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1 Advances in Environmental Biology, 5(5): , 2011 ISSN This is a refereed journal and all articles are professionally screened and reviewed ORIGINAL ARTICLE Production of Wheat Doubled Haploid Lines and Assessment of Their Tolerance to Yellow Rust 1 Bakhshi T., 2 Bozorgipour R., 2 Bakhtiar F., 2 Afshari F., 3 Sharif moghaddasi, M. 1 MSc. Student of Plant Biotechnology, Islamic Azad University, Science and Research Branch, Tehran. 2 Professor, Instructor and Associate professor, Seed and Plant Improvement Institute, Karaj, Iran. 3 Assist. Prof. Islamic Azad University/saveh branch Iran. Bakhshi T., Bozorgipour R., Bakhtiar F., Afshari F., Sharif moghaddasi, M.: Production of Wheat Doubled Haploid Lines and Assessment of Their Tolerance to Yellow Rust ABSTRACT This research was conducted to produce doubled haploid lines of wheat through chromosome elimination (wheat maize crosses ). Three wheat hybrids : DH-29 : MILAN/SH7//SHIROODI DH-30 : INIA//SW /STAR/3/MILAN/SHA7 DH-32 : NANJING8201/KAUZ//MILAN/SHA7 were pollinated with maize pollen of 2 genotypes H1 : KSC108, 403 and compund of pollen of them, to produce doubled haploid lines of wheat resistance to yellow rust disease. Overall 2015 florets pollinated. Fertilization frequencies ranged from 87.35% in H1 genotype of maize to 92.96% in 403 genotype of maize ( mean 90.15% ) was significantly different among the maize genotypes. Embryo formation in the dissected seeds range from 93.59% in DH-29 genotype of wheat to 27.18% in DH-32 genotype of wheat. Of the 517 cultured embryos ( mean 27.18% ), 80.69% greminated and 69.93% of them developed into plants. After colchicine treatment, 53.06% of plants survived and their seeds were harvested. Significant variation was found between genotypes of wheat and maize genotypes in the frequency of haploid plants. To evaluate wheat response to yellow rust, 64 doubled haploid lines, two resistant control cultivars ( parsi, sivand, morvarid ) and a susceptible control cultivar ( bolani), were tested as seedling in greenhouse conditions. Resistance was measured by infection type (I.T), latent period (L.P), postule size (P.S), postule density (P.D). In this study was used from 1 race of yellow rust : 6E150A+. Analysis of variance showed significant differences among of these genotypes, for all traits. Cluster analysis on the basis of four traits classified all the lines into two major groups ; resistant lines and susceptible lines. Overall, these preliminary results suggest that the doubled haploid system could be used as an effective means to produce desirable resistant lines in the shortest time. Key words: wheat, yellow rust, resistance, doubled haploid lines. Introduction Yellow rust disease by Puccini a striiformis fungus is one of the main wheat diseases in Iran and west and central Asia. Epidemics of this disease were accompanied by 1/5 million/ton damages in 1372 and 1374 [10]. Yellow rust is one the common diseases in Iran by considerable damages in wheat. Using resistant cultivars is the main prevention method, so different amendment methods are employed. Double haploid method can be used for production of the resistant cultivars according to the following advantages: 1) reduction in an amendment period, 2) Increase in silexion performance crossing of the species is valuable technique in plant genetic and plant amendment scientific plans, in some cross Corresponding Author Bakhshi T., MSc. Student of Plant Biotechnology, Islamic Azad University, Science and Research Branch, Tehran.

2 breeds, chromosome incompatibility leads to omission of the Y chromosome in the beginning of the embryo growth that after the saving of the embryo and transferring to the artificial culture a haploid embryo is developed. This system was suggested in barley in H. bulbosume H. vulgare by kasha and Kao in [3] for the first time. In 1986 a haploid was produced in wheat by this technique [3]. Laurie & Bennett [4] reported wheat haploid by crossing with corn. The main methods of production of the wheat haploid are : 1. Between specie crossing 2. Chromosome omission 3. In vitro culture. preventive factors of using stamen culture in amendment programs involve genetic dependencies (creation of the some genotypes), appearance of the albino plant and genetic changes [1]. Germination with H.bulbosume and omission of the H.bulbosume chromosomes were successful in wheat cultivars having recessive allele in crossing locus of krl, kr2 on chromosome 5A and 5B [8,9]. Recently, the best way to produc wheat haploid is crossing with corn reported with Laurie and bennet [6]. The aim of this research is to produce double haploid wheat lines and investigation of the embryonic resistance in green house condition. Materials and methods Three wheat genotypes of DH-32, DH-30, DH-29 and two corn genotypes H1, 403 and compund of pollen of them were used. Wheat and corn were transferred into green house after germination and transmitting embryo into vase. Disfertilization and germination were conducted according to Laurie and Bennet [6]. After pollination, the clusters were treated by 2,4-D days after pollination, they were transferred to the laboratory and haploid seeds were removed from the ear, in sterile condition embryos were removed and they were cultured in MS, M5, when the roots and shoots were prepared, haploid embryos were transferred into vase by grain sand and pit mass by 1:1. In appearing claws period the embryos were treated by solution of colchicines %2 for 5/5 hours. After treatment, the roots were exposed in flowing water for 24 hours for removing roots, and then the plants were transferred in to vase. Following factors were noted. The results were compared by x2 test and the graphs were plotted by excel. The percentage of the seed formation, embryo formation, germination, haploid embryo production, double haploid plant, products of 64 lines from double haploid method were used prepared from grain department of the research institute of the amendment, preparation of the seed and sapling with Bolani number (as a control) as a completely random method with three repetition 6E 150 A+ was the used breed. Spores of this pathotype 983 were provided by pathology section of grain department in karj seeds, sapling preparation and amendment research institute after infection of the embryos with mentioned pathotype the latent period, infection type, pustule density and pustule size were measured and compared. After germination of the seeds and transmission to soil and growth of the first leaf, they were treated by distilled water and Towin 20 oil in one drop/ liter. Then the bushes were mixed with spore and inoculated with powered in 1:4 by sprinkling spore method. The leaves were treated by distilled water and the bushes were covered by plastic lid. In order to germination of the spores, the vases were placed in dark room with 10 and humidity of 100% for 24h then appearing yellow pustules on the leaves is noted. This trend was continued for 25 minute, after inoculation lines infection type was determined by MC Neal and et al method [7]. In plants without pustule, number 25 was considered for statistical analysis. In order to measurement of the density and size of the pustules infected leaves were removed by scissors and they were transferred to solution lacto phenol for 48 hours for establishment and deco ovation, after 25 day of inoculation. The pustules were measured by plate micrometer and their length and width and area were calculated, for measurement of the pustules density parts of the leaf were placed on the plate and they were counted number of the pustules was calculated in m3. Software MSTATC, SAS and SPSS were employed for variance analysis and cluster analysis. Results and discussion 1. Comparison of the Seed Formation Percentage As you seen from figure 1, the percentage of the seed formation was variable by 100 (number of the germinated floret/ number of the formed seed) with arrange 89/37% from 83/72% in DH- 29 H1 till 95/03% in DH-30 by compund of pollen of them. There was a meaningful difference in and wheat genotype after x2 test. It seems that removing and appearing in disfertilization reduce seed yield because of dryness of stigma and nonpemeatibilty of them. 2. Comparison of the Embryo Formation Percentage According to the figure 2 the percentage of the embryo 100 ( number of the formed seeds/ number of the formed embryo) by average 26/84% from 6/77% in DH till 44/92% in DH was variable with a meaningful deference in combination of the wheat genotype with corn H1. It seems that these changes affected by corn and wheat genotype showing the importance of the hormone 2, 4- D.

3 3. Comparison of the Embryo Germination Percentage According to the figure 3 the percentage of the embryo germination 100 ( number of the formed embryo number of the germinated embryo) with average of 25/89% from 85/20% in DH-29 H1 till 40/92% in DH was variable, after meaningful difference. Based on above results and zhang and et al [11] experiments it can be said that concentration of the 2, 4-D affects on percentage of the embryo formation different embryo culture techniques affection germination. In case of incorrect disinfection laminar hood or culture areas most of the embryos will be infected avoiding wound when removing embryo from seed by sculpl is necessary. Manner of placing embryo in culture should be considered, thus the chlopetil of the embryo must be upward. 4. Yield of the haploid embryo production yield of haploid embryo o production 100 (number of the germinated floret/ number of the haploid embryo) indicates yield of wheat and corn. According to graph 4 level of the haploid embryo production yield was variable from 3/10% in DH- 29 H1 till 35/44% in DH In H1, 403 and wheat genotype a meaningful difference was observed. There are different variable in haploid embryo production step that affects on yield. The manner of using hormone 2, 4-D and time of cross- bread are important of course seasonal changes and greenhouse changes affection environmental conditions. 5. yield of double haploid plant production double haploid plant production yield 100 (number of germinated floret/ number of the double haploid plants) indicate wheat Corn yield in production of the double haploid plants. According to the graph 5 the level of double haploid plant production yield was variable from 2/19% DH- 32 H1 till 23/48% in DH Analysis of the Lines: In this research 64 lines besides parents and control cultivar were examined randomly. Based on table 1 results of the 64 double haploid lines and 6E150A+, the mean of the latent period, infection type, size and density of the pustule is different meaningfully. In other words, studied lines were different and they were resistant. As you seen from table 2, in lines 7, 9, 13, 16, 17 the first pustules on infected leaves were observer nine days after inoculation as the lowest latent period. Lines 8, 12, 19, 53, 63, lines 1, 22, 32, 39, 48, 52, 59, Lines 2, 3, 5, 10, 21, 24, 54, 56, 62, 65, Lines 29, 34, 34, 42, 45, 49, Lines 4, 15, 33, 40, 47, Lines 46, 50, 984 Line 44, Lines 61, 14, 18, 23, 25, 26, 27, 28, 36, 37, 38, 41, 55, 57, 58 had ten, eleven, twelve, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty- one, and twenty- five days latent periods respectively. According to Borers & Lope [2] longterm latent period has high correlation with low length of rust bands and low level infection in the field. Thus, by long- term latent period lines having resistance genes prevent yellow rust. According to table 2, lines 7, 9, 13, 16, 17 are sensitive lines from latent period since after nine days the first pistols were observed. The changes are affected by genotype and experiment environment. According lines 7, 8, 12, 13, 16, 17, 19 had infection type 9 as sensitive type, Lines 59, 53, 63 had infection type 8 and Lines 3, 5, 10, 21, 22, 24, 32, 39, 48, 52, 54, 56, 62, 102 had infection type 8 and the others were less than 6, that they are resistant lines according to [7]. Broers and lopez [2] showed high correlation between long- term latent period and low infection type. In these research lines 6, 14, 18, 26, 27, 28, 36, 37, 38, 41, 57, 58 had low infection type and long- term latent period. Line 48 and lines 3, 12, 13, 19, 39, 59, 64 had the biggest pustules respectively. Lines with more infection have big pustules. In this research lines 312, 13, 19, 39, 59, 64 had big pustules and high infection type. Line 6 had high density equally 3/65 and high infection type. Lines 9, 53, 50, 63 had the next most pustules density and Lines 9, 8, 7, 8 had the highest infection type density and size of the pustules have direct relationship between infection type. Results of Cluster Analysis: In order to measure and determine genetic distance farness and closeness period relativity and non- relativity and patterns of genetic diversity in resistance to yellow rust, a cluster method was used. Euclidean coefficient determines genotypes distance. The far distance leads to farness of the two clusters. In this research lines were clustered by latent period infection type, pustule size, pustule density. All lines are divided in to 6 main groups that reduce their senility from left to right. So lines 58, 56,47, 26, 36, 37, 38 is resistant line and Line 1, 52 is a sensitive line. According to dendrogram all resistance and semiresistance lines are in right cluster and they were selected by experiment.

4 985 Fig. 1: Comparison of the seed formation percentage between cross. Fig. 2: Comparison of the embryo formation between crosses. Fig. 3: Comparison of the embryo germination between crosses. Fig. 4: Yield of haploid embryo production among crosses.

5 986 Fig. 5: Double haploid plant production yield.

6 987 Table 1: Variance analysis of the completely difference plan for different trails relative to 6E 150A+. Meaningful in p= 1% S.O.V df MS Latent period Infection type Pustule size Pustule density Genotype 63 29/52** 3/67** 6/7** 103/63** Error 128 1/09 2/39 0/16 0/50 Table 2: Danken multicasts test for different traits in to examined lines in greenhouse condition relative to 6E 150A+ wheat yellow rust No of Lines Name of Lines Latent period Infection type Pustule size Pustule density 1 PWS-N AB 10.6 D 3.1 FGHIJK 2.8 CDEF 2 PWS-N ABC 11.3 D 3.7 BCDEFG 2.6 DEFGHI 3 PWS-N ABC 11.6 D 2.9 GHIJKL 2.5 EFGHIJ 4 PWS-N ABC 11.6 D 3.6 BCDEFGH 3 BCD 5 PWS-N ABC 10.6 D 3.7 BCDEFG 1.5 MNOP 6 PWS-N-11 0 E 25 A 0 O 0 Q 7 PWS-N AB 11 D 3.4 CDEFGHI 2.7 CDEFGH 8 PWS-N-13 8 A 10.6 D 3.6 BCDEFG 2.2 IJKL 9 PWS-N AB 10.6 D 2.8 IJKL 3 BCD 10 PWS-N ABC 11 D 2.8 IJKL 1.1 P 11 PWS-N-18 0 E 25 A 4.4 B 2.2 IJKL 12 PWS-N ABC 11 D 3.8 BCDEF 2.3 GHIJK 13 PWS-N-23 7 ABC 11.3 D 3.8 BCDEF 2.7 CDEFGH 14 PWS-N-24 0 E 25 A 0 O 0 Q 15 PWS-N ABC 11.3 D 2.4 KLM 1.9 KLM 16 PWS-N-26 8 A 10 D 3.1 GHIJK 3.6 A 17 PWS-N-29 6 ABC 15 C 2.2 LMN 2.9 BCDE 18 PWS-N-30 0 E 0 D 0 O 0 Q 19 PWS-N AB 10.6 D 4.2 B 2.2 IJKL 20 PWS-N-33 2 DE 25 A 3.7 BCDEFG 2.6 DEFGHI 21 PWS-N-34 7 ABC 11.6 D 3.6 BCDEFG 1.5 MNOP 22 PWS-N ABC 11.3 D 2.8 HIJKL 2.3 HIJK 23 PWS-N-40 0 E 25 A 0 O 0 Q 24 PWS-N ABC 11 D 5.3 A 2.5 EFGHIJ 25 PWS-N-43 0 E 25 A 0 O 0 Q 26 PWS-N-47 0 E 25 A 0 O 0 Q 27 PWS-N-48 0 E 25 A 0 O 0 Q 28 PWS-N-49 0 E 25 A 0 O 0 Q 29 PWS-N BC 15.6 C 3.1 FGHIJK 2.5 EFGHIJ 30 PWS-N BC 15.6 C 2.2 LMN 1.7 MNO 31 PWS-N-54 2 DE 21.6 B 3.3 DEFGHI 2.3 GHIJK 32 PWS-N ABC 11.3 D 2.5 JKLM 2.6 DEFGHI 33 PWS-N-56 6 ABC 15 C 1.6 N 1.6 MNO 34 PWS-N-57 6 ABC 15 C 2.8 HIJKL 2.1 JKL 35 DH ABC 11.6 D 3 BCDEF 2.2 IJKL 36 DH E 25 A 0 O 0 Q 37 DH E 25 A 0 O 0 Q 38 DH E 25 A 0 O 0 Q 39 DH ABC 11.3 D 3.4 CDEFGHI 2.7 CDEFGH 40 DH ABC 11.3 D 3.2 EFGHIJK 1.5 NOP 41 DH E 25 A 0 O 0 Q 42 DH ABC 12 D 3.4 CDEFGHI 2.4 EFGHIJ 43 DH D 20.6 B 1.9 MN 1.6 MNO 44 DH C 16.3 D 3.2 EFGHIJ 1.4 NOP 45 DH ABC 15 C 3.2 EFGHIJ 3.1 BC 46 DH ABC 10.6 D 3.4 CDEFGHI 2.7 CDEFGH 47 DH ABC 15 C 3.6 BCDEFGH 2.1 JKL 48 DH ABC 11D 3.5 CDEFGHI 2.8 BCDEF 49 DH ABC 15 C 3.7 BCDEFG 2.8 CDEFG 50 DH ABC 11.3 D 3.7 BCDEFG 2.2 CDEFG 51 DH AB 10.3 D 4 BCDE 2.8 CDEF 52 DH AB 11.3 D 3.4 CDEFGHI 2.6 DEFGHI 53 DH AB 11.3 D 3.6 BCDEFGH 3 BCD 54 DH ABC 11.3 D 2.4 JKLM 2.4 EFGHIJ 55 DH DE 21.6 B 3.2 EFGHIJK 2.5 EFGHIJ 56 DH AB 10.6 D 3.6 CDEFGHI 2.2 IJKL 57 DH E 25 A 0 O 0 Q 58 DH E 25 A 0 O 0 Q 59 DH ABC 15 C 4.1 BCD 3.1 BC 60 DH ABC 11.3 D 3.6 BCDEFGH 2.7 CDEFGH

7 988 Table 2: Continue. 61 DH DE 21.6 B 3 FGHIJK 2.8 BCDEF 62 DH ABC 11.6 D 2.4 KLM 2.4 FGHIJ 63 DH AB 11 D 3.7 BCDEFG 3.2 B 64 DH ABC 11.6 D 3 FGHIJK 1.8 LMN 65 susceptible 8 A 10 D 3.1 GHIJK 3.6 A Acknowledgment This work was helped by Mr. Azimi Kargar Ataullah, the Islamic azad university,karaj branch that i thanks him. Refrences 1. Bozorgipour, R., The use in vitro techniques for crop improvement in cereal. Ph.D. Thesis. The University of Cambridge. 2. Broers., L.H.M., Breeding for partial resistance in wheat to strip rust. In : Jacobs, Th. Andparlevliet, J.E. (eds) Durability of disease resistance. Kluwer Academic Publishers, pp: Kasha, K.J., K.N. Kao., High frequency haploid production in barley (Hordeum vulgare L.). Nature 225: Laurie, D.A and M.D. Bennet., Wheat maize hybridization. Can. J. of Genet : Cytol., 28: Laurie., D.A. and M.D. Bennet., The effect of crossability loci Kr1 and Kr2 on fertilization frequency in hexaploid wheat maize crosses. Thor. Appli. Genet., 73: Laurie, D.A. and M.D.Bennett, The production of haploid wheat plants from wheat maize crosses. Theor. Appl. Genet., 76: Mc Neal, F.H., C.F. Knozak, E.P. Smith, W.S. Tate, T.S. Russell, A uniform system for recording and processing cereal data. U. S. Dept. Res. Seru. ARS., pp: Stich, L.A. and J.W. Snap., Doubled haploid production in winter wheat and triticale genotypes, using the Hordeum bulbusome system. Euphtica, 35: Snape, J.W., Doubled haploid breeding. Theoretical basis and practical application. pp: In: Mujeebkazi, A and L. A. Sitch. (eds). Review of advance in plant biotechnology CIMMYT. Mexico. 10. Torabi, M., V. Mardoukhi, K. Nazari, F. Afshari, A.R. Forootan, M.A. Ramin, H. Golzar and A.S. Kashani, Effectiveness of wheat yellow rust resistance genes in different parts of Iran. Cereal Rust and Powdery Mildews Bulleting 23: Zhang., J.B., W.J. Friebe, S.A. Raupp., B.S. Harrison, Gill., Wheat embryogenesis and haploid production in wheat maize hybrids. Euphytica., 90: