Field pepperweed (<i>Lepidium campestre</i> 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 Alzheimers 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.<p> 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.<p> 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.

Field pepperweed (<i>Lepidium campestre</i> 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 Alzheimers 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.<p> 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.<p> 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.

In Lepidium campestre und Lepidium appelianum konnten insgesamt acht Fruchtentwicklungsgene isoliert werden. Diese untersuchten Gene sind wahrscheinlich allesamt Orthologe zu Fruchtentwicklungsgenen aus Arabidopsis thaliana. Ein isoliertes SHP-Gen aus Aethionema carneum ist wahrscheinlich ortholog zu SHP2 aus Arabidopsis thaliana. Dieses Gen (AecSHP2) könnte ein Abkömmling des letzten gemeinsamen Vorfahren von Aethionema und den restlichen Brassicaceen sein, welches mit Ausnahme der Gattung Aethionema in den Brassicaceen duplizierte. Allein aufgrund der Aminosäuresequenzen lassen sich keine direkten Rückschlüsse auf die Beteiligung einzelner Gene an der Evolution von Schließfrüchten ziehen. Wahrscheinlicher sind regulatorische Änderungen eines oder mehrerer Fruchtentwicklungsgene innerhalb der Früchte von L. appelianum, die zur Ausbildung von indehiszenten Früchten führen. Für die untersuchten Arten der Gattung Lepidium konnte die Methode der Expressionsanalysen (in situ Hybridisierung) etabliert werden. Expressionsstudien lassen eine ähnliche genetische Regulation der Fruchtöffnung von Lepidium campestre und Arabidopsis thaliana vermuten, diese Regulation scheint in Öffnungsfrüchten von Lepidium campestre konserviert zu sein. Aufgrund einer ähnlichen Regulation in der Gattung Brassica ist es möglich, dass die genetische Regulation von Öffnungsfrüchten innerhalb der Core Brassicaceae konserviert ist. Die genetische Regulation von Lepidium appelianum ist bislang nicht geklärt. Zum Verständnis der Evolution von Schließfrüchten in Lepidium sind umfangreiche heterologe Transformationsversuche notwendig. Diese heterologen Transformationsversuche sollten zunächst mit den für die Evolution von Schließfrüchten in Frage kommenden Kandidatengenen durchgeführt werden, nach den Ergebnissen der vorliegenden Arbeit sind dies die Gene FRUITFULL und REPLUMLESS. Die Fruchtanatomie heterokarper Früchte in verschiedenen Aethionema-Arten scheint einer ähnlichen genetischen Regulation zu folgen. Hierauf deuten anatomische Untersuchungen von Ae. carneum- und Ae. saxatile-Früchten hin.