Protocol for in vitro shoot multiplication of ancient pear cultivars and landraces

In 2016, a project was started to preserve the diversity of older pear cultivars grown in the Czech Republic. The purpose of this study was to develop an efficient in vitro system for rapid propagation of pear explants as initial plant material for cryopreservation experiments. The donor shoots were obtained in March from mature trees grown at the Research and Breeding Institute of Pomology (RBIP) Holovousy Ltd., Department of Genebanks. Selected pear genotypes ‘Avranches’, ‘Jakobibirne’, ‘Marillat’, and ‘Flamandka’ were successfully established in vitro using 0.15% mercuric chloride as disinfectant. Six MS-based media containing 1, 2 or 4 mg L^‑1 zeatin or 1, 2 or 4 mg L^‑1 BAP (6-benzylaminopurine) were used for multiplication experiments. Multiplication rate was defined as the number of newly formed shoots (>10 mm) per initial shoot tip after four weeks of culture. Cultivars in the study differed in their proliferation and development potential in MS medium according to hormone level between 1.0 and 3.8. Generally, the highest proliferation rate (3.8) in our experiments was obtained for cultivar ‘Marillat’ on MS medium with 2 mg L^‑1 BAP. Sufficient multiplication at a rate of 3.1 was obtained only for ‘Marillat’ on a medium with the highest concentration cytokinin zeatin of 4 mg L^‑1. Results obtained in our study confirmed preliminary findings that BAP was an important plant growth regulator for proliferation and growth in pear micropropagation. During the in vitro multiplication phase, for all tested cultivars, no morphological abnormalities such as excessive callus formation, hyperhydricity or production of abnormally narrow leaves were noted.