Protocols for Adventitious Regeneration of Amelanchier alnifolia var. cusickii and Lonicera kamtschatica ‘Jugana’

The aim of this work was to assess the regeneration capacity of Amelanchier alnifolia var. cusickii and Lonicera kamtschatica cv. ‘Jugana’ from different types of explants under various hormonal treatments. The whole leaves, petioles, and internodal segments of in vitro plants were examined as explants. Several plant growth regulators (cytokinins and auxins) were evaluated for their ability to induce adventitious regeneration. Direct and indirect organogenesis was achieved under certain culture conditions in both species. The frequency of shoot regeneration was strongly dependent on concentrations of plant growth regulators in the induction media (L.kamtschatica ‘Jugana’) or concentrations of plant growth regulators in the induction media and type of explant (A. alnifolia var. cusickii). Results showed that leaves were not suitable explants for A. alnifolia var. cusickii. Both species were able to regenerate shoots from internodal segments and petioles. The highest induction of shoots was obtained on Murashige and Skoog (MS) medium enriched with 2 mg/L thidiazuron (TDZ) and 0.5 mg/L indole-3-butyric acid (IBA) for Amelanchier alnifolia and with 1 mg/L TDZ and 0.2 mg/L indole-3-acetic acid (IAA) for L. kamtschatica ‘Jugana’. Obtained adventitious shoots were further proliferated in order to investigate their multiplication capacity. The multiplication of shoots was successful in all cultivars, with the best results reported in A. alnifolia var. cusickii (7.07 shoots/explant on average).

New Strategies to Overcome Present CRISPR/Cas9 Limitations in Apple and Pear: Efficient Dechimerization and Base Editing

Despite recent progress, the application of CRISPR/Cas9 in perennial plants still has many obstacles to overcome. Our previous results with CRISPR/Cas9 in apple and pear indicated the frequent production of phenotypic and genotypic chimeras, after editing of the phytoene desaturase (PDS) gene conferring albino phenotype. Therefore, our first objective was to determine if adding an adventitious regeneration step from leaves of the primary transgenic plants (T0) would allow a reduction in chimerism. Among hundreds of adventitious buds regenerated from a variegated T0 line, 89% were homogeneous albino. Furthermore, the analysis of the target zone sequences of twelve of these regenerated lines (RT0 for “regenerated T0” lines) indicated that 99% of the RT0 alleles were predicted to produce a truncated target protein and that 67% of RT0 plants had less heterogeneous editing profiles than the T0. Base editors are CRISPR/Cas9-derived new genome-editing tools that allow precise nucleotide substitutions without double-stranded breaks. Hence, our second goal was to demonstrate the feasibility of CRISPR/Cas9 base editing in apple and pear using two easily scorable genes: acetolactate synthase—ALS (conferring resistance to chlorsulfuron) and PDS. The two guide RNAs under MdU3 and MdU6 promoters were coupled into a cytidine base editor harboring a cytidine deaminase fused to a nickase Cas9. Using this vector; we induced C-to-T DNA substitutions in the target genes; leading to discrete variation in the amino-acid sequence and generating new alleles. By co-editing ALS and PDS genes; we successfully obtained chlorsulfuron resistant and albino lines in pear. Overall; our work indicates that a regeneration step can efficiently reduce the initial chimerism and could be coupled with the application of base editing to create accurate genome edits in perennial plants.