Study of Resistance Components at Seedling Stage to Stripe Rust in Some Doubled Haploid Wheat Lines

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1 Advances in Environmental Biology, 6(4): , 2012 ISSN This is a refereed journal and all articles are professionally screened and reviewed ORIGINAL ARTICLE Study of Resistance Components at Seedling Stage to Stripe Rust in Some Doubled Haploid Wheat Lines 1 Tayebeh Bakhshi, 2 Reza Bozorgipour, 3 Mohammad Sharrif Moghaddasi 1 Department of Agronomy and Plant Biotechnology, Science and Research Branch, Islamic Azad University, Tehran, Iran 2 Seed and Plant Improvement Institute, Karaj, Iran. 3 Agronomy and Plant Breeding Department,Saveh Branch,Islamic Azad University,Iran Tayebeh Bakhshi, Reza Bozorgipour, Mohammad Sharrif Moghaddasi; Study of Resistance Components at Seedling Stage to Stripe Rust in Some Doubled Haploid Wheat Lines ABSTRACT In order to evaluate the reaction to yellow rust, 64 wheat doubled haploid lines resulted from wheat and maize hybrid chromosome elimination method were tested relative to one wheat yellow rust disease pathogen pathotype in Sari region. These pathotype is 166E14A +. Wheat double haploid lines were tested in seedling stage in greenhouse condition by three resistant control cultivars (Parsi, Sivand and Morvarid) and sensitive Bolani cultivar. All lines and cultivars were planted in three replications in plastic pots in culture room. In one and two leaves stages, the seedlings were inoculated with urodiniospores of each pathotypes separately. After 14 to 25 days, the infection type of each line was recorded based on 0-9 scale. Resistance was measured by infection type, latent period, pustule size and pustule density. The analysis of variance showed a significant difference among genotypes. Accordingly the resistance lines were selected. These lines can be used as resistance resources relative to pathotypes in improvement programs. The results indicate that doubled haploid system can be useful method for production of optimal resistant lines in short term. Key words: Doubled haploid lines, Resistance, Wheat, Yellow Rust Introduction Yellow rust or strip rust caused by puccinia striiformis west. f. sp. tritici is a common wheat disease in Iran and west and central Asia countries. In Iran the epiphytotics have been seen since 1991.The severs epiphytotics was observed during 1993 and 1995 by optimal climate for disease. Several wheat cultivars were sensitive against these pathotypes [16]. Epiphytotics of yellow rust caused considerable damages equal to %15 of total production by 1.5 million ton [16]. Access to resistant resources against different types of yellow rust and cultivation of resistant cultivars is the reliable and principle way in order to control and reduce the damages [12,3]. It seems that haploid improvement method is the best method for planting resistance cultivars against yellow rust, since it accelerates improvement plans in addition to increase selection efficacy [13]. The main methods are 1- cultivation of anther 2- cultivation of microspore 3- germination with Hordeum bulbosum 4- germination with maize. The prohibiting factors in using stamen in improvement programs involve genetics dependence (weak reaction of some genotypes), appearance of albino plants and genetics changes [1]. Germination with H. bolbosum and omission of chromosomes H. bolbosum were only successful in wheat cultivars that there were recessive alleles in locus by crossing capability of Kr1, Kr2 on chromosomes 5B, 5A [14]. Recently, the best method is crossing with maize [5] that it can be obtain 4-6 florets for each germinated ear. The resistance is evaluated by resistance parts like infection type, latent period, pustule size and pustule density by different methods. Infection type is result of reciprocal effect of host and pathogen that it can be used as descriptor of resistance and severity of pathogenesis. Different scales have been offered for evaluation of infection types, Mc Neal et al [11] is common. The goal of this research is to produce wheat haploid lines and study of the resistance in seedling stage under greenhouse conditions. Materials and methods 64 lines produced in in cereal research center of seed, seed and plant improvement institute by their parents and Bolani sensitive cultivar (control) were tested in greenhouse in three duplications randomly Corresponding Author Tayebeh Bakhshi, Department of Agronomy and Plant Biotechnology, Science and Research Branch, Islamic Azad University, Tehran, Iran Adelehbakhshi@yahoo.com

2 block desighn against yellow rust. 166E14A + on Bolani cultivar from Sari region was research pathotype. The spores of these breeds were prepared from pathology section in cereal research center of seed, seed and plant improvement institute. In order to test the seedling resistance, the rust spores were proliferated on Bolani sensitive cultivar in greenhouse. In order to inoculation of the experiment materials, the spores were collected by suction system from Bolani cultivar and they were used immediately. After germination of the seeds and transfer to soil and growth of the first leaf of the seedling, distilled water involving Tween-20 oil was poured one drop per liter in order to obtain uniform distribution and leaf surface viscosity. Then the bushes were mixed by spores and inoculated uniformly by 1:4 ratios as adding spore by pounce box. Then the leaves were rinsed with distilled water and the bushes were covered with plastic lids. In order to germination of the spores,the pots were transferred to dark for 24 hours by 10.c and relative humidity of 100, then they were transferred to ordinary green house by 15.c. The mentioned traits were noted after appearance of the first pustule on the leaves. Traits of infection type, latent period, pustule size and pustule density were measured. For measuring of the latent period, the period between 1524 inoculation and appearance of the first rust pustules were noted until 25 days. Infection type was determined days after inoculation with Mc Neal et al [11] method. In order to measure the size and density of pustules, the infected leaves were removed by scissors after 25 days and they were transferred to bottles involving lacto phenol solution. The pustules size was measured by graded and eye micrometer by measuring length, width and area. For measuring pustule density the leaves were placed on lam and binocular and the number of pustules were counted and number of pustules were calculated in cm 2. In order to analysis of variance SAS and SPSS were employed. Results and Discussion Lines analysis of variance: In this research 64 produced lines by their parent and Bolani resistance cultivar were cultivated in balanced completely design block. In this experiment 166E14A + pathotype of Sari region was used. The results of analyses are summarized in table 1. The infection type, latent period, pustule size and pustule density were significant. In other words, there was a significant difference among traits. Table 1: Analysis of variance of unbalanced completely randomized design for different traits to race 166E14A MS S.O.V df Latent period Infection type Pustule size Genotype ** ** 14.42** Error **; Significant at α=0.0 Pustule density 12.32** 0.10 In column for evolution of resistance, infection types of 0-6 and types 7-9 were considered as resistance and sensitive types respectively in table 2 [11]. In Lines 1, 2, 3, 45, 49, 52, 53, 55, 57, 58, 61, 63, 64 and 65 the first pustules were observed 10 days after inoculation, it was the lowest latent period among experimental lines. The latent period of lines 5, 20, 29, 35, 39, 40, 47, 48 and 50 was 11days and for lines 13, 15, 17, 22, 25, 33, 34, 43, 46, 51 and 62 were 12 days. It was 13 days for line 4 and the remainder had latent period of days as resistant lines. According to Broers and lopez-atilano [2] the latent period has high correlation with short length of rust stripes and less infection in the field. So, the long latent period increases resistance gens, thus it prevents rapid appearance of yellow rust on the host. These changes are the result of the environmental condition in addition to the effect of genotype. Ma and Singh [9] investigated several wheat genotypes in different growth stages and places and they showed that the latent period is affected by greenhouse climate in addition to cultivar and growth stage. Lines 4, 5, 13, 15, 17, 20, 22, 25, 29, 33, 34, 39, 40, 43, 46, 48, 50, 51 and 62 had infection type of 7 and they showed the sensitive infection type. Infection type in lines 2, 35, 45, 7, 49, 53, 55, 57, 58 and 61 were 8 and in line 1, 3, 52, 63, 64 and 65 were 9 and in the remainder it was less than 6 as the resistant lines [11]. Broers and Lopez-Atilano (1993) suggested high correlation between long latent period and low infection type. In this research lines by less infection type had long latent period. From pustule size view point, line 1 produced the biggest pustules of 5.4 cm 2 for pathotype 166E14A +. From pustule density view point line 45 had the highest density of 4.9 pustules in a cm 2 for pathotype 166E14A +. According to experiments the size of pustules is reduced by increase of the number of the pustules and by reduction of the pustules their size increases. 2. 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.

3 The far distance leads to farness of the two clusters. In this research lines were clustered by latent period, infection type, pustule size and pustule density. All 1525 lines are divided in to 3 main groups that reduce their senility from right to left (fig 1). Table 2: Comparison of different traits in doubled haploid wheat lines in greenhouse conditions to race 166E14A + NO. Line name Infection type Latent period Pustule size Pustule density 1 PWS-N-3 9 A 10 I 1.4 BCDEF 5.4 AB 2 PWS-N ABC 10.6 GHI 4.8 B 4.9 BCDEF 3 PWS-N-7 9 A 10 I 4.8 B 5.3 BC 4 PWS-N-8 7 E 13.3 E 3.1 HI 3.4 HI 5 PWS-N DE 11.6 FG 3.8 DEFGH 4 FGHI 6 PWS-N-11 0 I 25 A 0 L 0 M 7 PWS-N-12 0 I 25 A 0 L 0 M 8 PWS-N-13 0 I 25 A 0 L 0 M 9 PWS-N-15 4 G 17 C 2 JK 2.3 JK 10 PWS-N-17 0 I 25 A 0 L 0 M 11 PWS-N-18 0 I 25 A 0 L 0 M 12 PWS-N-19 0 I 25 A 0 L 0 M 13 PWS-N-23 7 E 12 F 3.3 GH 3.8 GHI 14 PWS-N-24 0 I 25 A 0 L 0 M 15 PWS-N-25 7 E 12 F 3.9 CDEFGH 4.3 DEFGH 16 PWS-N-26 0 I 25 A 0 L 0 M 17 PWS-N-29 7 E 12 F 3.9 CDEFGH 4.3 DEFGH 18 PWS-N-30 0 I 25 A 0 L 0 M 19 PWS-N-31 6 F 15 D 2.4 J 1.7 JK 20 PWS-N DE 11.6 FG 3.8 EFGH 4.2 EFGHI 21 PWS-N-34 4 G 17 C 2.5 J 1.7 JK 22 PWS-N-36 7 E 12 F 4.5 BCDE 4.6 BCDEFG 23 PWS-N-40 0 I 25 A 0 L 0 M 24 PWS-N-42 0 I 25 A 0 L 0 M 25 PWS-N-43 7 E 12 A 4.2 BCDEF 4.4 CDEFG 26 PWS-N-47 0 I 25 A 0 L 0 M 27 PWS-N-48 0 I 25 A 0 L 0 M 28 PWS-N-49 0 I 25 A 0 L 0 M 29 PWS-N DE 11.6 FG 3.8 EFGH 4.6 BCDEFG 30 PWS-N-53 0 I 25 A 0 L 0 M 31 PWS-N-54 6 F 15 D 2.2 JK 2.3 JK 32 PWS-N-55 0 I 25 A 0 L 0 M 33 PWS-N-56 7 E 12 F 4.5 BCDE 4.3 DEFGH 34 PWS-N-57 7 E 12 F 4.3 BCDEF 4.7 BCDEFG 35 DH BCD 11 FGHI 4.7 BCD 5.2 BC 36 DH H 20 B 1.5 K 0.8 L 37 DH I 25 A 0 L 0 M 38 DH G 16.3 C 2.5 J 1.7 K 39 DH CDE 11.3 FGH 4.5 BCDE 4.8 BCDEF 40 DH CDE 11.3 FGH 4.1 BCDEFG 4.4 CDEFG 41 DH I 25 A 0 L 0 M 42 DH I 25 A 0 L 0 M 43 DH E 12 F 3.9 DEFGH 4 FGHI 44 DH F 15 D 2.5 J 2.1 JK 45 DH AB 10,3 HI 4.9 B 5.3 BC 46 DH E 12 F 3.8 EFGH 4.2 EFGHI 47 DH BCD 11 FGHI 4.2 BCDEF 4.9 BCDEF 48 DH CDE 11.3 FGH 3.9 CDEFGH 4.5 BCDEFG 49 DH AB 10.3 HI 4.7 BC 4.8 BCDEF 50 DH CDE 11.3 FGH 4.5 BCDE 4.9 BCDEF 51 DH E 12 F 4.3 BCDEF 4.3 DEFGH 52 DH F 15 D 5.1 BCDE 4.3 BCDEF 53 DH ABC 10.6 GHI 3.6 FGH 4.3 DEFGH 54 DH F 15 D 2.3 JK 2.5 IJ 55 DH AB 10.3 HI 4.5 BCDE 4.9 BCDEF 56 DH I 25 A 0 L 0 M 57 DH ABC 10.6 GHI 3.8 EFGH 5 BCDEF 58 DH AB 10.3 HI 4.7 BC 5 BCDEF 59 DH I 25 A 0 L 0 M 60 DH I 25 A 0 L 0 M 61 DH AB 10.3 HI 4.4 BCDE 4.5 BCDEFG 62 DH E 12 F 3.8 DEFGH 3.3 I 63 DH A 10 I 4.3 BCDEF 5 BCDEF 64 DH A 10 I 4.5 BCDE 5.2 BCD 65 susceptible 9 A 10 I 6.1 A 6.2 A

4 1526 So lines 6, 12, 14, 16, 18, 23, 24, 26, 27, 28, 30, 32, 34, 37, 41, 42, 56, 59 and 60 are resistant lines and Lines 1, 2, 3, 35, 47, 52, 53, 55, 57, 63 and 64 are a sensitive lines. According to dendrogram all resistance and semi- resistance lines are in left cluster and they were selected by experiment. For example lines 6, 12, 14, 16, 18, 23, 24, 26, 27, 28, 30, 32, 34, 37, 41, 42, 56, 59, 60 are resistant lines and observed in left cluster Fig. 1: Dendrogram of wheat doubled haploid lines based on their resistance to race of 166E14A +

5 1527 Conclusion: According to studies (Inagaki, 1997; Matzk and Mahn, 1994) it can be concluded that crossing of wheat and maize is an effective method for production of wheat haploid and doubled haploid. This method can be used instead of production of microspore and anther. Using different isolates of a pathotype in evaluation of wheat lines and cultivars resistance (multipathotypes screening) in controlled conditions and in seedling stage can be led to identification of resistance genes in developed lines and cultivars. By production of double haploid lines as new composed lines, this method can be used in plant improvement programs especially disease resistance. In this research the lines produced by this method were selected for evaluation of resistance in seedling stage. References 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. and R.M. Lopez-Atilano, Components of adult resistance in bread wheat to stripe rust. Proceding of the 6th International Congress of Plant Pathology, Johnson, R., Durability of resistance in crops: Some closing remarks about the topics and the symposium. pp In: Jacobs, TH and J. E. Parlevliet (eds). Durability of Disease resistance. Kluwer Academic Publisher. 4. Inagaki, M., Technical advances in wheat haploid production using ultra wide crosses. Jircas journal, 4: Laurie, D.A., M.D. Bennet, The production of haploid wheat from wheat maize crosses. Theoretical Applied Genetics, 76: Riera-Lizarazu and A. Mujeeb-Kazi, Maize (zea mays L.) mediated wheat (Triticum aestivum L.) polyhaploid production using various crossing method. Cereal Research Communication, 18: Riely, R. and V. Chspman, The inheritance in wheat of crossability with rye. Genet. Research, 9: Matzk, F., A. Mahn, Improved techniques for haploid production in wheat using chromosome elimination. Plant Breeding, 113: Ma, H. and R.P. Singh, Expression of adult resistance to stripe rust at different growth stages of wheat. Plant Disease, 80: Matzk, F. and A. Mahn, Improvement techniques for haploid production in wheat using chromosome elimination. Plant Breeding, 113: Mc Neal, F.H., C.F. Konzak, E.P. Smith, W.S. Tale and T.S. Russell, A uniform system for recording and processing cereal research data. U. S. Department of Agricultural Research Service, Article, ARS, pp: Parlevliet, J.E., Further evidence of polygenic inherthance of partial resistance in barley to leaf rust. Euphytica, 27: Snape, J.W., Doubled haploid breeding. Theoretical basis and practical application, pp: In: Mujeeb-kazi, A and L.A. Stich, (eds). Review of Advances in Plant Biotechnology CIMMYT. Mexico. 14. Sitch, L.A. and J.W. Snape, Factors affecting haploid lproduction in wheat (Triticum aestivum). Theoretical Applied Genetics, 70: Torabi, M., V. Mardoukhi, K. Nazari, F. Afshari, A.R. Forootan, M.A. Ramai, H. Golzar and A. Kashani, Effectiveness of wheat yellow rust resistance genes in different parts of iran. Cereal Rusts and Powdery Mildews Bulletin, 23: Torabi, M. and K. Nazari, Seedling and adult plant resistance to yellow rust in Iranian bread wheats. Euphytica, 100: