Optimisation of sucrose, inorganic nitrogen and abscisic acid levels for Santalum album L. somatic embryo production in suspension culture

Abstract

An efficient process for the production of somatic embryos of sandalwood was developed by statistical optimisation of important medium constituents (viz. sugar, inorganic nitrogen and abscisic acid). Higher embryogenesis was observed after optimisation. The optimal levels for the constituents were; 37.56 g/l sucrose, 11.6 mM nitrate, 7.89 mM ammonium and 1.31 mg/l abscisic acid. Embryogenesis efficiency of 57.35% was achieved from experiments with media containing optimal levels of these four constituents. The correlation coefficient was 0.92 ensuring a satisfactory adjustment of the model to the experimental values.

Introduction

Sandalwood (Santalum album L) is an important forest tree valued for its commercial and traditional social importance, and has found a place in the world perfumery market. The estimated global annual requirement is about 10 000 tonnes of wood (equivalent to 200 tonnes oil), involving a trade of about $125 million. Unfortunately this important tree species is under the threat of extinction due to illegal poaching and spike disease. Conventional propagation methods for this tree were ineffective to meet the huge demand of planting stock for the growing sandalwood market. Usual micropropagation through multiple shoot induction followed by rooting does not work well for most tree species, including sandalwood. To find an alternative and rapid propagation system, somatic embryogenesis was first established for this plant as early as 1965 [1]. To meet the challenge of commercial viability, a protocol with high and consistent production of somatic embryos was required. To this end, a bioreactorbased production system using liquid medium was developed where a scale-up study could be realised [2]. Studies on somatic embryogenesis, in many plants have established the importance of the levels of sugar, inorganic nitrogen and abscisic acid during the maturation and conversion stages [3], [4], [5], [6], [7]. Earlier studies in this laboratory [2], [8], [9] have shown that these four constituents in the production media influence the maturation and conversion stages in sandalwood somatic embryogenesis. Individual optimisation of critical factors that influence the process were carried out. This type of optimisation does not however, consider the interactions between constituents in the medium. Again, a large number of experiments are required to be performed to optimise each parameter separately. Even then the optimisation is far from accurate. In this work, three critical factors, sucrose, inorganic nitrogen and abscisic acid levels were subjected to statistical optimisation with a common response (embryogenesis efficiency). A model was also developed considering the interactions of these three factors. These studies are common for microbial bio-processes [10], [11], [12], [13], [14] but are rare in plant cell culture [15], [16].