High frequency somatic embryogenesis, regeneration and correlation of alkaloid biosynthesis with gene expression in Papaver somniferum

Abstract

Opium poppy (Papaver somniferum) remains the most important source for several pharmaceutical benzylisoquinoline alkaloids including the narcotic analgesic morphine, the anti-tussive drug codeine. Selection, breeding as well as other molecular practices have produced large number of germplasm with modulated biosynthesis and altered accumulation of specific alkaloids. However, there is need to modulate biosynthetic pathways of these alkaloids through transgenic approach, which requires development of an efficient regeneration system for genetic transformation. We studied response of different explants for somatic embryogenesis and developed an efficient protocol for P. somniferum somatic embryogenesis, regeneration as well as successful transplantation in fields. In our system, embryogenic callus from root explants was induced by 2,4-D and kinetin followed by maturation with gibberellin and abscisic acid and regeneration by hormone free half-strength MS media. Though root regeneration from somatic embryos has been shown as limiting factor in established protocols, we observed efficient root and shoot regeneration through somatic embryos in our study. We analyzed alkaloid content in in vitro callus and field grown plants and concluded developmental control of alkaloid biosynthesis. Absence of transcripts of some of the genes and specific alkaloids also suggests regulation at transcriptional level of alkaloid biosynthesis pathway.

Somatic Embryogenesis: Fundamental Aspects and Applications

“…A combination of auxin (NAA or 2,4-D) and cytokinin (BAP or Kin) is employed to induce the embryogenic cultures at doses ranging from 1 to 2.5 mg/L. Roots from developing seedlings can also be used as starting material with good results Pathak et al 2012). Although shoots readily developed from the mature embryos, root formation has shown to be a limiting step (Ovecka et al 1997).…”

Comparative Transcriptome Analysis Using High Papaverine Mutant of Papaver somniferum Reveals Pathway and Uncharacterized Steps of Papaverine Biosynthesis

“…Solvent containing extracted alkaloids was recovered and estimation of various alkaloids was carried out through separation using High Pressure Liquid Chromatography following the standard method [34], [35]. The average content of each alkaloid of at least three samples was calculated and data are expressed as mean +/−standard deviation (S.D.…”

Application of Somatic Embryogenesis to Secondary Metabolite-Producing Plants

“…Although shoots readily developed from the mature embryos, root formation has shown to be a limiting step (Ovecka et al 1997). Roots from developing seedlings can also be used as starting material with good results Pathak et al 2012). As for hypocotyl-derived calli, hormones should be removed to promote embryo maturation .…”

“…As for hypocotyl-derived calli, hormones should be removed to promote embryo maturation . ABA and GA (between 0.1 and 0.2 mg/L) can be added to the culture medium to promote maturation (Pathak et al 2012).…”