Callus Induction and Somatic Embryogenesis
in Nypa fruticans Wurmb Zygotic Embryo
Georgianna Kae R. Oguis1, Cyrose Suzie C. Silvosa1 and Gilda C. Rivero1,2*
1College of Science and Mathematics
University of the Philippines Mindanao, Davao City, Davao del Sur
1,2Institute of Biology, College of Science,
University of the Philippines Diliman, Quezon City
Nypa fruticans has been reported to be a potential source of ethanol. To date, there is no available protocol on tissue culture suitable for N. fruticans to produce superior and uniform planting materials for future wide-scale production. This paper investigated the influence on callus induction of various concentrations of 2,4-D and activated charcoal as well as the effects of three orientations of the explant on the media. Sixty-four percent of the explants cultured in full-strength MS media, supplemented with various combinations of different concentrations of 2,4-D and activated charcoal, produced protocorm-like structures in cultures with lower concentrations of 2,4-D combined with high concentrations of activated charcoal. Fifty-two percent of protocorm-like structures in T18 cultures significantly (p<0.05) developed white, compact, nodular calli and soft, white, friable calli eight weeks after inoculation. Twenty percent of the explants in the T18 cultures developed somatic embryos sixteen weeks after inoculation. Explants in cultures with higher 2, 4-D concentrations combined with activated charcoal significantly (p<0.05) formed brown, hard, compact calli. Treatments devoid of activated charcoal did not induce callus and did not produce somatic embryos. The absence of activated charcoal in the MS media also led to browning of explants within the eighth week of incubation. The orientation of explants on the culture media resulted in callus initiation only in the T18 cultures, although protocorm-like structures were observed in all cultures regardless of the explant orientation.
Nypa fruticans Wurmb, the nipa palm, remains uncultivated and grows mainly as natural stands near estuaries and rivers (Rasco 2010; Tsuji et al. 2011) throughout the Philippines. Its large pinnate leaves are traditionally harvested for use as roof thatches and stabilizers of coastal areas, while its sap is regularly harvested for small-scale vinegar and vodka production (Rasco 2010; Rasco et al. 2012). N. fruticans sap contains as much as 60% sugar and its annual yield was reported to be much more than those from sweet potatoes, coconut, tapioca, and sugarcane (Hamilton and Murphy 1988). The depleting petroleum resources and increasing demand for fuel brought on great interest . . . . . read more
ABOHATEM M, ZOUINE J, HADRAMI IE. 2011. Low concentrations of BAP and high rate of subcultures improve the establishment and multiplication of somatic embryos in date palm suspension cultures by limiting oxidative browning associated with high levels of total phenols and peroxidase activities. Sci Hortic 130: 344-348.
ADKINS SW, SAMOSIR Y, NIKMATULLAH A,OGLE HJ. 2005. Coconut (Cocos nucifera) in vitro ecology: Modifications of headspace and medium additives can optimize somatic embryogenesis. Acta Hort (ISHS) 692: 21-32.
BENSON EE, ROUBELAKIS-ANGELAKIS KA. 1993. Oxidative stress in recalcitrant tissue cultures of grapevine. Free Radic Biol Med 16(3): 355-362.
EBRAHIMIE E, NAGHAVI MR, HOSSEINZADEH A, BEHAMTA MR, DEHCHESHMEH MM, SARRAFI A, SPANGENBERG G. 2007. Induction and comparison of different in vitro morphogenesis pathways using embryo of cumin (Cuminum cyminum L.) as a model material. Plant Cell Tiss Organ Cult 90: 293-311.
HAMILTON L, MURPHY D. 1998. Use and management of Nipa palm (Nypa fruticans, Arecaceae): A review. Economic Bot 42: 206-213.
KARUNARATNE S, PERIYAPPERUMA K.1989. Culture of immature somatic embryos of coconut, Cocos nucifera L., callus proliferation and somatic embryogenesis. Plant Sci 62: 247‒253.
LOW ET, ALIAS H, BOON SH, SHARIFF EM, TAN CYA, OOI LC, CHEAH SC, RAHA AR, WAN KL, SINGH R. 2008. Oil palm (Elaeis guineensis Jacq.) tissue culture ESTs: Identifying genes associated with callogenesis and embryogenesis. BMC Plant Biol 8: 1‒19.
MAGNAVAL C, NOIROT M, VERDEIL JL, BLATTES A, HUET C, GROSDEMANGE F, BEULE T, BUFFARD-MOREL J. 1997. Specific nutritional requirements of coconut calli (Cocos nucifera L.) during somatic embryogenesis induction. J Plant Physiol 150: 719‒728.
MOURA EF, MOTOIKE SY, VENTRELLA MC, DE SA JUNIOR AQ, CARVALHO M. 2009. Somatic embryogenesis in macaw palm (Acrocomia aculeata) from zygotic embryos. Sci Hortic 119 (4): 447‒454.
MURASHIGE T, SKOOG F. 1962. A revised medium for rapid growth and bioassays with tobacco cultures. Physiol Plant 15: 473‒497.
POUDYAL BK, ZHANG D, LIU J, SHI Q. 2008. Studies on browning problem and phenols content on shoots of yali, Aikansui and Abbe Fetel pears for in vitro culture. Front Agric China 2(3): 321‒330.
RASCO ET JR. 2010. Biology of nipa palm. (Nypa fruticans Wurmb., Arecaceae) and its potential for alcohol production. Asia Life Sciences 19(2): 373-388.
RASCO ET JR, RAGAS RE, JUNIO RG. 2012. Morphological and sap yield variation in Nipa (Nypa fruticans Wurmb.). Asia Life Sciences 21(1): 123-132
SAMOSIR YMS, GODWIN ID, ADKINS SW. 1998. An improved protocol for somatic embryogenesis in coconut (Cocos nucifera). Acta Hort (ISHS) 467‒476.
SGHAIER B, BAHLOUL M, BOUZID RG, DRIRA N. 2008. Development of zygotic and somatic embryos of Phoenix dactylifera L. cv. Deglet Nour: Comparative study. Sci Hortic 116(2): 169-175.
TEIXEIRA J, SONDAHL M, KIRBY E. 1994. Somatic embryogenesis from immature inflorescence of oil palm. Plant Cell Rep 13: 247—250.
TSUJI K, GHAZALLI NF, ARIFFIN Z, NORDIN S, KHAIDIZAR MI, DULLOO ME, SEBASTIAN LS. 2011. Biological and ethnobotanical characteristics of nipa palm (Nypa fructicans Wurmb.): A review. Sains Malaysiana 40:1407-1412.