Development of plant regeneration and transformation techniques towards reducing glucosinalbin biosynthesis in field pepperweed (Lepidium campestre L.)

Abstract

Field pepperweed (Lepidium campestre L.) is a cruciferous winter annual wild edible plant. It has potential medicinal properties as it contains a considerable level of glucoraphanin, which is the precursor for sulforaphane, a phase 2 protein inducer. Phase 2 proteins either directly or indirectly promote the scavenging of strong oxidants, and thus decrease the risk of many complex disorders such as atherosclerosis and Alzheimer’s disease. However, field pepperweed plants also contain glucosinalbin, an antinutritional compound. For field pepperweed to become a green vegetable crop or source of functional food, it is desirable to reduce or eliminate glucosinalbin. The biosynthesis of glucosinalbin may be down-regulated through biotechnology. To that end, in the present studies, experimental protocols for plant regeneration and Agrobacterium-mediated transformation have been developed for field pepperweed. Establishment of such methods represents a vital first step in the process of engineering field pepperweed for enhanced nutritional value.

The effect of explant type and various combinations of growth regulators on regeneration were evaluated in three accessions of field pepperweed (Ames 13179, 13180 and 15718). Among the three genotypes, accession Ames 13179 had the highest regeneration frequency under several conditions. Regeneration from hypocotyl explants was more rapid and prolific than regeneration from either mature leaf or cotyledonary explants. Segments from the acropetal end of the hypocotyls were more regenerable than those from the basipetal end. Evaluation of different hormonal combinations and concentrations identified an optimal growth regulator combination (3 mg L-1 thidiazuron / 0.1 mg L-1 naphthalene acetic acid) for shoot induction.

The plant regeneration system established was adopted for field pepperweed transformation using the acropetal segments of hypocotyls as explants. Two plant expression constructs were tested for down-regulating by RNA interference with the expression of a field pepperweed cytochrome P450 gene named LcCYP79B2. This gene may be involved in biosynthesis of glucosinalbin. Conditions for transformation such as pre-culture, co-cultivation time, and antibiotic concentration were evaluated. Transgenic plants were obtained and confirmed by histochemical staining of the reporter â-glucuranidase activity and PCR (polymerase chain reaction) analysis of the NPTII gene. The current study has established efficient plant regeneration and transformation protocols for field pepperweed. They should be useful for future molecular biology studies and biotechnological applications in this species.