In vitro Induction and Phenotypic Variations of Autotetraploid Garlic (Allium sativum L.) With Dwarfism

Abstract

Garlic (Allium sativum L.) is a compelling horticultural crop with high culinary and therapeutic values. Commercial garlic varieties are male-sterile and propagated asexually from individual cloves or bulbils. Consequently, its main breeding strategy has been confined to the time-consuming and inefficient selection approach from the existing germplasm. Polyploidy, meanwhile, plays a prominent role in conferring plants various changes in morphological, physiological, and ecological properties. Artificial polyploidy induction has gained pivotal attention to generate new genotype for further crop improvement as a mutational breeding method. In our study, efficient and reliable in vitro induction protocols of autotetraploid garlic were established by applying different antimitotic agents based on high-frequency direct shoot organogenesis initiated from inflorescence explant. The explants were cultured on solid medium containing various concentrations of colchicine or oryzalin for different duration days. Afterward, the ploidy levels of regenerated plantlets with stable and distinguished characters were confirmed by flow cytometry and chromosome counting. The colchicine concentration at 0.2% (w/v) combined with culture duration for 20 days was most efficient (the autotetraploid induction rate was 21.8%) compared to the induction rate of 4.3% using oryzalin at 60 μmol L^-1 for 20 days. No polymorphic bands were detected by simple sequence repeat analysis between tetraploid and diploid plantlets. The tetraploids exhibited a stable and remarkable dwarfness effect rarely reported in artificial polyploidization among wide range of phenotypic variations. There are both morphological and cytological changes including extremely reduced plant height, thickening and broadening of leaves, disappearance of pseudostem, density reduction, and augmented width of stomatal. Furthermore, the level of phytohormones, including, indole propionic acid, gibberellin, brassinolide, zeatin, dihydrozeatin, and methyl jasmonate, was significantly lower in tetraploids than those in diploid controls, except indole acetic acid and abscisic acid, which could partly explain the dwarfness in hormonal regulation aspect. Moreover, as the typical secondary metabolites of garlic, organosulfur compounds including allicin, diallyl disulfide, and diallyl trisulfide accumulated a higher content significantly in tetraploids. The obtained dwarf genotype of autotetraploid garlic could bring new perspectives for the artificial polyploids breeding and be implemented as a new germplasm to facilitate investigation into whole-genome doubling consequences.

Keywords: autopolyploid; colchicine; dwarfness; garlic; in vitro; oryzalin; whole-genome duplication.