Selection for Sustainable Preservation through In Vitro Propagation of Mature Pyrus spinosa Genotypes Rich in Total Phenolics and Antioxidants

Abstract

Pyrus spinosa Forssk. (almond-leaved pear) is a wild-growing native tree of the hilly countryside of Greece, which has recently gained interest for its valuable endogenous substances. In the present work, the determination of the total phenol content (TPC) and antioxidant capacity (AC) was carried out during 2021 and 2022 in leaves from 32 genotypes of mature wild-growing trees at four different locations of Central Macedonia District, Greece: Agia Anastasia, Chalkidiki, Lagadas and Pieria. The measurements taken from the leaf samples of all genotypes revealed a strong positive linear correlation between TPC and AC (R2 = 0.772). Two genotypes from Agia Anastasia, coded as AA2 and AA3, were comparatively the richest in TPC (44.86 for AA2 and 46.32 mg GAE/g fresh weight for AA3) and AC (70.31 and 71.21 μg AAE/g fresh weight for the same genotypes). For these two genotypes of high TPC and AC, an efficient micropropagation protocol was developed to preserve and multiply this valuable germplasm. Newly emerged shoot tips were excised from winter shoots and, after disinfestation, they were established on a modified Murashige and Skoog (MS) nutrient medium with 5 μΜ of 6-Benzyloaminopurine (BAP). For shoot multiplication, explants from clean cultures were transferred to Pear Medium 1 with 5 μΜ of BAP. The effect of the nutrient medium was tested by using five different nutrient media (modified MS, Pear Medium 1, Pear Medium 2, DKW and WPM) supplemented with 5 μΜ of BAP. Pear Medium 1 was the most effective in shoot formation. Among the four different BAP concentrations (0, 5, 10 and 20 μΜ) used in Pear Medium 1, 5 μΜ of BAP resulted in the production of the significantly highest number of shoots (22.7 shoots per explant, 2.4 cm long). The exposure of cultures to 10 μmol·m−2·s−1 for one week under light irradiance followed by 35 μmol·m−2·s−1 for four weeks increased both the number and length of the shoots produced. A 20-s dip of the shoot bases into 49.0 μΜ of Indole-3-butyric acid (IBA) in EtOH equally affected rooting in a modified rooting MS (79%) and Pear Medium 1 (80%). The root system developed better in the modified rooting MS medium (mean number of roots of 2.7 with a length of 3.9 cm). The acclimatization of the plantlets was successfully carried out (high survival rates five weeks after their establishment) on a perlite substrate under controlled environmental conditions.