US2013333068A1PendingUtilityA1

Genes and uses for plant enhancement

64
Assignee: COFFIN MARIEPriority: Apr 29, 2008Filed: Apr 24, 2009Published: Dec 12, 2013
Est. expiryApr 29, 2028(~1.8 yrs left)· nominal 20-yr term from priority
Inventors:Marie Coffin
C12N 15/8261C12N 15/8241C12N 15/8271C12N 15/8251C12N 15/8247Y02A40/146C12N 15/8273C12N 15/8274C12N 15/8209C12N 15/8275
64
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Claims

Abstract

Transgenic seed for crops with enhanced agronomic traits are provided by trait-improving recombinant DNA in the nucleus of cells of the seed where plants grown from such transgenic seed exhibit one or more enhanced traits as compared to a control plant. Of particular interest are transgenic plants that have increased yield. The present invention also provides recombinant DNA molecules for expression of a protein, and recombinant DNA molecules for suppression of a protein.

Claims

exact text as granted — not AI-modified
What is claimed is: 
     
         1 . A plant cell nucleus with stably-integrated, recombinant DNA comprising a promoter that is functional in plant cells and that is operably linked to protein coding DNA encoding a protein having an amino acid sequence comprising a Pfam domain module NTP_transferase::Hexapep::Hexapep::Hexapep;
 wherein said plant cell nucleus is selected by screening a population of transgenic plants that have said recombinant DNA in its nuclei and express said protein for an enhanced trait as compared to control plants that do not have said recombinant DNA in their nuclei; and   wherein said enhanced trait is selected from group of enhanced traits consisting of enhanced water use efficiency, enhanced cold tolerance, enhanced heat tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein and enhanced seed oil.   
     
     
         2 . A plant cell nucleus with stably integrated, recombinant DNA, wherein
 (a) said recombinant DNA comprises a promoter that is functional in said plant cell and that is operably linked to a protein coding DNA encoding a protein having an amino acid sequence comprising a Pfam domain module selected from the group consisting of Syntaxin::SNARE, Pro_isomerase, Pkinase, ATP-synt_G, Carboxyl_trans, CDC50, GATase::GMP_synt_C, F-box::WD40::WD40::WD40, dsrm::dsrm, Pyr_redox — 2::Pyr_redox_dim, WAK::Pkinase, Pkinase_Tyr, PTPA, Biotin_lipoyl::E3_binding::2-oxoacid_dh, AAA, LRRNT — 2::LRR — 1::LRR — 1::LRR — 1, PRA1, TIM, YTH, ThiF, Hep — 59, Pkinase, PALP::Thr_dehydrat_C::Thr_dehydrat_C, zf-Tim10_DDP, CAF1, Pkinase, Pyr_redox — 2::Pyr_redox_dim, Response_reg, YIF1, NPH3, LRRNT — 2::LRR — 1::LRR — 1::LRR — 1::LRR — 1::Pkinase_Tyr, NOI, Fer2, AA_permease, Caleosin, IF4E, Pkinase, Ribul_P — 3_epim, F-box::WD40::WD40::WD40::WD40::WD40::WD40, MIP, p450, Ribosomal_S5::Ribosomal_S5_C, Hpt, TBC, Acyltransferase, Epimerase, Pkinase, Dus, Se1R, PH, Tyr-DNA_phospho, RRM — 1, Pkinase, ECH, SRPRB, DUF599, Pkinase::efhand::efhand::efhand::efhand, ORC2, PMEI, p450, GLTP, Suc_Fer-like, CBS::CBS, ADH_N::ADH_zinc_N, LRRNT — 2::LRR — 1::LRR — 1::LRR — 1::Pkinase, F-box, adh_short, polyprenyl_synt, GHMP_kinases_N::GHMP_kinases_C, PsbW, Na_H_Exchanger::TrkA_C, Ribosomal_L12, CDC50, DCP1, Pyr_redox — 2::Pyr_redox_dim, Steroid_dh, tRNA-synt — 1c:::tRNA-synt — 1c_C, Cupin — 1::Cupin — 1, peroxidase, Sugar_tr, Pro_isomerase, Iso_dh, PTS — 2-RNA, Aminotran — 5, ENOD93, KH — 1::KH — 1::KH — 1::KH — 1::KH — 1, Mito_carr::Mito_carr::Mito_carr, DUF569::DUF569, C1 — 2::C1 — 2::C1 — 3::C1 — 2::C1 — 3::C1 — 2, DnaJ::zf-C2H2, Aldedh, AAA::Rep_fac_C, DUF641, Aminotran — 1 — 2, TB2_DP1_HVA22, zf-C3HC4, RRM — 1::RRM — 2, Pkinase, PPR::PPR::PPR::PPR::PPR::PPR::PPR::PPR::PPR::PPR::PPR, Pyr_redox — 2, IQ::IQ, SMP::SMP::SMP, AWPM-19, CLP_protease, Pkinase::NAF, PRMT5, PPR::PPR::PPR::PPR::PPR::PPR, Aldedh, ATP-synt_C::ATP-synt_C, adh_short, RCC1::RCC1::RCC1::RCC1::FYVE::DZC, p450, PfkB, PB1, Hexapep::Hexapep::Hexapep::Hexapep, Aminotran — 1 — 2, DUF862, Aldedh, ADH_N::ADH_zinc_N, CDC48—N::AAA::AAA, AA_permease, UQ_con, Mem_trans, GFA, OPT, DUF887, Di19, U-box::Arm::Arm, DAO, G-alpha, Aminotran — 3, 2OG-FeII_Oxy, Lectin_legB::Pkinase, NAC::UBA, efhand_like::PI-PLC-X::PI-PLC-Y::C2, TLC, ATP-grasp — 2::Ligase_CoA, Pkinase, PRC::PRC, MFS — 1::Sugar_tr, Copine::zf-C3HC4, RRM — 1::RRM — 1::RRM — 1, Pribosyltran, AA_kinase, PhzC-PhzF, Gp_dh_N::Gp_dh_C, Sugar_tr, Pre-SET::SET, Alpha-amylase::Alpha-amyl_C2, Cyclin_N, FAE1_CUT1_RppA::ACP_syn_III_C, RPE65, efhand_like::PI-PLC-X::PI-PLC-Y::C2, Mito_carr::Mito_carr::Mito_carr, Invertase_neut, DSPc, LANC_like, Aminotran — 5, Glutaminase, PPR::PPR::PPR::PPR::PPR::PPR::PPR::PPR, PGI, SSB, p450, La::RRM — 1::RRM — 3, Methyltransf — 11, SBF, NTP_transferase::Hexapep::Hexapep::Hexapep, Mov34, Hydrolase, AAA, RRM — 1::RRM — 1, Pkinase::NAF, F-box::FBA — 1, p450, F-box::LRR — 2, zf-DHHC, TBP::TBP, DUF607, FMO-like, adh_short, ATP-synt_ab_N::ATP-synt_ab, RNase_PH, PP2C, UQ_con, Aminotran — 1 — 2, p450, FMO-like, Gp_dh_N::Gp_dh_C, Mito_carr::Mito_carr::Mito_carr, zf-CCCH::zf-CCCH::zf-CCCH::zf-CCCH::zf-CCCH, GST_C, GH3, PAP_fibrillin, ThiF, Ribul_P — 3_epim, DUF260, p450, MACPF, Steroid_dh, Response_reg, Carb_anhydrase, Aldedh, Pkinase, Methyltransf — 11, PK::PK_C, Pkinase, DEK_C::SWIB::SWIB, TATA_RF, Tryp_alpha_amyl, Y_phosphatase2, TATA_RF, NifU_N, ENT, Pkinase, F-box::Kelch — 2::Kelch — 2, WD40::WD40, MtN3_slv::MtN3_slv, TB2_DP1_HVA22, Pkinase, Aldedh, AAA, Pyridoxal_deC, zf-CCHC::zf-CCHC::zf-CCHC::zf-CCHC, Peptidase_M24, LRRNT — 2::LRR — 1::LRR — 1::LRR — 1::LRR — 1::Pkinase, Arf, DUF1279, UPF0185, Rhodanese, adh_short, Tryp_alpha_amyl, Pkinase, UPF0139, CBS, Glyco_transf — 29, iPGM_N::Metalloenzyme, Proteasome, Pkinase, UBX, ELFV_dehydrog_N::ELFV_dehydrog, p450, ADH_N::ADH_zinc_N, zf-C3HC4, NDK, NAD_Gly3P_dh_N::NAD_Gly3P_dh_C, GlutR_N::Shikimate_DH::GlutR_dimer, p450, SNF5, p450, Pribosyltran, AIG1, Response_reg::CCT, mTERF, DUF220, Pkinase::NAF, zf-CCCH::zf-CCCH, Pkinase, Pkinase, MATH::MATH, Asparaginase, Pkinase, Gp_dh_N::Gp_dh_C, Pkinase, KH — 1, Fcf1, PK::PK_C::PEP-utilizers, CRAL_TRIO_N::CRAL_TRIO, Raffinose_syn, DUF584, BT1, Aminotran — 1 — 2, C1 — 2::C1 — 3::C1 — 3::C1 — 2::C1 — 3::C1 — 3::C1 — 2, FMO-like, PLACE, F-box::Kelch — 2::Kelch — 1::Kelch — 2, XS::XH, STT3, ABC_tran::ABC_tran, Aldo_ket_red, NTF2::RRM — 1, DUF594, Biotin_lipoyl::2-oxoacid_dh, RRM — 1, FHA, Mov34, MT-A70, Brix, X8, Auxin_inducible, Peptidase_M18, Sec61_beta, F-box::Kelch — 1::Kelch — 1, Brix, TIM, CTP_transf — 1, Tetraspannin, PALP, DUF822, Pkinase, B3::B3, MFS — 1, SFT2, F-box::Kelch — 1::Kelch — 1, DUF588, Thioredoxin, Alba, Ion_trans — 2::Ion_trans — 2, Peptidase_C48, YDG_SRA::Pre-SET::SET, Aldedh, Abhydrolase — 3, p450, U-box::Arm::Arm::Arm::Arm, AA_permease, LAG1, peroxidase, PAD_porph, Pkinase, F-box, AIG2, p450, AARP2CN::DUF663, UPF0153, NAD_binding — 2::6 PGD, Pkinase, B_lectin::S_locus_glycop::PAN — 1::Pkinase, EMP24_GP25L, DUF6::DUF6, DUF163, Sina, adh_short, Rho_GDI, zf-CCHC::zf-CCHC::zf-CCHC::zf-CCHC::zf-CCHC::zf-CCHC::zf-CCHC, VHS::GAT, Pyrophosphatase, DMRL_synthase, Cyclin_N::Cyclin_C, NDK, TPP_enzyme_N::TPP_enzyme_M::TPP_enzyme_C, SH3 — 1, UDPG_MGDP_dh_N::UDPG_MGDP_dh::UDPG_MGDP_dh_C, UIM::efhand, DUF579, Pkinase, DHBP_synthase::GTP_cyclohydro2, Peptidase_C14, Glycolytic, Pkinase, Lipase — 3, LRRNT — 2::LRR — 1::LRR — 1::LRR — 1::LRR — 1, Snf7, BT1, PPDK_N::PEP-utilizers::PEP-utilizers_C, V-ATPase_G, RNA_pol_I_A49, Zip, WD40::WD40::WD40, Branch, RCC1::RCC1::RCC1::RCC1, TFIID-18kDa, Pkinase, Cullin, TFIID-31 kDa, Asp, Pkinase, Ubie_methyltran, PPR::PPR, PPR::PPR::PPR::PPR::PPR::PPR::PPR, Fructosamin_kin, PPR::PPR::PPR::PPR::PPR::PPR, Pkinase_Tyr, p450, AA_permease, Glycos_transf — 1, ECH::3HCDH_N::3HCDH, MIP, ADH_N::ADH_zinc_N, Aa_trans, DUF1517, Redoxin, RRS1, Pribosyltran, Sulfate_transp::STAS, DUF167, MMR_HSR1::DUF933, Aminotran — 1 — 2, AA_permease, DUF620, Aldedh, Aminotran — 1 — 2, LRR — 1, CDP-OH_P_transf, DHBP_synthase::GTP_cyclohydro2, NHL::NHL, WD40::U3_snoRNA_C, Na_sulph_symp, adh_short, Thioredoxin, Ribosomal_L7Ae, OTCace_N::OTCace, Mito_carr::Mito_carr::Mito_carr, Pescadillo_N::BRCT, PPR::PPR::PPR::PPR::PPR::PPR::PPR::PPR::PPR::PPR, mTERF, Chalcone, Per1, PPR::PPR, Na_H_Exchanger::TrkA_N, DEAD::Helicase_C, Cyclin_N::Cyclin_C, PP-binding, SRF-TF, SATase_N::Hexapep::Hexapep::Hexapep, PK::PK_C, adh_short, DUF6::DUF6, Pyr_redox — 2, G-patch, WD40::WD40::WD40::WD40, G-alpha, VQ, WD40::WD40::WD40::WD40::WD40, Aminotran — 3, Pribosyltran, LRRNT — 2::LRR — 1::LRR — 1::LRR — 1::LRR — 1::LRR — 1, elF3_subunit, H_PPase, Nodulin-like, Whi5, Tryp_alpha_amyl, NIR_SIR_ferr::NIR_SIR::NIR_SIR_ferr, RRM — 1::RRM — 1, Band — 7, Asp, MFS — 1, F420_oxidored, Pkinase, PRK, Na_Ca_ex::Na_Ca_ex, Bombesin, Pkinase, Epimerase, Pkinase, Inositol_P, Arm::Arm::Arm::Arm, PB1, Hin1, p450, RNA_pol_Rpb5_N::RNA_pol_Rpb5_C, Adap_comp_sub, Peptidase_M16::Peptidase_M16—C, Pkinase, PGK, Biotin_lipoyl::E3_binding::2-oxoacid_dh, Alg6_Alg8, F-box, MMR_HSR1, G6PD_N::G6PD_C, DAD, malic::Malic_M::PTA_PTB, ADH_N::ADH_zinc_N, LRR — 1::LRR — 1::LRR — 1::LRR — 1:LRR — 1::LRR — 1::LRR — 1, PfkB, PA, Pyr_redox — 2::Pyr_redox_dim, SIP1, PP2C, BT1, AhpC-TSA, GASA, Aminotran — 3, Sad1_UNC, Pkinase, zf-MYND::PDCD2—C, Pkinase, PGK, ArfGap, GATase::GMP_synt_C, p450, DUF1637, 2OG-FeII_Oxy, Aldedh, DUF231, Glyco_transf — 8, Sec1, DUF1475, Cellulose_synt, RRM — 1::RRM — 1::RRM — 1, Usp, AA_permease, Acetyltransf — 1::Bromodomain, Glyco_tran — 28_C, RNA_pol_N, NTP_transferase, malic::Malic_M, Histone, Epimerase, UPF0061, ClpS, LEA — 5, Auxin_inducible, PAP_fibrillin, Aldo_ket_red, Gln-synt_C, DREPP, p450, SFT2, eIF2A, Cenp-O, CPSase_sm_chain::GATase, WD40::WD40::WD40, Pkinase, Porin — 3, Pkinase, Aminotran — 1 — 2, GST_N::GST_C, F-box::Kelch — 1::Kelch — 1, IU_nuc_hydro, SYF2, PDT, Subtilisin_N::PA, Sugar_tr, WD40::WD40, p450, DUF829, Pyr_redox — 2::Pyr_redox_dim, Aminotran — 1 — 2, Cyclin_N::Cyclin_C, YgbB, Sugar_tr, SRF-TF, DUF231, DUF584, BT1, zf-C3HC4, Metallophos, FA_hydroxylase, p450, ACBP::Ank::Ank, LSM, SAM — 1, LRR — 1::LRR — 1::Pkinase, Cullin, F-box::FBA — 1, peroxidase, Rib — 5-P_isom_A, zf-A20::zf-AN1, Dimerisation::Methyltransf — 2, PGAM, PTR2, Copine, PALP, Exo_endo_phos, G6PD_N::G6PD_C, PLAC8, Aldo_ket_red, Ank::Ank, FBPase, zf-C3HC4, ACT::ACT, Suc_Fer-like, Pkinase_Tyr, G-alpha, RAMP4, Glutaredoxin, RRM — 1::RRM — 1, MT-A70, Response_reg::CCT, Arf, AP2, DUF177, 200-FeII_Oxy, LRRNT — 2::LRR — 1::LRR — 1::LRR — 1::LRR — 1::LRR — 1, LRR — 1, Aldedh, DUF1350, FAE1_CUT1_RppA::ACP_syn_III_C, Sybindin, zf-C3HC4, Nfu_N::NifU, Rho_GDI, PK::PK_C, NOSIC::Nop, PK::PK_C, PRK::Pribosyltran, PfkB, p450, DUF231, TFIID — 30 kDa, Aldedh, zf-AN1, GHMP_kinases_N::GHMP_kinases_C, DUF423, Pkinase, Cu-oxidase — 3::Cu-oxidase::Cu-oxidase — 2, PCI, RNase_PH::RNase_PH_C, DUF59, NTP_transferase, CH::EB1, Pkinase::Pkinase_C, DUF866, SMP::SMP::SMP, Aminotran — 3, Transket_pyr::Transketolase_C, Copine, His_biosynth, Tbf5, DUF543, p450, C2, DUF616, Gp_dh_N::Gp_dh_C, Smg4_UPF3, DUF231, DUF89, WD40::WD40, Aldedh, ACT::ACT::ACT::ACT, Pkinase, Pyridoxal_deC, Skp1_POZ::Skp1, NAP, FolB, p450, Pentapeptide::Pentapeptide, NTP_transferase::MannoseP_isomer, SQS_PSY, Cyclase, GASA, Rick — 17 kDa_Anti, FMO-like, B_lectin::S_locus_glycop::PAN — 2, Peptidase_C12, Ribosomal_S6e, Glyoxalase, Response_reg::CCT, PHD::SET, Redoxin, G_glu_transpept, Synaptobrevin, p450, RALF, PALP, Branch, DUF579, Aminotran — 3, Nramp, Enolase_N::Enolase_C, Str_synth, FAD_binding — 3, MOSC_N::MOSC, Spc97_Spc98, Glycolytic, F-box::WD40::WD40::WD40::WD40, AA_kinase::ACT::ACT, RALF, and ATP_bind — 1;   (b) said recombinant DNA comprises a promoter that is functional in said plant cell and that is operably linked to a protein coding DNA encoding a protein comprising an amino acid sequence with at least 90% identity to a consensus amino acid sequence selected from the group consisting of SEQ ID NO: 94617 through 94734;   (c) said recombinant DNA comprises a promoter that is functional in plant cells and that is operably linked to a protein coding DNA encoding a protein comprising an amino acid sequence selected from the group consisting of 819, 867, 906, 914, 967, 1009, 1040, 1086, 1107, 1227, 1233, 1250, 1293, 1311, 1320, 1342, 1364, 1365, 1373, 1398, 1402, 1439, 1443, 1473, 1495, 1539, 1596, and homologs thereof listed in table 19;   (d) said recombinant DNA comprises a promoter that is functional in said plant cell and that is operably linked to a protein coding recombinant DNA encoding a protein having an amino acid sequence having at least 70% identity to an amino acid sequence of SEQ ID NO 1227;   (e) said recombinant DNA suppresses comprises a promoter that is functional in said plant cell and operably linked to DNA that transcribe into RNA that suppresses the level of an endogenous protein wherein said endogenous protein has an amino acid sequence comprising a pfam domain module selected from the group consisting of DUF1475, DUF1350, Arm::Arm::Arm::Arm, NifU_N, ATP_bind — 1, DUF599, LRR — 1, Pyr_redox — 2::Pyr_redox_dim, malic::Malic_M, Glycolytic, IF4E, Pkinase, UPF0139, GlutR_N::Shikimate_DH::GlutR_dimer, PLAC8, PLAC8, DUF220, OPT, Glyco_transf — 8, Per1, Usp, mTERF, PB1, CAF1, Pkinase, F-box::Kelch — 1::Kelch — 1, PHD::SET, Nfu_N::NifU, RCC1::RCC1::RCC1::RCC1, X8, Pkinase::NAF, UIM::effiand, Glutaredoxin, DUF1517, Cupin — 1::Cupin — 1, ClpS, Spc97_Spc98, Subtilisin_N::PA, CBS, PTPA, p450, AA_permease, Sina, p450, Mov34, Thioredoxin, SATase_N::Hexapep::Hexapep::Hexapep, Epimerase, RRM — 1::RRM — 2, Synaptobrevin, Ribosomal_S6e, Redoxin, MFS — 1, PPR::PPR, NPH3, and DUF866; or   (f) said recombinant DNA comprises a promoter that is functional in said plant cell and operably linked to DNA that transcribe into RNA that suppresses the level of an endogenous protein wherein said endogenous protein has an amino acid sequence with at least 70% identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 811, 813, 819, 891, 893, 909, 911, 1439, 1584, and homologs thereof listed in table 16;
 and wherein said plant cell nucleus is selected by screening a population of transgenic plants that have said recombinant DNA and an enhanced trait as compared to control plants that do not have said recombinant DNA in their nuclei; and wherein said enhanced trait is selected from group of enhanced traits consisting of enhanced water use efficiency, enhanced cold tolerance, enhanced heat tolerance, enhanced resistance to salt exposure, enhanced shade tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein and enhanced seed oil. 
   
     
     
         3 . The plant cell nucleus of  claim 2  wherein said protein coding DNA encodes a protein having an amino acid sequence selected from the group consisting of SEQ ID NO: 804 through SEQ ID NO: 94613. 
     
     
         4 . The plant cell nucleus of  claim 2  further comprising DNA expressing a protein that provides tolerance from exposure to an herbicide applied at levels that are lethal to a wild type of said plant cell. 
     
     
         5 . The plant cell nucleus of  claim 4  wherein said herbicide is a glyphosate, dicamba, or glufosinate compound. 
     
     
         6 . A transgenic plant cell or plant comprising a plurality of plant cells with the plant cell nucleus of  claim 2 . 
     
     
         7 . The transgenic plant cell or plant of  claim 6  which is homozygous for said recombinant DNA. 
     
     
         8 . A transgenic seed comprising a plurality of plant cells with a plant cell nucleus of  claim 2 . 
     
     
         9 . The transgenic seed of  claim 8  from a corn, soybean, cotton, canola, alfalfa, wheat or rice plant. 
     
     
         10 . A transgenic pollen grain comprising a haploid derivative of a plant cell nucleus of  claim 2 . 
     
     
         11 . A method for manufacturing non-natural, transgenic seed that can be used to produce a crop of transgenic plants with an enhanced trait resulting from expression of recombinant DNA in a nucleus of  claim 2 , wherein said method for manufacturing said transgenic seed comprising:
 (a) screening a population of plants for said enhanced trait and said recombinant DNA, wherein individual plants in said population can exhibit said trait at a level less than, essentially the same as or greater than the level that said trait is exhibited in control plants which do not contain the recombinant DNA, wherein said enhanced trait is selected from the group of enhanced traits consisting of enhanced water use efficiency, enhanced cold tolerance, enhanced heat tolerance, enhanced high salinity tolerance, enhanced shade tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein and enhanced seed oil,   (b) selecting from said population one or more plants that exhibit said trait at a level greater than the level that said trait is exhibited in control plants, and   (c) collecting seeds from selected plant selected from step b.   
     
     
         12 . The method of  claim 11  wherein said method for manufacturing said transgenic seed further comprising
 (a) verifying that said recombinant DNA is stably integrated in said selected plants, and 
 (b) analyzing tissue of said selected plant to determine the expression or suppression of a protein having the function of a protein having an amino acid sequence selected from the group consisting of SEQ ID NO:804-1606. 
 
     
     
         13 . The method of  claim 11  wherein said seed is corn, soybean, cotton, alfalfa, canola wheat or rice seed. 
     
     
         14 . A method of producing hybrid corn seed comprising:
 (a) acquiring hybrid corn seed from a herbicide tolerant corn plant which also has stably-integrated, recombinant DNA in a nucleus of  claim 2 ;   (b) producing corn plants from said hybrid corn seed, wherein a fraction of the plants produced from said hybrid corn seed is homozygous for said recombinant DNA, a fraction of the plants produced from said hybrid corn seed is hemizygous for said recombinant DNA, and a fraction of the plants produced from said hybrid corn seed has none of said recombinant DNA;   (c) selecting corn plants which are homozygous and hemizygous for said recombinant DNA by treating with an herbicide;   (d) collecting seed from herbicide-treated-surviving corn plants and planting said seed to produce further progeny corn plants;   (e) repeating steps (c) and (d) at least once to produce an inbred corn line; and   (f) crossing said inbred corn line with a second corn line to produce hybrid seed.

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