10/25/2016. Doubled haploid technique in corn breeding. Production of doubled haploid plants using wide crosses. Gynogenesis
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1 10/27/2016 Dubled haplid technique in crn breeding Intrductin Prductin f Dubled Haplid Lines Inductin f Haplids Chrmsme Dubling and self fertilizatin Why (dubled) haplids? The Dubled Haplid Facility at ISU Ursula Frei (Dubled Haplid Facility) 1218 Agrnmy Hall ufrei@iastate.edu Prductin f haplid and dubled haplid plants Andrgenesis: Anther and micrspre culture crn plant (diplid) pllen egg cells (haplid) (haplid) Seed with diplid embry successful regeneratin f haplids first reprted in Datura in 1964 and tbacc (1969) highly effective and applicable t varius plant species (barley, wheat, maize, rice, triticale, rye, tbacc, rapeseed and ther species in the Brassica) prblem: high gentype dependency within species and recalcitrant agricultural species such as leguminus plants, wdy plants Maize anther culture paternal maternal In vitr Anther culture Micrspre culture Gyngenesis (nin, sugar beet) In viv Pllen f same species ig in maize irradiated pllen (sunflwer) bulbsum technique in barley haplid inducer lines in maize Wide hybridizatin (wheat, ptat) micrspres and immature pllen grains can revert frm the gametphytic t the sprphytic pathway initiated thrugh varius bitic and abitic factrs (pretreatment f flwering dnr plants, develpmental stage f micrspres/immature pllen at cllectin, media cmpsitin, temperature and light regime ) haplids plants either regenerate directly frm micrspres (direct embrygenesis) r frm callus (rgangenesis) embrygenesis is preferred. Barely Micrspre culture (Guasmi et al 2013) Gyngenesis Prductin f dubled haplid plants using wide crsses Allium cepa Fays et al Wheat: pllinatin with pllen f maize + tissue culture step: embry rescue Barley: pllinatin f Hrdeum vulgare with wilde relative H. bulbsum tissue culture step necessary Ptat: pllinatin f Slanum tubersum with wilde relative S. phureja n tissue culture step necessary culture f varius parts f the un-pllinated flwer: vules, placenta attached vules, varies r whle flwer buds althugh successful in several plant species, applied mainly in nin and sugar beet gentype dependent and influenced by grwing cnditins f the dnr plant in thery several cells f the embry sac culd develp int a haplid plant (egg cell, synergids, antipdal cells and nn-fused plar nuclei) Egg cells develp directly int haplid plantlets (haplid parthgenesis) withut an intermediate callus phase 1
2 Maternal inductin f haplids in crn RWS/RWK-76 1 st generatin Inductin and haplid selectin + dnr x inducer - early flwering - makes readily side shts - gd pllen shedder, ver several days - inductin rate: 6-10% haplids ( 6-10%) hybrids (90-94%) - nt adapted t Mid West grwing cnditins - susceptible t wind - lw germinatin rate - difficult maintenance f parental lines and F1 Haplid inducing lines in maize Stck 6 W23ig CAUHOI ZMS KMS KEMS W14 STOCK6: Ce, 1959 PHI-3 MHI RWS FIGH1 PK6 CAUHOI: Chinese high il inducer FIGH1: Brdes et al KEMS:Shatskaya et al., 1994 KMS: Krichnevy Marker Saratvsky MHI: Mldavian Haplid Inducer PHI-3: Rtarenc - Prcera PK6: Barrett, 2008 RWS: Reber et al.,2005 UH400: Chang & Ce 2009 W14: Lashernes & Beckert 1988 W23ig: NSL30060 with ig ZMS: Tyrnv 1984 UH400 Haplid inducing lines in maize Selectable Marker R1-nj reasnable high haplid inductin rate tassel traits: abundant pllen shed ver a lnger perid f time (hand-pllinatins: side-shts with additinal later tassels, medium height, easy bendable/ntbreaking tassel) (fr islatins: plant height) agrnmic traits: seed set upn self-pllinatin, self-inductin rate, germinatin rate, ldging, disease resistance ne r mre selectable marker fr haplid identificatin Frd, R. 2000: THE AMERICAN BIOLOGY TEACHER, 62:
3 Haplid selectin Haplid selectin Endsperm Embry Dnr Inducer ut crssed r self-pllinated Lethal H embry F 1 embry Haplid selectin Haplid selectin Alternative marker in haplid selectin additinal phentypical marker (PI1) red rts Rtarenc, 2010 Alternative marker in haplid selectin 3
4 Kernel Weight (mg) Kernel Oil (%) 10/25/2016 Alternatives in haplid selectin Alternatives in haplid selectin shrt turn-ver time between seasns visual haplid selectin based n R1-nj by hand is labr intensive time-cnsuming, errrprne selectin rate: 1000/h (higher when selecting directly frm the cb) Kernel Weight Percent Oil gal: autmatin f haplid selectin Haplid Hybrid Haplid Hybrid Discriminatin between haplid and diplid seed with the VidemeterLab 3 (Videmeter A/S, Hørshlm, DK) High-thrughput single-kernel NIR analyzer Alternatives in haplid selectin bichemical differences between haplids and hybrids nn-destructive NIRS 0.20 Data cllectin takes ~6 secnds per kernel Micrbalance cllects individual seed weights, ±1 mg Cllects NIR spectrum, nm at 1 nm intervals Seeds can be indexed in micrtiter plates Hybrid Class Distance P1-102 Haplids ( 10 3 ) P1-102 Hybrids ( 10 3 ) P1-103 Haplids ( 10 3 ) 0.15 P1-103 Hybrids ( 10 3 ) P2-20 Haplids P2-20 Hybrids Haplid Class Distance Rger et al Dubled Haplid Line prductin in crn Chrmsme Dubling in Haplid Maize 1 st generatin Inductin and haplid selectin + dnr x inducer Spntaneus dubling - rare event during the first cell divisins r later in the develpment f the plant - the later spntaneus dubling ccurs, the larger the percentage f plant tissue that is haplid (chimera). Dubling by chemical treatment 2 nd generatin Dubling and self pllinatin haplids ( 6-10%) hybrids (90-94%) - chemical treatment f germinated seed r yung seedling with substances that interfere with cell divisin - Substances: clchicine, antimicrtubule herbicides (amiprphsmethyl (APM), prnamide, ryzalin, and trifluralin) - There are different applicatin mehds fr chrsmsme dubglin agents: submersin and injectin 4
5 Chrmsme Dubling in Haplid Maize Submersin Chrmsme Dubling in Haplid Maize Injectin Partially fertile tassel ~ ¼- ½ Preparing the ear fr pllinatin Cllecting pllen frm single anthers. 5
6 Pllinatin WHY DOUBLED HAPLOIDS? time efficiency 100% pure inbred lines in 2 generatins cmpared t 6-7 generatins f subsequent inbreeding higher selectin intensity cmpared t selectin in still segregating families In 2010, Pineer generated mre crn inbred lines via dubled haplid technlgy than we had prduced in the 80 year histry f ur breeding prgram. Speed in Line Develpment QTL MAPPING: Speed, Accuracy Funder line 1 x Funder line 2 Type f Ppulatin Strength Weakness Selfing F 1 F 2:3 RIL Speed f prductin d and a estimates Hmgeneus families Pwer f QTL detectin Hetergeneus families Slw prductin Inbred line Speed f prducing hmgeneus families Pwer f QTL detectin Labrius prductin prcess Lwer recmbinatin (>RIL) BC Speed f prductin Hetergeneus families a and d cnfunded Inbred line Germplasm Enhancement in Maize (GEM) Mike Blanc, Candy Gardner Extic Maize Ppulatins BC1 Recurrent elite parent: B47 r Z51 Line 1 Line 2 X r Trait / Gene Stacking Prbability fr Fixatin f Target Genes N. f genes F Selfing (F 2 ) Inductin Isgenic BC1- lines - GEM Gal: Fixatin f target alleles 6
7 Service Sme numbers: Requested # f lines: L= 100 Inductin Rate: IR=0.06 Germinatin rate f dnr ppulatin: GRD=0.95 Pllinatin rate in nursery: PR=0.85 Average number f kernels/cb: K= dnr plants fr inductin crsses, kernels have t be selected Germinatin rate f haplids: Haplids surviving treatment: Haplids established in the field: Haplids shedding pllen: Pllinated haplids with seed set: Success rate: GRH=0.95 SV=0.8 F=0.95 P=0.15 S=0.95 SR= haplids have t be germinated, treated and planted ut in the field Research prjects Training Inducer Develpment: develpment f new inducer lines with better adaptin t the grwing cnditins in the Mid West develpment f inducer lines fr specialty crps like ppcrn and sweet crn develpment f inducer with additinal selectable marker genes Haplid selectin: autmated systems fr haplid selectin: NIRS fine mapping and gene islatin fr genes invlved in inductin ability Chrmsme dubling: the ability fr spntaneus dubling a heritable trait increase the efficiency f clchicine applicatin fr chrmsme dubling during summer seasn we have grups visiting plant breeder and scientists frm all ver the wrld jin us fr 2-3 weeks during summer t learn technlgy hands-n internships fr undergraduate students 7
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