Mihály Róbert

English

University of Szeged

Volume 46(3-4):9-10, 2002Acta Biologica Szegediensishttp://www.sci.u-szeged.hu/ABSCereal Research Non-profit Company, Szeged, HungaryR Mihály*, E Kótai, O Kiss,  J PaukABSTRACT                            The fate of transgene is an up-to-date question in plant genetic and breedingresearch. For a breeder, the ability to transfer foreign gene and its Mendelian inheritance is avery important aspect. In crossing experiments the homogenous T2 transgenic line 124 wascrossed with different non-transgenic females. The isolated hybrid embryos from the crosseswere tested in medium containing bialaphos. This experiment showed the dominant characterof bar gene. All the hybrid embryos germinated onto 5mg/l bialaphos medium expressedresistance, while the control variety (GK Góbé) showed sensitivity to the bialaphos as expected.The fertile T1 generation expressed 3:1 segregation ratio for bar +/bar- plants. The transformedforeign gene (bar) was stable in the wheat genome after two self pollinations and PAT asasays.It is therefore, possible to transform transgene successfully via sexual way into non-transgenicgenotypes. Cell level selection of bar gene was possible in anther culture. The regeneratedhaploids and spontaneous diploids were herbicide resistant. Using in vitro haploid technique(anther culture), it’s possible to produce homogenous bar resistant wheat lines by cell-levelselection. Acta Biol Szeged 46(3-4):9-10 (2002)KEY WORDStranformationwheatanther culture*Corresponding author. E-mail: robert.mihaly@gk-szeged.hu9In vitro selection of transformed foreign gene (bar) in wheat anthercultureProceedings of the 7th Hungarian Congress on Plant Physiology, 2002S1-03Genetically transformed bread wheat (Triticum aestivum L.)plants have been produced during the past few years (Vasilet al. 1993; Weeks et al. 1993; Becker et al. 1994; Nehra et.al 1994). At the end of 20th century the fundamental discov-eries made in the field of molecular biology and cell andtissue culture have initiated a biological revolution in plantbreeding and agricultural production. The first geneticallyengineered food crops have been introduced into the marketin some countries (Nehra et al. 1995), but several questionshave been under discussion. The fate of transgene is an up-to-date question in plant genetic and breeding research.Earlier, six independent fertile transgenic lines of springwheat bearing selectable marker gene bar had been obtained.This paper gives a report on the sexual transfer, selection anduse of the bar gene in non-transformed genotypes for thedevelopment of homozygosity in the offspring generations.Materials and MethodsPlant materialDonor plants of CY-45 spring wheat (Triticum aestivum L.)line which was previously selected for it’s high response tosomatic tissue culture was used. The wheat line was grownin fitotron chamber and in greenhouse and immature embryoswere excised, cultured, bombarded, bialaphos selected andregenerated as described earlier by Vasil et al. (1993).Crossing (emasculation, pollination) and embryo cultureswere made according to protocols in cell and tissue culturehandbooks. The microspore and anther culture were madeusing our previous published methods (Pauk et al. 1992).Transgenic experimentsTo optimize the transformation system, transient expressionexperiments were carried out using pAHC25 plasmid con-taining UidA reporter gene and bar marker gene. In the stabletransformation experiments, pAHC 20 plasmid containingthe selectable bar gene for resistance to the herbicide (Finale14 SL 150g/l ammonium glufosinate made by AgrEvoGmbh. was used. The selectable bar gene encodes theenzyme phosphinothricin acetyltransferase (PAT) thatinactivates phosphinothricin (PPT), the active ingredient ofthe herbicide by acetylation. Plasmid DNAs were precip-itated, absorbed to gold particles and delivered to target tissueusing a DuPont PDS-1000/He device.ResultsDevelopment of homogenous transgenic wheatlinesSix transgenic spring wheat lines (106-3a, 116, 117, 124,128, 129) were obtained from transformation experiments ofbombarded immature embryo-derived calli using pAHC20plasmid molecule, followed by bialaphos selection. All of theR0 regenerants were identified as transgenic plants using PATassay, RNA-RNA hybridazation and herbicide spray. Five ofthe R0 six lines produced complete fertile heads while oneline (106-3a) was partially fertile. The offsprings of thecomplete fertile transgenic (T) lines (116, 117, 124, 128, 129)were used in the sexual gene transfer experiments. The T1generation of 4 transgenic lines (116, 124, 128, 129) showeda 3:1 segregation in PAT assay. The line 117 showed closeto 3:1 segregation ratio. The PAT positive lines were self-pollinated and the subsequent generation was checked bydirect herbicide spray. In the T2 generation, the four completefertile lines (116, 124, 128, 129) expressed a homogenous10herbicide resistance. The line 117 was homogenous. The T2homogenous herbicide resistant plants were used as maleparent in the crossing experiments.Sexual transfer of the bar transgeneFor the breeder, the ability to transfer foreign gene and itsMendelian inheritance is a very important aspect. In crossingexperiments the homogenous T2 transgenic line 124 wascrossed with different non-transgenic females. The isolatedhybrid embryos from the crosses were tested in mediumcontaining bialaphos. The result of this experiment showedthe dominant character of bar gene. All the hybrid embryosgerminated onto 5mg/l bialaphos medium expressed resist-ance, while the control variety (GK Góbé) showed sensitivityto the bialaphos as expected. In the next experiment differenttransgenic lines (male) were tested on a wild type (GKGaraboly non tansgenic) variety. The sexual transfer of thetransgene in case of line 116, 124 and 128 was good, all thehybrid embryos expressed resistance on the bialaphosembryo culture. However, two thirds of the hybrid embryosof transgenic line 117 were sensitive to the bialaphos indi-cating that the line 117 was not homogenous.Selection of the transgene in anther andmicrospore cultureTo develop the cell-level selection system of the bar gene,anther and microspore culture experiments were made usingtransgenic lines and bialaphos selection. T2 PAT resistanttransgenic lines were used as donors in anther culture. Threetreatments were applied, in the first treatment, bialaphos wasnot used in the anther culture, in the second treatments themedium was supplemented with bialaphos added into liquidmedium at the time of isolation while in the third treatment,bialaphos supplement was added 2 weeks after isolation. Inall the treatments there was no significant difference inembryoid production. Similar results were obtained inmicrospore culture, but the 3 mg/l bialaphos gave the bestselection result.Production of transgenic haploids and doubledhaploids in anther cultureThe haploids and spontaneous diploids regenerated fromselective anther and microspore culture experiments ex-pressed total herbicide resistance to Finale 14SL. Via chro-mosome doubling of transgenic haploids, homogenousdiploid (DH) wheat lines were obtained. The stability of barthe transgene in doubled haploid wheat genome is understudy.DiscussionThe fertile T1 generation expressed 3:1 segregation ratio forbar+/bar- plants. The transformed foreign gene (bar) wasstable in the wheat genome after two self pollinations andPAT assays. It is therefore, possible to transform transgenesuccessfully via sexual way into non-transgenic genotypes.Cell-level selection of bar gene was possible in anther andmicrospore culture. The regenerated haploids and sponta-neous diploids are herbicide resistant. Finally, by using invitro haploid technique (anther and microspore culture), it’spossible to produce homogenous bar resistant wheat lines bycell-level selection.AcknowledgmentsThe authors wish to thank Wheat Consotium - involving thistransformation project - sponsored by Educational Ministry,Budapest.ReferencesBecker D, Brettschneider R, Lörz H (1994) Fertile transgenic wheat plantsfrom microprojectile bombardment of scutellar tissue. Plant Jour 5:299-307.Nehra NS, Chibbar RN, Leung N, Caswell K, Mallard C, Steinhauser L,Baga M, Kartha KK (1994) Self-fertile transgenic wheat plantsregenerated from isolated scutellar tissues following microprojectilebombardment with two distinct gene constructs. Plant Jour 5:285-297.Nehra NS, Chibbar RN, Kutty KK (1995) Wheat transformation: Methodsand prospects. Plant Breeding Abstracts 65:803-808.Pauk J, Manninen O, Mattila I, Salo Y, Pulli S (1991) Androgenesis inhexaploid spring wheat F2 populations and their parents using amultiple-step regeneration system. Plant Breeding 107:18-27.Vasil V, Srivastava V, Castillo, AM, Fromm M, Vasil IK (1993): Rapidproduction of transgenic wheat plants by direct bombardment ofcultured immature embryos. Bio/technol 11:1553-1558.Weeks JT, Anderson OD, Blechl AE (1993) Rapid production of multipleindependent lines of fertile transgenic wheat (Triticum aestivum). PlantPhysiol 102:1077-1084.

In vitro selection of transformed foreign gene (bar) in wheat anther culture

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In vitro selection of transformed foreign gene (bar) in wheat anther culture

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