Optimisation of sucrose, inorganic nitrogen and abscisic acid levels for Santalum album L. somatic embryo production in suspension culture
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].
Section snippets
Initiation and maintenance of callus culture
Callus culture was initiated by growing the hypocotyl part of aseptically grown seedling of sandalwood in MS medium [17] containing the growth regulator 2,4-dichloro phenoxy acetic acid (2,4-D) at 4.52 mM level and sucrose (30 g/l). The callus biomass so obtained was subcultured on the same medium every fortnight.
Embryogenesis induction medium
The callus biomass obtained as above was used for establishing suspension cultures in the same liquid medium except in place of 2,4-D, two other plant hormones, indole acetic acid
Somatic embryo morphology
After 14 days incubation in embryo production medium, mature embryos were produced (Fig. 3b) from suspended embryogenic cells (Fig. 3a). The mature embryos have 2–5 mm length.
Optimization
Using central composite rotatable design method, a total of 25 experiments with different combinations of sucrose, nitrate, ammonium and ABA levels were performed (Table 2).The response was taken at the maximum mature embryo production and thus maximum embryogenesis efficiency, after 14 days of culture. The results were
Conclusions
In this study, a clear and substantial significance for rotatable experimental design and statistical optimisation for somatic embryo production has been shown. A minimum number of experiments in CCRD provided a useful way for optimising critical factors in plant tissue culture related studies. Traditional single point optimisation methods as followed in tissue culture experiments has two serious limitations: firstly, it requires a large number of experiments and secondly, it does not consider
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