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https://doi.org/10.15407/agrisp6.02.003
IMPROVEMENT OF THE TECHNOLOGY OF OBTAINING STABLE (DI)HAPLOID REGENERANTS FROM EMBRYONIC CULTURE OF APOMICTIC SUGAR BEET (BETA VULGARIS) BREEDING MATERIAL WITHOUT THE USE OF COLCHICINE
N. S. Kovalchuk 1, M. V. Roik 1, Ya. M. Hadzalo 2 , T. M. Nediak 1, O. A. Zinchenko 1

1 Institute of Bioenergy Crops and Sugar Beet, NAAS 25, Klinichna Str., Kyiv, 03110, Ukraine

2 National Academy of Agrarian Sciences, 9, Omelianovych-Pavlenko Str., Kyiv, 01010 Ukraine

E-mail: sugarbeet@ukr.net, natalakovalcuk461@gmail.com

Received February 18, 2019 / Received April 22, 2019 / Accepted July 19 , 2019
Abstract
Aim. To evaluate the effi ciency of inducing generative, reduced parthenogenesis and to better use the differentiating potential of the embryo culture under apomictic seed production in selection materials of sugar beet with cytoplasmic male sterility (CMS), and B) to isolate homozygous lines (dihaploids) without the use of polyploidizing substances. Methods. Apomictic (agamosper- mous) seed production in apocarpous pollen sterile lines from B. vulgaris subsp. vulgaris var. altissima (sugar beet) using classi- cal so-called Owen sterile cytoplasm and sterile cytoplasm from Beta maritimа and Beta patula as sources, was conducted under pollen free conditions and spatial isolation in the greenhouse breeding complex of the Yaltushkivska experimental breeding station (Yaltushki, Ukraine). The specifi cities of embryonic development of apomictic embryos were studied with the purpose of effi cient regulation of the induction of explants in vitro as donors of the culture of immature embryos. Fluorescent fl ow cytophotometry in combination with the computer program of the Partec Ploidy Analyser PA-2 (Partec GmbH, Germany, now Sysmex), were used to determine the degree of ploidy, enabling the selection of haploid and dihaploid lines in vitro. A genetic method was developed using the expression of morphological marker indices of nuclear genes of anthocyanin coloring (R+ r–) of regenerant plants in vitro and ploidy determination for differentiation by generative (reduced) parthenogenesis. The sampling technique that took into account the hormonal composition of cultural media and the level of genome ploidy, sample frequency and statistical analysis of the results was determined using the appropriate statistics; the percentage of regenerants, induced by different types of morpho- genesis and ploidy in vitro, was determined along with the measurement error to control the accuracy of the selected sampling (number of seed embryos). Results. The selected cultural medium No. 3, based on the basal medium according to Gamberg et al., 1968 (21), contained 6 BAP – 2 mg/l, 2.4 D – 0.5 mg/l, gibberellic acid – 0.1 mg/l, which ensured a success rate of 4.4 to 23.3 % of direct regeneration of shoots from the embryo culture, depending on the genotype of donors, and 4–10 % for induction and proliferation of callus. In ten experimental numbers of alloplasmic lines of sugar beet, the incidence of haploids and mixoploids among the regenerants from the embryo culture fl uctuated within the wide range of 14.8 – 62.2 % and exceeded the indices, ob- tained by other known methods of haploid parthenogamy, which had the values of 3.79 – 6.25 %. Conclusions. The homozygous lines and dihaploids were determined and set apart/stabilized in the process of micropropagation, where the differentiation of clones was made on the basis of total DNA content in interphase nuclei, using information of histograms generated in fl uorescent fl ow cytometry with the Partec Ploidy Analyser PA-II instrumentation. The medium, based on macro- and microsalts according to Gamberg et al., 1968 (21) was found to be the most effi cient; it ensured at least partially successful direct regeneration in the culture of embryos within the range of 4.40 ± 1.29 to 23.3 ± 3.45 %. The success of direct regeneration of apomictic material depended on the composition of the cultural medium used fi rst and foremost, and to a lesser extent on the stages of embryogenesis from day 12 till day 32, differentiated by the fi xation period for seed embryos starting from the beginning of fl owering. Homozygous lines were created without polyploid-inducing substances due to spontaneous transfer of some cells of haploid regenerant plants to a higher level of ploidy, that can be used in the breeding of sugar beet. Genetic determination of apomictic seed reproduction in alloplasmic lines and pollen free lines of sugar beet and the technologies of inducing dihaploids allow reducing the period of inzucht-crossing considerably to obtain homozygous lines, creating unique material for chromosome engineering and marker-oriented selection with target combinations of genes in homozygous state.
Key words:apomixis (apozygoty), haploids, dihaploids, alloplasmic lines, Beta maritima, ploidy analyzer (PA) Partec.
References

1. Hand ML, Vít P, Krahulcová A, Johnson SD, Oelkers K, Siddons H, Chrtek JJR, Fehrer J, Koltunow AM. Evolution of apomixis loci in Pilosella and Hieracium (Asteraceae) inferred from the conservation of apomixislinked markers in natural and experimental populations. Heredity (Edinb). 2014;114(1):17-26. doi:10.1038/hdy. 2014.61.

2. Hojsgaard D, Klatt S, Baier R, Carman JG, Hörandl E. Taxonomy and biogeography of apomixis in Angiosperms and associated biodiversity characteristics. Crit. Rev. Plant Sci. 2014;33(5):414-27. doi:10.1080/07352689.2014.898488.

3. Rodriguez-Leal D, Vielle-Calzada J-P. Regulation of apomixis: learning from sexual experience. Curr. Opin. Plant Biol. 2012;15:549-55. http://dx.doi.org// 10.1016/j. pbi.2012.09.005.

4. Okada T, Ito K, Johnson SD., Oelkers K, Suzuki G, Houben A, Mukai Y, Koltunow AM. Chromosomes carrying meiotic avoidance loci in three apomictic eudicot Hieracium subgenus Pilosella species share structural features with two monocot apomicts. Plant Physiol. 2011;157:1327-41. doi: https://doi.org/10.1104/pp.111. 181164.

5. Yamashita K, Nakazawa Y, Namai K., Amagai M, Tsukazaki H, Wako T, Kojima A. Modes of inheritance of two apomixis components, diplospory and parthenogenesis, in Chinese chive (Allium ramosum) revealed by analysis of the segregating population generated by back-crossing between amphimictic and apomictic diploids. Breed. Sci. 2012;62(2):160-9. doi:10.1270/jsbbs.62.160.

6. Naumova TN, Hayward MD, Wagenvoort M. Apomixis and sexuality in diploid and tetraploid accession of Brachiaria decumbens. Sex. Plant Reprod. 1999;12(1):43-52. doi: https://doi.org/10.1007/s004970050170.

7. Szkutnik T. Apomixis in the sugar beet reproduction system. Acta Biol. Cracoviensia Series Botanica. 2011; 52(1):87-96. doi: 10.2478/v10182-010-0011-y.

8. Kashin AS, Tsvetova MI, Demochko YuA. Cytogenetic peculiarities of the genesis of apical meristem cells in case of gametophytic apomixis (with reference to autonomous Asteraceae apomicts). Cytol. Genet. 2011;45(2):85-96.

9. Naumova TN. Apomixis and amphimixis in fl owering plants. Cytol. Genet. 2008;42(3):179-88.

10. Khan YJ, Choudhary R, Tyagi H, Singh AK. Apomixis: The molecular perspectives and its utilization in crop breeding. J. AgriSearch. 2015;2(3):153-61.

11. Maletskii SI, Maletskaya EI, Yudanova SS. Apozygous seed production in the system of the genus Beta (Chenopodiaceae) and Vavilov’s homology series. Vavilovskiy zhurnal genetiki i selektsii, 2011;15(1):66-79. http:// www.bionet.nsc.ru/vogis/pict_pdf/2011/15_1/7.pdf

12. Bohomolov MA. Apomixis in sugar beet (Beta vulgaris L.). Review of domestic and foreign studies. Tekhnologia vysokikh urozhaev 2018;11:27-33. http://saharmag.com/ netcat_fi les/userfi les/other/Bogomolov.pdf

13. Yudanova SS. Mixoploidy and apozygoty in sugar beet. Sugar Tech, 2003;5:173-5. https://doi.org/10.1007/BF02943630.

14. Maletskaya EI, Yudanova SS, Maletskii SI. Haploids in apozygotic seed progenies of sugar beet (Beta vulgaris L.). Sugar Tech. 2009;11(1):60-64. do i:10.1007/s12355- 009-0010-z.

15. Bogomolov MA, Fedulova TP. Introgression of apomixis - a new way of creating sugar beet hybrids (Beta vulgaris L.). Sakharnaya svekla. 2018;(2):4-7. doi: 10.25802/SB.2018.02.13380.

16. Murovec J, Bohanec B. Haploids and doubled haploids in plant breeding. Plant Breed., 2012:87-106. doi: 10.5772/29982.

17. Zhuzhzhalova TP, Podvigina OA, Znamenskaya VV, Vasil’chenko EN, Karpechenko NA, Zemlyanukhina OA. Sugar beet (Beta vulgaris L.) haploid parthenogenesis in vitro: factors and diagnostic characters. Agric. Biol. 2016;51(5):636-44. doi: 10.15389/agrobiology. 2016.5.636eng

18. Roik N, Kovalchuk N, Yatseva O. Apozygoty as a method of creating initial materials of sugar beet. Bull. Agric. Sci. 2014;(6):45-7. http://agrovisnyk.com/oldpdf/visnyk_ 06_2014.pdf

19. Maletskii SI, Yudanova SS, Maletskaya EI. Analysis of epigenomic and epiplastome variability in the haploid and dihaploid sugar beet (Beta vulgaris L.) plants. Agric. Biol. 2015;50(5):579-89. doi:10.15389/agrobiology. 2015.5.579.

20. Levites E, Kirikovich S. The heteroallicity instead of heterozygosity in haploids of sugar beet Beta vulgaris L. Bull. Sci. Pract. 2017;(5):32-8. https://www.academia.edu/33122753/Bulletin_of_Science_and_Practice_5_2017.pdf.

21. Gamborg OL, Miller RA, Ojima K. Nutrient requirement of suspension cultures of soybean root cells. Exp. Cell Res. 1968;50(1):151-8. doi: 10.1016/0014- 4827(68)90403-5.

22. Roik M, Kovalchuk N, Lysenk V., Himich N. Apozygotism as a new method of enrichment of gene pool of sugar beet. Bull. Agric. Sci. 2015;(11): 49-52. http://agrovisnyk.com/pdf/ua_2015_11.pdf. (in Ukrainian).

23. Kovalchuk N, Roik N, Potapovich O. New sterile cytoplasms from wild beet Beta vulgaris SSP maritima L. from Greece and Turkey. Sakharnaya svekla, 2013;4:31-5. http://sugarbeet.ru/pdf/2013/042013.pdf#page=31

24. Shyryaeva EI. Methodological instructions regarding cytoembryological studies in breeding of sugar beet. Kyiv: VNIS, 1984:62 p.

25. Bilous VO, Ilenko II, Oliynyi NA, Pavlovska LL, Berdyshev OG. To the method of obtaining haploid plants of sugar beet from isolated unfertilized seed bud. Sugar beet. 2006;(6):12-3.

26. Pat. No. 104295. Ukraine. MPK (2016.01) A01H 4/0. Method of obtaining haploid and dihaploid lines of sugar beet based on apozygoty and cytoplasmatic male sterility/ Roik MV, Yatseva OA, Nediak TM, Potapovych OA, Kachalovska SO. - No. u 2015 06234; appl. 24.06.2015 publish. 25.01.2016.

27. Lukaszewska E, Sliwinska E. Most organs of sugarbeet (Beta vulgaris L.) plants at the vegetative and reproductive stages of development are polysomatic. Sex Plant Reprod. 2007;20:99-107. https://doi.org/10.1007/ s00497-007-0047-7.

28. Gmurman VE. Theory of probabilities and mathematical statistics. M.: Vysshaya shkola, 1999:479 p.

29. Kunakh VA, Adonin VI, Ozheredov SP, Blume YaB. Mixoploidy in wild and cultivated species of Cruciferae capable of hybridizing with rapeseed Brassica napus. Cytol. Genet. 2008;42(3):204-9. doi: 10.3103/ S0095452708030079.