Abstract
Potato (Solanum tuberosum L.), belonging to Solanaceae family, globally ranks as the fourth-largest food (vegetable) crop. In order to sustain the supply of quality propagules, a large-scale in vitro shoot, microtuber and root development protocol was successfully established for potato cv. Badami alu, for the first time. Initially, the tuber sprout explants were inoculated in Murashige and Skoog (MS) basal medium individually with 0.5–1.5 mg/l 6-benzyladenine, kinetin, meta-topolin (mT) and zeatin (Zea) for regeneration. The earliest (~ 3 days) shoot initiation with maximum number (~ 9) and length (81 mm) shoots were recorded in MS medium fortified with 1 mg/l mT after 4 weeks of growth period. The earliest (44 days) microtuber formation with maximum number (4.3) was recorded in MS medium fortified with 1 mg/l Zea after 8 weeks growth stage. On the other hand, the hierarchical clustering heat map, network plot, and principal component analyses illustrated the association among cytokinins (sources and concentrations) and the different growth attributes under study. In vitro-regenerated shootlets were then cultured in MS medium individually with 1–3 mg/l indole-3-acetic acid (IAA) and indole-3-butyric acid. The earliest (~ 7 days) root induction was recorded in MS medium with 1 mg/l IAA, whereas the maximum number (47) with most elongated (28.3 mm) roots were recorded in 2 mg/l IAA after 4 weeks culture period. The micropropagated plantlets were grown in cocopeat (for 1 month) followed by soil (for 2 months) under field conditions (shade net house) that resulted in 95% survival rate. Stability of ploidy level of micropropagated and successively field-grown plantlets with their mother plant was affirmed by flow cytometry, likewise, genetic fidelity was assessed for the same via inter simple sequence repeats, directed amplification of minisatellite-region DNA, start codon targeted polymorphism, and conserved DNA-derived polymorphism markers, and a 100% monomorphism was found, proving that there was no genetic variation among them. Since there is no report of non-conventional mass propagation of this cultivar to date, the present protocol will be immensely helpful for its accelerated large-scale propagation and its genetic improvement.
Key message
First report on usage of meta-Topolin and zeatin for micropropagation and microtuberization followed by acclimatization and field performance of true-to-type propagules (ensured by flow cytometry and molecular markers) in potato.
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Data Availability
All data generated or analysed during this study are included in this published article.
Abbreviations
- AFLP:
-
Amplified fragment length polymorphism
- BA:
-
6-benzyladenine
- BAP:
-
6-benzylaminopurine
- CDDP:
-
Conserved DNA-derived polymorphism
- DAMD:
-
Directed amplification of minisatellite-region DNA
- FCM:
-
Flow cytometry
- IAA:
-
Indole-3-acetic acid
- IBA:
-
Indole-3-butyric acid
- ISSR:
-
Inter simple sequence repeats
- Kn:
-
Kinetin
- MS:
-
Murashige and Skoog
- mT:
-
meta-Topolin
- NPA:
-
Network plot analysis
- PCR:
-
Polymerase chain reaction
- PGR:
-
Plant growth regulator
- RAPD:
-
Random amplified polymorphic DNA
- SCoT:
-
Start codon targeted
- Zea:
-
Zeatin
References
Abd ElHamid R, Abd ElTawab FM, Abdel Razik AB, Allam AI, ELDoliefy AEA (2018) Micropropagation and start codon targeted characterization of four stevia cultivars in Egypt. Arab Univ J Agric Sci 26:635–645. https://doi.org/10.21608/AJS.2018.15968
Astarini IA, Margareth D, Febryanti NPK, Defiani MR, Temaja IGRM (2023) Production of quality early-generation seed potatoes in Bali, Indonesia. IOP Conf Ser: Earth Environ Sci 1133:012013. https://doi.org/10.1088/1755-1315/1133/1/012013
Azad MAK, Khatun Z, Eaton TEJ, Hossen MI, Haque MK, Soren EB (2020) Generation of virus free potato plantlets through meristem culture and their field evaluation. Am J Plant Sci 11:1827–1846. https://doi.org/10.4236/ajps.2020.1111131
Bano A, Shah AH, Shah SA, Khan I, Attacha S, Khan W, Anjum S, Bibi A, Masood R, Jan SA (2020) An efficient cost-effective protocol for micropropagation of potato using natural mentha extract. Fresenius Environ Bull 29:4253–4261
Bertsouklis K, Tsopela S (2023) In vitro propagation of three populations of the endangered, greek endemic Cerastium candidissimum and short-term storability of alginate-encapsulated shoot explants for exploitation and conservation. Horticulturae 9:273. https://doi.org/10.3390/horticulturae9020273
Bhattacharyya P, Kumar S, Lalthafamkimi L, Sharma R, Kumar D, Singh D, Kumar S (2023) Molecular and phytomedicinal stability of long term micropropagated Malaxis acuminata: an endangered terrestrial orchid of biopharmaceutical importance. S Afr J Bot 155:372–382. https://doi.org/10.1016/j.sajb.2023.01.051
Buckseth T, Sharma AK, Sharma S, Moudgil V, Saraswati A (2018) Influence of node number and orientation of explants on in vitro growth and development of potato. Cytologia 83:19–22. https://doi.org/10.1508/cytologia.83.19
Burgos G, Zum Felde T, Andre C, Kubow S (2020) The potato and its contribution to the human diet and health. In: Campos H, Ortiz O (eds) The potato crop: its agricultural, nutritional and social contribution to humankind. Springer, Cham, Switzerland, pp 37–74. https://doi.org/10.1007/978-3-030-28683-5
Chiipanthenga M, Maliro M, Demo P, Njoloma J (2012) Potential of aeroponics system in the production of quality potato (Solanum tuberosum L.) seed in developing countries. Afr J Biotechnol 11:3993–3999. https://doi.org/10.5897/AJB10.1138
Collard BC, Mackill DJ (2009) Start codon targeted (SCoT) polymorphism: a simple, novel DNA marker technique for generating gene-targeted markers in plants. Plant Mol Biol Rep 27:86–93. https://doi.org/10.1007/s11105-008-0060-5
da Silva LI, Magalhães HM (2023) Growth regulators alter the development and metabolism of lemon balm seedlings cultured in vitro. Rev Caatinga 36:372–380. https://doi.org/10.1590/1983-21252023v36n214rc
Dhiman N, Devi K, Bhattacharya A (2021) Development of low cost micropropagation protocol for Nardostachys jatamansi: a critically endangered medicinal herb of Himalayas. S Afr J Bot 140:468–477. https://doi.org/10.1016/j.sajb.2021.04.002
Duncan DB (1955) Multiple range and multiple F test. Biometrics 11:1–42
Ejaz B, Mujib A, Mamgain J, Malik MQ, Syeed R, Gulzar B, Bansal Y (2022) Comprehensive in vitro regeneration study with SCoT marker assisted clonal stability assessment and flow cytometric genome size analysis of Carthamus tinctorius L.: an important medicinal plant. Plant Cell Tissue Organ Cult 148:403–418. https://doi.org/10.1007/s11240-021-02197-x
El-Dessoky S, Attia AO, Ismail IA, El-Hallous EI (2016) In vitro propagation of potato under different hormonal combinations. Int J Adv Res 4:684–689
Ellis D, Salas A, Chavez O, Gomez R, Anglin N (2020) Ex situ conservation of potato [Solanum Section Petota (Solanaceae)] genetic resources in genebanks. In: Campos H, Ortiz O (eds) The potato crop: its agricultural, nutritional and social contribution to humankind. Springer, Cham, pp 109–138. https://doi.org/10.1007/978-3-030-28683-5_4
Galbraith DW, Harkins KR, Maddox JM, Ayres NM, Sharma DP, Firoozabady E (1983) Rapid flow cytometric analysis of the cell cycle in intact plant tissues. Science 220:1049–1051. https://doi.org/10.1126/science.220.4601.1049
Gantait S, Mitra M (2021) Role of meta-topolin on in vitro shoot regeneration: an insight. In: Ahmad N, Strnad M (eds) Meta-topolin: a growth regulator for plant biotechnology and agriculture. Springer, Singapore, pp 143–168. https://doi.org/10.1007/978-981-15-9046-7_12
Gantait S, Mukherjee E (2021) Induced autopolyploidy—a promising approach for enhanced biosynthesis of plant secondary metabolites: an insight. J Genet Eng Biotechnol 19:4. https://doi.org/10.1186/s43141-020-00109-8
García-Fortea E, Lluch-Ruiz A, Pineda-Chaza BJ, García-Pérez A, Bracho-Gil JP, Plazas M, Gramazio P, Vilanova S, Moreno V, Prohens J (2020) A highly efficient organogenesis protocol based on zeatin riboside for in vitro regeneration of eggplant. BMC Plant Biol 20:6. https://doi.org/10.1186/s12870-019-2215-y
Gautam N, Bhattacharya A (2021) Molecular marker based assessment of genetic homogeneity within the in vitro regenerated plants of Crocus sativus L.–a globally important high value spice crop. South Afr J Bot 140:461–467. https://doi.org/10.1016/j.sajb.2021.03.038
Hajare ST, Chauhan NM, Kassa G (2021) Effect of growth regulators on in vitro micropropagation of potato (Solanum tuberosum L.) Gudiene and Belete varieties from Ethiopia. Sci World J 2021:5928769. https://doi.org/10.1155/2021/5928769
Hammer Ø, Harper DAT, Ryan PD (2001) PAST: paleontological statistics software package for education and data analysis. Palaeontol Electron 4:9
Hirsch CN, Hirsch CD, Felcher K, Coombs J, Zarka D, Van Deynze A, De Jong W, Veilleux RE, Jansky S, Bethke P, Douches DS, Buell CR (2013) Retrospective view of North American potato (Solanum tuberosum L.) breeding in the 20th and 21st centuries. G3 Genes Genomes Genet 3:1003–1013. https://doi.org/10.1534/g3.113.005595
Karmakar B, Miya FU, Chakraborty R, Roy S (2022) Comparative analyses of the starch quality isolated from a local red potato and a commercial non-pigmented potato cultivar. Vegetos 35:723–735. https://doi.org/10.1007/s42535-021-00331-w
Kaur A, Reddy MS, Kumar A (2017) Efficient, one step and cultivar independent shoot organogenesis of potato. Physiol Mol Biol Plants 23:461–469. https://doi.org/10.1007/s12298-017-0418-y
Khanam MN, Javed SB, Anis M, Alatar AA (2020) meta-topolin induced in vitro regeneration and metabolic profiling in Allamanda cathartica L. Ind Crops Prod 145:111944. https://doi.org/10.1016/j.indcrop.2019.111944
Khan A, Riaz N, Hassan I, Farooq U, Manzoor R, Rasool A, Ullah K, Erum NIS (2021) Effect of growth regulators on micropropagation of exotic potato (Solanum tuberosum L.) under in vitro condition. Agrociencia 55:2–9
Krishna Vrundha CP, Aswathi NV, Thomas TD (2021) The role of meta-topolin in plant morphogenesis in vitro. In: Ahmad N, Strnad M (eds) Meta-topolin: a Growth Regulator for Plant Biotechnology and Agriculture. Springer, Singapore, pp 93–118. https://doi.org/10.1007/978-981-15-9046-7_10
Lalthafamkimi L, Bhattacharyya P, Bhau BS, Wann SB, Banik D (2021) Direct organogenesis mediated improvised mass propagation of Pogostemon cablin: a natural reserve of pharmaceutical biomolecules. S Afr J Bot 140:375–384. https://doi.org/10.1016/j.sajb.2020.08.018
Lekamge D, Sasahara T, Yamamoto SI, Hatamoto M, Yamaguchi T, Maki S (2021) Effect of enhanced CaCl2, MgSO4, and KH2PO4 on improved in vitro growth of potato. Plant Biotechnol 38:401–408. https://doi.org/10.5511/plantbiotechnology.21.0830a
Mahanta M, Gantait S, Mukherjee E, Bhattacharyya S (2023a) meta-topolin-induced mass propagation, acclimatization and cyto-genetic fidelity assessment of gerbera (Gerbera jamesonii Bolus ex Hooker f). S Afr J Bot 153:236–245. https://doi.org/10.1016/j.sajb.2022.11.032
Mahanta M, Gantait S, Sarkar S, Sadhukhan R, Bhattacharyya S (2023b) Colchicine-mediated in vitro polyploidization in gerbera hybrid. 3 Biotech 13:74. https://doi.org/10.1007/s13205-022-03457-z
Mamgain J, Mujib A, Ejaz B, Gulzar B, Malik MQ, Syeed R (2022) Flow cytometry and start codon targeted (SCoT) genetic fidelity assessment of regenerated plantlets in Tylophora indica (Burm. f.) merrill. Plant Cell Tissue Organ Cult 150:129–140. https://doi.org/10.1007/s11240-022-02254-z
Masekesa TR, Gasura E, Ngadze E, Icishahayo D, Kujeke GT, Chidzwondo F, Robertson I (2016) Efficacy of zeatin, kinetin and thidiazuron in induction of adventitious root and shoot from petiole explants of sweetpotato cv. Brondal. S Afr J Bot 104:1–5. https://doi.org/10.1016/j.sajb.2015.11.001
Mohapatra P, Ray A, Sandeep IS, Parida R, Mohanty S (2021) Genetic and biochemical stability of in vitro raised and conventionally propagated Centella asiatica-A valuable medicinal herb. S Afr J Bot 140:444–453. https://doi.org/10.1016/j.sajb.2021.01.004
Molla MMH, Nasiruddin KM, Al-Amin M, Khanam D, Salam MA (2011) Effect of growth regulators on direct regeneration of Potato. International Conference on Environment and Industrial Innovation, vol.12, IACSIT Press, Singapore
Mungai SW, Wainaina CM, Kavoo AM, Mwajita MR (2024) Effect of in vitro calcium fortification on regeneration and microtuberization of three selected irish potato varieties. Int J Hortic Sci Technol 11:47–58. https://doi.org/10.22059/ijhst.2022.347334.583
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
Neri JC, Meléndez-Mori JB, Tejada-Alvarado JJ, Vilca-Valqui NC, Huaman-Huaman E, Oliva M, Goñas M (2022) An optimized protocol for micropropagation and acclimatization of Strawberry (Fragaria × ananassa Duch.) Variety ‘Aroma’. Agronomy 12:968. https://doi.org/10.3390/agronomy12040968
Nowicki M, Foolad MR, Nowakowska M, Kozik EU (2012) Potato and tomato late blight caused by Phytophthora infestans: an overview of pathology and resistance breeding. Plant Dis 96:4–17. https://doi.org/10.1094/PDIS-05-11-0458
Ochatt SJ (2008) Flow cytometry in plant breeding. Cytom: J Int Soc Anal Cytol 73A:581–598. https://doi.org/10.1002/cyto.a.20562
Pawar BD, Markad NR, Wagh RS, Neumann M, Kale AA, Chimote VP (2023) Proline and silver nitrate promotes multiple shoot induction from mature embryo and shoot tip explants of sorghum. Sugar Tech 25:1187–1195. https://doi.org/10.1007/s12355-023-01277-w
Pramanik B, Sarkar S, Bhattacharyya S, Gantait S (2021) meta-topolin-induced enhanced biomass production via direct and indirect regeneration, synthetic seed production, and genetic fidelity assessment of Bacopa monnieri (L.) Pennell, a memory-booster plant. Acta Physiol Plant 43:107. https://doi.org/10.1007/s11738-021-03279-1
Rai MK (2023) Start codon targeted (SCoT) polymorphism marker in plant genome analysis: current status and prospects. Planta 257:34. https://doi.org/10.1007/s00425-023-04067-6
Redhwan A, Acemi A, Özen F (2023) Effects of plant growth regulators on in vitro seed germination, organ development and callogenesis in Pancratium maritimum L. Plant Cell Tissue Organ Cult 154:97–110. https://doi.org/10.1007/s11240-023-02514-6
Rout BM, Bhatia AK, Kajla S, Singh T, Chandel R, Kumar V (2022) Micropropagation and assessment of genetic fidelity of tissue-cultured plants in potato (Solanum tuberosum L.‘Kufri Lima’). Eur J Hortic Sci 87:1-9. https://doi.org/10.17660/eJHS.2022/013
Roy B (2019) Characterization of farmers’ varieties of potato (Solanum tuberosum L.) of Cooch Behar district of West Bengal. Indian J Plant Genet Resour 32:314–317. https://doi.org/10.5958/0976-1926.2019.00033.0
Saeiahagh H, Mousavi M, Wiedow C, Bassett HB, 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 Tissue Organ Cult 138:1–10. https://doi.org/10.1007/s11240-019-01597-4
Saidi A, Jabalameli Z, Ghalamboran M (2018) Evaluation of genetic diversity of carnation cultivars using CDDP and DAMD markers and morphological traits. The Nucleus 61:129–135. https://doi.org/10.1007/s13237-018-0238-7
Saker MM, Moussa TAA, Heikal NZ, ELLil AHA, Abdel-Rahman RM (2012) Selection of an efficient in vitro micropropagation and regeneration system for potato (Solanum tuberosum L.) cultivar Desiree. Afr J Biotechnol 11:16388–16404
Shahriyar S, Akram S, Khan K, Miya MF, Sarkar MAR (2015) In vitro plant regeneration of potato (Solanum tuberosum L.) at the rate of different hormonal concentration. Asian J Med Biol Res 1:297–303
Sharma SK, Bryan GJ, Winfield MO, Millam S (2007) Stability of potato (Solanum tuberosum L.) plants regenerated via somatic embryos, axillary bud proliferated shoots, microtubers and true potato seeds: a comparative phenotypic, cytogenetic and molecular assessment. Planta 226:1449–1458. https://doi.org/10.1007/s00425-007-0583-2
Song G, Chen Q, Callow P, Mandujano M, Han X, Cuenca B, Bonito G, Medina-Mora C, Fulbright DW, Guyer DE (2021) Efficient micropropagation of chestnut hybrids (Castanea spp.) using modified woody plant medium and zeatin riboside. Hortic Plant J 7:174–180. https://doi.org/10.1016/j.hpj.2020.09.006
Song J, Bradeen JM, Naess SK, Raasch JA, Wielgus SM, Haberlach GT, Liu J, Kuang H, Austin-Phillips S, Buell CR, Helgeson JP, Jiang J (2003) Gene RB cloned from Solanum bulbocastanum confers broad spectrum resistance to potato late blight. Proc Nat Acad Sci 100:9128–9133. https://doi.org/10.1073/pnas.1533501100
Subrahmanyeswari T, Gantait S, Kamble SN, Singh S, Bhattacharyya S (2023) meta-topolin-induced regeneration and ameliorated rebaudioside-A production in genetically uniform candy-leaf plantlets (Stevia rebaudiana Bert). S Afr J Bot 159:405–418. https://doi.org/10.1016/j.sajb.2023.05.045
Subrahmanyeswari T, Gantait S, Sarkar S, Bhattacharyya S (2022a) Accelerated mono-phasic in vitro mass production of banana propagules and their morpho-cyto-genetic stability assessment. S Afr J Bot 146:794–806. https://doi.org/10.1016/j.sajb.2022.02.011
Subrahmanyeswari T, Verma SK, Gantait S (2022b) One-step in vitro protocol for clonal propagation of Dendrobium Yuki White, a high value ornamental orchid hybrid. S Afr J Bot 146:883–888. https://doi.org/10.1016/j.sajb.2022.03.036
Sudheer WN, Praveen N, Al-Khayri JM, Jain SM (2022) Role of plant tissue culture medium components. In: Rai AC, Kumar A, Modi A, Singh M (eds) Advances in plant tissue culture. Elsevier, Academic Press, pp 51–83. https://doi.org/10.1016/B978-0-323-90795-8.00012-6
Tiwari JK, Chandel P, Gupta S, Gopal J, Singh BP, Bhardwaj V (2013) Analysis of genetic stability of in vitro propagated potato microtubers using DNA markers. Physiol Mol Biol Plants 19:587–595. https://doi.org/10.1007/s12298-013-0190-6
Tripathi D, Rai KK, Rai SK, Rai SP (2018) An improved thin cell layer culture system for efficient clonal propagation and in vitro withanolide production in a medicinal plant Withania coagulans Dunal. Ind Crops Prod 119:172–182. https://doi.org/10.1016/j.indcrop.2018.04.012
Vilariño S, Florido MDC, García JL, Cantos M (2023) Effects of culture system and substrate composition on micropropagated plantlets of two varieties of Stevia rebaudiana Bert. Physiologia 3:74–85. https://doi.org/10.3390/physiologia3010006
Vylíčilová H, Bryksová M, Matušková V, Doležal K, Plíhalová L, Strnad M (2020) Naturally occurring and artificial N9-cytokinin conjugates: from synthesis to biological activity and back. Biomolecules 10:832. https://doi.org/10.3390/biom10060832
Yadav NR, Sticklen MB (1995) Direct and efficient plant regeneration from leaf explants of Solanum tuberosum l. cv. Bintje Plant Cell Rep 14:645–647. https://doi.org/10.1007/BF00232730
Yaseen FK, Toma RS, Carbonera D (2017) The effects of vitamins on micropropagation of Desiree and Mozart potatoes (Solanum tuberosum L). Sci J Univ Zakho 5:53–56. https://doi.org/10.25271/2017.5.1.301
Zaheer K, Akhtar MH (2016) Potato production, usage, and nutrition—a review. Crit Rev Food Sci Nutr 56:711–721. https://doi.org/10.1080/10408398.2012.724479
Zahid NA, Jaafar HZ, Hakiman M (2021) Micropropagation of ginger (Zingiber officinale Roscoe) ‘Bentong’ and evaluation of its secondary metabolites and antioxidant activities compared with the conventionally propagated plant. Plants 10:630. https://doi.org/10.3390/plants10040630
Zaib-Un-Nisa, Shah AH, Shah SH, Farooq G, Sajad MA, Khan MAS (2020) Effects of natural growth regulators on micropropagation of potatoes. Pure Appl Biol 9:1442–1459. https://doi.org/10.19045/bspab.2020.90150
Acknowledgements
The authors acknowledge the experimental facilities from Plant Tissue Culture and Molecular Biology laboratories at Regional Nuclear Agriculture Research Centre, Bidhan Chandra Krishi Viswavidyalaya, West Bengal, India. Authors express their gratefulness to Dr. Sayantan De and Dr. Joyashree Mallick from Zonal Adaptive Research Station (Krishnanagar), Govt. of West Bengal, India for providing the potato germplasm and also for the assistance during the acclimatization process.
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MC : Methodology, Investigation, Data curation, Formal analysis, Writing – original draft. TS : Methodology, Investigation, Data curation, Formal analysis, Writing – original draft, review & editing. SB : Conceptualization, Supervision, Writing – review & editing. SG : Conceptualization, Supervision, Resources, Methodology, Investigation, Data curation, Formal analysis, Software, Validation, Writing – original draft, review & editing.
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Char, M., Subrahmanyeswari, T., Bhattacharyya, S. et al. meta-Topolin-induced in vitro propagation, field evaluation, flow cytometry and molecular marker-based genetic stability assessment of potato cv. Badami alu. Plant Cell Tiss Organ Cult 155, 809–826 (2023). https://doi.org/10.1007/s11240-023-02601-8
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DOI: https://doi.org/10.1007/s11240-023-02601-8