In 2016, a project has been started to preserve the diversity of older plum cultivars grown on the territory of the Czech Republic. The purpose of this study was to develop an efficient in vitro system for rapid propagation of plum explants as an initial plant material for sanitation from viral contamination by chemotherapy and long term storage by cryopreservation experiments. Due to the fact that different species and subspecies of genus Prunus contributed to the diverse gene constitution of existing plum cultivars, the response of particular genotypes can vary in in vitro culture environment. Despite increasing numbers of reports of successful micropropagation more research is still needed to identify appropriate in vitro media compositions and concentrations of phytohormones for particular cultivars. The donor shoots were obtained in March from mature trees grown at the Research and Breeding Institute of Pomology (RBIP) Holovousy Ltd., Department of Fruit Genebanks. Selected plum genotypes ‘Svestka domaci’, ‘Hamanova’ and ‘Chrudimska’ were successfully established in vitro using 0.15% mercuric chloride as a disinfection solution. Developing shoots were serially subcultured onto fresh media for six consecutive 4-week passages. This provided a stock collection of shoots for proliferation studies. To assess the effect of plant growth regulators on multiplication, in vitro shoots were assigned to the basal MS medium supplemented with 1, 2 and 4 mg L-1 BAP (6-benzylaminopurine) or 0.5 and 1 mg L-1 TDZ (thidiazuron). All cultures were incubated in a growth room under 16 h of cool-white fluorescent light provided by Sylvania/Germany tubular lamps (F18W/840-TB) at 22 ± 1°C. The irradiance was 40 mmol m-2 s-1 at plant height. Multiplication rate was defined as the number of newly formed shoots (>10 mm) per initial shoot tip after four weeks of culture. Each combination of genotype and treatment involved 25 shoot tips and each experiment was repeated four times. Data from four independent experiments were pooled and expressed as the mean. Treatment means were compared with the standard error (SE) of the mean. Cultivars in the study differed in their proliferation and development potential in MS medium according to hormone level between 1.1 and 5.9. Generally, the highest proliferation rate (5.9) in our experiments was obtained for cultivar ‘Hamanova’ on MS medium with the highest concentration of BAP 4 mg L-1. Concerning cytokinin TDZ, sufficient multiplication rate 5.1 was obtained only for ‘Hamanova’ on medium with higher concentration 1 mg L-1. Results obtained in our study confirmed preliminary findings that BAP was an important plant growth regulator for proliferation and growth in plum micropropagation. During in vitro multiplication phase, for all tested cultivars, any morphological abnormalities such as excessive callus formation, hyperhydricity or production of abnormally narrow leaves were not noted. The observed differences in multiplication and morphology among plum genotypes under the influence of an exogenous BAP or TDZ could result from the genetic control of different auxin and cytokinin metabolisms of plant tissue. Examination of rooting of in vitro propagated plants is now in progress.
Take Away Notes:
• The described technique enabled us to multiply and maintain sufficient amount of in vitro plants of three plum cultivars for research experiments with in vitro virus elimination and cryopreservation. Moreover a successful in vitro culture technique would provide an alternative method, which can potentially multiply selected plum genotypes including rootstocks more rapidly than traditional nursery systems. Micropropagation techniques described in this paper may also be applied to other Prunus domestica cultivars. Advanced vocational schools and universities could also use obtained results to expand their teaching or research.