Biomics

The rol-genes of Agrobacterium rhizogenes play a key role in the formation of hairy roots but their integration into the plant genome disrupts the normal development of the plant due to hormonal changes, which in turn leads to various phenotypic abnormalities. Hairy roots culture have specific characteristics such as unlimited and high speed growth on a medium without phytohormones, the ability to actively form lateral roots due to lack of apical dominance and loss of a geotropic orientation. There are a large number of studies showing that the content of secondary metabolites in hairy roots culture can far exceed their concentration in wild-type plants, which is associated with the stimulating effect of rol-gene products. In addition to hairy roots as prospective producers of bioactive molecules, such as secondary metabolites and recombinant proteins transgenic plants are also considered. Thanks to the high regenerative ability of the hairy roots culture, whole rol-genes expressed plants can be generated. Such plants can potentially possess economically valuable traits, or these plants can be used as producers of primary and secondary metabolites. The purpose of our work was to refine the main stages of the biotechnology of regenerated transgenic plants of tobacco Nicotiana tabacum L. from the hairy roots culture. Hairy roots of tobacco were made by inoculating leaf explants. Callus formation in hairy roots cultures was induced on MS medium supplemented with naphthaleneacetic acid and kinetin. For the shoot regeneration, the calluses were transplanted into MS medium with 6- benzylaminopurine and indole-3-acetic acid. Rooting of regenerated shoots was carried out on the hormone-free MS medium. 15 transgenic tobacco plants were transplanted into the soil conditions, which will be used to study the effect of the products of these genes on growth and stress resistance, and also on the content of secondary metabolites compared to wild-type plants.

Nicotiana tabacum, hairy roots, Agrobacterium rhizogenes, rol-genes, shoot regeneration, transgenic plants

1. Авраменко Т.В. Активность и продукция пероксидаз III класса в клеточных культурах растений, трансформированных генами rolB и rolC: автореф. дис. канд. биол. наук / Т. В. Авраменко; Биол.-почв. ин-т ДВО РАН. Владивосток, 2015. 24 с. [Avramenko T.V Activity and production of Class III peroxidases in cell cultures of plants transformed with rolB and rolC genes: Abstract. dis. cand. biol. sciences / T.V Avramenko; Biol.-soil. institute of Far Eastern Branch of RAS. Vladivostok, 2015. 24 P. In Russian].
2. Дрейпер Д.С., Армитидж Ф., Дьюри Г., Джэкоб Л., Уолден Р., Кумар А., Джефферсон Р., Хэмил Д. Генная инженерия растений. - М.: Мир, 1991. С. 408. [Draper J., Armitage F., Dury G., Jacob L., Walden R., Kumar A., Jefferson R., Hamil D. Plant genetic transformation and gene expression. A laboratory manual. Oxford; Boston: Blackwell Scientific Publications 366 p].
3. Кузовкина И.Н., Вдовитченко М.Ю. Генетически трансформированные корни как модельная система для изучения физиологических и биохимических процессов корневой системы целого растения / В кн.: Молекулярно-генетические и биохимические методы в современной биологии растений. Под ред. Кузнецова Вл. В., Кузнецова В.В., Романова Г.А. Москва, 2012. БИНОМ, Лаборатория знаний, C. 137–153. [Kuzovkina I.N., Vdovitchenko M.Yu. Genetically transformed roots as a model system for studying the physiological and biochemical processes of the root system of an entire plant / In: Molecular genetic and biochemical methods in modern plant biology. Kuznetsov Vl.V., Kuznetsov V.V., Romanov G.A. (eds.) Moscow, 2012. BINOM, Laboratory of Knowledge, P. 137–153. In Russian].
4. Кулаева О.А., Матвеева Т.В., Лутова Л.А. Горизонтальный перенос генов от агрообактерий к растениям // Экологическая генетика. 2006. Т. IV. С. 10–19. [Kulaeva O.A., Matveeva T.V., Lutova L.A Horizontal transfer of genes from agroobacteria to plants // Ecological Genetics. V. IV. P. 10–19. In Russian].
5. Михайлова Е.В., Кулуев Б.Р., Ясыбаева Г.Р., Чемерис А.В. Создание культур бородатых корней Withania somnifera и оценка параметров их роста при выращивании на твердых и жидких питательных средах // Вестник биотехнологии и физико-химической биологии им. Ю.А. Овчинникова. 2017. Т. 13. С. 40–45. [Mikhailova E.V., Kuluev B.R., Yasibaeva G.R., Chemeris A.V. Creation of cultures of hairy roots Withania somnifera and assessment of parameters of their growth at cultivation on firm and liquid nutritious environments // Journal “Yu.A. Ovchinnikov Bulletin of Biotechnology and Physical and Chemical Biology”. V. 13. P. 40–45. In Russian].
6. Мусин Х.Г., Якупова А.Б., Михайлова Е.В., Кулуев Б.Р. Особенности роста культур генетически трансформированных (бородатых) корней табака и витании при изменении объема питательной среды // Вестник биотехнологии и физико-химической биологии им. Ю.А. Овчинникова. 2017. Т. 13. С. 46–50. [Musin Kh.G., Yakupova A.B, Mikhailova E.V., Kuluyev B.R. Peculiarities of the growth of cultures of genetically transformed (hairy) roots of tobacco and vitania with a change in the volume of nutrient medium // Journal “Yu.A. Ovchinnikov Bulletin of Biotechnology and Physical and Chemical Biology”. 2017. V. 13. P. 46–50. In Russian].
7. Павлова О.А., Матвеева Т.В., Лутова Л.А. Rol-гены Agrobacterium rhizogenes // Экологическая генетика. 2013. Т. XI. С. 59–68. [Pavlova O.A., Matveeva Т.V., Lutova L.A. Rol-genes of Agrobacterium rhizogenes // Ecological Genetics. 2013. V. XI. P. 59–68. In Russian].
8. Эрст А.А., Зибарева Л.Н., Железниченко Т.В., Ковзунова О.В. Культура генетически трансформированных корней (hairy roots) Silene roemeri Friv. – источник получения фитоэкдистероидов // Вестник Томского государственного университета. Биология. 2017. № 37. С. 17–30. [Erst A.A., Zibareva L.N., Zheleznichenko T.V., Kovzunova O.V. Culture of genetically transformed roots (hairy roots) Silene roemeri Friv is source of phytoecdysteroids production // Tomsk State University Journal of Biology. 2017. №. 37. P. 17–30. In Russian].
9. Ясыбаева Г.Р., Вершинина З.Р., Кулуев Б.Р., Чемерис А.В. Безбактериальное получение косматых корней // Вестник защиты растений. Т. 89. С. 187–188. [Yasibaeva G.R., Vershinina Z.R., Kuluev B.R., Chemeris A.V. Induction of hairy roots without Agrobacterium transformation // Plant Protection News. 2016. V. 89. P. 187–188. In Russian].
10. Altamura M.M. Agrobacterium rhizogenes rolB and rolD genes: regulation and involvement in plant development // Plant Cell, Tissue and Organ Culture. 2004. V. 77. Р. 89–101. doi:10.1023/B:T ICU.0000016609.22655.33
11. Bonhomme V., Laurain Mattar D., Fliniaux M.A. Effects of the rolC gene on hairy root: induction development and tropane alkaloid production by Atropa belladonna // Nat. Prod. 2000. V. 63. P. 1249–1252. doi:10.1021/np990614l
12. Bulgakov V.P., Tchernoded G.K., Mischenko N.P., Khodakovskaya M.V., Glazunov V.P., Radchenko S.V., Zvereva E.V., Fedoreyev S.A., Zhuravlev Y.N. Effect of salicylic acid, methyl jasmonate, ethephon and cantharidin on anthraquinone production by Rubia cordifolia callus cultures transformed with the rolB and rolC genes // Biotechnol. 2002. V. 97. P. 213–221. doi:10.1016/ S0168-1656(02)00067-6
13. Bulgakov V.P., Shkryl Y.N., Veremeichik G.N., Gorpenchenko T.Y., Vereshchagina Y.V. Recent advances in the understanding of Agrobacterium rhizogenes-derived genes and their effects on stress resistance and plant metabolism // Adv. Biochem. Eng. Biotechnol. 2013. V. 134. P. 11–22. doi:10.1007/10_2013_179
14. Carneiro M., Vilaine F. Differential expression of the rolA plant oncogene and its effect on tobacco development // Plant J. 1993. V. 3. P. 785–792. doi:10.1111/j.1365-313X.1993.00785.x
15. Hatamoto H., Boulter M.E. Shirsat A.H., Croy E.J., Ellis J.R. Recovery of morphologically normal transgenic tobacco from hairy roots co-transformed with Agrobacterium rhizogenes and a binary vector plasmid // Plant Cell Reports. 1990. V. 9. P. 88–92. doi:10.1007/BF00231556
16. Kiselev K.V., Dubrovina A.S., Veselova M.V., Bulgakov V.P., Fedoreyev S.A., Zhuravlev Y.N. The rolB gene-induced overproduction of resveratrol in Vitis amurensis transformed cells // J. Biotechnol. 2007. V. 128. P. 681–692. doi:10.1016/j.jbiotec.2006.11.008
17. Mauro M.L., Trovato M., Paolis A.D., Gallelli A., Costantino P., Altamura M.M. The plant oncogene rolD stimulates flowering in transgenic tobacco plants // Dev. Biol. 1996. V. 180. P. 693–700. doi:10.1006/dbio.1996.0338
18. Mauro M.L., Costantino Р.P., Bettini P. The never ending story of rol genes: a century after // Plant Cell, Tissue and Organ Culture. V. 131. P. 201–212. doi:10.1007/s11240-017-1277-5
19. Mohajjel-Shoja H. Contribution to the study of the Agrobacterium rhizogenes plast genes, rolB and rolC, and their homologs in Nicotiana tabacum. Thesis of University of Strasbourg. 2010.
20. Nilsson O., Tumineh H., Sundberg B., Olsson O. The Agrobacterium rhizogenes rolB and rolC promoters are expressed in pericycle cells competent to serve as root initials in transgenic hybrid aspen // Physiologia Plantarum. 1997. V. 100. P. 456–462. doi:10.1111/j.1399-3054.1997.tb03050.x
21. Palazon J., Cusido R.M., Roig C., Piñol M.T. Expression of the rolC gene and nicotine production in transgenic roots and their regenerated plants // Plant Cell Rep. 1998. V. 17. P. 384–390. doi:10.1007/s002990050411
22. Trovato M., Mauro M.L., Costantino P., Altamura M.M. The rolD gene from Agrobacterium rhizogenes is developmentary regulated in transgenic tobacco // Protoplasma. 1997. V. 197. P. 111–120. doi:10.1007/BF01279889
23. Schmülling T., Schell J., Spena A. Single genes from Agrobacterium rhizogenes influence plant development // EMBO J. 1988. V. 7. P. 2621–2629.
24. Shkryl Y.N., Veremeichik G.N., Bulgakov V.P., Tchernoded G.K., Mischenko N.P., Fedoreyev S.A., Zhuravle Y.N. Individual and combined effects of the rolA, B and C genes on anthraquinone production in Rubia cordifolia transformed calli // Biotechnol. Bioeng. 2008. V. 1. P. 118–125. doi:10.1002/bit.21727
25. Spena A., Schmulling T., Koncz C., Shell J.S. Independent and synergistic activity of rolA, B and C loci in stimulating abnormal growth in plants // EMBO J. 1987. 6. Р 3891–3899.
26. Veremeichik G.N, Shkryl Y.N, Bulgakov V.P, Avramenko T.V, Zhuravlev Y.N. Molecular cloning and characterization of seven class III peroxidases induced by overexpression of the agrobacterial rolB gene in Rubia cordifolia transgenic callus cultures // Plant Cell Rep. V.6. P.1009–1019. doi:10.1007/s00299-011-1219-3