Establishment of a novel tissue culture method, stem-disc culture, and its practical application to micropropagation of garlic (Allium sativum L.)

Abstract

A restricted part of the undeveloped stem of the garlic clove, called the “stem disc”, which is just under the basement of the immature foliage leaves, proved to be a very potent explant for the micropropagation of garlic. Twenty to thirty tissue-cultured shoots consistently were differentiated from a single clove during 1 month of culture on phytohormone-free Linsmaier and Skoog medium. In addition, more than 90% of the shoots formed bulblets in vitro during an additional 1 month of culture. Pretreatment of the garlic bulbs at 4 °C for approximately 8 weeks before preparing the stem discs enhanced both shoot development and bulblet formation. This novel method for culturing garlic was designated the stem-disc culture method. Shoot development in this type of in vitro culture apparently is divided into four stages: expansion of tissue zones surrounded by the basal parts of the immature foliage leaves, formation of dome-shaped structures, bud differentiation directly from each dome, and development into shoots and bulblets. The dome-shaped structures appeared within 5 days of the onset of culture and had developed independently into shoots approximately 1 cm high 3 weeks later. Histological observations showed that both the internal cell organization and formation process of the dome-shaped structures were similar to those in the meristem. In addition, events leading to the formation of these dome-shaped structures appeared to be initiated by vigorous cell division in the epidermis of concentric tissue zones surrounded by the basements of immature foliage leaves. The results of several field trials showed that the stem-disc culture method is useful for the production of garlic seed plants, including virus-free plantlets. Furthermore, it is a novel field cultivation system for garlic in that the seedlings produced by in vitro-induced bulblets are used as seed instead of the usual cloves.