Towards developing effective decontamination procedures for in vitro culture of embryonic axes excised from recalcitrant seeds.

Abstract

Control of seed-associated micro-organisms is vital in reducing losses of plants of economic importance. Recalcitrant seeds being metabolically active and able to be stored only under conditions of high relative humidity makes it more difficult to control contaminants. Nevertheless, means need to be developed to eliminate, or at least curtail, seed-associated fungi and bacteria. The use of biological control is a highly recommended alternative to chemical control for reducing the risk of killing beneficial organisms, as well as in terms of health and environmental hazards. Furthermore, when working with seed-derived tissues, it is extremely important to optimise a method or methods to control contamination without compromising the viability or further development of the explants. The original aim of the present study was to determine whether the biocontrol agents, EcoT® and Eco77® (commercial products of the spores of Trichoderma harzianum) would effectively control/eliminate micro-organisms from the embryonic axes of Trichilia dregeana, while promoting growth under in vitro conditions. Other means were also tested for their efficacy in controlling contaminants; these were application of Benlate®, Nipastat® (a mixture of parabens), anodic water (the anodic fraction of an electrolysed dilute solution of calcium and magnesium chloride), sodium dichloro-isocyanurate (Medi-Chlor®[NaDCC]) and alginate gel encapsulation of the embryonic axes. Prior to the experiments, fungal contaminants from the embryonic axes were isolated on potato dextrose medium and identified using light microscopy. EcoT and Eco77 were initially individually tested by co-culture as conidial suspensions with the embryonic axes. A further approach used liquid culture (potato dextrose broth) as well as solid culture medium (based on sugarcane bagasse) in/on which the strains of Trichoderma harzianum had been grown. This was aimed at testing for the possible presence of compounds released by T. harzianum into the media, which might prove to be effective in curtailing/eliminating the axis-associated microflora. Among the different treatments tested, the best method was utilised to decontaminate the embryonic axes prior to minimal-growth storage (hydrated axes encapsulated in alginate gel ‘beads’). Penicillium spp. were predominant among the different fungi isolated, which included Fusarium spp., Rhizopus spp., Aspergillus niger and Aspergillus flavus. Co-culturing with T. harzianum for 24 h was successful in terms of the survival of the embryonic axes, although the roots produced were shorter than when axes were cultured alone, but had no effect in eliminating the contaminants. Longer periods of co-culturing with T. harzianum affected the germination of the embryonic axes of T. dregeana compared with axes germinated in the absence of the biocontrol agent (control). The culture filtrate negatively affected germination of the T. dregeana embryonic axes, although it was effective against the associated contaminants. Nipastat was effective in reducing the contamination, and, depending on the concentration, did not affect germination adversely. Medi-Chlor was highly effective in eliminating all the contaminants from axes in vitro. Both these treatments were therefore used to decontaminate axes before minimal-growth storage. All the NaDCC-treated, encapsulated axes examined after 14 d hydrated storage [in Magenta boxes] and after 14-42 d in polythene bags survived; however the axes stored in aluminium foil-lined bags and Eppendorf tubes soon lost viability. The recommendation is therefore made that the decontamination treatment based on use of NaDCC (or other preparations of sodium dichloro-isocyanurate) be tested on embryonic axes of a range of recalcitrant-seeded species, and, if successful, the procedure be introduced into cryopreservation protocols. The use of NaDCC has emerged as a promising method of eliminating contaminating microflora which otherwise compromise in vitro procedures, from seed-derived explants. Furthermore, containment of decontaminated encapsulated axes in sealed polythene bags offers an apparently ideal means of temporary storage and dissemination. The results should find considerable applicability when excised embryonic axes representing the germplasm of recalcitrant seeds, are cryoconserved.