOROGENOUS CELLS1), IDN2 (INVOLVED IN DE NOVO 2), MET1 (METHYL TRANSFERASE 1), NPRD1a (NUCLEAR POLYMERASE D 1a), NRPD1b (NUCLEAR POLYMERASE D 1b), NRPD2 (NUCLEAR POLYMERASE D 2), NRPE1 (NUCLEAR RNA POLYMERASE E 1), NRPE2 (NUCLEAR RNA POLYMERASE E 2), RDR2 (RNA-DEPENDENT RNA POLYMERASE 2), RDR6 (RNA-DEPENDENT RNA POLYMERASE 6), SGS3 (SUPPRESSOR OF GENE SILENCING 3), SUVH2 (SUPPRESSOR OF VARIEGATION 3-9 HOMOLOG 2), and SUVH9 (SUPPRESSOR OF VARIEGATION 3-9 HOMOLOG 9). 
     
     
         20 . The method of  claim 15 , wherein the plant defective for RdDM activity is maize, rice, sorghum, sugarcane, barley, oat, wheat, turfgrass, soybean, canola, cotton, tobacco, sunflower, safflower, or alfalfa.