Nutrient Media Formulation for Micropropagation and Acclimatization of Herbaceous Perennials Using Turmeric As a Model Plant

Murashige and Skoog (MS) medium was formulated 50 years ago for in vitro growth of tobacco callus using a “one factor at a time” (OFAT) method.  OFAT confounds interactive effects that are often important in mineral nutrition.  This current work uses Curcuma longa L. (turmeric) as a model plant and simultaneously characterizes the interactive effects of multiple nutrient factors on in vitro plant growth in liquid media, and ex vitro acclimatization as influenced by in vitro plant nutrition.  Two separate experiments were conducted; each examined five factors (macronutrient and mesonutrient experiments, respectively).  A response surface method was used to partition variation in responses by the five main factor effects with quadratic resolution and second order interactive effects.  The macronutrient experiment altered the main components of the observational units: media volume (25-45 ml), plant density (3-9 divisions), sucrose concentration (1.5-6% m/v), macronutrient concentration (20-100 mM [NO₃^-]=[NH₄⁺]+[K⁺]), and [NH₄⁺]:[K⁺] ratio (0 to 0.5).  Vessels started with the fewest divisions, the most medium, and 4% sucrose had the highest multiplication ratio.  Plantlets with the largest fresh mass grew most quickly in the greenhouse.  These large, fast-growing plantlets came from vessels started with the fewest divisions, most medium, greatest sucrose concentration, and 75 mM macronutrients with 0.12 [NH₄⁺]:[K⁺].  Plantlets with the greatest mass were likely deficient in P and Mg.  Although few plants per vessel yielded a high multiplication ratio (5.2x), the greatest number of new plantlets (25) came from vessels with the greatest number of initial divisions (9).  The mesonutrient experiment fixed [NH₄⁺]=5mM, media volume=40 ml, and sucrose=5%; and altered plant density (3-9 divisions), P (1.25-6.25 mM), Ca (3-9 mM), Mg (1.5-4.5mM) and macronutrients ([NO₃^-]+[K⁺] = 40-120mM).  The greatest multiplication ratio came in vessels with the fewest divisions and 5 mM P.  Vessels with the largest plantlets also started with fewest divisions, P was 6.25 mM (or greater), macronutrients were 120 mM (or greater), Mg was 3mM, and Ca was 4.5 mM.  Maximization of the numbers of plantlets produced and their subsequent greenhouse growth will be presented for these independent factors.  The D-optimal designs used in both experiments used small numbers of vessels compared to factorial designs.  D-optimal is useful to establish ranges and significant interactions in multifactor experiments.  Computer-generated sample spaces, calculations, and graphical visualizations of multidimensional responses enable more comprehensive combinations of environmental factors, compared with the prior generation, when most biotechnology media were originally formulated.