DOI: https://doi.org/10.21498/2518-1017.15.2.2019.173558

Callus formation, organogenesis and microclonal reproduction in different species of the genus Linum L. in vitro

С. В. Міщенко, Л. М. Кривошеєва

Abstract


Purpose. To reveal the frequency and intensity of callus formation and organogenesis, the effectiveness of microclonal reproduction of various species of the genus Linum L. (Linaceae) in vitro.

Methods. For in vitro induction of callus formation and organogenesis, hypocotyl segments of species Linum usitatissimum L. convar. elongatum and convar. usitatissimum, L. tenue Desf., L. bienne Mill., L. corymbulosum Pchb., L. nervosum Waldst. & Kit., L. flavum L., L. campanulatum L., L. perenne L., L. austriacum L., L. grandiflorum Desf., L. strictum L. were cultivated on Murashige and Skoog medium supplementedwith 0.05 mg/l 1-naphthylacetic acid and 1.0 mg/l 6-benzyl aminopurine at 22–24 °C, relative humidity of 60–80%,with 16 hours photoperiod (2500 flux). For microclonal reproduction Murashige and Skoog, White, Gamborg and Eveleigh media and their modifications were used. The measurement results were interpreted by the arithmetic mean, standard error for the sample mean, the leastsignificantdifference and ranked.

Results. Different species of the genus Linum to a large extend are capable of forming callus and regenerating shoots under the specified cultivation conditions. The frequency of callus formation for the studied samples on the 35th day of cultivation varied within 81.25–100%, the mass of callus from one explant – 0.21–1.64 g, the frequency of organogenesis – 12.50–100%, the number of shoots – 1.8–7.6 pcs. and the height of the shoots was 0.82–2.12 cm. The following species: L. usitatissimum convar. elongatum, L. tenue, L. bienne and L. strictum were distinguished by a high intensity of callus formation. Intensive organogenesis was pecular to L. tenue, L. bienne, L. flavum, L. austriacum and L. grandiflorum. The efficiency of somaclone obtaining was quite low in L. nervosum and L. campanulatum. In total, for the microclonal reproduction of species of the genus Linum Murashige and Skoog, Gamborg and Eveleighmedia supplementedwith 12.5 g/l glucose were optimal. At the final stages of microclonal propagation, before transferring microclones in vivo, it is advisable to use White medium, which contributes to a high frequency of rhizogenesis. Varieties of L. usitatissimum convar. elongatum and convar. usitatissimum had diffe­rent responses to in vitro culture.

Conclusions. The frequency and intensity of callus formation and organogenesis, the effectiveness of microclonal reproduction depended on the genotype of a particular species; therefore it is advisable to select the composition of the nutrient medium and growth regulators for each of them. Some species of the genus Linum have not yet been studied in vitro, so the obtained results allow expanding the scope of their use in practice, in particular in breeding as a new source material with somaclonal variation, interspecific crosses, and ornamental floriculture.


Keywords


Linum L.; in vitro; nutrient medium; phytohormones; shoot

References


Optasiuk, O. M., & Shevera, M. V. (2011). Rid Linum L. u flori Ukrainy [The genus Linum L. in the flora of Ukraine]. Кyiv: Alterpres. [in Ukrainian]

Shisha, E. N., Emets, A. I., Guzenko, E. V., Lemesh, V. A., Kartel’, N. A., & Blyum, Ya. B. (2011). Study of the regeneration capability and root formation in Ukrainian and Belarusian flax cultivars. Fiziol. Biokhim. Kul’t. Rast. [Physiology and Biochemistry of Cultivated Plants], 43(1), 57–64. [in Russian]

Janowicz, J., Niemann, J., & Wojciechowski, A. (2012). The effect of growth regulators on the regeneration ability of flax (Linum usitatissimum L.) hypocotyl explants in in vitro culture. BioTechnologia, 93(2), 135–138. doi: 10.5114/bta.2012.46578

Siegień, I., Adamczuk, A., & Wróblewska, K. (2013). Light affects in vitro organogenesis of Linum usitatissimum L. and its cyanogenic potential. Acta Physiol. Plant., 35(3), 781–789. doi: 10.1007/s11738‑012‑1118-4

Mundhara, R., & Rashid, A. (2006). TDZ-induced triple-response and shoot formation on intact seedlings of Linum, putative role of ethylene in regeneration. Plant Sci., 170(2), 185–190. doi: 10.1016/j.plantsci.2005.06.015

Seta‑Koselska, A., & Skórzyńska‑Polit, E. (2017). Optimization of in vitro culture conditions for obtaining flax (Linum usitatissimum L. cv. Modran) cell suspension culture. BioTechnologia, 98(3), 183–188. doi: 10.5114/bta.2017.70796

Tsygankova, V. A., Bayer, O. O., Andrusevich, Ya. V., Galkin, A. P., Brovarets, V. S., Yemets, A. I., & Blume, Ya. B. (2016). Screening of five and six-membered nitrogen-containing heterocyclic compounds as new effective stimulants of Linum usitatissimum L. organogenesis in vitro. Int. J. Med. Biotechnol. Genetics, S2:001, 1–9. doi: 10.19070/2379-1020-SI02001

Yildiz, M., & Özgen, M. (2004). The effect of a submersion pretreatment on in vitro explant growth and shoot regeneration from hypocotyls of flax (Linum usitatissimum). Plant Cell Tiss. Organ. Cult., 77(1), 111–115. doi: 10.1023/B:TICU.0000016493.03592.c3

Yildiz, M., Sağlik, C., Telci, C., & Erkilich, E. G. (2011). The effect of in vitro competition on shoot regeneration from hypocotyl explants of Linum usitatissimum. Turk. J. Bot., 35(2), 211–218. doi: 10.3906/bot-1005-26

Beyaz, R., & Yildiz, M. (2019). The effect of inter-plantal com­petition on in vitro seed germination and seedling growth in flax (Linum usitatissimum L.). Eskişehir Technical Univ. J. of Sci. and Tech. C – Life Sci. and Biotech., 8(1), 61–68. doi: 10.18036/aubtdc.427128

Rutkowska‑Krause, I., Mankowska, G., Lukaszewicz, M., & Szo­pa, J. (2003). Regeneration of flax (Linum usitatissimum L.) plants from anther culture and somatic tissue with increased resistance to Fusarium oxysporum. Plant Cell Rep., 22(2), 110–116. doi: 10.1007/s00299-003-0662-1

Burbulis, N., Blinstrubienė, A., Sliesaravičius, A., & Vensku­to­nienė, E. (2005). Influence of genotype, growth regulators, sucrose level and preconditioning of donor plants on flax (Linum usitatissimum L.) anther culture. Acta Biol. Hung., 56(3–4), 323–331. doi: 10.1556/ABiol.56.2005.3-4.15

Burbulis, N., & Blinstrubienė, A. (2011). Genotypic and exo­genous factors affecting linseed (Linum usitatissimum L.) anther culture. J. Food Agricult. Environ., 9(3–4), 364–367. doi: 10.1234/4.2011.2285

Burbulis, N., Blinstrubienė, A., Masienė, R., & Jonytiene, V. (2012). Influence of genotype, growth regulators and sucrose concen­tration on linseed (Linum usitatissimum L.) anther culture. J. Food Agricult. Environ., 10(3–4), 764–767. doi: 10.1234/4.2012.3509

Millam, S., Davidson, D., & Powell, W. (1992). The use of flax (Linum usitatissimum) as a model system for studies on organogenesis in vitro: the effect of different carbohydrates. Plant Cell Tiss. Organ Cult., 28(2), 163–166. doi: 10.1007/BF00055512

Chen, Y., & Dribnenki, P. (2002). Effect of genotype and medium composition on flax Linum usitatissimum L. anther culture. Plant Cell Rep., 21(3), 204–207. doi: 10.1007/s00299-002-0500-x

Soroka, A. I. (2010). Peculiarities of donor plant preparation and flax anther cultivation in vitro for haploid plant production. Vìsnik Zaporìzʹkogo nacìonalʹnogo unìversitetu. Bìologìčnì nauki [Visnyk of Zaporizhzhya National University. Biological Sciences], 2, 13–19. [in Russian]

Sakhare, S. P., & Mendhulkar, V. D. (2016). Embryo excised cal­lus induction and rhizogenesis in Linum usitatissimum L. Int. J. Pharm. Bio. Sci., 7(3), 507–511.

Blinstrubienė, A., Burbulis, N., & Masienė, R. (2017). Genotypic and exogenous factors affecting linseed ovary culture. Zemdirbyste-Agriculture, 104(3), 243–248. doi: 10.13080/z-a.2017.104.031

Bonell, M., & Lassaga, S. L. (2002). Genetic analysis of the res­ponse of linseed (Linum usitatissimum L.) somatic tissue to in vitro cultivation. Euphytica, 125(3), 367–372. doi: 10.1023/A:1016013609068

The Plant List. (n.d.). Retrieved from http://www.theplantlist.org

Diederrichsen, A., & Richards, K. (2003). Cultivated flax and the genus Linum L.: Taxonomy and germplasm conservation. In A. D. Muir, & N. D. Westcott (Eds.), Flax: The genus Linum (pp. 39–42). Boca Raton: CRC Press.

Zelentsov, S. V., Zelentsov, V. S., Moshnenko, E. V., & Ryaben­ko, L. G. (2016). Modern understanding of the phylogeny and taxonomy of genus Linum L. and flax (Linum usitatissimum L.). Maslichnye kul’tury. Nauchno-tekhnicheskiy byulleten’ VNIIMK [Oil Crops. Scientific and technical bulletin of All-Russia Research Institute of Oil Crops], 1, 106–121. [in Russian]

Murashige, T., & Skoog, F. (1962). A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol. Plant., 15(3), 473–497. doi: 10.1111/j.1399‑3054.1962.tb08052.x

Gamborg, O. L., & Eveleigh, D. E. (1968). Culture methods and detection of glucanases in suspension cultures of wheat and barley. Can. J. Biochem. Cell B., 46(5), 417–421. doi: 10.1139/o68-063


GOST Style Citations


Оптасюк О. М., Шевера М. В. Рід Linum L. у флорі України. Київ : Альтерпрес, 2011. 276 с.

Шиша Е. Н., Емец А. И., Гузенко Е. В. и др. Изучение регенерационной способности и корнеобразования у сортов льна-долгунца украинской и белорусской селекции. Физиология и биохимия культ. растений. 2011. Т. 43, № 1. С. 57–64.

Janowicz J., Niemann J., Wojciechowski A. The effect of growth regulators on the regeneration ability of flax (Linum usitatissimum L.) hypocotyl explants in in vitro culture. BioTechnologia. 2012. Vol. 93, Iss. 2. P. 135–138. doi: 10.5114/bta.2012.46578

Siegień I., Adamczuk A., Wróblewska K. Light affects in vitro organogenesis of Linum usitatissimum L. and its cyanogenic potential. Acta Physiol. Plant. 2013. Vol. 35, Iss. 3. P. 781–789. doi: 10.1007/s11738‑012‑1118-4

Mundhara R., Rashid A. TDZ-induced triple-response and shoot formation on intact seedlings of Linum, putative role of ethylene in regeneration. Plant Sci. 2006. Vol. 170, Iss. 2. P. 185–190. doi: 10.1016/j.plantsci.2005.06.015

Seta‑Koselska A., Skórzyńska‑Polit E. Optimization of in vitro culture conditions for obtaining flax (Linum usitatissimum L. cv. Modran) cell suspension culture. BioTechnologia. 2017. Vol. 98, Iss. 3. P. 183–188. doi: 10.5114/bta.2017.70796

Tsygankova V. A., Bayer O. O., Andrusevich Ya. V. et al. Screening of five and six-membered nitrogen-containing heterocyclic compounds as new effective stimulants of Linum usitatissi­mum L. organogenesis in vitro. Int. J. Med. Biotechnol. Genetics. 2016. S2:001. P. 1–9. doi: 10.19070/2379-1020-SI02001

Yildiz M., Özgen M. The effect of a submersion pretreatment on in vitro explant growth and shoot regeneration from hypocotyls of flax (Linum usitatissimum). Plant Cell Tiss. Organ. Cult. 2004. Vol. 77, Iss. 1. P. 111–115. doi: 10.1023/B:TICU.0000016493. 03592.c3

Yildiz M., Sağlik C., Telci C., Erkilich E. G. The effect of in vitro competition on shoot regeneration from hypocotyl explants of Linum usitatissimum. Turk. J. Bot. 2011. Vol. 35, Iss. 2. P. 211–218. doi: 10.3906/bot-1005-26

Beyaz R., Yildiz M. The effect of inter-plantal competition on in vitro seed germination and seedling growth in flax (Linum usitatissimum L.). Eskişehir Technical Univ. J. of Sci. and Tech. C – Life Sci. and Biotech. 2019. Vol. 8, Iss. 1. P. 61–68. doi: 10.18036/aubtdc.427128

Rutkowska-Krause I., Mankowska G., Lukaszewicz M., Szopa J. Regeneration of flax (Linum usitatissimum L.) plants from anther culture and somatic tissue with increased resistance to Fusarium oxysporum. Plant Cell Rep. 2003. Vol. 22, Iss. 2. P. 110–116. doi: 10.1007/s00299-003-0662-1

Burbulis N., Blinstrubienė A., Sliesaravičius A., Venskutonienė E. Influence of genotype, growth regulators, sucrose level and preconditioning of donor plants on flax (Linum usitatissimum L.) anther culture. Acta Biol. Hung. 2005. Vol. 56, Iss. 3–4. P. 323–331. doi: 10.1556/ABiol.56.2005.3-4.15

Burbulis N., Blinstrubienė A. Genotypic and exogenous factors affecting linseed (Linum usitatissimum L.) anther culture. J. Food Agricult. Environ. 2011. Vol. 9, Iss. 3–4. P. 364–367. doi: 10.1234/4.2011.2285

Burbulis N., Blinstrubienė A., Masienė R., Jonytienė V. Influence of genotype, growth regulators and sucrose concentration on linseed (Linum usitatissimum L.) anther culture. J. Food Agricult. Environ. 2012. Vol. 10, Iss. 3–4. P. 764–767. doi: 10.1234/4.2012.3509

Millam S., Davidson D., Powell W. The use of flax (Linum usi­ta­tissimum) as a model system for studies on organogenesis in vitro: the effect of different carbohydrates. Plant Cell Tiss. Organ. Cult. 1992. Vol. 28, Iss. 2. P. 163–166. doi: 10.1007/BF00055512

Chen Y., Dribnenki P. Effect of genotype and medium compo­si­tion on flax Linum usitatissimum L. anther culture. Plant Cell Rep. 2002. Vol. 21, Iss. 3. P. 204–207. doi: 10.1007/s00299-002-0500-x

Сорока А. И. Особенности подготовки материала и культивирования in vitro пыльников льна при получении гаплоидных растений. Вісн. Запорізького нац. ун-ту. Біологічні науки. 2010. № 2. C. 13–19.

Sakhare S. P., Mendhulkar V. D. Embryo excised callus induction and rhizogenesis in Linum usitatissimum L. Int. J. Pharm. Bio. Sci. 2016. Vol. 7, Iss. 3. P. 507–511.

Blinstrubienė A., Burbulis N., Masienė R. Genotypic and exo­genous factors affecting linseed ovary culture. Zemdirbyste-Agriculture. 2017. Vol. 104, No. 3. P. 243–248. doi: 10.13080/z-a.2017.104.031

Bonell M., Lassaga S. L. Genetic analysis of the response of linseed (Linum usitatissimum L.) somatic tissue to in vitro cultivation. Euphytica. 2002. Vol. 125, Iss. 3. P. 367–372. doi: 10.1023/A:1016013609068

The Plant List. URL: http://www.theplantlist.org

Diederrichsen A., Richards K. Cultivated flax and the genus Li­num L.: Taxonomy and germplasm conservation. Flax: The genus Linum / A. D. Muir, N. D. Westcott (Eds.). Boca Raton : CRC Press, 2003. P. 39–42.

Зеленцов С. В., Зеленцов В. С., Мошненко Е. В., Рябенко Л. Г. Современные представления о филогенезе и таксономии рода Linum L. и льна обыкновенного (Linum usitatissimum L.). Масличные культуры. Науч.-техн. бюл. ВНИИ масличных культур. 2016. Вып. 1. С. 106–121.

Murashige T., Skoog F. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol. Plant. 1962. Vol. 15, Iss. 3. P. 473–497. doi: 10.1111/j.1399‑3054.1962.tb08052.x

Gamborg O. L., Eveleigh D. E. Culture methods and detection of glucanases in suspension cultures of wheat and barley. Can. J. Biochem. Cell B. 1968. Vol. 46, Iss. 5. P. 417–421. doi: 10.1139/o68-063







Creative Commons License
This work is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License.

DOI: 10.21498/2518-1017

Flag Counter