In vitro stable regeneration of onion and garlic from suspension culture and chromosomal instability in solid callus culture

Abstract

An efficient regeneration system from bulb-derived callus tissues in suspension of onion ( Allium cepa L.) and garlic ( A. sativum L.) was established. Callus culture was induced in Gelrite ®-solidified Murashige and Skoog's (MS) modified basal medium with 9.05 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.93 μM kinetin supplements. After 4 weeks of induction, the callus tissue was partly transferred to liquid MS media containing different levels of α-naphthaleneacetic acid (NAA) and kinetin for plant regeneration and the rest was maintained in the same medium for chromosome analysis and nuclear DNA quantification following in situ microspectrophotometry. The cultures in suspension, maintained in agitated condition for 8 weeks, showed a high frequency of rapidly regenerated plants after transferring to Gelrite ®-solidified one half strength of MS basal medium. Chromosome analysis of the regenerated plants, transferred to the field with 90% survival rate, revealed stable chromosome number (2 n = 16) in both species. On the other hand, callus tissues maintained in solid induction medium for long period showed abnormality in chromosome behavior leading to the formation of both hypo- and hyper-diploid cells along with the diploid cells. The frequency of aneuploid cells (2.2–48.9%) increased with callus age in both species with high and statistically significant number of hyperdiploid cells. The role of endoreduplication as well as non-disjunction of chromosomes resulting in instability in chromosome number has been suggested. This was also supported by the nuclear DNA value in successive passages with statistically significant increase.