Propagation of asparagus (Asparagus officinalis L.) via somatic embryogenesis

Abstract

This thesis reports the development of somatic embryogenesis system for asparagus (Asparagus officinalis L.). Somatic embryogenesis is important as an efficient system for clonal propagation. To achieve this technique, the first step is the initiation of callus followed by the induction of the embryos and their developments into plants.

The optimized concentration of cytokinin and kinetin for callus formation from in vitro green stem explants of two asparagus genotypes (CRD 168 and CRD 100) was assessed. Maximum callus growth was observed at 1 mg/l of both naphthalene acetic acid (NAA) and kinetin. This optimized medium provided an important base for the selection of the best callus forming genotypes of asparagus.

Based on the optimized callus initiation medium, effect of different light intensities on callus formation was determined. High frequency of calli initiation was observed in darkness. A soft, translucent and highly friable callus was also produced in darkness while the green and compact callus was observed in light condition. To investigate the importance of genotypic effects on callus initiation, twenty asparagus genotypes which are under evaluation as clonal cultivars or as the parents of new hybrid cultivars in breeding programme were used. Among the 20 asparagus genotypes tested, ASC 26, ASC 69 and CRD 168 were all highly amenable to cell culture as indicated by high callus initiation growth in both dark and light conditions. Some genotypes did not form callus in low or high light intensity.

A protocol for somatic embryogenesis system for asparagus was established. A soft, translucent and highly friable callus was used to regenerate somatic embryos. Embryogenic calli were initiated to form somatic embryos in 0.2 mg/l NAA and 3 % sucrose concentration followed by transferring to hormone-free medium (hfm) with the same sucrose concentration. Somatic embryogenesis was investigated in four genotypes (ASC 69, ASC 26, CRD 168 and CRD 100). The development of somatic embryogenesis could be induced in all genotypes however their responses varied markedly. Embryos were induced to germinate and develop into complete plantlets by immersing the embryos in 1 mg/l gibberellic acid (GA₃) for 10 minutes and transferring to hfm with 3 % sucrose. The plantlets obtained appeared to be uniform in size.

The results and findings from this research demonstrated that callus is important in asparagus genetic manipulation. The protocols established in this thesis provided a rapid and reliable clonal propagation of some asparagus genotypes.