Isolation and characterization of gametic microprotoplasts from developing microspores of Lilium longiflorum for partial genome transfer in the Liliaceous ornamentals

Abstract

We have established a system for isolating gametic microprotoplasts from developing microspores of Lilium longiflorum 'Hinomoto' (2n =2× =24) as a first step toward the production of intergeneric hybrid plants with one or a few alien chromosomes via microprotoplast fusion in the Liliaceous ornamentals. Anthers of this cultivar containing microsporocytes at mainly diakinesis to metaphase I were cultured for 3–4 days in a medium containing half-strength MS salts, double-strength MS vitamins, 1 g l–1 casamino acids, 100 g l–1 sucrose, and one of four spindle toxins: amiprophos-methyl, isopropyl N-(3-chlorophenyl)carbamate (CIPC), colchicine or propyzamide. Of the four spindle toxins examined, CIPC at concentrations of 5 or 10 µM efficiently induced micronucleation with the mean number of nuclei per meiocyte being 7.5. CIPC treatment also efficiently induced micronucleation in the other five Lilium genotypes evaluated (L. regale, L. longiflorum 'Georgia', L. speciosum 'Uchida', the Asiatic hybrid lily 'Connecticut King' and the Aurelian hybrid lily 'Golden Splendor') with the mean number of nuclei per meiocyte falling within the range 5.4–11.7. In 'Hinomoto', each meiocyte nucleus formed a microcell 4–5 days after initiation of CIPC treatment. Following isolation of such meiocytes from the anthers and their incubation in a cell-wall-digesting enzyme solution, gametic (micro)protoplasts of less than 10 µm, 10–20 µm and 20–50 µm in diameter were obtained with yields of 5.5×104, 6.6×104 and 4.9×104 per anther, respectively. Smaller microprotoplasts with DNA content below the 2C level, as indicated by flow cytometric analysis, were enriched by sequential filtration through nylon sieves of decreasing pore size (50, 20 and 10 µm). The relative fluorescence intensity in some of their nuclei corresponded to that of one or only a few chromosomes.