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.

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.

Field cress genome mapping: Integrating linkage and comparative maps with cytogenetic analysis for rDNA carrying chromosomes

Field cress (Lepidium campestre L.), despite its potential as a sustainable alternative oilseed plant, has been underutilized, and no prior attempts to characterize the genome at the genetic or molecular cytogenetic level have been conducted. Genetic maps are the foundation for anchoring and orienting annotated genome assemblies and positional cloning of candidate genes. Our principal goal was to construct a genetic map using integrated approaches of genetic, comparative and cytogenetic map analyses. In total, 503 F2 interspecific hybrid individuals were genotyped using 7,624 single nucleotide polymorphism markers. Comparative analysis demonstrated that ~57% of the sequenced loci in L. campestre were congruent with Arabidopsis thaliana (L.) genome and suggested a novel karyotype, which predates the ancestral crucifer karyotype. Aceto-orcein chromosome staining and fluorescence in situ hybridization (FISH) analyses confirmed that L. campestre, L. heterophyllum Benth. and their hybrids had a chromosome number of 2n = 2x = 16. Flow cytometric analysis revealed that both species possess 2C roughly 0.4 picogram DNA. Integrating linkage and comparative maps with cytogenetic map analyses assigned two linkage groups to their particular chromosomes. Future work could incorporate FISH utilizing A. thaliana mapped BAC clones to allow the chromosomes of field cress to be identified reliably.

Rorippa sylvestris, Creeping Fieldcress: A Threat to Production Ornamentals and Its Control

The perennial Brassicaceae specie Rorippa sylvestris has been described as a weed in Scandinavian forest nurseries and in Irish, Canadian, and American nurseries. It was first introduced into Philadelphia, Pennsylvania in about 1818 and has been reintroduced repeated since. It produces little seed unless there are two different introductions together because they are self incompatible. Propagation is principally by rhizome segments from soil or ornamental propagation material. Greenhouse studies to evaluate the depth of emergence of rhizome pieces indicated that 3-cm segments would routinely emerge from depths of at least 24 cm. Allocation to shoot, roots, stems or shoots form rhizomes varied greatly by depth. The greatest shoot weight was from shallow depths with increasing new shoots from rhizomes and stem weights from deeper depths. Preemergence control was excellent with dichlobenil granules at 3 or 6 lb/A, isozaben at 1 or 2 lb/A and the geotextile/herbicide (Biobarrier). The geotextile (Typar) fitted as collars alone were not effective. Trifluralin incorporated into the surface 2 inches at 2 lb/A was effective but did allow some emergence. Trifluralin plus isoxaben or oryzalin plus isoxaben were also effective at rates of 2 plus 0.5, 4 plus 1, or 6 plus 1.5 lb/A, or 3 plus 1, 4.5 plus 1.5, or 6 plus 2 lb/A, respectively, of the two herbicide combinations. Metolachlor at 3, 4.5, or 6 lb/A was ineffective for preemergence control of 3 cm rhizome pieces. Post emergence control was not commercial with 2,4-D, triclopyr, clopyralid or a combination of the latter two, when treated in the 6 to 8 leaf stage with 0.25% or 0.5% solutions. Once creeping field cress is established in ornamentals it is very difficult to control.