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Optimization process for de-novo organogenesis and regeneration of transgenic papaya plants using leaf tissue as explants

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Abstract

Papaya (Carica papaya L.) is widely grown in tropical and subtropical regions. The destructive disease caused by emerging strains of Papaya ringspot virus (PRSV) demands effective transgenic resistance to target atypical virus strains threatening the crop. Papaya transformations are mainly conducted on the explants of immature zygotic or somatic embryos, which are difficult to obtain and proceed, and are largely affected by seasonal factors. Here, we attempted to develop an efficient process for organogenesis using various tissues of ex-vitro or in-vitro grown papaya seedlings. Leaf lamina, hypocotyl and stem sections of seedlings of the papaya variety Sindhi were used for establishment of callus culture through 12 different callus induction treatments (CIT 1–12), with various combinations of plant growth regulators (PGRs). Our results revealed that CIT-11 and CIT-10 enhanced callus induction from ex-vitro leaf discs with midrib, with 86% and 80% efficiency respectively, superior to 53% of CIT-10 using in-vitro leaf discs with midrib. The expression of binary vector construct pSN-PRSV CP in Agrobacterium strain GV3101 was verified in Nicotiana benthamiana and papaya by RT-PCR analysis. Calli derived from leaf tissues (with midrib) of papaya, transformed with the binary vector were successfully regenerated on the shoot induction treatment SIT-13 (Gamborg B5 medium + 0.5 mg/L TDZ + 0.01 mg/L NAA) and were subsequently rooted on root induction treatment RIT-4 (Gamborg B5 medium + 1 mg/L IBA). The transformed explants were regenerated with an efficiency of 26%. The whole process is unique in term of explant selection, source of explant (ex-vitro grown papaya plants) and media formulations as, the leaf tissue from ex-vitro grown papaya showed highest callusing and regeneration efficiency overall.

Key message

Successful transgenic papaya regeneration is possible from transformed calli, obtained from in-vitro and ex-vitro leaf explants.

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Data availability

The data will be available on request.

Abbreviations

CP:

Coat protein

PRSV:

Papaya ringspot virus

RT–PCR:

Reverse transcription polymerase chain reaction

CIT:

Callus induction treatment

SIT:

Shoot induction treatment

RIT:

Root induction treatment

DTT:

Dithiothreitol

dNTP:

Deoxynucleotide triphosphate

NAA:

1-Naphthaleneacetic acid

BAP:

6-Benzylaminopurine

TDZ:

Thidiazuron

IAA:

Indole-3-acetic acid

Kan:

Kanamycin

LB:

Luria-Bertani Broth

PDR:

Pathogen derived resistance

PGRs:

Plant growth regulators

Hectares:

Ha

CaMV:

Cauliflower mosaic virus

CMV:

Cucumber mosaic virus

IBA:

Indole-3-butyric acid

nptII:

Neomycin phosphotransferase II

GOI:

Gene of interest

GA3:

Gibberellic acid

2,4-D, 2,4:

Dichlorophenoxyacetic acid

Kin:

Kinetin

CH:

Caseine hydrolysate

MgCl2 :

Magnesium Chloride

KCL:

Potassium Chloride

ddH2O:

Double distilled water

References

  • Ahmad N, Anis M (2007) Rapid plant regeneration protocol for cluster bean (Cyamopsis tetragonoloba L. Taub.). J Hortic Sci Biotechnol 82:585–589. https://doi.org/10.1080/14620316.2007.11512277

    Article  CAS  Google Scholar 

  • Ali A, Natsuaki T, Okuda S (2004) Identification and molecular characterization of viruses infecting cucurbits in Pakistan. J Phytopathol 152:677–682. https://doi.org/10.1111/j.1439-0434.2004.00915.x

    Article  CAS  Google Scholar 

  • Al-Shara B, Taha RM, Rashid K (2018) Biotechnological methods and limitations of micropropagation in Papaya (Carica Papaya L.) production: A review. J Anim Plant Sci 28:1208–1226

    CAS  Google Scholar 

  • Anandan R, Thirugnanakumar S, Sudhakar D, Balasubramanian P (2011) In vitro organogenesis and plantlet regeneration of (Carica papaya L.). J Agric Technol 7:1339–1348

    Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • Anandan R, Deenathayalan T, Kumar NS, Deepak KV (2018) An alternative in vitro plant regeneration system in papaya ( Carica papaya L.) through callus derived nodular cultures. Meta Gene 17:147–152. https://doi.org/10.1016/j.mgene.2018.06.007

    Article  Google Scholar 

  • Arora IK, Singh RN (1978) In vitro plant regeneration in papaya. Curr Sci 47:867–868

    Google Scholar 

  • Ascencio-Cabral A, Gutiérrez-Pulido H, Rodríguez-Garay B, Gutiérrez-Mora A (2008) Plant regeneration of Carica papaya L. 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

    Article  CAS  Google Scholar 

  • Azad MAK, Amin L, Sidik NM (2014) Gene technology for papaya ringspot virus disease management. Sci, World J

    Book  Google Scholar 

  • Bhattacharya J, Khuspe SS, Renukdas NN, Rawal SK (2002) Somatic embryogenesis and plant regeneration from immature embryo explant of papaya. Biologia Plantarum 46(3):327–331

    Article  Google Scholar 

  • Bhattacharya J, Renukdas NN, Khuspe SS, Rawal SK (2003) Multiple shoot regeneration from immature embryo explants of papaya. Biol Plant 47:327–331

    Article  Google Scholar 

  • Cai W, Gonsalves C, Tennant P et al (1999) A protocol for efficient transformation and regeneration of Carica papaya L. Vitr Cell Dev Biol - Plant 35:61–69. https://doi.org/10.1007/s11627-999-0011-3

    Article  CAS  Google Scholar 

  • Carlos-Hilario LR, Christopher DA (2015) Improved Agrobacterium-mediated transformation of Carica papaya cultivar ‘Kapoho’ from embryogenic cell suspension cultures. Vitr Cell Dev Biol - Plant 51:580–587. https://doi.org/10.1007/s11627-015-9719-4

    Article  CAS  Google Scholar 

  • Campos NA, da Silva GJ, de Paula MFB, Rodrigues TB, Rodrigues LAZ, Paiva LV (2016) Direct organogenesis protocol from shoot segments of’Solanum tuberosum’cv. Monalisa. Aust J Crop Sci 10(7):964–968

    Article  CAS  Google Scholar 

  • Chen MH, Wang PJ, Maeda E (1987) Somatic embryogenesis and plant regeneration in Carica papaya L. tissue culture derived from root explants. Plant Cell Rep 6:348–351. https://doi.org/10.1007/BF00269557

    Article  CAS  PubMed  Google Scholar 

  • Chen G, Ye CM, Huang JC et al (2001) Cloning of the papaya ringspot virus (PRSV) replicase gene and generation of PRSV-resistant papayas through the introduction of the PRSV replicase gene. Plant Cell Rep. https://doi.org/10.1007/s002990000311

    Article  PubMed  Google Scholar 

  • Cheng YH, Yang JS, Yeh SD (1996) Efficient transformation of papaya by coat protein gene of papaya ringspot virus mediated byAgrobacterium following liquid-phase wounding of embryogenic tissues with caborundum. Plant Cell Rep 16:127–132

    Article  CAS  PubMed  Google Scholar 

  • Clarindo WR, de Carvalho CR, Araújo FS et al (2008) Recovering polyploid papaya in vitro regenerants as screened by flow cytometry. Plant Cell Tissue Organ Cult 92:207–214. https://doi.org/10.1007/s11240-007-9325-1

    Article  Google Scholar 

  • Davis MJ, Ying Z (2004) Development of papaya breeding lines with transgenic resistance to Papaya ringspot virus. Plant Dis 88:352–358

    Article  CAS  PubMed  Google Scholar 

  • de Melo APC, Seleguini A (2013) Stages of maturity and removal of physical sarcotesta on seedling emergence and development of papaya/Estadio de maturacao de frutos e remocao fisica da sarcotesta na producao de mudas de mamao. Comunicata Scientiae 4(1):20–26

    Google Scholar 

  • Evans EA, Ballen FH, Crane JH (2012) An overview of US papaya production, trade, and consumption. EDIS 9:1–8

    Google Scholar 

  • Fan H, Yan X, Fu M et al (2022) Interactive Effect of Biological Agents Chitosan, Lentinan and Ningnanmycin on Papaya Ringspot Virus Resistance in Papaya (Carica papaya L.). Molecules 27(21):7474. https://doi.org/10.3390/molecules27217474

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • FAO (2021) FAO. http://www.fao.org/faostat/en/#rankings/countries_by_commodity. Accessed 20 Mar 2023 

  • Farzana ARF, Palkadapala PGVN, Meddegoda KMMN, Samarajeewa PK (2008) Somatic embryogenesis in papaya (Carica papaya L. cv. Rathna). J Natl Sci Found Srilanka 36:41–50

    Google Scholar 

  • Fermin G, Inglessis V, Garboza C et al (2004) Engineered resistance against Papaya ringspot virus in Venezuelan transgenic papayas. Plant Dis 88:516–522

    Article  PubMed  Google Scholar 

  • Ferreira SA, Pitz KY, Manshardt R et al (2002) Virus Coat Protein Transgenic Papaya Provides Practical Control of Papaya ringspot virus in Hawaii. Plant Dis. https://doi.org/10.1094/PDIS.2002.86.2.101

    Article  PubMed  Google Scholar 

  • Fitch MM (1993) High frequency somatic embryogenesis and plant regeneration from papaya hypocotyl callus. Plant Cell Tissue Organ Cult 32:205–212

    Article  CAS  Google Scholar 

  • Fitch MMM, Manshardt RM, Gonsalves D et al (1990) Stable transformation of papaya via microprojectile bombardment. Plant Cell Rep 9:189–194. https://doi.org/10.1007/BF00232177

    Article  CAS  PubMed  Google Scholar 

  • Fitch MM, Manshardt RM, Gonsalves D et al (1992) Virus resistant papaya plants derived from tissues bombarded with the coat protein gene of papaya ringspot virus. Biotechnology 10:1466–1472

    CAS  Google Scholar 

  • Gamborg OL, Miller R, Ojima K (1968) Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res 50:151–158

    Article  CAS  PubMed  Google Scholar 

  • Giang DTT, Van PT, Tanaka M, Teixeira da Silva JA (2011) Sterilization and Germination of Papaya ( Carica papaya L.) Seed and Response to LEDs. Seed Sci Biotechnol 5:56–58

    Google Scholar 

  • Gonsalves D (1998) Control of Papaya ringspot virus in papaya: A Case Study. Annu Rev Phytopathol 36:415–437. https://doi.org/10.1146/annurev.phyto.36.1.415

    Article  CAS  PubMed  Google Scholar 

  • Guerra MP, Vesco LLD (2010) Strategies for the micropropagation of bromeliads. Protocols for in vitro propagation of ornamental plants. pp 47–66

    Chapter  Google Scholar 

  • Karam NS, Al-Majathoub M (2000) Direct shoot regeneration and microtuberization in wild Cyclamen persicum Mill. using seedling tissue. Sci Hortic 86:235–246

    Article  CAS  Google Scholar 

  • Kępczyńska E, Kępczyński J (2023) Hormonal regulation of somatic embryogenesis in Medicago spp. Plant Cell Tissue Organ Cult. https://doi.org/10.1007/s11240-023-02593-5

    Article  Google Scholar 

  • Koehler AD, Usuario CCR, Abreu IS (2013) Somatic embryogenesis from leaf explants of hermaphrodite Carica papaya: a new approach for clonal propagation. Afr J Biotechnol 12:2386–2391. https://doi.org/10.5897/AJB12.1939

    Article  Google Scholar 

  • Kozai T, Xiao Y, Nguyen QT et al (2005) Photoautotrophic (sugar-free medium) micropropagation systems for large-scale commercialization. Propag Ornam Plants 5:23–34

    Google Scholar 

  • Kumar PP, Dimps Rao C, Goh CJ (1998) Influence of petiole and lamina on adventitious shoot initiation from leaf explants of Paulownia fortunei. Plant Cell Rep 17:886–890. https://doi.org/10.1007/s002990050503

    Article  CAS  PubMed  Google Scholar 

  • Kumari S, Trivedi M, Mishra M (2015) PRSV resistance in papaya (Carica papaya L.) through genetic engineering: A review. J Appl Hortic 17:243–248

    Article  Google Scholar 

  • Kung YJ, Yu TA, Huang CH et al (2010) Generation of hermaphrodite transgenic papaya lines with virus resistance via transformation of somatic embryos derived from adventitious roots of in vitro shoots. Transgenic Res 19:621–635. https://doi.org/10.1007/s11248-009-9344-2

    Article  CAS  PubMed  Google Scholar 

  • Lai CC, Yu TA, Yeh SD, Yang JS (1998) Enhancement of in vitro growth of papaya multishoots by aeration. Plant Cell Tissue Organ Cult 53:221–225

    Article  Google Scholar 

  • Litz RE, Conover RA (1977) Tissue culture propagation of papaya. Proc Florida State Hortic Soc 90:245–246

    Google Scholar 

  • Litz RE, Conover RA (1982) In vitro somatic embryogenesis and plant regeneration from Carica papaya L. ovular callus. Plant Sci Lett 26:153–158

    Article  CAS  Google Scholar 

  • Litz RE, Stephen KO, Conover RA (1983) In vitro growth of Carica papaya L. cotyledons. Sci Hortic 19:287–293

    Article  CAS  Google Scholar 

  • Lius S, Manshardt RM, Maureen M et al (1997) Pathogen-derived resistance provides papaya with effective protection against papaya ringspot virus. Mol Breed 3:161–168. https://doi.org/10.1023/A:1009614508659

    Article  Google Scholar 

  • Malabadi RB, Kumar SV, Mulgund GS, Nataraja K (2011) Induction of somatic embryogenesis in Papaya (Carica papaya). Res Biotechnol 2:40–55

    Google Scholar 

  • Manshardt RM, Wenslaff TF (1989) Zygotic polyembryony in interspecific hybrids of Carica papaya and C. cauliflora. J Am Soc Hortic Sci 114:684–689

    Article  Google Scholar 

  • Marri MY, Memon A, Lashari MI (2013) Profitability analysis of minor fruit in Sindh. Life Sci Int J 7:2809–2813

    Google Scholar 

  • Mishra M, Shukla N, Chandra R (2007) Micropropagation of papaya (Carica papaya L.). In Protocols for micropropagation of woody trees and fruits. Springer Netherlands, Dordrecht, pp 437–441

    Chapter  Google Scholar 

  • Mumo NN, Mamati GE, Ateka EM, et al (2020) Metagenomic Analysis of Plant Viruses Associated With Papaya Ringspot Disease in Carica papaya L. in Kenya. Front Microbiol 11:. https://doi.org/10.3389/fmicb.2020.00205

  • Murashige T, Skoog F (1962) A Revised Medium for Rapid Growth and Bio Agsays with Tohaoco Tissue Cultures. Physiol Plant 15:437–497

    Article  Google Scholar 

  • Nadeem A, Mehmood T, Tahir M et al (1997) First Report of Papaya Leaf Curl Disease in Pakistan. Plant Dis 81:1333–1333. https://doi.org/10.1094/PDIS.1997.81.11.1333B

    Article  CAS  PubMed  Google Scholar 

  • Naseem S, Roofi A, Zafar Y, Hafeez FY (2013) Detection and phylogenetic relationship of Papaya ringspot vitus-P in Pakistan. J Plant Pathol 95:643–645

    Google Scholar 

  • Noshad QQ, Zafar Y, Khan MA et al (2015) First record of papaya ring spot virus (PRSV) strain in Malir district Sindh and in Islamabad Pakistan. Int J Agric Biol 17:399–402

    Google Scholar 

  • Oad FC, Lakho AA, Khan A et al (2001) Economics of Papaya in Malir District, Karachi-Pakistan. Int J Agric Biol 3:477–481

    Google Scholar 

  • Palei S, Dash DK, Rout GR (2019) Standardization of in vitro protocol for plant regeneration of Carica papaya cv. Co8 through indirect organogenesis. J Pharmacogn Phytochem 8:1954–1956

    CAS  Google Scholar 

  • Prahardini PER, Sudaryono T (1992) The effect of additional naphtalene acetic acid and benzil adenine to the growth of papaya cv. Dampit using MS in vitro culture, J Hortik

    Google Scholar 

  • Preece JE (2008) Stock Plant Physiological Factors Affecting Growth and Morphogenesis. In: George EF, Hall MA, De Klerk G-J (eds) Plant Propagation by Tissue Culture, vol 1, 3rd edn. The Background, Springer, Neth, pp 403–422

    Google Scholar 

  • Premchand U, Mesta RK, Devappa V et al (2023) Survey, Detection, Characterization of Papaya Ringspot Virus from Southern India and Management of Papaya Ringspot Disease. Pathogens 12:1–25. https://doi.org/10.3390/pathogens12060824

    Article  CAS  Google Scholar 

  • Saeed W, Naseem S, Gohar D, Ali Z (2019) Efficient and reproducible somatic embryogenesis and micropropagation in tomato via novel structures - Rhizoid Tubers. PLoS ONE 14:1–24. https://doi.org/10.1371/journal.pone.0215929

    Article  CAS  Google Scholar 

  • Saleem A, Yeh SD, Saeed W, et al (2021) Genetic variability and evolutionary dynamics of atypical Papaya ringspot virus infecting Papaya. PLoS One 1–22. https://doi.org/10.1371/journal.pone.0258298

  • Santos SAD, Silva RFD, Pereira MG et al (2009) Estudos morfo-anatômicos de sementes de dois genótipos de mamão (Carica papaya L.). Rev Bras Sementes 31:116–122

    Article  Google Scholar 

  • Schöb H, Kunz C, Meins F (1997) Silencing of transgenes introduced into leaves by agroinfiltration: A simple, rapid method for investigating sequence requirements for gene silencing. Mol Gen Genet 256:581–585. https://doi.org/10.1007/s004380050604

    Article  PubMed  Google Scholar 

  • Shah DM, Rommens CM, Beachy RN (1995) Resistance to diseases and insects in transgenic plants: progress and applications to agriculture. Trends Biotechnol 13:362–368

    Article  CAS  Google Scholar 

  • Solórzano-Cascante P, Sánchez-Chiang N, Jiménez VM (2018) Explant type, culture system, 6-benzyladenine, meta-topolin and encapsulation affect indirect somatic embryogenesis and regeneration in Carica papaya L. Front Plant Sci 9:1769. https://doi.org/10.3389/fpls.2018.01769

    Article  PubMed  PubMed Central  Google Scholar 

  • Souza Júnior MT, Nickel O, Gonsalves D (2005) Development of virus resistant transgenic papayas expressing the coat protein gene from a Brazilian isolate of Papaya ringspot virus. Fitopatol Bras 30:357–365

    Article  Google Scholar 

  • Tennant P, Fermin G, Fitch MM et al (2001) Papaya ringspot virus resistance of transgenic rainbow and SunUp is affected by gene dosage, plant development, and coat protein homology. Eur J Plant Pathol 107:645–653. https://doi.org/10.1023/A:1017936226557

    Article  CAS  Google Scholar 

  • Vyas MB, Shah SK (2016) Review on nutritional and medicinal values of “ Carica. J Pharmacogn Phytochem 5:284–286

    CAS  Google Scholar 

  • Yang JS, Yu TA, Cheng YH, Yeh SD (1996) Transgenic papaya plants from Agrobacterium-mediated transformation of petioles of in vitro propagated multishoots. Plant Cell Rep 15:459–464

    Article  CAS  PubMed  Google Scholar 

  • Yeh SD, Gonsalves D (1985) Translation of papaya ringspot virus RNA in vitro: Detection of a possible polyprotein that is processed for capsid protein, cylindrical-inclusion protein, and amorphous-inclusion protein. Virology 143:260–271. https://doi.org/10.1016/0042-6822(85)90113-8

    Article  CAS  PubMed  Google Scholar 

  • Yeh SD, Gonsalves D, Wang HL et al (1988) Control of papaya ringspot virus by cross protection. Plant Dis 72:375–380

    Article  Google Scholar 

  • Yie ST, Liaw SI (1977) Plant regeneration from shoot tips and callus of papaya. In-Vitro 13:564–568

    Article  CAS  PubMed  Google Scholar 

  • Zhou C, Yang M, Luo X, Kuang R, Yang H, Yao J, Huang B, Wei Y (2021) Transcriptomic analysis of papaya (Carica papaya L.) shoot explants obtained by leaf-and stem-inoculation methods for adventitious roots induction. Scientia Horticulturae 276:109762

    Article  CAS  Google Scholar 

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Funding

We are greatly thankful to Higher Education Commission Pakistan (HEC) for funding the research work under HEC-NRPU project no. 3551.

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  1. Department of Biosciences, COMSATS University Islamabad, Islamabad, Pakistan

    Anam Saleem, Zahid Ali & Saadia Naseem

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  1. Anam Saleem
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Contributions

AS: Performed experiments, Data curation, Methodology, Investigation, Writing – original draft.

ZA: Resources, lab expertise, Methodology, Data curation, Formal analysis, review & editing.

SN: Resources, Conceptualization, Funding acquisition, Data curation, review & editing.

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Correspondence to Saadia Naseem.

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Saleem, A., Ali, Z. & Naseem, S. Optimization process for de-novo organogenesis and regeneration of transgenic papaya plants using leaf tissue as explants. Plant Cell Tiss Organ Cult 157, 49 (2024). https://doi.org/10.1007/s11240-024-02728-2

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