Regeneration of Carica papaya “Rathna” Variety through Somatic Embryogenesis from Immature Zygotic Embryos
A. D. R. P. Aluthge *
Plant Virus Indexing Center, Gabadawaththa, Homagama, Sri Lanka.
S. M. S. Nagahawaththa
Plant Virus Indexing Center, Gabadawaththa, Homagama, Sri Lanka.
B. M. V. S. Basnayake
Plant Virus Indexing Center, Gabadawaththa, Homagama, Sri Lanka.
C. Hettiarachchi
Department of Chemistry, University of Colombo, Colombo 03, Sri Lanka.
D. M. J. B. Senanayake
Rice Research and Development Institute, Batahlegoda, Ibbagamuw, Sri Lanka.
*Author to whom correspondence should be addressed.
Abstract
Papaya (Carica papaya L.), in family; caricaceae is an important fruit crop all over the world. Papaya Ring Spot Virus (PRSV) causes one of the most destructive diseases of papaya, lowering its yield. Genetic engineering has been proven to be a useful tool to introduce genes to plants where conventional breeding methods have failed. Most of the successful plant genetic engineering applications are involved with transforming of genes using Agrobacterium mediated transformation where regeneration of plants using tissue culture-based techniques. Hence, the present study was conducted to develop tissue culture-based C. papaya through somatic embryogenesis from zygotic embryos of papaya for subsequent genetic engineering to obtain PRSV resistance. Immature fruits were randomly selected from self-pollinated plants in the field. Zygotic embryos were directly excised from sterilized seeds and successfully initiated on half strength Murashige and skoog (MS) medium. The medium supplemented with 400 mg/L glutamine and different concentrations of 2, 4 Di- chlorophenoxyaciatic acid (2,4-D) (3 ml/L to 12 ml/L) and different amounts of vitamins (5 ml/L to 10 ml/L) were tested as calli induction medium. The medium supplemented with 10 mg/L 2,4-D and 5 ml/L vitamin consisted of significantly higher number of calli (60%). The inducted pre-embryogenic calli were matured on hormone free medium. The matured somatic embryos were introduced into the germination medium containing full strength MS salts supplemented with different concentrations of kinetin (1 ml/L to 6 ml/L). The medium supplemented with 2 mg/L of kinetin recorded the significantly highest number (75%) of plants compared to the other media within the stipulated time duration. Germinated embryos were introduced into full strength MS media without hormones. The fully grown plants with high density root system were acclimatized gradually for a period of three months.
Keywords: Papaya Ring Spot Virus, Zygotic embryo, Somatic embryogenesis, 2, 4 Di- chlorophenoxyaciatic acid, Kinetin
How to Cite
Downloads
References
Evans EA, Ballen FH. Economic analysis coordinator II, UF/IFAS Tropical Research and Education Center, Homestead; 2012.
FAO. Production year. Organization of United Nations for Agriculture and the food (FAO) Rome Italy. 2011;50:187-190.
Fitch M, Manshardt R. Somatic embryogenesis and plant regeneration from immature zygotic embryos of papaya (Carica papaya L.). Plant Cell Reports, Berlin. 1990;9:320-324.
Rezukdas NN, Khuspe SS, Rawal SK. Multiple shoot regeneration from immature embryo explants of papaya. Biologia-plantarum, Dordrecht. 2003;47(3):327- 331.
Razali RM, Drew R. A refined protocol for embryogenesis to transfer PRSV-P resistance genes from Vasconcella pubescens to Carica papaya. Acta Horticulturae, The Hague. 2014;1022:47-53.
Bruijne E, Langhe E, De, Van Rijck R. Actions of hormones and embryoid formation in callus cultures of Carica papaya. International Symposium over Fytofarmacie en Fytiatrie, Gent. 1978;26: 637-645.
Yie ST, Liaw SI. Plant regeneration from shoot tips and callus of papaya. In vitro, Columbia. 1977;13:564-568.
Arora IK, Singh RN. Growth hormones and in vitro callus formation of papaya. Scientiahorticulturae, New York. 1978;8: 357-361.
Anandan R, Sudhakara D, Balasubramaniana O, Gutiérrez- Morab A. In vitro somatic embryogenesis from suspension cultures of Carica papaya L. Scientiahorticulturae, New York. 2012;136: 43-49.
Chen MH, Wang PJ, Maeda E. Somatic embryogenesis and plant regeneration in Carica papaya L. Tissue culture derived from root explants. Plant Cell Reports, Berlin. 1987;11:348-351.
Clarindo WR, Carvalho CR, De, Araujo FS, Abreu IS, De, Otoni WC. Recovering polyploid papaya in vitro regenerants as screened by flow cytometry. Plant Cell, Tissue and Organ Culture, Dordrecht. 2008;92:207-214.
Litz RE, Conover RA. Somatic embryogenesis in cell cultures of Carica stipulata. Hort Science. 1980;15:733-735.
Tsay HS, Sue CY. Anther culture of papaya (Carica papaya L.). Plant Cell Reports, Berlin. 1985;4:28-30.
Rimberia FK, Sunagawa H, Urasaki N, Ishimine Y, Adaniya S. Embryo induction via anther culture in papaya and sex analysis of the derived plantlets. Scientia-horticulturae. New York. 2005;103:199-208.
Cabrera-Ponce JL, Vegas-Garcia, Herrera-Estrella L. Regeneration of transgenic papaya plants via somatic embryogenesis induced by Agrobacterium rhizogenes. In Vitro Cell & Developmental Biology – Plant, Cambridge. 1996;32:86–90.
Homhuan S, Kijwijan B, Wangsomnuk P, Bodhipadma K, Leungc DWM. Variation of plants derived from indirect somatic embryogenesis in cotyledon explants of papaya. Sciencia Asia, Banbgok. 2008;34: 347-352.
Teixeira Da Silva JA. Callus induction in papaya (Carica papaya L.), and synseed production for low temperature storage and cryopreservation. Folia Horticulture, Krakow. 2014b;26(2):155-162.
Vilasini P, Latipah Z, Salasiah A. Induction of somatic embryogenesis and plant regeneration from immature embryos of Eksotika papaya (Carica papaya). Journal of Tropical and Food Sciences, Kuala Lumpur. 2000;28(2):121-126.
Abreu IS, Carvalho CR, Clarindo WR. Massal induction of Carica papaya L. ‘Golden’ somatic embryos and somaclone screening by flow cytometry and cytogenetic analysis. Cytologia, Tokyo. 2014;79(4):475–484.
White PR. The cultivation of animal and plant cells, 2nd edn. Ronald Press, New York; 1963.
Gamborg OL, Miller RA, Ojima K. Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res. 1968;50: 151-158.
Murashige T, Skoog F. A revised medium for rapid growth and bio-assays with tobacco tissue cultures. Physiol Planta. 1962;15:473–497.
Evans DA, Sharp WR, Flick CE. Growth and behaviour of cell culture: embryogenesis and organogenesis. Newyork, Academic Press; 1981.
George EF. Plant propagation by tissue culture part1 the technology. England, Exegetics Limited; 1993.
Bajaj YPS. Somatic embryogenesis and its applications for crop improvement. In: Biotechnology in Agriculture and Forestry 30, Somatic Embryogenesis and Synthetic Seed I. (Y. P. S. Bajaj-Ed.). Verlag Berlin Heidelberg- Germany, Springger-Verlag. 1995;30:105-125.
Junior S, Níckel O, Gonsalves D. Development of transgenic papayas expressing the coat protein gene from a Brazilian isolate of Papaya ringspot virus (PRSV) = Desenvolvimento de mamoeiros transgênicos resistentes a vírus expressando o gene da capa protéica de um isolado brasileiro de Papaya ringspot virus. Fitopatologia Brasileira. 2005;30: 114-120.
Cai WQ, Gonsalves C, Tennant P, Fermin G, Souza M, Sarindu N, Jan FJ, Zhu HY, Gonsalves D. A protocol for efficient transformation and regeneration of Carica papaya L. In Vitro Cellular & Developmental Biology- Plant, Cambridge. 1999;35(1):61-69.
Fitch M, Manshardt R. High frequency somatic embryogenesis and plant regeneration from papaya hypocotyls callus. Plant Cell, Tissue and Organ Culture, Dordrecht. 1993;32:205-212.
Mallikarjun YK. Molecular characterization, cloning of coat protein gene, epidemiology and management of Papaya ringspot virus, Ph.D. Thesis, University of Agricultural Sciences, Dharwad, India; 2003.
Farzana ARF, Palkadapala PGVN, Meddegoda KMMN, Samarajeewa PK, Eeswara JP. Somatic embryogenesis in papaya (Carica papaya L. Cv. Rathna). J. Natn. Sci. Foundation Sri Lanka. 2008; 36:41-50.
Chang WC, Hsing WI. In vitro flowering of embryoids derived from mature root callus of ginseng (Panax ginseng). Nature 1983;284:341–342.
Ammirato PV. Embryogenesis. In: Handbook of Plant Cell Culture. Vol. 1. D.A. Evans, W. R. Sharp. P. V. Ammirato, and Y. Yamada (ed.). Macmillan, New York, in press; 1983.
Kavathekar A, Ganapathi P, Johri B. In vitro response of embryoids of Eschscholzia. Biologia Plantarum. 1978; 20:98-106.
Fujimura T, Komamine A. Mode of action of 2,4-D and zeatin on somatic embryogenesis in a carrot cell suspension culture. Zeitschrift für Pflanzenphysiologie. 1980;99:1-8.
Ramesh M, Murugiah V, Gupta AK. Efficient in vitro plant regeneration via leaf base segments of Indica rice (Oryza sativa L.) Indian Journal of Experimental Biology. 2009;47(1):68-74.