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Type :thesis
Subject :SB Plant culture
Main Author :Muhammad Al-Amin Mazlan
Title :Plant tissue culture of pomegranate, Punica granatum L.
Place of Production :Tanjong Malim
Publisher :Fakulti Teknikal dan Vokasional
Year of Publication :2018
Corporate Name :Universiti Pendidikan Sultan Idris
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Abstract : Universiti Pendidikan Sultan Idris
The purpose of this study was to examine the propagation ability of Punica granatum L. through tissue culture system. In addition, callus induction was also carried out in the study  to observe the rate of cell differentiation on explants. The experimental design that was used in  this research is Completely Randomized Design (CRD). The complete regeneration of the Punica  granatum L. plant and callus production was successfully produced through the tissue culture  technique. Explants used in this study were leaves, stem  and  roots  from  8-week-old  aseptic   seedlings.  All  cultures  were  stored  at  a temperature of 25 ± 1ºC and light 16 hours of light,  8 hours dark. Plant acclimatization was done on three different medium, garden soil, coco peat and  vermiculite. All data was  recorded  and  analyzed  using  ANOVA.  Leaf  explants  were  found  to   be  very responsive   and   Murashige   and   Skoog   (MS)   medium   added   with   2.0   mg/l  Benzylaminoupurine  (BAP)  +  1.5  mg/l  Napthalene  Acetic  Acid  (NAA)  has  been identified as  the optimum medium for shoot regeneration.  Leaf  explant managed to produce  the  number  of   shoots  in  vitro  with  1.3333±0.3228  per  explant  at  week  8. Whereas from stem explant, MS  media added with 0.5 mg/l BAP and 2.0 mg/l NAA gave  the  highest  shoot  development  with  1.0000   ±  0.2837  shoots  per  explant. Combination  of  NAA  and  BAP,  at  1.5  mg/l  respectively  was   used  for  complete regeneration  of  Punica  granatum  L.  in  vitro.  In  addition,  artificial   seeds  of  Punica granatum were also produced. As a conclusion, this method was proven to be  successful in producing new generation of Punica granatum L. and its features are preserved. This  proves that plant tissue culture technology could be an alternative solution to achieve high quality of Punica granatum L, therefore could increase the crop production.  


Abd El-Gawad, N.A., N.S. Zaied and M.A. Saleh, 2010. A comparative study on different

carbon source concentrations and gelling agent on in vitro proliferation of pineapple (Ananas 

comosus). The Journal of Nat we and Science, 8(2): 1-5


Acheapong   E.   (1982).   Multiplication   and   Conservation   of   Cocoyam   (Xarthosoma 

sajiltifoljum)  germplasm  by  application  of  tissue  culture  methods  Ph.D  Thesis. University 

of Burmingham, UK


Akoroda M. O. (1990). Ethno botany of Telfaria Occidtendalic Among Igbos of Nigeria.

Econ. Bot. 44 (1): 29-39.


Aladele S. E. and Kuta D. (2008). Environmental and genotypic effects on the rowth rate of in vitro 

cassava plantlet (Manihot esceulenta) African Journal of Biotechnology Vol. 7 (4), pp. 381-385.


Al-Khateeb,  A.A.,  (2008).  Regulation  of  in  vitro  bud  formation  of  date  palm  (Phoenix 

dactylifera L.) cv. Khanezi by different carbon sources. Bioresource Technology, 99(14): 6550-6555.


Al-Khateeb,  A.A.,  (2008).  Regulation  of  in  vitro  bud  formation  of  date  palm  (Phoenix 

dactylifera L.) cv. Khanezi by different carbon sources. Bioresource Technology, 99(14): 6550-6555.


Al-Khayri, (2003). In vitro germination of somatic embryos in date palm : Effect of auxin 

concentration and strength of MS salts. Current Science, 84(3): 680-683.


Amin  MN,  Islam  MN,  Azad  MAK  (1999).  Regeneration  of  plantlets  in  vitro  from  the 

seedling explants of pomegranate (Punica granatum L.). Plant Tissue Cult. 9(1): 53-61.


Andreu,  P.,  J.A.  Marín  (2005).  In  vitro  culture  establishment  and  multiplication  of  the 

Prunus  rootstock  Adesoto  101’  (P.  insititia  L.)  as  affected  by  the  type  of propagation 

of the donor plant and by the culture medium composition. Scientia Horticulturae, 106(2): 258-267.


Ara H, Jaiswal Uma, Jaiswal VS ( 2000 ) Synthetic seed: Prospects and limitations, Current Science 

78, 1438 – 1444


Attree, S.M.; Pomeroy, M.K.; Fowke, L.C. ( 1994 ) Production of vigoous, dessication tolerant white 

spruce (Picea-glauca (moench) voss) synthetic seeds in a bioreactor.

Plant Cell Rep., 13, 601–606.


Baaya, M.H.M. (2002). Studies on propagation of some fruit species by using tissue culture

techniques.  M.  Sci.  Thesis,  Hort.  Dept.,  Fac.  of  Agric.,  Moshtohor,  Zagazig University.


Babak V.K, Ahmad E. And Masoud T. ( 2013 ) In Vitro Propagation of Pomegranate ( Punica  granatum  

)  Cv.  ‘Males  Yazdi’.  Deparments  of  Horticulture  Science, Faculty of Agriculture, University 

of Bu-Ali Sina, Hamedan, Iran.


Babbar, S.B. and S.C. Gupta (1986) . Effect of Carbon Source on Datura metel Microspore 

Embryogenesis   and   the   Growth   of   Callus   Raised   from   Microspore-derived Embryos. 

Biochemie und Physiologie der Pflanzen, 181(5): 331-338.


Bhatia  P,  Ashwath  N  (2005).  Effect  of  medium  pH  on  shoot  regeneration  from  the 

cotyledonary explants of Tomato. Biotechnol., 4: 7-10.


Bridgen, Mark P. (1994). "A Review of Plant Embryo Culture". Hortscience 29: 1243– 1246.


Brown DCW, Leung DWM, Thorpe TA (1979). Osmotic requirement for shoot formation in tobacco callus. 

Physiol. Planta, 46: 36-41.


Chang, P.Y, Fujita, S., Kohno, K., Ashrafuzzaman, M.D., Nakamura, N. And Hayashi, N. (2000) 

Purification and characterization of polyphenol oxidase from banana ( Musa sapientum L.) 

pulp.Journal of Agricultural and Food Chemistry 48: 2732-2735.


Christian  Raquin  (1983).  Utilization  of  Different  Sugars  as  Carbon  Source  for  in  vitro 

Anther Culture of Petunia. Zeitschrift für Pflanzenphysiologie, 111(5): 453-457.


Conger, B. V. (1980). Cloning Agricultural Plants via In-vitro Techniques, CRC Press Inc, Boca 

Raton, Florida, pp; 1-50


Dalmacio  I.  F.  (1992).  Biotechnology  for  sustainable  Agriculture  National  Institute  of 

Biotechnology and Applied Microbiology, University of the Philippines.Dalziel, J.

M. 1937. The useful plants of West Tropical Africa. Whitefriars Press, London, 612 pp.


Dalziel J.M. (1937) The useful plants of West Tropical Africa. Whitefriars press,London.



Fagbemi   T.N.F.,   Eleyinmi   A.F.,   Atum   H.   N.,   Akpambang   O.   (2005)   Nutritional 

composition   of   fermented   fluted   pumpkin   (Telfairiaoccidentalis)   seeds   for 

productionof  “ogiriugu”.  IFT  Annual  Meeting,  July  15-20  –  New  Orleans,



Frusciante, L.,   Barone, A.,Carputo,D. Ercolano, M.R., Della Rocca, F., & Esposito, S.

(2010).  Evaluation  and  use  of  Plant  Biodiversity  for  Food  and  Pharmaceutical. Fitoterpia. 

71 : 66 – S72.


Fuentes, S.R.L., M.B.P. Calheiros, J. Manetti-Filho and L.G.E. Vieira (2000). The effect of silver 

nitrate and different carbohydrate sources on somatic embryogenesis in Coffea canephora. Plant Cell 

Tiss. Org. Cult., 60: 5-13.


Gamborg,  O.L.,  T.  Murshige,  T.A.  Thorpe  and  I.K.  Vasil  (1976).  Plant  tissue  culture 

media. In vitro, 12(7): 473- 478.


Gautheret, R. J. (1934). Culture du tissus cambial. Comptes Rendus Hebdomadaires des Séances de 

l'Académie des Sciences, 198, 2195–2196.


Gautheret, R. J. (1935). Recherches sur la culture des tissus végétaux. Ph.D. Thesis, Paris


Gautheret,  R.  J.  (1939).  Sur  la  possibilité  de  réaliser  la  culture  indéfinie  des  

tissus  de tubercules de carotte. Comptes Rendus Hebdomadaires des Séances de l'Académie des 

Sciences, 208, 118–120.


Gbile Z.O.(1998). Collection, Conservation and Utilization of medicinal plants. In Robert PA,  &  

Janice  EA  (eds.).  Conservation  of  Plant  Genes  III:  Conservation  and Utilization of African 

Plants. Missouri Botanical Gardens Press. pp. 163-174.


Gibson IS (2000). Plant sugar-response pathways. Part of a complex regulatory web. Plant Physiol., 

124: 1532-1539.


Gurel S, Gulsen Y (1998). The effects of different sucrose, agar and pH levels on in vitro shoot 

production of Almond (Amygdalus communis L.). Tr. J. Bot., 22: 363-373.


Haberlandt, G. (1902) Kulturversuche mit isolierten Pflanzenzellen. Sitzungsber.  Akad.

Wiss. Wien. Math.-Naturwiss. Kl., Abt. J. 111, 69–92.


Hassan, S., All. M. (2012). Studies on improving flowering and fruiting of lecont e pears.

Ph.D. Hort., Dept., Fac. of Cairo Univ.


IUCN (2006). IUCN Red List of Threatened Species. IUCN, Gland, Switzerland.


IUCN (2006). IUCN Red List of Threatened Species. IUCN, Gland, Switzerland.


Jain, N., M.W. Bairu, W.A. Stirk, J. Van Staden (2009). The effect of medium, carbon source   and   

explant   on   regeneration   and   control   of   shoot-tip   necrosis   in Harpagophytum 

procumbens. South African Journal of Botany, 75(1): 117-121.


M., K. Imizu and T. Hirano (2001). Double-phase in vitro culture using sorbitol increases shoot   

proliferation   and   reduces   hyperhydricity   in   Japanese   pear.   Scientia

Horticulturae, 89(3,4): 207-215.


Kumar HGA Murthy HN and Paek KY (2003 ). Embryogenesis and plant regeneration

from anther cultures of Cucumis sativus L. Scientia Hort , 98: 213-222


Levin, R., Stav, R., Alper, Y. and Watad, A. A. (1998). A technique for repeated nonaxenic 

subculture of plant tissues in a bioreactor on liquid medium containing sucrose. Plant Tissue 

Culture Biotechnology. (3):41–45.


Lloyd, G. and B.H. McCown 1(981). Commerciallyfeasiblemicropropagation of Mountain Laurel, Kalmia 

latifolia, by shoot tip culture. Proc. Int. Plant Prop. Soc., 30: 421- 427.


Lyam  P.T.,Musa  M.  L.,  Jamaleddine,  Z.O.,  Okere,  A.U.,  Carlos  A.  and  Odofin,  W.T. 

(2012). The potentials of Temporary immersion bioreactor TIBS in meeting crop production demand in 

Nigeria. Journal of biology and life science ISSN 2157 -6076 Vol. 3:1.


Mantel  A.H.  and  Smith  H.  Culture  factors  that  influence  secondary  metabo  lism 

accumulation in plat cell and tissue culture. In: Mantell. A., Smith H(Eds). Plant biotechnology, 

Cambridge University Press, Cambridge, 1983; 75-110.


Mantel    S.H.,    Mathews    J.A.    and    McKee    R.A.    (1985).    Principles    of    plant 

Biotechnology,Blackwell Scientific Publications, London.


Miyajuma, D. (2006). "Ovules that failed to form seeds in zinnia (Zinnia violacec Cav)".

Sci Hortic 107 (2): 176–182. doi:10.1016/j.scienta.2005.06.014.


Mukherjee, P., N. Husain, S.C. Misra and V.S. Rao (2010). In vitro propagation of a grape 

rootstock, deGrasset (Vitis champinii Planch.): Effects of medium compositions and plant growth 

regulators. Scientia Horticulturae, 126(1): 13-19.


Murashige, T. and F. Skoog (1962). A revised medium for rapid growth and bioassays with tobacco 

tissue cultures. Physiol. Plant, 15: 473-497.


Murashige, T. (1973). Nutrition of plant cells and organs in vitro. In Vitro, 9: 81-85.


Murkute, A., Patil, S., Patil, B.N., Mayakumari, M.S., (2002) Micropropagation in pomegranate, 

callus induction and differentiation. South Indian Hort. 50:49–55.


Musa  M.  L.,  Lyam  P.T.,  Jamaleddine,  Z.O.,  Okere,  A.U.,  Carlos  A.  and  Odofin,  W.T. 

(2012). The potentials of Temporary immersion bioreactor TIBS in meeting crop production demand in 

Nigeria. Journal of biology and life science ISSN 2157 -6076 Vol. 3:1.


Naik, S., Pattnaik, S., Chand, P., (2000) High frequency auxiliary shoot proliferation and plant 

regeneration from cotyledonary nodes of pomegranate. Sci. Hort. 79:175- 83.


Nehanjali P., Kamlesh K. Ajay Kumar T. (2012 ) In Vitro Organogenesis from Cotyledon

Derived  Callus  Cultures  of Punica  granatum L.  cv.  Kandhari  Kabuli.  National Academy of 

Science, India.


Nehanjali  P.,  Kamlesh  K.  Ajay  Kumar  T.  (2013  )  Direct  Organogenesis  in Punica granatum 

L. cv. Kandhari Kabuli from Hypocotyl Explants. National Academy of Science, India


Nowak B, Milczzynski K, Hudy L (2004). Sugar uptake and utilization during adventitious bud  

differentiation  on  in  vitro  leaf  explants  of  Wegierka  Zwykla  plum  (Prunus domestica). 

Plant Cell, Tissue Organ Cult., 76: 255-260.


Okere, A. U. and Adegeye, A. (2011). In vitro propagation of an endangered medicinal timber species 

Khaya grandifoliola C. Dc. African Journal of Biotechnology Vol. 10(17), pp. 3335-3339.


Omura, M., Matsuta, N., Moriguchi, T., Kuzaki, I., Sanada, T., (1987) Establishment of tissue 

culture methods in dwarf pomegranate (Punica granatum L. var. ?Nana?) and application for the 

induction of variants. Bull. Fruit Tree Res. 14:17-44.


Pajimi O. and Fashola T. R. (2010). In vitro micro-propagation of Plukenetia  conophora Mull.Arg. 

Journal of South Pacific Agriculture. 14(1&2):1-5


Petersen, K.K., J. Hansen, A. Bouvet, R. Galvayrac, M. Paques (2001). Significance of different  

carbon sources  and sterilization methods on callus induction  and plant regeneration of Miscanthus 

X ogiformis Honda Giganteus. Plant Cell Tiss. Org. Cult., 58: 189-197.


Redenbaugh,  K.;  Paasch,  B.D.;  Nichol,  J.W.;  Kossler,  M.E.;  Viss,  P.R.;Walker,  K.A.  ( 

1986 ) Somatic seeds-encapsulation of asexual plant embryos. Nat. Biotechnol, 4, 797–801. 



Redenbaugh, K.; Fujii, J.A.; Slade, D ( 1988 ). Encapsulated plant embryos. In Advances in 

Biotechnological Processes; Mizrahi, A., Ed.; Alan R. Liss Inc.: New York, NY, USA; pp. 225–248.


Redenbaugh, K.; Fujii, J.A.; Slade, D.( 1993 ) Hydrated coating for synthetic seeds. In Synseeds: 

Application of the Synthetic Seeds to Crop Improvement; CRC Press: Boca Raton, FL, USA, ; pp. 



Raj D. And Kamlesh K. ( 2010 ). In Vitro Regeneration of Punica granatum plants from different   

Juvenile   Explants.   Department   of   Biotechnology,   University   of Horticulture and 

Forestry, India.


Reed, Sandra (2005). Robert N. Trigiano and Dennis J. Gray, ed. Plant Development and

Biotechnology. CRC Press. pp. 235–239. ISBN 0-8493-1614-6.


Reinert, J. and P.R. White (1956). The cultivation in vitro of tumor tissues and normal

tissues of Picea glauca. Physiol. Plant., 9: 117-189.


Reynolds T.L. Am. J. Bot. 1982; 73: 914-918


Risser, P.G. and P.R. White (1964). Nutritional requirements of spruce tumor cellsin vitro.

Physiol. Plant. 17: 620-635.


Sakano  K  (1990).  Proton/phosphate  stoichiometry in  uptake  of  inorganic  phosphate  by 

cultured cells of Catharanthus roseus (L.) G. Don. Plant Physiol., 93: 479-483.


Shah  MI,  Jabeen  M,  Ilahi  I.  2003.  In  v?tro  callus  induction,  its  proliferation  and 

regeneration in seed explants of wheat (Triticum aestivum L.) variety Lu-26S. Plant Journal of 

Botany 35(2), 209- 217.


Sanusi I. S., Odofin W. T,, Aladele S. E, , Olayode M. N., Gamra E. O., Fajimi O. (2008) In  vitro  

culture  of  Telferia  occidentalis  under  different  cytokinins  and  auxin combination. African 

journal of biotechnology. 7 (14):2407-2408


Singh N.V., Singh S.K., Patel V.B. 2007. In vitro axillary shoot proliferation and clonal 

propagation of ‘G-137’ pomegranate (Punica granatum). INDIANJ. AGRIC. SCI. 77(8): 505-508.


Stavarek  SJ,  Croughan  TP,  Rains  DW  (1980).  Regeneration  of  plants  from  long-term 

cultures of alfalfa cells. Plant Sci. Lett., 19: 253-261.


Steward, F.C., Mapes, M.O., and Mears, K. (1958). Growth and organized development of cultured  

cells.  II.  Organization  in  cultures  grown  from  freely  suspended  cells, American Journal of 

Botany. 45:705-708


Tange,  H.,  Y.  Low  and  C.  Lui  (2008).  Plant  regenerate  from  in  vitro  leaves  of  four 

commercial pyrus species. Plant Soil Environs, 54(4): 140-148.


Teisson, C. and Alvard, D. (1995). A new concept of plant in vitro cultivation in liquid medium: 

Temporary Immersion. In: Current Issues in Plant Molecular and Cellular Biology.Terzi,  M.,  Cella, 

 R.  and  Falavigna,  A.  (Editors).  Kluwer  Academic Publisher pp


Thomas, D.T & Philip B. (2005). Thidiazuron-induced high frequency shoot organogenesis from   

leaf-derived   callus   of   a   medicinal   climber,Tylophora  indica   (Burm.F) Merrill.In Vitro 

Cell .Dev.Biol- Plant. 41:124-128.


Vespasiano, B.d.N. and W.C. Otoni (2003). Carbon sources and their osmotic potential in

plant tissue culture: does it matter?. Scientia Horticulturae, 97(3-4): 193-202.


White, P.R. (1963). The Cultivation of Animal and Plant Cells. 2nd ed. Ronald Press,

New York.


Yang ZH, Ludders P (1993).Organogenesis of Punica granatum L. var.Nana.

Angewandte Botanik, 67(5-6): 151-156.


Zhu LW, Zhang SM, Song FS, Gong XM, Fang WJ, Sun J, Li SW (2003).

Regeneration system of pomegranate by in vitro culture. Acta. Hort. Sinica,

430(2): 207-208.










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