ISSN 1313 - 8820Volume 6, Number 2

June 2014

2014

Scope and policy of the journalAgricultural Science and Technology /AST/ – an International Scientific Journal of Agricultural and Technology Sciences is published in English in one volume of 4 issues per year, as a printed journal and in electronic form. The policy of the journal is to publish original papers, reviews and short communications covering the aspects of agriculture related with life sciences and modern technologies. It will offer opportunities to address the global needs relating to food and environment, health, exploit the technology to provide innovative products and sustainable development. Papers will be considered in aspects of both fundamental and applied science in the areas of Genetics and Breeding, Nutrition and Physiology, Production Systems, Agriculture and Environment and Product Quality and Safety. Other categories closely related to the above topics could be considered by the editors. The detailed information of the journal is available at the website. Proceedings of scientific meetings and conference reports will be considered for special issues.

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Radoslav SlavovFaculty of AgricultureTrakia University, Stara ZagoraBulgaria

Editors and Sections

Genetics and Breeding

Atanas Atanasov (Bulgaria)Nikolay Tsenov (Bulgaria)Max Rothschild (USA)Ihsan Soysal (Turkey)Horia Grosu (Romania)Bojin Bojinov (Bulgaria)Stoicho Metodiev (Bulgaria)

Nutrition and Physiology

Nikolai Todorov (Bulgaria)Peter Surai (UK)Zervas Georgios (Greece)Ivan Varlyakov (Bulgaria)

Production Systems

Dimitar Pavlov (Bulgaria)Bogdan Szostak (Poland)Dimitar Panaiotov (Bulgaria)Banko Banev (Bulgaria)Georgy Zhelyazkov (Bulgaria)

Agriculture and Environment

Georgi Petkov (Bulgaria)Ramesh Kanwar (USA)Martin Banov (Bulgaria)

Product Quality and Safety

Marin Kabakchiev (Bulgaria)Stefan Denev (Bulgaria)Vasil Atanasov (Bulgaria)

English Editor

Yanka Ivanova (Bulgaria)

2014

ISSN 1313 - 8820 Volume 6, Number 2June 2014

Anther culture response of winter barley (Hordeum vulgare L.)

B. Dyulgerova*, D. Vulcheva, D. Dimova

Institute of Agriculture, 8400 Karnobat, Bulgaria

Abstract. The potential of anther culture to produce double-haploid lines was evaluated for Bulgarian winter barley (Hordeum vulgare L.) breeding applications. Тwo 6-row and two 2-row F hybrids were studied. Anthers were culture on two different induction media – N6 and MS, both supplemented with 60 2

g/L maltose, 40 g/L starch, 3 g/L Gelrite. Composition of the culture media, genotype and their interaction showed significant effect on the frequency of responsive anthers and regenerated plants. The response ranged from 1.33 to 60.67 green regenerants per 100 anthers cultured. The number of green plants regenerated on N6 based media was significantly higher than on MS based media in the genotypes – F Lambic x KT 287 and F PG 4348 x KT 2168. Changes 2 2

in the composition of the culture media and the identification of responsive genotypes makes anther culture an efficient procedure for Bulgarian barley breeding program.

Keywords: anther culture, doubled haploids, Hordeum vulgare L.

AGRICULTURAL SCIENCE AND TECHNOLOGY, VOL. 6, No 2, pp , 2014162 - 164

Introduction contained 60 g/L maltose, 40 g/L wheat starch, 3 g/L Gelgite, 2 mg/L naphthaleneacetic acid (NAA) and 1 mg/L 6-benzylaminopurine

o(BA). Anthers were incubated in the dark, at 25 ± 1 C, for 30 days After the first protocol for barley anther culture was developed and the Petri dishes were then transferred to the light (16 h). The by Clapham (1973), plant breeders paid attention to anther culture frequency of responsive anthers, green and albino plantlets was and its possible application in breeding. The advantages of anther assessed. Green plantlets were rooted in test tubes containing culture in barley breeding are: shortening the breeding cycle by modified MS medium (Olsen, 1987), with 30 g/L sucrose, 7 g/L agar, immediate fixation of homozygosity, increased selection efficiency and 0.5 mg/L indole-3-acetic acid (IAA).and allowing the early expression of recessive genes. Numerous

Plantlets with well-developed roots were transferred to pots doubled haploid lines have been produced and field tested, including containing standard potting mix. Pots were covered with a beaker some resistant to barley yellow mosaic virus (Foroughi-Wehr and which was gradually removed until exposing the plants to ambient Friedt, 1984; Gheorghe, 2010). Nevertheless, most of the studies conditions. Self-fertilization and formation of grains were attained in have been done using a small number of responsive cultivars which the greenhouse. Seeds of spontaneously doubled barley plants have an extraordinary androgenetic capacity. The use of highly were collected. responsive genotypes may adapt the technique to genotypes which

The parameters observed were number of responsive anthers, are not very useful for practical purposes and restrict the genetic number of total plants regenerated and number of green and albino variability available to the breeder (Luckett and Smithard, 1992). In plants regenerated per 100 anthers cultured. Statistical analysis was order to prevent that, it is recommended to develop protocols and performed using Generalized Linear Model (GLM) procedure in the culture media applicable to a broader spectrum of genotypes.SPSS. Mean separations were tested by Duncan's Multiple Range The objective of this study was to evaluate androgenetic Test at 5% probability level. capacity of hybrid genotypes in two culture media for obtaining

doubled-haploids for Bulgarian winter barley breeding program.

Results and discussionMaterial and methods

Variance analysis showed a significant effect of culture media and genotype on the frequency of responsive anthers and The work was performed by using F PG 4365 x K-99/23-2, PG 2

regenerated plants (Table 1). The effects of the two factors are not 4348 x KT 2168, Lambic x KT287, Merian x KT 287 population independent, which means that genotypes respond differently to the derived from crosses made at the Institute of Agriculture, Karnobat. culture medium and both culture media induce different effects Donor plants were grown in the field. Spikes were collected when depending on the genotype.anthers were in the early-mid and mid-uninucleate stage. They were

Percentage of responsive anthers on MS medium ranged preselected on the basis of interligule length between the flag and between 7.00 and 51.00%, with an average of 26.17% (Table 2). An the uppermost leaf. Before removing spikes they were surface-average of 38.67% of the anthers cultured on N6 medium were sterilized with an aerosol of 96% ethanol. One or two anthers from responsive, with a minimum of 5.00% and a maximum of 74.00%. the middle floret were detached, stained with 4% acetocarmine and The superiority of N6 medium was statistically significant for 3 of 4 examined under the microscope to check the initial stage.genotypes by the rate of responsive anthers. Only the hybrid F PG 2 Anthers were cultured in two different culture media: modified 4348 x KT 2168 produced a similar number of responding anthers on MS (Murashige and Skoog, 1962) or N6 (Chu, 1981). Both media

162

* e-mail: bdyulgerova@abv.bg

the two media. The highest rate of responding anthers was green plantlets of the total number of plants regenerated. According manifested by F Lambic x KT 287 – 51.33% on MS media and to Logue et al. (1993), the ability to regenerate a great number of 2

green plants depends on the reduction of albinism. Knudsen et al. 74.00% on N6 media. The higher plant regeneration rate on N6 (1989) reported that the proportion of green plants and the total media was recorded for two of four hybrids. The frequency of green frequency of regeneration are determined by different genetic traits. plants per 100 anthers plated in F Lambic x KT 287 was almost 2

This explains why regeneration of green plants is rather influenced 13–fold higher on N6 based medium than on MS based medium – by the ratio green/albino than by the total frequency of regeneration. 60.67% on N6 medium compared with 4.67% on MS medium. The F2

According to Knudsen et al. (1989) and Larsen et al. (1991), the hybrid PG 4348 x KT 2168 showed 6.00 in contrast with 3.33 green proportion of green plants is strongly genotype dependent and has plants regenerated per 100 anthers for the media N6 and MS, great heritability. Logue et al. (1993) showed that the ratio respectively. green/albino remained constant even when donor plants were Compared with N6, MS medium has a greater amount of total cultivated under completely different environments.nitrogen and ammonium (Grimes and Hodges, 1990). Modhorst and

The two hybrids F Merian x KT 287 and F Lambic x KT 287, 2 2 Lörz (1993) found that the poor development of microspores in + + - which had a common parent demonstrated different androgenetic media containing just NH or with a high ratio NH /NO is caused by 4 4 3

potential. According to Larsen et al. (1991) it is common for hybrids ammonium toxicity. It is also possible that the effects of the addition to present levels of response equal or superior to their parents. of ammonium to the culture medium may be caused by a change in Therefore, the higher androgenetic capacity of F Lambic x KT 287 is 2 cytokinin biosynthesis (Weissman, 1972a, 1972b; Darral and probably due to the cultivar Lambic. Wareing, 1981; Mercier and Kerbauy, 1991).

In our study the frequency of spontaneous diploids was No relationship between rate of anther responded and rate of 72.67%. This result confirmed that barley anther culture usually plant regeneration in some of hybrids was observed. On the medium shows a high percentage of spontaneous doubling of the MS F Lambic x KT 287 produced 51.33 responsive anthers and 2

chromosomes making it unnecessary to treat plants with colchicine 18.67 total plants per 100 anthers and F Merian x KT 287 produced 2 in order to obtain doubled haploids (Foroughi-Wehr and Friedt, 32.00 responsive anthers and 22.67 total plants per 100 anthers. 1984; Olsen, 1987; Luckett and Smithard, 1992). A total of 117 These results confirm the hypothesis that androgenetic capacity doubled haploid lines were obtained from different crosses. These may result from two independent processes: induction and lines set seed and were multiplied for agronomic testing in the field.regeneration. These processes are controlled by different genetic

The efficiency of the anther culture method is largely dependent mechanisms that may not be present together. The best genotypes on plant genotype and cultivation conditions (Bojanova and are those combining high frequency of induction with a reasonable Gramatikova, 1996; Jacquard et al., 2006). This usually makes frequency of regeneration.comparison of the work of various laboratories difficult as genotypes, The greater capacity of some genotypes to develop green media and culture conditions differ considerably. The results of the plantlets could be caused by differences in the ratio of green/total current study indicate the importance of considering such plantlets. All hybrids on N6 medium showed higher frequency of

Table 1. Mean square from analysis of variance for frequency of responsive anthers, green and albino plant regeneratedper 100 anthers cultured in barley anther culture.

*, *** - significant at p = 0.05 and p = 0.001; RA – percentage of responsive anthers; GP – percentage of green plantsregenerated; AP – percentage of albino plants regenerated; TP – percentage of total plants regenerated.

Source of Variation RA GP AP TP

Genotype

Culture medium

Genotype X medium

Error

11365.000***

3125.000***

610.926*

160.679

3858.333***

4400.556***

3781.296***

225.802

2768.657***

823.472***

816.065***

88.966

12469.028***

9031.250***

8085.324***

346.065

Table 2. Percentage of responsive anthers, percentage of total, green and albino plant regeneration in F2 hybrids ofwinter barley

a, b, c, d – Values followed by the same letter in columns do not differ significantly (p < 0.05)

Genotype Media

F2 PG 4365 x K-99/23-2

F2 PG 4348 x KT 2168

F2 Lambic x KT 287

F2 Merian x KT 287

MS

N6

MS

N6

MS

N6

MS

N6

Responsiveanthers, %

Totalplantregeneration, %

Green plantregeneration, %

Albinoplantregeneration, %

d14.33c33.66d 7.00d5.00b51.33a74.00c32.00

bc42.00

c 5.00cd 9.67c 4.33c 7.00cd 18.67a100.00b 22.67cd 19.00

c 1.33c 3.33c 3.33bcd 6.00cd 4.67a60.67b17.33b16.00

c 3.67bc 6.33c 1.00c 1.00b14.00a39.33bc 5.33c 3.00

163

interactions in the development of protocols with locally adopted recombinant barley yellow mosaic virus resistant Hordeum vulgare genotypes and methodology. lines by anther culture. Theoretical and Applied Genetics, 67, 377-

According to Luckett and Smithard (1992), a productivity of 10 382.plants/100 plated anthers allows the production of 100 doubled Gheorghe B, 2010. Results regarding the yield potential, haploid lines from 50 different crosses by a single worker during one morphologic and physiologic characters of some double haploid year. In our study using different F populations an average of 14.08 inbreed barley lines in Western Romania. Analele Universităţii din 2

Oradea, Fascicula, Protecţia Mediului, XV, 46-52.and a maximum of 60.67 green plants/100 plated anthers were Grimes HD and Hodges TK, 1990. The inorganic NO3-:NH4+ ratio obtained. These results are similar or superior to those obtained by influences plant regeneration and auxin sensitivity in primary callus Finnie et al. (1989), Knudsen et al. (1989), Kühlmann and Foroughi-derived from immature embryos of indica rice (Oryza sativa L.). Wehr (1989), Luckett and Smithard (1992). Obtaining a reasonable Journal of Plant Physiology, 136, 362-367.number of doubled haploids makes anther culture an useful tool for

Jacquard C, Asakaviciute R, Hamalian AM, Sangwan RS, the Bulgarian barley breeding program.Devaux P and Clement C, 2006. Barley anther culture: effects of annual cycle and spike position on microspore embryogenesis and albinism. Plant Cell Reports, 25, 375-381.

Conclusion Knudsen S, Due IK and Andersen SB, 1989. Components of response in barley anther culture. Plant Breeding, 103, 241-246.

Composition of the culture media (induction media – N6 and Kühlmann U and Foroughi-Wehr B, 1989. Production of doubled MS), genotype (two 6-row and two 2-row F hybrids) and their 2 haploid lines in frequencies sufficient for barley breeding programs. interaction showed a significant effect on the frequency of Plant Cell Reports, 8, 78-81.responsive anthers and regenerated plants. The number of green Larsen ET, Tuvesson IKD and Andersen SB, 1991. Nuclear genes plants regenerated on N6 based media was significantly higher than affecting percentage of green plants in barley (Hordeum vulgare L.) on MS induction media in the genotypes – F Lambic x KT 287 and F anther culture. Theoretical and Applied Genetics, 82, 417-420.2 2

Logue SJ, Giles LC and Sparrow DHB, 1993. Genotype and PG 4348 x KT 2168. The response in our study ranged from 1.33 to environment strongly influence barley anther culture response using 60.67 green regenerants per 100 anthers cultured. Changes in the Australian genotypes. Australian Journal of Botany, 41, 227-236.composition of the culture media and the identification of responsive Luckett DJ and Smithard RA, 1992. Doubled haploid production by genotypes makes anther culture an efficient procedure for Bulgarian anther culture for Australian barley breeding. Australian Journal of barley breeding program.Agricultural Research, 43, 67-68.Mercier H and Kerbauy GB, 1991. Effects of nitrogen source on growth rates and levels of endogenous cytokinins and chlorophyll in Referencesprotocorms of Epidendrum fulgens. Journal of Plant Physiology, 138, 195-199.

Bojanova V and Gramatikova M, 1996. Androgenetic induction in Modhorst AP and Lörz H, 1993. Embryogenesis and development

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(Bg).by the amount and composition of nitrogen sources in culture media.

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Reviews

Genetics and Breeding

Nutrition and Physiology

Production Systems

Selection for linear traits for legs and feet and its significance for dairy cattle breedingI. Marinov

Morphological analysis of spikes and grouping of accessions of Triticum turgidum ssp. dicocconH. Stoyanov

Crops from Cucurbitaceae in collection of the Maritsa Vegetable Crops Research Institute, Plovdiv – local cultivars and their application in breeding programmeN. Velkov, V. Petkova

Study of chickpea seed size heredity (Cicer arietinum L.)R. Sturzu, T. Nistor, F. Bodescu, C. Melucă

Egg quality traits in layers from different production types H. Lukanov

Evaluation of Bulgarian winter common wheat varieties of yield stability in South BulgariaZ. Uhr, G. Rachovska, G. Delchev

Study of opportunities for effective use of varieties from Ukraine for creating early winter wheat lines II.Grain qualityN. Tsenov, D. Atanasova, I. Stoeva, E. Tsenova

Anther culture response of Winter Barley (Hordeum vulgare L.)B. Dyulgerova, D. Vulcheva, D. Dimova

Evaluation of grain yield in advanced lines two-rowed winter barleyM. Dimitrova-Doneva, D. Valcheva, D. Vulchev, B. Dyulgerova, M. Gotcheva, T. Popova

Haematology and some serum parameters of broilers fed decorticated fermented Prosopis africana seed mealD. Hassan, N. Yusuf, M. Musa, I. Musa-Azara, R. Barde, D. Ogah, A. Yakubu, M. Ari

Comparative age – related weight and morphometrical investigation of the intraorbital glands in the common bronze turkey (Meleagris Meleagris Gallopavo)D. Dimitrov

Effect of the dietary supplement Zarnela on some duodenal chyme parameters in yearling rams V. Radev, I. Varlyakov, T. Slavov, R. Mihaylov

13. Variations of maize yield and some quality indices of grain depending on the type of main soil tillageP. Yankov, M. Drumeva, D. Plamenov

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Behavior, sexual maturity and productive traits in gilts reared under different floor areaM. Kirov, V. Doychev.

Influence of organic fertilizers Aminobest and Greenfol on the initial stages of growth of wheat (Triticum aestivum L.)М. Pashev, D. Yakimov, V. Budаeva

Algorithm for determining the type of turns carried out by the unit in shuttle movement in the field while using precision farming systemsK. Trendafilov

Correlations between morphological and productive parameters in Burley tobaccoY. Dyulgerski, Tz. Radoukova

Growth and fruiting of the apple sort Braeburn 7926 grafted on M9 Pajam2 and MM106G. Dobrevska

Theoretical model of biogas production from cattle slurry with different additive of maize silageK. Peychev, R. Georgiev

New data about Agaricus (Section Minores, Agaricaceae) in BulgariaM. Lacheva, Tz. Radoukova

Changes of the humus status of Pellic Vertisol (FAO) soil type under long-term growing of filed crops in a stationary trial without fertilizationV. Koteva, S. Kostadinova

Non-fish marine resources and their exploitation along the Bulgarian Black Sea coastE. Petrova – Pavlova

Productivity and grass density of the main types of pastures in Sakar and Strandja regionV. Vateva

Automatic measurement of Loin eye urea with computer vision system Z. Zlatev, S. Ribarski, M. Mladenov

Influence of the kinetics of salting for Bulgarian white brined cheese, produced by mechanized and traditional technology on the starter and adjunct microflora during ripeningP. Panayotov, V. Mihailov, D. Rakov

Effect of season, lactation period and number of lactation on mastitis incidence and milk yields in dairy cowsT. Penev, Zh. Gergovska, I. Marinov, V. Kirov, K. Stankov, Y. Mitev, Ch. Miteva

Agriculture and Environment

Product Quality and Safety

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thmance in dairy cows,IX International Conference on Production Diseases in Farm Animals, September 11–14, Berlin, Germany.Thesis:Hristova D, 2013. Investigation on genetic diversity in local sheep breeds using DNA markers. Thesis for PhD, Trakia University, Stara Zagora, Bulgaria, (Bg).

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