Effects of plant growth regulators, carbon sources and pH values on callus induction in Aquilaria malaccensis leaf explants and characteristics of the resultant calli

Abstract

The endangered tropical tree, Aquilaria malaccensis, produces agarwood for use in fragrance and medicines. Efforts are currently underway to produce valuable agarwood compoundsn tissue culture. The purpose of this study was to develop an optimal growth medium, specifically, the best hormone combination for callus suspension culture. Using nursery-grown A. malaccensis, sterilized leaf explants were first incubated on basic Murashige and Skoog (MS) gel medium containing 15g/L sucrose and at pH 5.7. Different auxin types including 1-naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), and indole-3-butyric acid (IBA), were tested at various concentrations (0.55, 1.1 and 1.65 μM) using the basic medium. Leaf explants were incubated for 30 days in the dark. Callus induced by 1.1 μM NAA had the highest biomass dry weight (DW) of 17.3 mg; however the callus was of a compact type. This auxin concentration was then combined with either 6-benzylaminopurine (BAP) or kinetin at 0.55, 1.1, 2.2 or 3.3 μM to induce growth of friable callus. The 1.1μM NAA + 2.2μM BAP combination produced friable callus with the highest biomass (93.3mg DW). When testing the different carbon sources and pHs, sucrose at 15g/L and pH at 5.7 yielded highest biomasses at 87.7mg and 83mg DW, respectively. Microscopic observations revealed the arrangement of the friable cells as loosely packed with relatively large cells, while for the compact callus, the cells were small and densely packed. We concluded that MS medium containing 15 g/L sucrose, 1.1 μM NAA + 2.2 μM BAP hormone combination, and a pH of 5.7 was highly effective for inducing friable callus from leaf explants of A. malaccensis for the purpose of establishing cell suspension culture.

Keywords

Agarwood / endangered tree / in vitro / plant growth regulators / Thymelaeaceae

References

[1] Akaneme FI, Eneobong EE. Tissue culture in Pinus caribaea Mor. var. Hondurensis barr. and golf. II: Effects of two auxins and two cytokinins on callus growth habits and subsequent organogenesis. African Journal of Biotechnology, 2008, 7(6): 757-765.

[2] Bhatia P, Ashwath N. Effect of medium pH on the shoot regeneration from the cotyledonary explant of tomatoes. Biotechnology, 2005, 4(1): 7-10.

[3] Bonaventure G, Baldwin IT. New insights into the early biochemical activation of jasmonic acid biosynthesis in leaves. Plant Signal Behavior, 2010, 5: 287-289.

[4] Che P, Gingerich DJ, Lall S, Howell SH. Global and hormone induced gene expression changes during shoot development in Arabidopsis. Plant Cell Reproduction, 2002, 14: 2771-2785.

[5] Collin HA, Edwards S. Plant cell culture. 1998, Oxford, UK: Bios Scientific Publisher

[6] Dash M, Patra JK, Panda PP. Phytochemical and antimicrobial screening of extracts of Aquilaria agallocha Roxb. African Journal of Biotechnology, 2008, 7(20): 3531-353.

[7] Declerk V, Korban SS. Influence of growth regulators and carbon sources on callus induction, growth and morphogenesis from leaf tissues of peach (Prunus persica L. Batsch). Journal of Horticultural Science, 1996, 71(1): 49-55.

[8] Gracia JL, Troncoso J, Sarmiento R, Troncoso A. Influence of carbon source and concentration on the in vitro development of olive zygotic embryos and explants raised from them. Plant Cell Tissue Organ Culture, 2002, 69: 95-100.

[9] IUCN International Union for Conservation of Nature and Natural Resources, 2011

[10] Karam NS, Jawad FM, Arikat NA, Shibli RA. Growth and rosmarinic acid accumulation in callus, cell suspension and root cultures of wild Salvia fructicosa. Plant Cell, Tissue and Organ Culture, 2003, 73: 117-121.

[11] Li JR, Liu MX, Chen HR, Wu ZB, Wang JJ. Callus initiation and subculture of Taxus chinensis. Journal of Forestry Research, 1999, 10(1): 11-14.

[12] Lima EC, Paiva R, Nogeira C, Soares FP, Emrich EB, Silva AN. Callus induction in leaf segments of Croton urucurana Baill. Journal of Agrotechnology, 2008, 32: 17-22.

[13] Ling APK, Tan KP, Hussein S. Comparative effects of plant growth regulators on leaf and stem explants of Labisa pumila var. Alata. Journal of Zhejiang University Science B, 2013, 14(7): 621-631.

[14] Luna LC. Manual of histological screening methods of armed forces institute of pathology. 1960, New York: McGraw Hill Book Co.

[15] Murashige T, Skoog E. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiology Plant, 1962, 15: 473-97.

[16] Na H, Kim KW, Kwack Y, Kim SK, Chun C. Comparative anatomy of embryogenic and non-embryogenic calli from Pimpinella brachycarpa. Journal of Plant Biology, 2007, 50(3): 344-350.

[17] Naef R. The volatile and semi-volatile constituents of agarwood, the infected heartwood of Aquilaria species: a review. Flavour and Fragrance Journal, 2011, 26: 73-87.

[18] Nurazah Z, Radzali M, Syahida A, Maziah M. Effects of plant growth regulators on callus induction from Cananga odorata flower petal explants. African Journal of Biotechnology, 2009, 8(12): 2740-2743.

[19] Nowak BK, Miczynski K, Hudy L. Sugar uptake and utilization during adventitious bud differentiation on in vitro leaf explant of Wegierka zwykla plum (Prunus domestica). Plant cell Tissue Organ Culture, 2004, 76: 255-260.

[20] Okudera Y, Ito M. Production of agarwood fragrant constituent in Aquilaria calli and cell suspension culture. Plant Biotechnology, 2009, 26: 307-315.

[21] Omamor IB, Asemota AO, Eke CR, Ezia EI. Fungal contaminants of the oil palm tissue culture in Nigerian Institute for Oil Palm Research (NIFOR). African Journal of Biotechnology, 2007, 2(10): 534-537.

[22] Rosli N, Maziah M, Chan KL, Sreeramanan S. Factors affecting the accumulation of 9-methoxycanthin-6-one in callus cultures of Eurycoma longifolia. Journal of Forestry Research, 2009, 20: 54-58.

[23] Sanavy SAM, Moeini MJ. Effects of Different pH Levels of Medium on Growth and Rooting of Single Nodes Resulted from Potato Meristem Culture. Plant Tissue Culture, 2003, 13(2): 151-154.

[24] Shahnewaj S, Bari MA. Effect of concentration of sucrose on the frequency of callus induction and plant regeneration in another culture of rice (Oryza sativa L.). Plant Tissue Culture, 2004, 14(1): 37-43.

[25] Shu YQ, Meng LH, Li DL, Chuan HZ, Lan JH, Hui ZZ. Production of 2-(2-phenylethyl) chromones in cell suspension cultures of Aquilaria sinensis. Plant Cell, Tissue and Organ Culture, 2005, 83: 217-221.

[26] Sugiyama M, Imamura K. Dose-, time- and tissue-dependent effects of 5-bromo-2′-deoxyuridine on the in-vitro organogenesis of Arabidopsis thaliana. Plant Cell Tissue Organ Culture, 2006, 87: 17-25.

[27] Vanisree M, Lee CY, Lo SF, Nalawade SM, Lin CH, Tsay HS. 2004. Studies on the production of some important secondary metabolites from medicinal plants by plant tissue cultures. Botanical Bulletin of Academia Sinica, Vol. 45.