Abstract
Papaya is an important crop with a short juvenile phase among the fruit crops. Direct and indirect regeneration protocols were developed for papaya cultivar Red Lady 786, by using apical, nodal, petiole, leaf and root segments. For direct regeneration, plant growth regulators (PGRs) i.e. BAP, Zeatin and NAA and indirect regeneration BAP, Kinetin, TDZ and NAA were investigated. The study showed that apical and nodal segments can undergo direct regeneration with distinct shoot differentiation without the callus phase, resulting in a high success rate of shoot development (65–88%). The indirect method involves the initiation of callus tissues, followed by morphogenesis of somatic embryogenesis significantly influenced by PGRs. A high success rate of somatic embryogenesis (ranging from 75–85%) was observed in cultures derived from multiple explants. The presence of somatic embryogenesis at specific differential stages, namely globular, heart, torpedo and cotyledonary had confirmed through stereo-microscopic analysis. The result significantly indicated that the supplementation of MS media with TDZ (0.3 mg/l), BAP (1.0 mg/l), NAA (0.10 mg/l), and 30 mg/l sucrose as the most efficient medium for somatic embryogenesis. Somatic embryos were sub-cultured on BAP, Kinetin and NAA resulted in a high frequency (75–80%) of complete plantlet regeneration except for root segment explant derived somatic embryos. The in-vitro regenerated plants via direct and indirect regeneration were successfully acclimatized in greenhouse with 88.89% survival rate. The genetic fidelity was checked by using 22 RAPD and 18 ISSR markers. This developed protocol may be utilized for commercial production of ‘Red Lady 786’ papaya plantlets.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Data availability
All data used in this research are included in this published article and its supplementary information files.
Change history
09 March 2024
The original version of this article has been revised: The doi link in the reference Al-Drisi et al. (2022) has been corrected.
Abbreviations
- PGRs :
-
Plant Growth Regulators
- BAP :
-
6-Benzylaminopurine
- NAA :
-
Naphthalene Acetic Acid
- TDZ :
-
Thidiazuron
- IBA :
-
Indole-3-acetic Acid
- IAA :
-
Indole-3-butyric Acid
- FYM :
-
Farm Yard Manure
- CTAB :
-
Cetyl Trimethyl Ammonium Bromide
- RAPD :
-
Random Amplified Polymorphic DNA
- SSR :
-
Simple Sequence Repeats
- MS :
-
Murashige and Skoog medium
- HgCl 2 :
-
Mercuric Chloride
- LAF :
-
Laminar Air flow
- LED :
-
Light-Emitting Diode
- LAS :
-
Leica Application Suite
- PAU :
-
Punjab Agricultural University
- TE :
-
Tris-EDTA
- PCR :
-
Polymerase Chain Reaction
- DNA :
-
Deoxyribonucleic Acid
- PAGE :
-
Polyacrylamide Gel Electrophoresis
- CRD :
-
Complete Randomized Design
- LSD :
-
Least Significant Difference
- SAS :
-
Statistical Analysis Software
- NTSYS :
-
Numerical Taxonomy and Multivariate Analysis System
- PCA :
-
Principal Component Analysis
- UPGMA :
-
Unweighted Pair Group Method with Arithmetic Mean
- ANS :
-
Average Number of Shoot
- ASL :
-
Average Shoot Length
- CM :
-
Centimetre
- MM :
-
Milometer
- ANRS :
-
Average Number of Roots per Shoot
- RTI :
-
Root Induction
- ARL :
-
Average Root Length
- AND :
-
Average Number of Days
- RAF :
-
Random Amplified Fragments
- AFLP :
-
Amplified Fragment Length Polymorphism
References
Agarwal M, Shrivastava N, Padh H (2008) Advances in molecular marker techniques and their applications in plant sciences. Plant Cell Rep 27:617–631. https://doi.org/10.1007/s00299-008-0507-z
Ahuja S, Mandal BB, Dixit S, Srivastava PS (2002) Molecular, phenotypic and biosynthetic stability in Dioscorea floribunda plants derived from cryopreserved shoot tips. Plant Sci 163:971–977. https://doi.org/10.1016/S0168-9452(02)00246-7
Al-Drishi E, Ibrahim M, Jasim A (2022) Explant type influences callus induction and shoots organogenesis in papaya under in vitro conditions. DYSONA – Appl Sci 4:15–20. https://doi.org/10.30493/das.2022.363580
Aluthge ADRP, Nagahawaththa SMS, Basnayake BMVS, Hettiarachchi C, Senanayake DMJB (2021) Regeneration of Carica papaya “Rathna” Variety through Somatic Embryogenesis from Immature Zygotic Embryos. Asian Jour Res Review Agri 3(1):185–192
Ammirato PV (1983) Embryogenesis. Handbook of plant cell culture
Anandan R, Thirugnanakumar S, Sudhakar D, Balasubramanian P (2011) In vitro organogenesis and plantlet regeneration of (Carica papaya L.). J Agric Tech 7:1339–1348
Anandan R, Sudhakar D, Balasubramanian P, Gutiérrez-Mora A (2012) In vitro somatic embryogenesis from suspension cultures of Carica papaya L. Sci Hortic 136:43–49. https://doi.org/10.1016/j.scienta.2012.01.003
Anonymous (2021) Package of practices for fruit crops. Punjab Agricultural University, Ludhiana, pp 115–118
Arora IK, Singh RN (1978) In vitro plant regeneration in papaya. Curr Sci 47:867–868
Ascencio-Cabral A, Rodríguez-Garay B, Gutiérrez-Pulido H, Gutiérrez-Mora A (2008) Plant regeneration of Carica papaya through somatic embryogenesis in response to light quality, gelling agent and phloridzin. Sci Hortic 118:155–160. https://doi.org/10.1016/j.scienta.2008.06.014
Bahrami Sirmandi H, Vahdati K (2008) Effect of carbohydrate source and polyethylene glycol on maturation and germination of somatic embryos in walnut (Juglans regia L.). In I International Symposium on Biotechnology of Fruit Species: BIOTECHFRUIT2008 839:165–172
Bertan I, Carvalho FIF, Oliveira AC, Benin G, Vieira EA, Valério IP (2009) Morphological, pedigree, and molecular distances and their association with hybrid wheat performance. Pesq Agropec Bras 44:155–163. https://doi.org/10.1590/S0100-204X2009000200007
Bhat KV, Jarret RL (1995) Random amplified polymorphic DNA and genetic diversity in Indian Musa germplasm. Genet Res Crop Evol 42:107–118. https://doi.org/10.1007/BF02539514
Bhattacharya J, Khuspe SS (2001) In vitro and in vivo germination of papaya (Carica papaya L.) seeds. Sci Hortic 91:39–49. https://doi.org/10.1016/S0304-4238(01)00237-0
Bhojawani SM, Rajadan K (1996) Plant Tissue Culture: Theory and Practice, a, Revised. Elsevier, North Holland
Bindu B (2015) Micro Propagation of Papaya Variety CO-5. Int J Res 2:46–49
Biswas Mk, Dutt M, Roy U, Islam R, Hossain M (2009) Development and evaluation of in vitro somaclonal variation in strawberry for improved horticultural traits. Sci Hortic 122:409–416. https://doi.org/10.1016/j.scienta.2009.06.002
Damasco OP, Graham GC, Henry RJ, Adkins SW, Godwin ID (1996) Random amplified polymorphic DNA (RAPD) detection of dwarf off-types in micropropagated Cavendish (Musa spp. AAA) bananas. Plant Cell Rep 16:118–123. https://doi.org/10.1007/BF01275464
De Klerk GJ (2002) Rooting of micro cuttings: Theory and practice. In Vitro Cell Dev Biol 38:415–422. https://doi.org/10.1079/IVP2002335
De Winnaar W (1988) Clonal propagation of papaya in vitro. Plant Cell Tissue Organ Cult 12:305–310
De Bruijne E, Langhe DE, Rick VR (1974) Actions of hormones and embryoid formation in callus cultures of Carica papaya International Symposium on Crop Protection. Fytopharmacie En Fytiatrie Rijkslandsbouwhoogeskool Medelingen 39:637–645
Debnath SC, Goyali JC (2020) In vitro propagation and variation of Antioxidant properties in micro propagated Vaccinium berry plants-A review. Molecules 25:788. https://doi.org/10.3390/molecules25040788
Ahmad N, Faisal M (2018) Thidiazuron: From urea derivative to plant growth regulator. Springer, Singapore, p 491. https://doi.org/10.1007/978-981-10-8004-3
Devarumath RM, Nandy S, Rani V, Marimuthu S, Muraleedharan N, Raina SN (2002) RAPD, ISSR and RFLP fingerprints as useful markers to evaluate genetic integrity of micropropagated plants of three diploid and triploid elite tea clones representing Camellia sinensis (China type) and C. assamica ssp. assamica (Assam-Indian Type). Plant Cell Rep 21:166–173. https://doi.org/10.1007/s00299-002-0496-2
DeVerno LL, Park YS, Bonga JM, Barrett JD (1999) Somaclonal variation in cryopreserved embryogenic clones of white spruce [Picea glauca (Moench) Voss.]. Plant Cell Rep 18:948–953. https://doi.org/10.1007/s002990050689
Dixit S, Mandal BB, Ahuja S, Srivastava PS (2003) Genetic stability assessment of plants regenerated from cryopreserved embryogenic tissues of Dioscorea Bulbifera L. using RAPD, biochemical and morphological analysis. CryoLetters 24:77–84
Dotto JM, Abihudi SA (2021) Nutraceutical Value of Carica Papaya: A Review Sci Afric 13:00933. https://doi.org/10.1016/j.sciaf.2021.e00933
Encina CL, Granero ML, Regalado JJ (2023) In Vitro Long-Term Cultures of Papaya (Carica papaya L. cv. Solo). Horti 9:671. https://doi.org/10.3390/horticulturae9060671
Estrella-Maldonado H, Posada-Pérez L, Talavera MC, Barredo FP, Kosky GR, Samtamaria JM (2018) The Expression of CpAUX1/LAXs and Most of the Long-distance CpPINs Genes Increases as the Somatic Embryogenesis Process Develops in C. papaya cv. “Red Maradol.” J Plant Growth Regul 37:502–516. https://doi.org/10.1007/s00344-017-9746-y
Fazili MA, Bashir I, Ahmad M, Yaqoob U, Geelani SN (2022) In vitro strategies for the enhancement of secondary metabolite production in plants: A review. Bulletin of the National Research Centre 46:1–12. https://doi.org/10.1186/s42269-022-00717-z
Victor MLV, Neftali OA (2016) Somatic embryogenesis: Fundamental aspects and applications. Springer, Cham, p 506. https://doi.org/10.1007/978-3-319-33705-0
Fitch MMM, Manshardt RM (1990) Somatic embryogenesis and plant regeneration from immature zygotic embryos of papaya (Carica papaya L.). Plant Cell Rep 9:320–324. https://doi.org/10.1007/BF00232860
Fitch MMM (1995) Somatic embryogenesis in papaya (Carica papaya L.). Springer, Berlin, Heidelberg, p 260–279. https://doi.org/10.1007/978-3-662-03091-2_18
Fonseca RM, Lopes R, Barros WS, Lopes MTG, Ferreira FM (2008) Morphologic Characterization and genetic diversity of Capsicum chinense Jacq. accessions along the upper Rio Negro - Amazonas. Crop Breed Appl Biotechnol 8:187–194
Fourre JL, Berger P, Niquet L, Andre P (1997) Somatic embryogenesis and somaclonal variation in Norway spruce: morphogenetic, cytogenetic and molecular approaches. Theor Appl Genet 94:159–169. https://doi.org/10.1007/s001220050395
Gagliardi RF, Pacheco GP, Carneiro LA, Valls JFM, Vieira MLC, Mansur E (2003) Cryopreservation of Arachis species by vitrification of in vitro-grown shoot apices and genetic stability of recovered plants. CryoLetters 24:103–110
García-Fortea E, Lluch-Ruiz A, Pineda-Chaza BJ, García-Pérez A, Bracho-Gil JP, Plazas M, Gramazio P, Vilanovo 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
George EF (1993) Plant propagation by tissue culture: a practical approach. Exegetics
Ghahremani R, Daylami SD, Mirmasoumi M, Askari N, Vahdati K (2021) Refining a protocol for somatic embryogenesis and plant regeneration of Phalaenopsis amabilis cv. Jinan from mature tissues. Turk J Agric 45(3):356–364. https://doi.org/10.3906/tar-2004-107
Ghazaeian M, Hossein Davarynejad G, Vahdati K, Ghasemi Bezdi K, Nemati H, Zeinalinejad K (2023) Assessment of Hybrids Between Carya and Juglans Via ISSR Markers. Erwerbs-Obstbau 65(2):289–292. https://doi.org/10.1007/s10341-022-00821-4
Gimenez C, Garcia ED, Enrech NXD, Blanca I (2001) Somaclonal variation in banana: cytogenetic and molecular characterization of somaclonal variant CIEN BTA)03. In Vitro Cell Dev Biol Plant 37:217–222. https://doi.org/10.1007/s11627-001-0038-6
Gonçalves LSA, Rodrigues R, Amaral Júnior AD, Karasawa M, Sudré CP (2008) Comparison of multivariate statistical algorithms to cluster tomato heirloom accessions. Genet Mol Res 7:1289–1297. https://doi.org/10.4238/vol7-4gmr526
Haggman HM, Ryynanen LA, Aronen TS, Krajnakova J (1998) Cryopreservation of embryogenic cultures of Scots pine. Plant Cell Tissue Organ Cult 54:45–53. https://doi.org/10.1023/A:1006104325426
Hartmann HT, Kester DE, Davies FT, Geneve RL (2002) Plant propagation: principles and practice, 7th edn. Prentice-Hall, Englewood Cliffs
Helliot B, Madur D, Dirlewanger E, De Boucaud MT (2002) Evaluation of genetic stability in cryopreserved Prunus. In Vitro Cell Dev Biol Plant 38:493–500. https://doi.org/10.1079/IVP2002325
Howell EC, Newbury HJ, Swennen RL, Withers LA, Ford Lloyd BV (1994) The use of RAPD for identifying and classifying Musa germplasm. Genome 37:328–332
Hu W, Fagundez S, Katin-Grazzini L, Li Y, Li W, Chen Y, Li Y (2017) Endogenous auxin and its manipulation influence in vitro shoot organogenesis of citrus epicotyl explants. Hort Science 4:15–18. https://doi.org/10.1038/hortres.2017.71
Hunková J, Kleman J, Gažo J, Gajodosova A (2023) Adventitious regeneration of blackberry, blueberry, and kiwiberry and assessment of genetic stability by ISSR markers. Biologia 78:349–359. https://doi.org/10.1007/s11756-022-01211-7
Isabel N, Tremblay L, Michaud M, Tremblay FM, Bousquet J (1993) RAPDs as an aid to evaluate the genetic integrity of somatic embryogenesis derived populations of Picea mariana (Mill.) B.S.P. Theor Appl Genet 86:81–87. https://doi.org/10.1007/BF00223811
Jariteh M, Ebrahimzadeh H, Niknam V, Vahdati K, Amiri R (2011) Antioxidant enzymes activities during secondary somatic embryogenesis in Persian walnut (Juglans regia L.). Afr J Biotechnol 10(20):4093–4099
Jariteh M, Ebrahimzadeh H, Niknam V, Mirmasoumi M, Vahdati K (2015) Developmental changes of protein, proline and some antioxidant enzymes activities in somatic and zygotic embryos of Persian walnut (Juglans regia L.). Plant Cell Tissue Organ Cult 122:101–115. https://doi.org/10.1007/s11240-015-0753-z
Jat R, Singh VP, Kumar V (2020) Greenhouse cultivation of fruit crops with special reference to India: An overview. Jour Appl Nat Sci 12:252–260. https://doi.org/10.31018/jans.vi.2276
Kaity A, Ashmore SE, Drew RA (2008) Assessment of genetic and epigenetic changes following cryopreservation in papaya. Plant Cell Rep 27:1529–1539. https://doi.org/10.1007/s00299-008-0558-1
Kaur K, Kaur A (2017) Papaya performance under various growing conditions cv. Red Lady 786. Agricult Sci Dig Res Jour 37:290–293. https://doi.org/10.18805/ag.D-4681
Koul B, Pudhuvai B, Sharma C, Kumar A, Sharma V, Yadav D, Jin JO (2022) Carica papaya L.: A Tropical Fruit with Benefits beyond the Tropics. Diversity 14:683. https://doi.org/10.3390/d14080683
Krishnanayak L, Kotiyal A, Koubouris G (2024) Optimized seed germination and adaptation of plantlets to a new environment of papaya cv. ‘Red Baby’ using organic media and plant growth regulators. Vegetos 37:355–362. https://doi.org/10.1007/s42535-023-00597-2
Kumar P, Srivastava DK (2015) High frequency organogenesis in hypocotyl, cotyledon, leaf and petiole explants of broccoli (Brassica olerace a L. var. italica), an important vegetable crop. Physiol Mol Bio Plants 21:279–285. https://doi.org/10.1007/s12298-015-0282-6
Kumari R, Kundu M, Mir H, kumar P, (2023) In Vitro Regeneration Technique of Papaya (Carica papaya L.) cv. Pusa Dwarf Through Shoot Tip Culture. Natl Acad Sci Lett 46:1–5. https://doi.org/10.1007/s40009-022-01186-8
Litz RE, Conover RA (1980) Somatic embryogenesis and plantlet formation from immature embryos of Carica papaya L. Hort Sci 15:703–704
Ludwig-Muller J (2003) Peroxidase isoenzymes as markers for the rooting ability of easy-to-root and difficult-to-root Grevillea species and cultivars of Protea obstusifolia (Proteaceae). In Vitro Cell Dev Biol Plant 39:377–383. https://doi.org/10.1079/IVP2003423
Malabadi RB, Kumar SV, Mulgund GS, Nataraja K (2011) Induction of somatic embryogenesis in papaya (Carica papaya). Res Biotechnol 2(5):40–55
Mali AM, Chavan NS (2016) In vitro rapid regeneration through direct organogenesis and ex-vitro establishment of Cucumis trigonus Roxb. -An underutilized pharmaceutically important cucurbit. Ind Crops Prod 83:48–54. https://doi.org/10.1016/j.indcrop.2015.12.036
Malik N, Sengar RS, Yadav MK, Singh SK, Singh G, Kumar M (2019) Effect of different plant growth regulators on in-vitro callus Induction in Carica papaya (cv. Pusa Nanha). Int J Curr Microbiol App Sci 8:1217–1225. https://doi.org/10.20546/ijcmas.2019.807.145
Maneesh M, Ramesh Ch, Sangeeta S (2007) Papaya. Genome Mapping and Molecular Breeding in Plants, vol 4. Springer, Fruits and Nuts, pp 343–351
Marić S, Bolarić S, Martinčić J, Pejić I, Kozumplik V (2008) Genetic diversity of hexaploid wheat cultivars estimated by RAPD markers, morphological traits and coefficients of parentage. Plant Breed 123:366–369. https://doi.org/10.1111/j.1439-0523.2004.00956.x
Martins M, Tenreiro R, Oliveira MM (2003) Genetic relatedness of Portuguese almond cultivars assessed by RAPD and ISSR markers. Plant Cell Rep 22:71–78. https://doi.org/10.1007/s00299-003-0659-9
Mattos LA, Amorim EP, Amorim VBO, Cohen KO, Ledo CADS, Silva SDO (2010) Agronomical and molecular characterization of banana. Germoplasm Pesq Agropec Bras 45:146–154. https://doi.org/10.1590/S0100-204X2010000200005
Mk B, Dutt M, Roy U, Islam R, Hossain M (2009) Development and evaluation of in vitro somaclonal variation in strawberry for improved horticultural traits. Sci Hortic 122:409–416. https://doi.org/10.1016/j.scienta.2009.06.002
Mohsenipoor S, Vahdati K, Amiri R, Mozaffari MR (2009) Study of the genetic structure and gene flow in Persian walnut (Juglans regia L.) using SSR markers. In VI International Walnut Symposium 861:133–142. https://doi.org/10.17660/ActaHortic.2010.861.17
Mondal M, Gupta S, Mukherjee BB (1990) In vitro propagation of shoot buds of Carica papaya L. (Caricaceae) var. Honey Dew Plant Cell Rep 8:609–612. https://doi.org/10.1007/BF00270065
Moradi S, Dianati Daylami S, Arab M, Vahdati K (2017) Direct somatic embryogenesis in Epipactis veratrifolia, a temperate terrestrial orchid. J Hortic Sci Biotechnol 92(1):88–97. https://doi.org/10.1080/14620316.2016.1228434
Morgante M, Hanafey M, Powell W (2002) Microsatellites are preferentially associated with nonrepetitive DNA in plant genomes. Nat Genet 30:194–200. https://doi.org/10.1038/ng822
Morton JF (1987) Papaya. Fruits of warm climates. FL, USA, Miami, pp 336–346
Mumo NN, Rimberia FK, Mamati GE, Kihurani AW (2013) In vitro regeneration of selected Kenyan papaya (Carica papaya L.) lines through shoot tip culture. Afric J Biotechnol 12:6826–6832
Mumo N, Rimberia F, Mamati G, Kihurani A (2014) In vitro Grafting of Selected Papaya (Carica Papaya L.) Lines in Kenya. Annu Res Rev Bio 4(14):2337–2346. https://doi.org/10.9734/ARRB/2014/9494
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
Nagel M, Pence V, Ballesteros D, Lambardi M, Popova E, Panis B (2024) Plant cryopreservation: principles, applications, and challenges of banking plant diversity at ultralow temperatures. Biotechnol. Annu Rev 75. https://doi.org/10.1146/annurev-arplant-070623-103551
Nguyen V, Yen C, Hsieh C (2018) Effect of nutritional and growth hormonal factors on in vitro regeneration of papaya (Carica papaya L. cv. Red Lady). J Nat Sci Found Sri Lanka 46:559–568. https://doi.org/10.4038/jnsfsr.v46i4.8631
Nickravesh MH, Vahdati K, Amini F, Di Pierro EA, Amiri R, Woeste K, Arab MM (2023) Reliable propagation of Persian walnut varieties using SSR marker-based true-to-type validation. HortScience 58(1):64–66. https://doi.org/10.21273/HORTSCI16851-22
Otsuki N, Dang NH, Kumagai E, Kondo A, Iwata S, Morimoto C (2010) Aqueous extract of Carica papaya leaves exhibits anti-tumor activity and immunomodulatory effects. Jour Ethno 127:760–767. https://doi.org/10.1016/j.jep.2009.11.024
Palei S, Dash DK, Rout GR (2019) Standardization of in vitro protocol for plant regeneration of Carica papaya cv. Co 8 through indirect organogenesis. J Pharm Phytochem 8:1954–1956
Palombi M, Damiano C (2002) Comparison between RAPD and SSR molecular markers in detecting genetic variation in kiwifruit (Actinidia deliciosa A. Chev). Plant Cell Rep 20:1061–1066. https://doi.org/10.1007/s00299-001-0430-z
Pang SZ, Sanford JC (1988) Agrobacterium-mediated gene transfer in papaya. J Am Soc Horti Sci 113:287–291. https://doi.org/10.21273/JASHS.113.2.287
Pérez LP, Montesinos YP, Olmedo JG, Rodriguez RB, Sánchez RR, Montenegro ON, Escriba RCR, Daniels D, Gómez-Kosky R (2016) Effect of phloroglucinol on rooting and in vitro acclimatization of papaya (Carica papaya L. var. Maradol Roja). In Vitro Cell Dev Biol Plant 52:196–203. https://doi.org/10.1007/s11627-015-9733-6
Perrier X, Jacquemoud-Collet JP (2006) DARwin. Dissimilarity analysis and representation for windows, Version, 6. http://darwin.cirad.fr/darwin. Accessed 20 Nov 2023
Radhakrishnan R, Ramachandran A, Kumari RBD (2009) Rooting and shooting: dual function of thidiazuron in in vitro regeneration of soybean (Glycine max. L). Acta Physiol Plant 31:1213–1217. https://doi.org/10.1007/s11738-009-0356-6
Rani V, Raina SN (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
Rani V, Singh KP, Shiran M, Nandy S, Goel S, Devarumanth RM, Sreenath HL, Raina SN (2000) Evidence for new nuclear and mitochondrial genome organizations among high frequency somatic embryogenesis-derived plants of allotetraploid Coffea arabica L. (Rubiaceae). Plant Cell Rep 19:1013–1020. https://doi.org/10.1007/s002990000228
Ratjens S, Mortensen S, Kumpf A, Bartsch M, Winkelmann T (2018) Embryogenic callus as target for efficient transformation of Cyclamen persicum enabling gene function studies. Front Plant Sci 9:1035. https://doi.org/10.3389/fpls.2018.01035
Ray T, Dutta I, Saha P, Das S, Roy SC (2006) 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 Tissue Organ Cult 85:11–21. https://doi.org/10.1007/s11240-005-9044-4
Razali RM, Drew R (2014) A refined protocol for embryogenesis to transfer PRSV-P resistance genes from Vasconcellea pubescens to Carica papaya. In; III Int Symposium on Papaya 1022:47–54. https://doi.org/10.17660/ActaHortic.2014.1022.4
Reuveni O, Shlesinger DR, Lavi U (1990) In vitro clonal propagation of dioecious Carica papaya. Plant Cell Tissue Organ Cult 20:41–46. https://doi.org/10.1007/BF00034755
Rival A, Bertrand L, Beale T, Combes MC, Trouslot P, Leshermes P (1998) Suitability of RAPD analysis for detection of somaclonal variation in oil palm (Elaeis guineensis Jacq.). Plant Breed 117:73–76
Rocha MC, Gonçalves LSA, Rodrigues R, Silva PRAD, Carmo MGFD, Abboud ACDS (2010) Uso do algoritmo de Gower na determinação da divergência genética entre acessos de tomateiro do grupo cereja. Acta Sci 32:423–431. https://doi.org/10.4025/actasciagron.v32i3.4888
Rohlf FJ (1998) NTSYSpc numerical taxonomy and multivariate analysis system version 2.0 user guide. Accessed 15 Nov 2023
Rose RJ, Mantiri FR, Kurdyukov S, Chen SK, Wang XD, Nolan KE, Sheahan, MB, Pua EC, Davey MR, (2010) Developmental biology of somatic embryogenesis, Springer, Berlin Heidelberg, p 3–26. https://doi.org/10.1007/978-3-642-04670-4
Roy PK, Roy SK, Hakim ML (2012) Propagation of papaya (Carica papaya L.) cv. Shahi through in vitro culture. Bangla J Bot 41:191–195
Ryavalad S, Malabasari TA, Shantappa T, Uppar DS, Biradar BD, Mantur SM (2019) Micropropagation Studies in Papaya (Carica papaya L.) cv.‘Surya.’ Int J Curr Microbiol App Sci 84:2362–2367. https://doi.org/10.20546/ijcmas.2019.804.275
Sadat-Hosseini M, Vahdati K, Leslie CA (2019) Germination of Persian walnut somatic embryos and evaluation of their genetic stability by ISSR fingerprinting and flow cytometry. HortScience 54(9):1576–1580. https://doi.org/10.21273/HORTSCI13787-18
Sahijram L, Soneji JR, Bollamma KT (2003) Analyzing somaclonal variation in micropropagated bananas (Musa spp.). In Vitro Cell Dev Biol Plant 39:51–556. https://doi.org/10.1079/IVP2003467
Schuster C, Gaillochet C, Medzihradszky A, Busch W, Daum G, Krebs M, Kehle A, Lohmann JU (2014) A regulatory framework for shoot stem cell control integrating metabolic, transcriptional, and phytohormone signals. Develop Cell 28:438–449. https://doi.org/10.1016/j.devcel.2014.01.013
Setargie A, Mekbib F, Abraha E (2015) In vitro propagation of papaya (Carica papaya L.). World J Agric Sci 11:84–88
Shivakumara NR, Ahlawt TR, Pandey AK, Patel D, Jena S, Chaudhary AD (2021) Comparative Morphology, Phenology and Productive Potential of Different Plant Types in Papaya cv. Red Lady Taiwan. Int J Curr Microbiol App Sci 10:2050–2059
Sirmandi HB, Vahdati K, Kalantari S (2009) Enhancement of maturation and germination of somatic embryos of Persian walnut (Juglans regia L) by Gellan gum, cold and PGR. In VI Int Walnut Symposium 861:333–344. https://doi.org/10.17660/ActaHortic.2010.861.46
Sudré CP, Gonçalves LSA, Rodrigues R, Amaral Júnior AD, Riva-Souza EM, Bento CDS (2010) Genetic variability in domesticated Capsicum spp as assessed by morphological and agronomic data in mixed statistical analysis. Genet Mol Res 9:283–294. https://doi.org/10.4238/vol9-1gmr698
Tatineni S, Hein GL (2023) Plant viruses of agricultural importance: Current and future perspectives of virus disease management strategies. Phytopathology 113:117–141. https://doi.org/10.1094/PHYTO-05-22-0167-RVW
Tegen H, Mohammed W (2016) The role of plant tissue culture to supply disease free planting materials of major horticultural crops in Ethiopia. Glob J Biol Agric 6:122–129
Touchell D, Turner SR, Senaratna T, Bunn E, Dixon KW (2002) Cryopreservation of australian species — the role of plant growth regulators. Springer, Berlin, Heidelberg, pp 373–390. https://doi.org/10.1007/978-3-662-04674-6_26
Tupke AH, Akhare AA, Gahukar SJ, Bahatkar BP, Zadokar AR, Gaikwad PN, Katkade RR (2021) DNA fingerprinting: a novel technique for identification of important species of citrus. Ann. Phytomed 10(1):202–207. https://doi.org/10.21276/ap.2021.10.1.-21
Turner S, Krauss SL, Bunn E, Senaratna T, Dixon K, Tan B, Touchell D (2001) Genetic fidelity and viability of Anigozanthos viridis following tissue culture, cold storage and cryo-preservation. Plant Sci 161:1099–1106. https://doi.org/10.1016/S0168-9452(01)00519-2
Tyagi M, Singh H, Jawandha SK (2015) Performance of papaya cultivars grown under protected conditions. Indian J Hortic 72:334–337. https://doi.org/10.5958/0974-0112.2015.00065.1
Vahdati K, Jariteh M, Niknam V, Mirmasoumi M, Ebrahimzadeh H (2004) Somatic embryogenesis and embryo maturation in Persian walnut. In V Int Walnut Symposium 705:199–205. https://doi.org/10.17660/ActaHortic.2005.705.24
Vendrame WA, Kochert G, Wetzstein HY (1999) AFLP analysis of variation in pecan somatic embryos. Plant Cell Rep 18:853–857. https://doi.org/10.1007/s002990050673
Venkatachalam L, Sreedhar RV, Bhagyalakshmi N (2007) Micropropagation in banana using high levels of cytokinins does not involve any genetic changes as revealed by RAPD and ISSR markers. Plant Growth Regul 51:193–205. https://doi.org/10.1007/s10725-006-9154-y
Verdeil JL, Alemanno L, Niemenak N, Tranbarger TJ (2007) Pluripotent versus totipotent plant stem cells: dependence versus autonomy? Trends Plant Sci 12:245–252. https://doi.org/10.1016/j.tplants.2007.04.002
Vidal JR, Rama J, Taboada L, Martin C, Ibanez M, Segura A, González-Benito ME (2009) Improved somatic embryogenesis of grapevine (Vitis vinifera) with focus on induction parameters and efficient plant regeneration. Plant Cell Tissue Organ Cult 96:85–94. https://doi.org/10.1007/s11240-008-9464-z
Vieira EA, Carvalho FIF, Bertan I, Kopp MM, Zimmer PD, Benin G, Silva JAGD, Hartwig I, Malone G, Oliveira ACD (2007) Association between genetic distances in wheat (Triticum aestivum L.) as estimated by AFLP and morphological markers. Genet Mol Biol 30:392–399. https://doi.org/10.1590/S1415-47572007000300016
Vij T, Prashar Y (2015) A review on medicinal properties of Carica papaya Linn. Asian Pac J Trop Dis 5:1–6. https://doi.org/10.1016/S2222-1808(14)60617-4
Manchanda P, Sidhu GS, Mankoo RK (2024) Bioactive compounds and antioxidant potential determination in callus tissue as compared to leaf, stem and root tissue of Carica papaya cv. Red Lady 786. J Food Meas Charact 37:1–14. https://doi.org/10.1007/s11694-024-02366-4
Vuong QV, Hiun S, Roach PD, Bowyer MC, Phllips PA, Scarlett CJ (2013) Effect of extraction conditions on total phenolic compounds and antioxidant activities of Carica papaya leaf aqueous extracts. Jour Herb Med 3:104–111. https://doi.org/10.1016/j.hermed.2013.04.004
Waidyaratne SM, Samanmalie LG (2022) In vitro Propagation of Carica papaya L. Variety ‘Horana Papaya Hybrid 01’Using Shoot Tip. World 10:15–19
Wallner E, Weising K, Rompf R, Kahl G, Kopp B (1996) Oligonucleotide and RAPD analysis of Achillea species: characterization and long-term monitoring of micropropagated clones. Plant Cell Rep 15:647–652. https://doi.org/10.1007/BF00232470
Wang Z, Weber JL, Zhong G, Tanksley SD (1994) Survey of plant short-tandem DNA repeats. Theor Appl Genet 88:1–6. https://doi.org/10.1007/BF00222386
Williams JGK, Kubelik AR, Livak KJ, Rafalski JA, Tingey SV (1990) DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res 18:6531–6535. https://doi.org/10.1093/nar/18.22.6531
Winkelmann T (2016) Somatic Versus Zygotic Embryogenesis: Learning from Seeds. Humana Press, New York. pp 25–46. https://doi.org/10.1007/978-1-4939-3061-6_2
Wu K, Zeng S, Chen Z, Duan J (2012) In vitro mass propagation of hermaphroditic Carica papaya cv. Meizhonghong Pak Jour Bot 44:1
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
Yu TA, Yeh SD, Cheng YH, Yang JS (2000) Efficient rooting for establishment of papaya plantlets by micropropagation. Plant Cell Tissue Organ Cult 61:29–35. https://doi.org/10.1023/A:1006475901439
Zietkiewicz E, Rafalski A, Labuda D (1994) Genome fingerprinting by simple sequence repeats (SSR)-anchored polymerase chain reaction amplification. Genomics 20:176–183. https://doi.org/10.1006/geno.1994.1151
Zimmerman JL (1993) Somatic embryogenesis: a model for early development in higher plants. Plant Cell 5:1411–1423. https://doi.org/10.1105/tpc.5.10.1411
Zhai Z, Wu Y, Engelmann F, Chen R, Zhao Y (2003) Genetic stability assessments of plantlets regenerated from cryopreserved in vitro cultured grape and kiwi shoot-tips using RAPD. CryoLetters 24:315–322
Acknowledgements
We are grateful to the Department of Fruit Science, College of Horticulture and Forestry for maintain and providing papaya explants and Dr. GS Kalkat Laboratory, School of Agricultural Biotechnology for providing us with the Molecular Biology Lab facilities, Punjab Agricultural University. Ludhiana.
Author information
Authors and Affiliations
Contributions
Conceptualization and supervision: Gurupkar Singh Sidhu; experiment designing- Gurupkar Singh Sidhu; maintaining and providing plant material- Mandeep Singh Gill; culturing and hardening- Vishal; molecular characterization: Vishal, Popat Nanaso Gaikwad; data analysis: Popat Nanaso Gaikwad, Sukhjinder Singh Mann, and Vishal; writing and original draft preparation- Vishal, and Popat Nanaso Gaikwad; review and editing- Gurupkar Singh Sidhu, Popat Nanaso Gaikwad and Pooja Manchanda: All authors read and approved the final manuscript.
Corresponding author
Ethics declarations
Conflict of interest
The authors state that the publishing of this research article does not create a conflict of interest for them.
Additional information
Communicated by Ming-Tsair Chan.
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
Below is the link to the electronic supplementary material.
Rights and permissions
Springer Nature or its licensor (e.g. a society or other partner) 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
Vishal, Sidhu, G.S., Gaikwad, P.N. et al. Optimized protocol for high-frequency papaya propagation: morpho-stereomicroscopic analysis and genetic fidelity assessment. Plant Cell Tiss Organ Cult 156, 81 (2024). https://doi.org/10.1007/s11240-024-02704-w
Received:
Accepted:
Published:
DOI: https://doi.org/10.1007/s11240-024-02704-w