Protoplast Isolation, Culture, and Regeneration in Common and Tartary Buckwheat.

Techniques based on the use of plant protoplasts are a convenient model for better understanding and observing developmental changes in the cells. The establishment of research tools based on protoplasts consists of many steps needed for optimization. Here, we describe the culture of morphogenic callus (MC)- and hypocotyl-derived protoplasts of common (Fagopyrum esculentum Moench) and Tartary (F. tataricum (L.) Gaertn.) buckwheat. Protoplasts embedding in agarose matrix and application of plant hormones, including phytosulfokine (PSK), enable the development of protoplast cultures and plant regeneration.

Promotive effect of phytosulfokine - peptide growth factor - on protoplast cultures development in Fagopyrum tataricum (L.) Gaertn.

BACKGROUND: Fagopyrum tataricum (Tartary buckwheat) is a valuable crop of great nutritional importance due to its high level of bioactive compounds. Excellent opportunities to obtain plants with the high level or the desired profile of valuable metabolites may be provided by in vitro cultures. Among known in vitro techniques, protoplast technology is an exciting tool for genetic manipulation to improve crop traits. In that context, protoplast fusion may be applied to generate hybrid cells between different species of Fagopyrum. To apply protoplast cultures to the aforementioned approaches in this research, we established the protoplast-to-plant system in Tartary buckwheat. RESULTS: In this work, cellulase and pectinase activity enabled protoplast isolation from non-morphogenic and morphogenic callus (MC), reaching, on average, 2.3 × 106 protoplasts per g of fresh weight. However, to release protoplasts from hypocotyls, the key step was the application of driselase in the enzyme mixture. We showed that colony formation could be induced after protoplast embedding in agarose compared to the alginate matrix. Protoplasts cultured in a medium based on Kao and Michayluk supplemented with phytosulfokine (PSK) rebuilt cell walls, underwent repeated mitotic division, formed aggregates, which consequently led to callus formation. Plating efficiency, expressing the number of cell aggregate formed, in 10-day-old protoplast cultures varied from 14% for morphogenic callus to 30% for hypocotyls used as a protoplast source. However plant regeneration via somatic embryogenesis and organogenesis occurred only during the cultivation of MC-derived protoplasts. CONCLUSIONS: This study demonstrated that the applied protoplast isolation approach facilitated the recovery of viable protoplasts. Moreover, the embedding of protoplasts in an agarose matrix and supplementation of a culture medium with PSK effectively stimulated cell division and further development of Tartary buckwheat protoplast cultures along with the plant regeneration. Together, these results provide the first evidence of developing a protoplast-to-plant system from the MC of Fagopyrum tataricum used as source material. These findings suggest that Tartary buckwheat's protoplast cultures have potential implications for the species' somatic hybridization and genetic improvement.

Combining genome size and pollen morphology data to study species relationships in the genus Daucus (Apiaceae).

BACKGROUND: The genus Daucus (Apiaceae) comprises about 40 wild species and the cultivated carrot, a crop of great economic and nutritional importance. The rich genetic diversity of wild Daucus species makes them a valuable gene pool for carrot improvement breeding programs. Therefore, it is essential to have good knowledge of the genome structure and relationships among wild Daucus species. To broaden such knowledge, in this research, the nuclear DNA content for 14 Daucus accessions and four closely related species was estimated by flow cytometry and their pollen morphology was analyzed by light and scanning electron microscopy (SEM). RESULTS: The flow cytometric analysis showed a 3.2-fold variation in the mean 2C values among Daucus taxa, ranging from 0.999 (D. carota subsp. sativus) to 3.228 pg (D. littoralis). Among the outgroup species, the mean 2C values were 1.775-2.882 pg. The pollen grains of Daucus were tricolporate, mainly prolate or perprolate (rarely) in shape, and mainly medium or small (rarely) in size (21.19-40.38 µm), whereas the outgroup species had tricolporate, perprolate-shaped, and medium-sized (26.01-49.86 µm) pollen grains. In the studied taxa, SEM analysis revealed that exine ornamentation was striate, rugulate, perforate, or the ornamentation pattern was mixed. At the time of shedding, all pollen grains were three-celled, as evidenced by DAPI staining. We also found high positive correlations between the length of the polar axis (P) and the length of the equatorial diameter (E) of pollen grains, as well as between P and P/E. However, when comparing cytogenetic information with palynological data, no significant correlations were observed. CONCLUSIONS: This study complements the information on the nuclear DNA content in Daucus and provides comprehensive knowledge of the pollen morphology of its taxa. These findings may be important in elucidating the taxonomic relationships among Daucus species and can help in the correct identification of gene bank accessions. In a broader view, they could also be meaningful for the interpretation of evolutionary trends in the genus.

Composition of the Reconstituted Cell Wall in Protoplast-Derived Cells of Daucus is Affected by Phytosulfokine (PSK).

Phytosulfokine-α (PSK), a peptidyl plant growth factor, has been recognized as a promising intercellular signaling molecule involved in cellular proliferation and dedifferentiation. It was shown that PSK stimulated and enhanced cell divisions in protoplast cultures of several species leading to callus and proembryogenic mass formation. Since PSK had been shown to cause an increase in efficiency of somatic embryogenesis, it was reasonable to check the distribution of selected chemical components of the cell walls during the protoplast regeneration process. So far, especially for the carrot, a model species for in vitro cultures, it has not been specified what pectic, arabinogalactan protein (AGP) and extensin epitopes are involved in the reconstruction of the wall in protoplast-derived cells. Even less is known about the correlation between wall regeneration and the presence of PSK during the protoplast culture. Three Daucus taxa, including the cultivated carrot, were analyzed during protoplast regeneration. Several antibodies directed against wall components (anti-pectin: LM19, LM20, anti-AGP: JIM4, JIM8, JIM13 and anti-extensin: JIM12) were used. The obtained results indicate a diverse response of the used Daucus taxa to PSK in terms of protoplast-derived cell development, and diversity in the chemical composition of the cell walls in the control and the PSK-treated cultures.

Raman, AFM and SNOM high resolution imaging of carotene crystals in a model carrot cell system.

Three non-destructive and complementary techniques, Raman imaging, Atomic Force Microscopy and Scanning Near-field Optical Microscopy were used simultaneously to show for the first time chemical and structural differences of carotenoid crystals. Spectroscopic and microscopic scanning probe measurements were applied to the released crystals or to crystals accumulated in a unique, carotenoids rich callus tissue growing in vitro that is considered as a new model system for plant carotenoid research. Three distinct morphological crystal types of various carotenoid composition were identified, a needle-like, rhomboidal and helical. Raman imaging using 532 and 488 nm excitation lines provided evidence that the needle-like and rhomboidal crystals had similar carotenoid composition and that they were composed mainly of ß-carotene accompanied by α-carotene. However, the presence of α-carotene was not identified in the helical crystals, which had the characteristic spatial structure. AFM measurements of crystals identified by Raman imaging revealed the crystal topography and showed the needle-like and rhomboidal crystals were planar but they differed in all three dimensions. Combining SNOM and Raman imaging enabled indication of carotenoid rich structures and visualised their distribution in the cell. The morphology of identified subcellular structures was characteristic for crystalline, membraneous and tubular chromoplasts that are plant organelles responsible for carotenoid accumulation in cells.