Establishment of an Efficient Protoplast Regeneration and Transfection Protocol for Field Cress (Lepidium campestre)

Field cress (Lepidium campestre) is a potential oilseed crop that has been under domestication in recent decades. CRISPR/Cas9 is a powerful tool for rapid trait improvement and gene characterization and for generating transgene-free mutants using protoplast transfection system. However, protoplast regeneration remains challenging for many plant species. Here we report an efficient protoplast regeneration and transfection protocol for field cress. Important factors such as type of basal media, type/combination of plant growth regulators, and culture duration on different media were optimized. Among the basal media tested, Nitsch was the best for protoplast growth in MI and MII media. For cell wall formation during the early stage of protoplast growth, relatively high auxin concentrations (0.5 mg L−1 NAA and 2,4-D), without addition of cytokinin was preferred for maintaining protoplast viability. After cell wall formation, 1.1 mg L−1 TDZ combined with either 0.05 mg L−1 NAA or 2,4-D was found to efficiently promote protoplast growth. On solid shoot induction medium, 1.1 mg L−1 TDZ without any auxin resulted in over 80% shoot generation frequency. A longer culture duration in MI medium would inhibit protoplast growth, while a longer culture duration in MII medium significantly delayed shoot formation. Using this optimized protoplast regeneration protocol, we have established an efficient PEG-mediated transfection protocol using a vector harboring the GFP gene, with transfection efficiencies of 50–80%. This efficient protoplast protocol would facilitate further genetic improvement of field cress via genome editing, and be beneficial to development of protoplast regeneration protocols for related plant species.

THE AUTOLYSIS PRODUCTS OF GLUCOSINOLATES IN „LEPIDIUM CAMPESTRE“ (L.) W. T. AITON (BRASSICACEAE)

Plant samples of Lepidium campestre (L.) W. T. Aiton (pepperwort) were analyzed in detail by GC and GC-MS. The analysis of the autolysates obtained from inflorescences, stems, leaves, and underground parts and the essential oil obtained by hydrodistillation allowed the identification of a series of glucosinolate degradation products, mainly isothiocyanates and nitriles. Besides previously identified ones in this species, the analyses resulted in the identification of degradation metabolites identified in pepperwort for the first time, and suggested the presence of heptyl glucosinolate, 3- phenylpropyl glucosinolate, glucoiberverin, glucoiberin, sinalbin, glucoerucin, glucoberteroin, gluconasturtiin, glucolepigramin, glucolesquerellin, glucobrassicanapin, and glucotropaeolin in this species as the most likely glucosinolate precursors. The results showed the localized accumulation of glucoberteroin, glucoerucin, and glucolesquerellin in the roots, the plant organ most exposed to pathogens, whereas sinalbin and glucobrassicanapin were accumulated in the reproductive organs and the organs most exposed to herbivores, i.e. in the aerial parts of the plant.

HOST STATUS OF PLANT SPECIES WITH NEMATICIDAL ACTIVITY AGAINST MELOIDOGYNE GRAMINICOLA (GOLDEN & BIRCHFIELD)

The rice root-knot nematode, Meloidogyne graminicola (Golden & Birchfield), is a nematode first described in 1965 from grasses and oats in Louisiana (US), and currently spread in rice crops in Asia, parts of the Americas and Africa. It can be detected in a wide range of more than 98 host plant species. In 2016 M. graminicola was detected for the first time in some rice fields of Northern Italy. In this paper, two greenhouse experiments (the first at 21-22 °C and the second one at 26-28 °C) are reported and discussed, in which three species containing bioactive compounds, Lepidium campestre (L.) R. Br., Eruca sativa Mill. cv. Nemat, and Crotalaria juncea L., were compared with Cucumis sativus L., a good host of M. graminicola, as control. Seedlings of each plant species were transplanted in pots containing mean 50 J2s 100 cm−3 soil. Three assessments were carried out about 30, 60, 90 days after transplantation, on both soil and roots. Temperature influenced M. graminicola life cycle, which was never completed at 21-22 °C; conversely, at 26-28 °C, both E. sativa and C. juncea reached a reproduction rate (R) = 0.01, confirming to be poor hosts, effective in decreasing the nematode infestation on the roots and in the soil, while L. campestre (R = 4.01) demonstrated to be a good host of M. graminicola more than the control C. sativus (R = 2.12), increasing considerably the nematode population after about 90 days.

QTL Mapping for Domestication-Related Characteristics in Field Cress (Lepidium campestre)—A Novel Oil Crop for the Subarctic Region

Domestication of a new crop requires identification and improvement of desirable characteristics Field cress (Lepidium campestre) is being domesticated as a new oilseed crop, particularly for northern temperate regions.. In the present study, an F2 mapping population and its F3 progenies were used to identify quantitative trait loci (QTLs) for plant height (PH), number of stems per plant (NS), stem growth orientation (SO), flowering habit (FH), earliness (ER), seed yield per plant (SY), pod shattering resistance (SHR), and perenniality (PE). A highly significant correlation (p < 0.001) was observed between several pairs of characteristics, including SY and ER (negative) or ER and PE (positive). The inclusive composite interval mapping approach was used for QTL mapping using 2330 single nucleotide polymorphism (SNP) markers mapped across the eight field cress linkage groups. Nine QTLs were identified with NS, PH, SO, and PE having 3, 3, 2, and 1 QTLs, explaining 21.3%, 29.5%, 3.8%, and 7.2% of the phenotypic variation, respectively. Candidate genes behind three of the QTLs and favorable marker alleles for different classes of each characteristic were identified. Following their validation through further study, the identified QTLs and associated favorable marker alleles can be used in marker-aided breeding to speed up the domestication of field cress.