Multiplex Site-Directed Gene Editing Using Polyethylene Glycol-Mediated Delivery of CRISPR gRNA:Cas9 Ribonucleoprotein (RNP) Complexes to Carrot Protoplasts.

The aim of this work was to show an efficient, recombinant DNA-free, multiplex gene-editing method using gRNA:Cas9 ribonucleoprotein (RNP) complexes delivered directly to plant protoplasts. For this purpose, three RNPs were formed in the tube, their activity was confirmed by DNA cleavage in vitro, and then they were delivered to carrot protoplasts incubated with polyethylene glycol (PEG). After 48 h of incubation, single nucleotide deletions and insertions and small deletions at target DNA sites were identified by using fluorescent-PCR capillary electrophoresis and sequencing. When two or three RNPs were delivered simultaneously, long deletions of 33-152 nt between the gRNA target sites were generated. Such mutations occurred with an efficiency of up to 12%, while the overall editing effectiveness was very high, reaching 71%. This highly efficient multiplex gene-editing method, without the need for recombinant DNA technology, can be adapted to other plants for which protoplast culture methods have been established.

Spectroscopic studies on bioactive polyacetylenes and other plant components in wild carrot root.

Polyacetylenes and other common plant components, such as starch, pectin, cellulose, and lignin, were studied in roots of the wild carrot (Daucus carota) subspecies D. carota subsp. gummifer and D. carota subsp. maximus by Raman spectroscopy. The components were measured in situ, directly in the plant tissue and without any preliminary sample preparation. The analysis was performed on the basis of the intense and characteristic key bands observed in the Raman spectrum. The two main carrot polyacetylenes falcarinol (1) and falcarindiol (2) have similar molecular structures, but their Raman spectra exhibit a small band shift in the symmetric -C≡C-C≡C- mode from 2258 cm⁻¹ to 2252 cm⁻¹. Quantum chemical calculations confirmed that the differences observed between the samples may be due to conformational and environmental changes. The polyacetylenes were also detected by Raman mapping, which visualized the distribution of the compounds across sections of carrot roots. The mapping technique was also applied to assess the distribution of lignin and polysaccharide compounds. The results showed the tissue-specific accumulation of starch and cell wall components such as lignin, pectin, and cellulose.

In situ simultaneous analysis of polyacetylenes, carotenoids and polysaccharides in carrot roots.

This paper presents an approach to simultaneously analyze polyacetylenes, carotenoids, and polysaccharides in carrot (Daucus carota L.) roots by means of Raman spectroscopy. The components were measured in situ in the plant tissue without any preliminary sample preparation. The analysis is based on the intensive and characteristic key bands observed in the Raman spectrum of carrot root. The molecular structures of the main carrot polyacetylenes, falcarinol and falcarindiol, are similar, but their Raman spectra exhibit specific differences demonstrated by the shift of their -C[triple bond]C- mode from 2258 to 2252 cm(-)(1), respectively. Carotenoids can be identified by -C=C- stretching vibrations (about 1520 and 1155 cm(-)(1)) of the conjugated system of their polyene chain, whereas the characteristic Raman band at 478 cm(-)(1) indicates the skeletal vibration mode of starch molecule. The other polysaccharide, pectin, can be identified by the characteristic band at 854 cm(-)(1), which is due to the -C-O-C- skeletal mode of alpha-anomer carbohydrates. The Raman mapping technique applied here has revealed detailed information regarding the relative distribution of polyacetylenes, carotenoids, starch, and pectin in the investigated plant tissues. The distribution of these components varies among various carrot cultivars, and especially a significant difference can be seen between cultivated carrot and the wild relative D. carota ssp. maritimus.