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The first double haploid lines were obtained in the anther culture more than 50 years ago, but so far their production remains more art than a well-established technology. There are a huge number of articles with conflicting information, both on preliminary processing of anthers and medias that determine the highest yield of regenerants, despite the fact that the relevance of research in this area is difficult to overestimate. Double haploid (DH) lines are used: For increasing efficiency of traditional breeding; obtaining populations of DH lines used in several stages of fixing heterosis effect (to remove semi-lethal genes, as well as homozygotization of material) and so on. The objective of the study is to investigate the efficiency of media for producing DH lines in anther culture of hybrids between Russian and foreign varieties. The anthers of 67 hybrid combinations, both interspecific and intraspecific hybrids, were used. 30-50 anthers were planted on Petri dish (6 сm). After formation of callus (size varied from 2 to 10 mm) on the medium for dedifferentiation, they were transferred to MS medium for regeneration. Plants from 5 cm in size were transferred to the root formation medium. We study the effectiveness of nutrient media, which are derivatives of N6, but with a different hormonal composition; and С, RZ media, the salt content was also changed. Medias RZ and C did not reliably differ in callus yield; the rest were reliably inferior to them in this trait. Therefore, to increase the yield of callus, a higher content of salts of KNO3, KH2PO4, MgSO4 • 7H2O, MnSO4 • 4H2O, ZnSO4 • 7H2O, H3BO3, CaCl2 • 2H2O vitamins is necessary. However, their hormonal composition is different. If the RZ medium contains naphthylacetic (NAA) and dichlorophenoxyacetic acids ( 2,4-D), then C is only 2,4-D. N6-based media, N6a and N6б contain only one auxin as well as C. In the study, the best results were shown by option with content of both hormones at a concentration 2 mg / l NAA and 1 mg/l 2,4-D. The results showed a significant positive effect of increasing iron (FeSO4 x 7 H2O) concentration (55.6 mg / L) both on callusogenesis and plant regeneration. In standard recipe of all the media used in the work, its concentration is 27.8 mg / l. To stimulate somatic embryogenesis, the media were modified also by the addition of abscisic acid (ABA) and silver nitrate AgNO3. Studies have shown that somatic embryogenesis is stimulated in many genotypes when using ABA in combination with cytokinins. Callusogenesis and regeneration in the considered combinations had maximum values ​​at AgNO3 concentration 2 mg/l and; 1 mg/l ABA for the of most of the studied hybrids.

Rice, anther culture, hybrids, silver nitrate, double haploid, naphthylacetic (NAA), dichlorophenoxyacetic acids (2.4 – D), abscisic acid (ABA), iron concentration (FeSO4 x 7 H2O).

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Kiviharju E, Pehu E. The effect cold and heat pretreatments on anther culture response of Avena sativa and A. sterilis. Plant Cell Tiss. Org. Cult. 1998;54:97- 104.

Sen C, Singh RP, Singh MK, Singh HB. Effect of cold pretreatment on anther culture of boro rice hybrids. International Journal of Plant Reproductive Biology. 2011;3:69-73.

Ruwani DM, Mayakudua G, Silva TD. Anther culture as a supplementary tool for rice breeding. In: Rice Crop - Current Developments. 2018;1-15.
DOI: 10.5772/intechopen.76157

Seldimirova OA. Embryology of plants-regenerants of wheat of the “photos” line of androcline origin. Genetics in the 21st century: Current status and development prospects. 2004;269.

Lee ST, Huang WL. Osmotic stress stimulates shoot organogenesis in callus of rice (Oryza sativa L.) via auxin signaling and carbohydrate metabolism regulation. Plant Growth Regulation. 2014;73:193-204.

Singh BD, Singh AK. Mapping populations. In: Marker-Assisted Plant Breeding: Principles and Practices. Part III. Springer, New York. USA. 2015;125-150.

Chen CC. In vitro development of plant from microspores of rice. In Vitro. 1977; 13:484-489.

Nabors MW, Heyser JW, Dykes TA, Mott KD. Long-duration, high-frequency plant regeneration from cereal tissue cultures. Planta. 1983;157(5):385.

Zhuo LS, Si HM, Cheng SH, Sun ZX. Phenylacetic acid stimulation of direct shoot formation in anther and somatic tissue cultures of rice (Oryza sativa L.). Plant Breeding. 1996;115:295-300.

Sasaki T. Rice cultivars having a high capacity for regeneration from the anther callus and their genealogies. Japan. J. Breed. 1986;36:64-65.

Goncharova YK. Selective elimination of alleles in rice anther cultures. Russian Journal of Genetics. 2013;49(2):170-177.

Strunnikov VA, Strunnikova LV. The nature of heterosis, methods for its enhancement and consolidation in subsequent generations without hybridization. Izvestiya A.N. Biological Series. 2000;6:679-687.

Goncharova Yu. K, Kharitonov EM. Increasing the productivity of inter specific rice hybrids. Vavilovsky Journal of Genetics and Breeding. 2012;16(4):556-565.

Goncharova Yu. K. Method of fixing the heterotic effect-implementation on plants (on the hundredth anniversary of the birth of V.A. Strunnikov). Russian Journal of Developmental Biology. 2014;11(45):367-370.

Chen Y. Anther and pollen culture of rice. Haploids of higher plants in vitro. China Academic Publishers. Beijing. 1986;3-25.

Lentini ZP, Martinez RC, Rosa WM. Androgenesis of highly recalcitrant rice genotypes with maltose and silver nitrate. Plant Science. 1995;110:127-138.

Silva TD. Indica rice anther culture: can the impasse be surpassed? Plant Cell Tiss. Organ Cult. 2010;100:1-11.

Goncharova Yu. K, Kharitonov EM. RU 2465771 C1, 11.10. A method of fixing heterosis of hybrids in subsequent generations. (GNU VNII rice of the Russian Agricultural Academy). Inventions. Useful models. 2012;31.

Raina SK, Zapata FJ. Enhanced anther culture efficiency of indica rice (Oryza sativa L.) through modification of the culture media. Plant Breed. 1997;116:305-315.

Glazyrina VA, Savenko EG, Shundrina LA. An in vitro anther culture method for creating doubled rice haploids from varieties resistant to low positive temperatures. In the book: Current status, problems and prospects for the development of agricultural science. Materials of the IV international scientific and practical conference. 2019;222-224.

Savenko EG. Improving the technology for producing doubled haploids in vitro for use in breeding rice of japonica subspecies. Krasnodar. Thesis Ph.D. 2017;146.

Raldugin GN, Sobolkova GI. Genotypic differences in the effect of abscisic acid on callus cultures of Brassica napus L. Plant Physiology. 1994;41:702-706.

Ismagul A, Elibay S, Bashabaeva BM, Abugalieva AI. Analysis of methods of material homozygization in breeding and development of culture protocols of isolated microspores of Kazakhstan wheat varieties. Vestnik Kaz NU. Biological Series. 2012;2(54):17-23.

Savenko EG, Kostryukova EN, Glazyrina VA, Shundrina LA. Callusogenesis and regeneration of rice hybrids at various concentrations of abscisic acid (ABA) . Rice growing. Krasnodar. 2017;2(35):46-50.

Shayakhmetov IF. Culture of cells and tissues of wheat in vitro and somatic embryogenesis. Thesis Doctor of Biological Sciences. St. Petersburg; 2001.

Goncharova Yu. K, Kharitonov EM, Bushman N. Yu, Vereshchagina SA. Comparative analysis of the efficiency of nutrient media for the induction of callus formation for rice hybrids. Russian Agricultural Sciences. 2014;40(1):5.

Niroula RK, Bimb HP. Effect of genotype and callus induction medium on green plant regeneration from anthers of Nepalese rice cultivars. Asian Journal of Plant Sciences. 2009;8:368-374.

Thuan OT, Tuan VD, Ba Bong B. Study on anther culture of F1 plants from crosses between aromatic and improved rice cultivars. Omonrice. 2001;9:41-45.

Weigt D, Kiel A, Nawracala J, Pluta M, Lacka A. Solid-stemmed spring wheat cultivars give beter androgenic response than hollow-stemmed cultivars in anther culture. In Vitro Cellular & Developmental Biology: Plant. 2016;52:619-625.

Tripathy SK. Anther culture for double haploid breeding in rice - A way forward. Rice Genomics & Genetics. 2018;9(1):1- 6.

Sharma A. Morphology response of growth regulators on callus of Japonica rice through anther culture. Clinical Biotechnology and Microbiology. 2018;2:424-442.

Naik N, Rout P, Umakanta N, Verma RL, Katara JL, Sahoo KK, Singh ON, Samantaray S. Development of doubled haploids from an elite indica rice hybrid (BS6444G) using anther culture. Plant Cell. Tissue and Organ Culture. 2017;1(28):679.

Herath HM, Bandara DC. Anther culture performance in selected high yielding indica (of Sri Lanka) and japonica rice varieties and their inter sub-speciic hybrids. Journal of the National Science Foundation of Sri Lanka. 2011;39(2):149-154.

Xa TT, Lang NT. Rice breeding for high grain quality through anther culture. Omonrice. 2011;18:68-72.

Germana MA. Anther culture for haploid and doubled haploid production. Plant Cell, Tissue and Organ Culture. 2011;104:283-300.
DOI: 10.1007/s11240-010-9852-z