The in vitro propagation of virus-free Amygdalus communis Linnaeus var. amara : a rootstock for peach and nectarine in the Maltese Islands

Abstract

The bitter almond (Amygda/us communis var. amara), due to its high resistance to physical and biotic stress is widely used as a rootstock for peaches and nectarines in the Maltese Islands. Nevertheless, the utilisation of almond as a rootstock is limited by the scarce availability of elite clones. At the moment the only method of propagation of bitter almond is through seeds, producing seedling populations with non-homogeneous characteristics. This study was thus undertaken aiming at establishing an effective in vitro propagation system for selected 'virus-free' local bitter almond genotypes. Research work was divided into two different parts, namely: • The phytosanitary assessment of local bitter almond with respect to virus and virus-like diseases, and • The definition of a suitable in vitro culture technique. Field surveys together with laboratory diagnostic assays (ELISA, biological indexing) were carried out to identify almond trees and rootstocks to be used as mother plants for in vitro culture studies. A total of about 400 trees were identified and the plant health status with respect to plum pox virus (PPV), prune dwarf virus (PDV), apple mosaic virus (ApMV), prunus necrotic ringspot virus (PNRSV), and apple chlorotic leafspot virus (ACLSV) was studied. 71.43% and 6.36% of the tested grafted and nongrafted bitter almonds respectively, proved to be infected with at least one of the five viruses for which they were tested. All the samples tested resulted negative for PPV. Ten potential 'virus-free' mother plants for in vitro culture were further tested for peach latent mosaic viroid (PLMVd) and hop stunt viroid (HSVd). None of the two viroids was detected. In vitro axillary and adventitious propagation of the ten selected genotypes of Amygdalus communis var. amara were then attempted. Different explants and different surface sterilisation protocols were used to initiate the in vitro culture of bitter almond. Micropropagation was achieved using axillary propagation starting from meristematic explants (nodes and buds) and to a lesser extent by adventitious morphogenesis through an intermediate callus stage on leaf segments. Attempts to induce direct adventitious propagation and/ or embryogenesis failed. The interaction and effects of plant growth regulator combinations, basal nutrient media, agar brand and concentration, sucrose concentration and light intensity were studied on the various stages of micropropagation. A simple and reliable procedure with moderate regeneration frequencies in indigenous A. communis var. amara genotypes was devised through adventitious propagation. To my knowledge, this is the first report on adventitious propagation of bitter almonds from leaf explants. Phenotypically true-to-type propagated plantlets were obtained when caulogenesis was induced on leaf segments cultured on Murashige and Skoog (MS) medium supplemented with 1.0 mg/I kinetin and 2.0 or 3.0 mg/I 2,4-D. A shoot regeneration index of 2.25 was achieved on MS medium supplemented with 1.5 mg/I kinetin and 0.05 mg/I NM. After inducing elongation on hormone-free MS medium, a 55% root induction was achieved on half-strength MS medium supplemented with 1.0 mg/ NAA. 56% of the micropropagated plants through indirect morphogenesis were fully acclimatised. A second in vitro propagation method for Amygdalus communis var. amara was achieved through axillary shoot proliferation. By changing different factors, the proliferation medium was optimised and a scheme which proved to be the most efficacious for axillary propagation of local bitter almond genotypes was drawn up. This protocol advocates the use of MS medium supplemented with 1.0 mg/I BAP, 0.05 mg/I NAA, 20 g/I sucrose and 0.65% agar (Agar-agar, Sigma) for shoot induction and proliferation and MS basal medium supplemented with 0.1 mg/I BAP and 0.05 mg/I NAA for elongation. An average proliferation rate of 4.9 every 4 weeks was achieved on the multiplication medium. Highest root induction (67%) was obtained on MS basal medium supplemented with 1.5 mg/I IBA, 20 g/I sucrose and 6.5 g/I agar and incubation for about 10 days at 2500 lux. A survival rate of 53% was achieved on weaning the plants. Proposals have been made regarding further studies that need to be carried out to improve the in vitro propagation scheme for Amygda/us communis var. amara to be feasible for use in mass commercial production.