Initiation pretreatment with Plant Preservative Mixture™ increases the percentage of aseptic walnut shoots

Abstract

Walnut (Juglans regia L.) is one of the most economically important nut crops and one of the more difficult to micropropagate. Initiation and maintaining contamination-free shoot cultures is difficult for many species especially for woody plants. Cultivars of walnut from field collections exhibited severe microbial and fungal contamination when standard initiation procedure was used. An alternative disinfection procedure was designed and tested to reduce the microbial contamination. One-yr-old shoots of walnut cultivars Kogylnichanu, Milotai 10, Peschansky, and Xin 2 were cut in the field in February 2021. Cuttings of 30 to 40 cm long were surface disinfested and were put into jars with water to force bud expansion. After 2 wk, the swollen dormant buds were excised from the stem, surface disinfected in 0.1% mercuric chloride for 10 min, washed with sterile water, and placed into test tubes with Driver and Kuniyuki medium (DKW) with and without Plant Preservative Mixture (PPM). Addition of PPM (0.2% v/v) to the initial medium reduced the number of contaminated explants on average for all cultivars from 67.8 to 37.3% and increased the percentage of green shoots from 2.2 to 12.6%. With the combined treatment (shaking explants in 5% (v/v) PPM followed by 0.2% (v/v) PPM in the initial medium), 21.7% visually clean shoots were obtained. These shoots were indexed on 523 detection medium, with 87.5% found to be bacteria-free and suitable for cryopreservation and for producing healthy walnut planting material.