Optimized tetraploidization strategies in tissue culture for Lolium, Festuca, and Festulolium

Abstract

In plant breeding, polyploidization is an established technique to obtain superior phenotypic characteristics. In seed propagated agricultural crops, seed treatments with antimitotic agents are often used to obtain chromosome doubling. Here, we developed a method to induce polyploidization in clonally propagated fodder grasses Lolium, Festuca, and the intergeneric hybrid Festulolium. The aim was to obtain specific genotypes at both the diploid and tetraploid levels. We evaluated different types of plant explants, and the effects of the type, concentration, and application mode of three antimitotic agents (oryzalin, colchicine, and trifluralin) on the survival rate and the polyploidization efficiency. The treatment of greenhouse-grown tillers with antimitotics only resulted in mixoploids, while tissue culture propagated plants were successfully polyploidized. A shock pretreatment, using a high concentration of an antimitotic agent during a short period, successfully induced tetraploids in all three genera. Additionally, supplementing the tissue culture medium with a lower dosage of an antimitotic agent during minimal four weeks after the shock pretreatment further promoted polyploidization. By our methods, we were able to generate diploid and tetraploid plants with an identical genomic constitution but different ploidy allowing investigation of the effects of polyploidization on plant physiology and gene regulatory networks.

Keywords

• artificial polyploidization
• chromosome doubling
• colchicine
• fodder grass
• in vitro culture