Abstract
Traditional cultivation methods used for domestic apples are not effective when applied for cultivation of the vulnerable wild apple Malus sieversii. However, this apple can be effectively grown using micropropagation, which is an established ex situ conservation tool. The study aimed to grow in vitro M. sieversii cultures using axillary buds as explant. To optimize the media and plant growth regulator combinations for successful in vitro culture, three different media were used: Quoirin and Lepoivre (QL), woody plant medium (WPM), and Murashige and Skoog (MS) containing four different concentrations of 6‑benzylaminopurine (BAP) combined with 0.2 mg/l or 0.5 mg/l of gibberellic acid (GA). Different concentrations of indole-3-butyric acid (IBA) and naphthalene acetic acid (NAA) added to half-strength QL medium were used for optimizing the rooting medium. The combination of QL medium with 1.5 mg/l BAP and 0.01 mg/l IBA resulted in 100% shoot regeneration. The number of shoots (17.20 ± 0.64) and shoot length (2.80 ± 0.10 cm) were greatest when QL medium with 0.75 mg/l BAP and 0.2 mg/l GA was used. One hundred percent root development with the greatest number of roots per explant (8.13 ± 0.44) and longest root length (3.77 ± 0.23 cm) were achieved when half-strength QL medium with 0.5 mg/l of IBA was used. Simple sequence repeat analysis confirmed the reliability of this protocol for efficient large-scale micropropagation of M. sieversii. Through this study, an efficient micropropagation protocol that can be used for large-scale cultivation as well as germplasm conservation of M. sieversii was developed.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Allex C, Shavlik J, Blattner F (1999) Neural network input representations that produce accurate consensus sequences from DNA fragment assemblies. Bioinformatics 15:723–728. https://doi.org/10.1093/bioinformatics/15.9.723
Amirchakhmaghi N, Hosseinpour B, Yousefzadeh H (2019) Development of a micropropagation protocol for Malus orientalis using axillary buds. In Vitro Cell Dev Biol Plant 55(5):625–634. https://doi.org/10.1007/s11627-019-09992-4
Amiri S, Mohammadi R (2021) Establishment of an efficient in vitro propagation protocol for Sumac (Rhus coriaria L.) and confirmation of the genetic homogeneity. Sci Rep 11:1–9. https://doi.org/10.1038/s41598-020-80550-4
Aremu A, Plačková L, Bairu M et al (2014) How does exogenously applied cytokinin type affect growth and endogenous cytokinins In micropropagated Merwilla plumbea? Plant Cell Tissue Organ Cult 118:245–256. https://doi.org/10.1007/s11240-014-0477-5
Baraldi R, Malavasi F, Predieri S, Castagneto M (1991) Effect of potassium humate on apple cv. ‘golden delicious’ cultured in vitro. Plant Cell Tissue Organ Cult 24:187–191. https://doi.org/10.1007/BF00033475
Bo H, Zheng X (2005) Relationship of the occurences and evolutions of wild-fruit forests with climatic factors in the Tianshan Mountain. Acta Bot Boreal Occident Sin 25:2266–2271
Brito G, Lopes T, Loureiro J, Rodriguez E, Santos C (2010) Assessment of genetic stability of two micropropagated wild olive species using flow cytometry and microsatellite markers. Trees 24(4):723–732. https://doi.org/10.1007/s00468-010-0442-9
Chokheli V, Dmitriev P, Rajupt V et al (2020) Recent development in micropropagation techniques for rare plant species. Plants 9:1733. https://doi.org/10.3390/plants9121733
Cornille A, Gladieux P, Giraud T (2013) Crop-to-wild gene flow and spatial genetic structure in the closest wild relatives of the cultivated apple. Evol Appl 6:737–748. https://doi.org/10.1111/eva.12059
Cornille A, Giraud T, Smulders M et al (2014) The domestication and evolutionary ecology of apples. Trends Genet 30:57–65. https://doi.org/10.1016/j.tig.2013.10.002
Costion C, Ford A, Cross H et al (2011) Plant DNA barcodes can accurately estimate species richness in poorly known floras. Plos One 6:e26841. https://doi.org/10.1371/journal.pone.0026841
Dobránszki J, da Silva JAT (2010) Micropropagation of apple—a review. Biotechnol Adv 28(4):462–488. https://doi.org/10.1016/j.biotechadv.2010.02.008
Doyle J, Doyle J, Brown A (1990) Chloroplast DNA phylogenetic affinities of newly described species in Glycine (Leguminosae: Phaseoleae). Syst Bot 15:466. https://doi.org/10.1002/j.1537-2197.1990.tb14467.x
Dzhangaliev A (1977) Dikaja jablonja Kazahstana. Nauka, Alma-Ata, p 280
Dzhangaliev A, Salova T, Turekhanova P (2010) The wild fruit and nut plants of Kazakhstan. Hortic Rev 29:305–371. https://doi.org/10.1002/9780470650868.ch3
Faisal M, Alatar AA, Ahmad N, Anis M, Hegazy AK (2012) An efficient and reproducible method for in vitro clonal multiplication of Rauvolfia tetraphylla L. and evaluation of genetic stability using DNA-based markers. Appl Biochem Biotechnol 168(7):1739–1752. https://doi.org/10.1007/s12010-012-9893-3
Forsline P, Aldwinckle H, Dickson E et al (2010) Collection, maintenance, characterization, and utilization of wild apples of Central Asia. Hortic Rev. https://doi.org/10.1002/9780470650868.ch1
Forte A, Ignatov A, Ponomarenko V et al (2002) Phylogeny of the Malus (apple tree) species, inferred from the morphological traits and molecular DNA analysis. Russ J Genet 38:1150–1161. https://doi.org/10.1023/a:1020648720175
Gómez-Kosky R, Armas P, Calimano M et al (2020) Effect of Phloroglucinol on in vitro rooting of sugarcane (Saccharum spp. Cv c90–469). Sugar Tech 23:466–471. https://doi.org/10.1007/s12355-020-00906-y
Gonzalez-Benito M, Martín C (2010) In vitro preservation of spanish biodiversity. In Vitro Cell Dev Biol Plant 47:46–54. https://doi.org/10.1007/s11627-010-9333-4
Grigoriadou K, Krigas N, Sarropoulou V et al (2019) In vitro propagation of medicinal and aromatic plants: the case of selected Greek species with conservation priority. In Vitro Cell Dev Biol Plant 55:635–646. https://doi.org/10.1007/s11627-019-10014-6
Guo Y‑X, Zhao Y‑Y, Zhang M, Zhang L‑Y (2019) Development of a novel in vitro rooting culture system for the micropropagation of highbush blueberry (Vaccinium corymbosum) seedlings. Plant Cell Tiss Organ Cult 139:615–620. https://doi.org/10.17129/botsci.2135
Harris S, Robinson J, Juniper B (2002) Genetic clues to the origin of the apple. Trends Genet 18:426–430. https://doi.org/10.1016/s0168-9525(02)02689-6
IUCN (2007) Red list of threatened species 2007 (Malus sieversii). https://www.iucnredlist.org/species/32363/9693009. Accessed 4 Mar 2022
Ji X, Wang Y, Zhang R et al (2008) Effect of auxin, cytokinin and nitrogen on anthocyanin biosynthesis in callus cultures of red-fleshed apple (Malus sieversii f.niedzwetzkyana). Plant Cell Tiss Organ Cult 120:325–337. https://doi.org/10.1007/s11240-014-0609-y
Kanungo S, Pradhan C, Sahoo SL, Sahu RK (2012) Role of auxins in the in vitro rooting and micropropagation of Holarrhena antidysenterica Wall., a woody aromatic medicinal plant, through nodal explants from mature trees. J Med Plants Res 6(31):4660–4666. https://doi.org/10.5897/JMPR12.171
Keresa S, Bosnjak AM, Baric M, Jercic IH, Sarcevic H, Bisko A (2012) Efficient axillary shoot proliferation and in vitro rooting of apple cv.‘topaz. Not Bot Horti Agrobot Cluj Napoca 40(1):113–118. https://doi.org/10.15835/nbha4017211
Kher MM, Nataraj M, Teixeira da Silva JA (2016) Micropropagation of Crataeva L. species. Rend Lincei 27(2):157–167. https://doi.org/10.1007/s12210-015-0478-2
Kher MM, Nataraj M, Arun Kumar A, Sitther V, Shekhawat M, Warrier R, Teixeira da Silva JA (2021) Tissue culture of Indian rosewood (Dalbergia latifolia Roxb.). Biologia. https://doi.org/10.1007/s11756-021-00914-7
Kovalchuk I, Lyudvikova Y, Volgina M, Reed B (2008) Medium, container and genotype all influence in vitro cold storage of apple germplasm. Plant Cell Tiss Organ Cult 96:127–136. https://doi.org/10.1007/s11240-008-9468-8
Krishna H, Alizadeh M, Singh D, Singh U, Chauhan N, Eftekhari M, Sadh R (2016) Somaclonal variations and their applications in horticultural crops improvement. 3 Biotech 6:54. https://doi.org/10.1007/s13205-016-0389-7
Land Code of the Republic of Kazakhstan Code of the Republic of Kazakhstan dated 20 June, 2003 No.442.
Lata H, Chandra S, Techen N, Khan I, ElSohly M (2016) In vitro mass propagation of Cannabis sativa l.: a protocol refinement using novel aromatic cytokinin meta-topolin and the assessment of eco-physiological, biochemical and genetic fidelity of micropropagated plants. J Appl Res Med Aromat Plants 3:18–26. https://doi.org/10.1016/j.jarmap.2015.12.001
Li X, Zhang D, Li JA (2018) A quick method for acquiring Malus sieversii’s tissue culture seedlings. North Hortic 14:31–37
Lizárraga A, Fraga M, Ascasíbar J, González ML (2017) In vitro propagation and recovery of eight apple and two pear cultivars held in a germplasm bank. AJPS 8(9):2238–2254. https://doi.org/10.4236/ajps.2017.89150
Lloyd G, McCown B (1980) Commercially-feasible micropropagation of mountain laurel, Kalmia latifolia, by use of shoot-tip culture. Commercially-feasible micropropagation of mountain laurel, Kalmia latifolia, by use of shoot-tip culture. Comb Proc Int Plant Propag Soc 30:421–427
Luby J, Forsline P, Aldwinckle H et al (2001) Silk road apples—collection, evaluation, and utilization of Malus sieversii from central asia. HortScience 36:225–231. https://doi.org/10.21273/hortsci.36.2.225
Magyar-Tábori K, Dobránszki J, Teixeira da Silva J, Bulley S, Hudak I (2010) The role of cytokinins in shoot organogenesis in apple. Plant Cell Tiss Organ Cult 101:251–267. https://doi.org/10.1155/2021/9931574
Marin J, Jones O, Hadlow W (1993) Micropropagation of columnar apple trees. J Hortic Sci 68:289–297. https://doi.org/10.1080/00221589.1993.11516354
Modgil M, Mahajan K, Chakrabarti S, Sharma D, Sobti R (2005) Molecular analysis of genetic stability in micropropagated apple rootstock mm106. Sci Hortic 104:151–160. https://doi.org/10.3906/bot-1312-61
Modgil M, Gupta R, Thakur M (2008) In vitro rooting and hardening in apple rootstock EMLA111-influence of some factors. Acta Hortic 865:339–344
Moriya S, Iwanami H, Okada K, Yamamoto T, Abe K (2010) A practical method for apple cultivar identification and parent-offspring analysis using simple sequence repeat markers. Euphytica 177:135–150. https://doi.org/10.1007/s10681-010-0295-8
Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497. https://doi.org/10.1111/j.1399-3054.1962.tb08052.x
Negishi N, Nakahama K, Urata N et al (2014) Hormone level analysis on adventitious root formation in Eucalyptus globulus. New For 45:577–587. https://doi.org/10.1007/s11056-014-9420-1
Nurtaza A, Magzumova G, Yessimseitova A et al (2021) Micropropagation of the endangered species Malus niedzwetzkyana for conservation biodiversity in Kazakhstan. In Vitro Cell Dev Biol Plant. https://doi.org/10.1007/s11627-021-10174-4
Omasheva M, Flachowsky H, Ryabushkina N et al (2017) To what extent do wild apples in Kazakhstan retain their genetic integrity? Tree Genet Genomes. https://doi.org/10.1007/s11295-017-1134-z
On approval of the Lists of rare and endangered species of animals and plants. Decree of the Government of the Republic of Kazakhstan dated October 31, 2006 N1034
Quoirin M, Lepoivre P (1977) Improved media for in vitro culture of prunus sp. Acta Hortic 78:437–442. https://doi.org/10.17660/actahortic.1977.78.54
Rani V, Raina S (2000) Genetic fidelity of organized meristem-derived micropropagated plants: a critical reappraisal. In Vitro Cell Dev Biol Plant 36:319–330. https://doi.org/10.1007/s11627-000-0059-6
Reed B (2011) Choosing and applying cryopreservation protocols to new plant species or tissues. Acta Hortic. https://doi.org/10.17660/actahortic.2011.908.48
Saeiahagh H, Mousavi M, Wiedow C, Bassett H, Pathirana R (2019) Effect of cytokinins and sucrose concentration on the efficiency of micropropagation Of ‘Zes006’ Actinidia chinensis var. chinensis, a red-fleshed kiwifruit cultivar. Plant Cell Tiss Organ Cult 138:1–10. https://doi.org/10.1007/s11240-019-01597-4
Shakina TN (2020) Opyt adaptacii rastenij-regenerantov k uslovijam ex vitro nekotoryh dekorativnyh i plodovo-jagodnyh kul’tur v Uchebno-nauchnom centre “Botanicheskij sad” Saratovskogo gosudarstvennogo universiteta im. NG Chernyshevskogo. Probl Bot Juzhnoj Sibiri Mongolii 19:315–320 (in Russian)
Sharma T, Modgil M, Thakur M (2007) Factors affecting induction and development of in vitro rooting in apple rootstocks. Indian J Exp Biol 45(9):824–829
Silfverberg-Dilworth E, Matasci C, Van de Weg W et al (2006) Microsatellite markers spanning the apple (Malus x domestica Borkh.) genome. Tree Genet Genomes 2:202–224. https://doi.org/10.1007/s11295-006-0045-1
Singh M, Sonkusale S, Niratker CH, Shukla P (2014) Micropropagation of Shorea robusta: an economically important woody plant. J For Sci 60(2):70–74. https://doi.org/10.17221/80/2013-JFS
de Souza Ferrari M, da Cruz R, dos Santos Queiroz M et al (2020) Efficient ex vitro rooting, acclimatization, and cultivation of Curcuma longa L. from mycorrhizal fungi. J Crop Sci Biotechnol 23:469–482. https://doi.org/10.1007/s12892-020-00057-2
Tasheva K, Kosturkova G (2010) Bulgarian golden root in vitro cultures for micropropagation and reintroduction. Cent Eur J Biol 5(6):853–863. https://doi.org/10.2478/s11535-010-0092-3
Teixeira da Silva J, Kher M, Soner D, Nataraj M (2019) Red sandalwood (Pterocarpus santalinus L. f.): biology, importance, propagation and micropropagation. J For Res 30(3):745–754. https://doi.org/10.1007/s11676-018-0714-6
Vasava D, Kher M, Nataraj M, Teixeira da Silva JA (2018) Bael tree (Aegle marmelos (L.) Corrêa): importance, biology, propagation, and future perspectives. Trees 32(5):1165–1198. https://doi.org/10.1007/s00468-018-1754-4
Volk G, Richards C, Reilley A et al (2005) Ex situ conservation of vegetatively propagated species: development of a seed-based core collection for Malus sieversii. J Am Soc Hortic Sci 130:203–210. https://doi.org/10.21273/jashs.130.2.203
Wang M‑R, Chen L, Teixeira da Silva JA, Volk G, Wang Q‑C (2018) Cryobiotechnology of apple (Malus spp.): development, progress and future prospects. Plant Cell Rep 37:689–709. https://doi.org/10.1007/s00299-018-2249-x
White T, Bruns T, Lee S (1990) PCR protocols—a guide to methods and applications. Trends Genet 6:229. https://doi.org/10.1016/0307-4412(91)90165-5
Xu J, Wang Y, Zhang Y, Chai T (2008) Rapid in vitro multiplication and ex vitro rooting of Malus zumi (Matsumura) Rehd. Acta Physiol Plantarum 30(1):129–132. https://doi.org/10.1007/s11738-007-0075-9
Yan G, Long H, Song W, Chen R (2007) Genetic polymorphism of Malus sieversii populations in Xinjiang, China. Genet Resour Crop Evol 55:171–181. https://doi.org/10.1007/s10722-007-9226-5
Yepes L, Aldwinckle H (1994) Micropropagation of thirteen Malus cultivars and rootstocks, and effect of antibiotics on proliferation. Plant Growth Regul 15:55–67. https://doi.org/10.1007/BF00024677
Zeng Q, Han Z, Kang X (2019) Adventitious shoot regeneration from leaf, petiole and root explants in triploid (Populus alba × P. glandulosa)× P. tomentosa. Plant Cell Tiss Organ Cult 138:121–130. https://doi.org/10.1007/s11240-019-01608-4
Zhang H, Zhang M, Wang L (2014) Genetic structure and historical demography of Malus sieversii in the Yili Valley and the western mountains of the Junggar Basin, Xinjiang, China. J Arid Land 7:264–271. https://doi.org/10.1007/s40333-014-0044-2
Zhang Y, Bozorov T, Li D, Zhou P, Wen X, Ding Y, Zhang D (2020) An efficient in vitro regeneration system from different wild apple (Malus sieversii) explants. Plant Methods 16:1–10. https://doi.org/10.1186/s13007-020-00599-0
Funding
This work was supported by the Ministry of Education and Science, Republic of Kazakhstan, IRN number AP09563185 for 2021, for the project “Development of cryobiotechnology of endangered plant species of Malus sieversii and Malus niedzwetzkyana for conservation and reproduction.”
Author information
Authors and Affiliations
Contributions
All authors contributed to the study conception and design. Material preparation, data collection, and analysis were performed by Dyussembekova Damira, Nurtaza Aidana, Yessimseitova Assel, Shevtsov Alexandr, Lutsay Viktoriya, Yerlan Ramankulov, and Kakimzhanova Almagul. The first draft of the manuscript was written by Kakimzhanova Almagul and Dyussembekova Damira, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Conceptualization: Kakimzhanova Almagul; methodology: Dyussembekova Damira, Nurtaza Aidana, Yessimseitova Assel, Shevtsov Alexandr, Lutsay Viktoriya, Yerlan Ramankulov, and Kakimzhanova Almagul; formal analysis and investigation: Kakimzhanova Almagul, Dyussembekova Damira; writing—original draft preparation: Kakimzhanova Almagul; writing—review and editing: Kakimzhanova Almagul; funding acquisition: Kabieva Saltanat, Kakimzhanova Almagul; resources: Saltanat Kabieva, Kakimzhanova Almagul, Shevtsov Alexandr; supervision: Kakimzhanova Almagul, project manager: Kabieva Saltanat.
Corresponding author
Ethics declarations
Conflict of interest
D. A. Kakimzhanova, D. Dyussembekova, A. Nurtaza, A. Yessimseitova, A. Shevtsov, V. Lutsay, Y. Ramankulov, and S. Kabieva declare that they have no competing interests.
Additional information
Data availability
The datasets generated during the current study are available from the corresponding author on reasonable request.
Rights and permissions
Springer Nature or its licensor holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
Kakimzhanova, A., Dyussembekova, D., Nurtaza, A. et al. An Efficient Micropropagation System for the Vulnerable Wild Apple Species, Malus sieversii, and Confirmation of Its Genetic Homogeneity. Erwerbs-Obstbau 65, 621–632 (2023). https://doi.org/10.1007/s10341-022-00720-8
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10341-022-00720-8