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Zheleznichenko T. V., Muraseva D. S., Asbaganov S. V., Voronkova M. S., Zaushintsena A. V., Novikova T. I. Study of the Genetic Variability of Adventive Microshoots of Picea pungens Engelm., Using DNA Markers and Flow Cytometry

Keywords:
in vitro culture, microshoots regeneration, thidiazuron, ISSR and RAPD analysis, flow cytometry, blue spruce
Pages:
45–54

Abstract

UDC 581.143.6

How to cite: Zheleznichenko T. V.1, Muraseva D. S.1, Asbaganov S. V.1, Voronkova M. S.1, Zaushintsena A. V.2, Novikova T. I.Study of the genetic variability of adventive microshoots of Picea pungens Engelm., using DNA markers and flow cytometry // Sibirskij Lesnoj Zurnal (Sib. J. For. Sci.). 2020. N 4. P. 45–54 (in Russian with English abstract and references).

DOI: 10.15372/SJFS20200406

© Zheleznichenko T. V., Muraseva D. S., Asbaganov S. V., Voronkova M. S., Zaushintsena A. V., Novikova T. I., 2020

For the first time, the genetic variability/stability of blue spruce Picea pungens Engelm. microshoots obtained in vitro by direct regeneration from zygotic embryos under the influence of a synthetic growth regulator with cytokinin activity – N-phenyl-N′-1, 2, 3-thidiazurol-5-urea or thidiazuron (TDZ) using ISSR and RAPD analysis, as well as by flow cytometry, was studied. Shoot formation was induced at ½ LV medium using two concentrations of TDZ (0.5 or 1 μM) for 28 days, then plant material was transferred to a hormone-free nutrient medium ½ LV for 35 days. The formed microshoots were separated from the explant’s tissues and cultured on the same hormone-free medium for another 35 days, than their genetic stability was evaluated. To evaluate the effect of each concentration of the growth regulator on the genome’s stability, microshoots formed by three different genotypes of donor plants were used. ISSR analysis revealed a slight polymorphism between the blue spruce microshoots formed by the same genotype. The genetic variability of regenerants in individual genotypes ranged from 1.29 to 1.58 %, while other genotypes have 100 % genetically stable microshoots. At the same time, RAPD analysis did not show differences between the analyzed samples; all studied primers initiated monomorphic spectra. Flow cytometry found that all the studied samples were diploid. The DNA content (2C) in microshoots ranged from 38.86 ± 0.55 – 40.35 ± 0.30 pg, and the genome size (1C) was 18961.79 ± 256.91–19933.27 ± 526.35 Mbp. The obtained results indicate a low somaclonal variability of blue spruce microshoots formed in vitro under as a result of direct regeneration from zygotic embryos under the influence of the studied concentrations in the TDZ.  

Article


СПИСОК ЛИТЕРАТУРЫ (REFERENCES)

Богданова E. B. Кариологическое изучение Picea pungens Engelm. в условиях интродукции // Бюлл. Бот. сада Саратов. гос. ун-та. 2009. № 8. С. 219–223 [Bogdanova E. B. Kariologicheskoe izuchenie Picea pungens Engelm. v usloviyakh introduktsii (Karyological study of Picea pungens Engelm. under conditions of introduction) // Byull. Bot. sada Saratov. gos. un-ta (Bull. Bot. garden Saratov St. Univ.). 2009. N. 8. P. 219–223 (in Russian)].

Владимирова О. С., Муратова Е. Н., Карпюк Т. В. Числа хромосом некоторых видов Picea и Larix // Бот. журн. 2007. Т. 92. № 5. С. 781–782 [Vladimirova O. S., Muratova E. N., Karpyuk T. V. Chisla khromosom nekotorykh vidov Picea i Larix (Chromosome numbers of some Picea and Larix species) // Bot. zhurn. (Bot. J.). 2007. V. 92. N. 5. P. 781–782 (in Russian with English abstract)].

Гамалей Ю. В., Шереметьев С. Н. Направления эволюции генома наземных и вторично-водных трав // Цитология. 2012. Т. 54. № 6. С. 449–458 [Gamaley Yu. V., Sheremet’ev S. N. Napravleniya evolyutsii genoma nazemnykh i vtorichno-vodnykh trav (Trends of genome evolution in land and secondary-water herbs) // Tsitologiya (Cytology). 2012. V. 54. N. 6. P. 449–458 (in Russian with English abstract)].

Железниченко Т. В., Новикова Т. И. Влияние аскорбиновой кислоты и глутатиона на индукцию соматического эмбриогенеза Picea pungens Engelmann // Turczaninowia. 2017. Т. 20. № 3. С. 27–35 [Zheleznichenko T. V., Novikova T. I. Vliyanie askorbinovoy kisloty i glutationa na induktsiyu somaticheskogo embriogeneza Picea pungens Engelmann (Effect of ascorbic acid and glutathione on somatic embryogenesis induction in Picea pungens Engelmann) // Turczaninowia. 2017. V. 20. N. 3. P. 27–35 (in Russian with English abstract)].

Железниченко Т. В., Мурасева Д. С., Стасова В. В., Новикова Т. И. Морфогенез Picea pungens Engelm. в культуре in vitro под действием тидиазурона // Сиб. лесн. журн. 2019. № 1. С. 57–64 [Zheleznichenko T. V., Muraseva D. S., Stasova V. V., Novikova T. I. Morfogenez Picea pungens Engelm. v kulture in vitro pod deystviem tidiazurona (Morphogenesis of Picea pungens Engelm. in vitro under the influence of thidiazuron) // Sib. lesn. zhurn. (Sib. J. For. Sci.). 2019. N. 1. P. 57–64 (in Russian with English abstract)].

Муратова Е. Н. В-хромосомы голосеменных // Усп. совр. биол. 2000. Т. 120. № 5. С. 452–465 [Muratova E. N. V-khromosomy golosemennykh (B chromosomes of gymnosperms) // Usp. sovr. biol. (Succes. contemp. biоl.). 2000. V. 120. N. 5. P. 452–465 (in Russian with English abstract)].

Седельникова Т. С. Изменчивость размера генома хвойных в экстремальных условиях произрастания // Усп. совр. биол. 2015. Т. 135. № 5. С. 514–528 [Sedel’nikova T. S. Izmenchivost’ razmera genoma khvoynykh v ekstremalnykh usloviyakh proizrastaniya (Variability of genome size in conifers under extreme environmental conditions) // Usp. sovr. biol. (Succes. contemp. biоl.). 2015. V. 135. N. 5. P. 514–528 (in Russian with English abstract)].

Седельникова Т. С., Муратова Е. Н., Пименов А. В. Изменчивость хромосомных чисел голосеменных растений // Усп. совр. биол. 2010. Т. 130. № 6. С. 557–568 [Sedel’nikova T. S., Muratova E. N., Pimenov A. V. Izmenchivost’ khromosomnykh chisel golosemennykh rasteniy (Variability of chromosome numbers in gymnosperms) // Usp. sovr. biol. (Succes. contemp. biоl.). 2010. V. 130. N. 6. P. 557–568 (in Russian with English abstract)].

Скапцов М. В., Смирнов С. В., Куцев М. Г., Шмаков А. И. Проблемы стандартизации в проточной цитометрии растений // Turczaninowia. 2016. Т. 19. № 3. С. 120–122 [Skaptsov M. V., Smirnov S. V., Kutsev M. G., Shmakov A. I. Problemy standartizatsii v protochnoy tsitometrii rasteniy (Problems of a standardization in plant flow cytometry) // Turczaninowia. 2016. V. 19. N. 3. P. 120–122 (in Russian with English abstract)].

Хемлебен В., Беридзе Т. Г., Бахман Л., Коварик Я., Торрес Р. Сателлитные ДНК // Усп. биол. хим. 2003. Т. 43. С. 267–306 [Khemleben V., Beridze T. G., Bakhman L., Kovarik Ya., Torres R. Satellitnye DNK (Satellite DNA) // Usp. biol. khim. (Adv. biol. chem.). 2003. V. 43. P. 267–306 (in Russian)].

Эрст А. А., Звягина Н. С., Новикова Т. И., Дорогина О. В. Клональное микроразмножение редкого вида Hedysarum theinum Krasnob. (Fabaceae) и оценка генетической стабильности регенерируемых растений с помощью ISSR маркеров // Генетика. 2015. Т. 51. № 2. С. 188–193 [Erst A. A., Zvyagina N. S., Novikova T. I., Dorogina O. V. Klonal’noe mikrorazmnozhenie redkogo vida Hedysarum theinum Krasnob. (Fabaceae) i otsenka geneticheskoy stabil’nosti regeneriruyemykh rasteniy s pomoshchyu ISSR markerov (Clonal micropropagation of a rare species Hedysarum theinum Krasnob. (Fabaceae) and assessment of the genetic stability of regenerated plants using ISSR markers) // Genetika (Genetics). 2015. V. 51. N. 2. P. 188–193 (in Russian with English abstract)].

Afele J. C., Saxena P. K. Somatic embryogenesis in blue spruce (Picea pungens Engelmann) // Somatic Embryogenesis in Woody Plants. For. Sci. Springer Sci. + Business Media Dordrecht, 1995. V. 44–46. P. 99–109.

Ali M., Mujib A., Tonk D., Zafar N. Plant regeneration through somatic embryogenesis and genome size analysis of Coriandrum sativum L. // Protoplasma. 2016. V. 254. N. 1. P. 343–352.

Bhojwani S. S. Plant tissue culture and its relevance to mulberry breeding In: Brainstorming meeting on genetics and biotechnology of silkworm and mulberry. India: CSRTI, Mysore, 1992. P. 1–11.

Bornet B., Branchard M. Nonanchored inter simple sequence repeat (ISSR) markers: reproducible and specific tools for genome fingerprinting // Plant Mol. Biol. Rep. 2001. V. 19. N. 3. P. 209–215.

Clarindo W. R., Carvalho C. R., Arauojo F. S., Abreu I. S., Otoni W. C. Recovering polyploid papaya in vitro regenerants as screened by flow cytometry // Plant Cell Tiss. Organ. Cult. 2008. V. 92. P. 207–214.

Cram W. H. Needle color and vigor of inbred progenies of Picea pungens // Hort. Sci. 1984. V. 19. N. 1. P. 125–126.

Doležel J. Flow cytometric analysis of nuclear DNA content in higher plants // Phytochem. Analysis. 1991. V. 2. Iss. 4. P. 143–154.

Doležel J., Bartoš J., Voglmayr H., Greilhuber J. Nuclear DNA content and genome size of trout and human // Cytometry. Part A. 2003. V. 51. Iss. 2. P. 127–128.

Doležel J., Greilhuber J., Suda J. Estimation of nuclear DNA content in plants using flow cytometry // Nature Protocols. 2007. V. 2. N. 9. P. 2233–2244.

Erst A. A., Zvyagina N. S., Novikova T. I., Dorogina O. V. Clonal micropropagation of a rare species Hedysarum theinum Krasnob. (Fabaceae) and assessment of the genetic stability of regenerated plants using ISSR markers // Rus. J. Gen. 2015. V. 51. N. 2. P. 158–162 (Original Rus. text © A. A. Erst, N. S. Zvyagina, T. I. Novikova, O. V. Dorogina, 2015, publ. in Genetika. 2015. V. 51. N. 2. P. 188–193).

Hizume M. Karyomorphological studies in the family Pinaceae // Mem. Fac. Educ. Ehime Univ. Ser. 3. Nat. Sci. 1988. V. 8. P. 1–108.

Jerico B. B., Lourdes I.-A., Lazaro S.-V., Jose C.-M., Nancy S.-B. In vitro regeneration of Pinus brutia Ten. var. eldarica (Medw.) through organogenesis // Afr. J. Biotechnol. 2012. V. 11. N. 93. P. 15982–15987.

Kaeppler S. M., Kaeppler H. F., Rhee Y. Epigenetic aspects of somaclonal variation in plants // Plant Mol. Biol. 2000. V. 43. P. 179–188.

Kirdar E., Ertekin M., Gökyer E., Çorbaci Ö. L. Mavi ladinin (Picea pungens Engelm.) Aşı ile Üretimi Üzerine Araştırmalar (Investigations on propagation by grafting of blue spruce (Picea pungens Engelm.)) // Kastamonu Üni. Orman Fakültesi Dergisi (Kastamonu Univ. J. For. Fac.). 2009. V. 9. N. 1. P. 35–41 (in Turkish with English summary).

Kumar P. S., Mathur V. L. Chromosomal instability in callus culture of Pisum sativum // Plant Cell, Tiss. Org. Cult. 2004. V. 78. P. 267–271.

Kurup S. S., Purayil F. T., Alkhaili M. M., Tawfik N. H., Cheruth A. J., Kabshawi M., Subramaniam S. Thidiazuron (TDZ) induced organogenesis and clonal fidelity studies in Haloxylon persicum (Bunge ex Boiss & Buhse): an endangered desert tree species // Physiol. Mol. Biol. Plants. 2018. V. 24. P. 683–692.

Larkin P. J., Scowcroft W. R. Somaclonal variation – a novel source of variability from cell cultures for plant improvement // Theor. Appl. Genet. 1981. V. 60. P. 197–214.

Litvay J. D., Verma D. C., Jonson M. A. Influence of loblolly pine (Pinus taeda L.). Culture medium and its components on growth and somatic embryogenesis of the wild carrot (Daucus carota L.) // Plant Cell Rep. 1985. V. 4. N. 6. P. 325–328.

Losada J. M., Leslie A. B. Why are the seed cones of conifers so diverse at pollination? // Annals Bot. 2018. V. 121. Iss. 7. P. 1319–1331.

Lyon L. Winter grafting of cedar, spruce, and ornamental cherry // Int. Plant Propagators’ Soc. Proc. 1983. V. 33. P. 54–55.

Mishiba K.-I., Tawada K.-I., Mii M. Ploidy distribution in the explant tissue and the calluses induced during the initial stage of internode segment culture of Asparagus officinalis L. // In Vitro Cell. Dev. Biol. – Plant. 2006. V. 42. N. 1. P. 83–88.

Murthy B. N., Murch S. J., Saxena P. K. Thidiazuron: a potent regulator of in vitro plant morphogenesis // In Vitro Cell. Dev. Biol. – Plant. 1998. V. 34. N. 4. P. 267–275.

Nehra N. S., Kartha K. K., Stushnott C., Giles K. L. The influence of plant growth regulator concentrations and callus age on somaclonal variation in callus culture regenerants of strawberry // Plant Cell, Tiss. Org. Cult. 1992. V. 29. N. 3. P. 257–268.

Novikova T. I., Asbaganov S. V., Ambros E. V., Zaytseva Y. G. TDZ-induced axillary shoot proliferation of Rhododendron mucronulatum Turcz and assessment of clonal fidelity using DNA-based markers and flow cytometry // In Vitro Cell. Dev. Biol. – Plant. 2020. V. 56. P. 307–317.

Obermayer R., Leitch I. J., Hanson L., Bennett M. D. Nuclear DNA C-values in 30 species double the familial representation in pteridophytes // Annals Bot. 2002. V. 90. N. 2. P. 209–217.

Ohri D., Khoshoo T. N. Genome size in gymnosperms // Plant Syst. Evol. 1986. V. 153. N. 1. P. 119–132.

Purayil F. T., Kurup S. S., Alkhaili M. M., Tawfik N. H., Al Dhaheri S. M., Cheruth A. J., Al Dhaheri S. S., Subramaniam S. ISSR-assisted analysis of clonal fidelity supported with SEM and histology using in vitro propagated plants of Moringa peregrina (Forssk.) Fiori – An endangered desert tree // South Afr. J. Bot. 2018. V. 114. P. 163–170.

Ray T., Dutta I., Saha P., Das S., Roy S. C. Genetic stability of three economically important micropropagated banana (Musa spp.) cultivars of lower Indo-Gangetic plains, as assessed by RAPD and ISSR markers // Plant Cell, Tiss. Org. Cult. 2006. V. 85. P. 11–21.

Rees H., Teoh S. B., Jones L. M. Heterochromatization and the possibility of gene inactivation in B chromosomes of Picea glauca // Heredity. 1977. V. 38. N. 2. P. 272.

Saker M. M., Bekheet S. A., Taha H. S., Fahmy A. S., Moursy H. A. Detection of somaclonal variations in tissue culture-derived date palm plants using isoenzyme analysis and RAPD fingerprints // Biol. Plant. 2000. V. 43. N. 3. P. 347–351.

Takagi H., Sugawara S., Saito T., Tasaki H., Yuanxue L., Guan K., Han D.-S., Godo T., Nakano M. Plant regeneration via direct and indirect adventitious shoot formation and chromosome-doubled somaclonal variation in Titanotrichum oldhamii (Hemsl.) Solereder // Plant Biotechnol. Rep. 2011. V. 5. N. 2. P. 187–195.

Tang W., Newton R. J. Plant regeneration from callus cultures derived from mature zygotic embryos in white pine (Pinus strobus L.) // Plant Cell Rep. 2005. V. 24. N. 1. P. 1–9.

Teoh S. B., Rees H. B chromosomes in white spruce // Proc. Roy. Soc. London. Ser. Biol. Sci. 1977. V. 198. N. 1133. P. 325–344.

Nieuwkirk J. P van, Zimmerman R. H., Fordham I. Thidiazuron stimulation of apple shoot proliferation in vitro // Hort. Sci. 1987. V. 21. N. 3. P. 516–518.

Zaytseva Y. G., Poluboyarova T. V., Novikova T. I. Effects of thidiazuron on in vitro morphogenic response of Rhododendron sichotense Pojark. and Rhododendron catawbiense cv. Grandiflorum leaf explants // In Vitro Cell. Dev. Biol. Plant. 2016. V. 52. N. 1. P. 56–63.

Zaytseva Y. G., Novikova T. I. TDZ-induced morphogenesis pathways in woody plant culture // Thidiazuron: From urea derivative to plant growth regulator. Chapter 3. Singapore: Springer Nature, 2018. P. 61–91.


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