Isolation and Characterization of Microprotoplasts from Propyzamide-treated Cell Suspension Cultures of Hemerocallis hybrida.

Abstract

To produce intergeneric hybrid plants with one or a few chromosomes via microprotoplast fusion in the Liliaceous ornamentals, we developed a system for isolating microprotoplasts from micronucleated cells in suspension cultures of Hemerocallis hybrida cv. Stella d'Oro (2n = 2x = 22). Micronucleation was efficiently induced with a micronucleus index up to 19.1 % by the following sequential treatment of suspension cultures: initially with 2 mM hydroxyurea for 24 h and then with 8 µM propyzamide for 60 h with the addition of 20 µM cytochalasin-B to the cultures 20 h after the initiation of the propyzamide treatment. Following the enzyme treatment of the micronucleated cells and ultra-centrifugation with a continuous iso-osmotic gradient of Percoll solution, microprotoplasts were isolated, each of which had a small nucleus surrounded by a thin rim of cytoplasm. Microprotoplasts less than 10 µm in diameter were obtained with yield of 2.9 × 10 4 cells per 1 ml packed cell volume of suspension cells through sequential filtration with nylon sieves with decreasing pore sizes (50, 20 and 10 µm). The DNA content of the microprotoplasts was less than the 2C level as indicated by flow cytometric analysis, and the relative fluorescence intensity in some of their nuclei corresponded to one or a few chromosomes.

Preparation of Microprotoplasts for Partial Genome Transfer via Microprotoplast Fusion in Liliaceous Ornamental Plants

“…The highest MNI of 19.1% was obtained by the following sequential treatments of the cultures: initially with 2 mM hydroxyurea for 24 h and then with 8 µM propyzamide for 60 h with the addition of 20 µM cytochalasin B at 20 h after the initiation of the propyzamide treatment (Fig. 2D) 22 . 3.…”

“…2B) derived from root segments of in vitro-grown plantlets ( Fig. 2A) 19 . They consisted of fine clumps with 20 to 50 cells (Fig.…”

“…3. Isolation of micronucleated protoplasts 22 At 78 h after the initiation of the sequential treatments described above, suspension cells were incubated for 6 h in a cell wall-digesting enzyme solution consisting of MS medium, 2% Cellulase Onozuka RS, 0.5% Macerozyme R-10, 10 mg/L picrolam, 5 mM 2-morpholinoethanesulfonic acid (MES), 0.5 M sorbitol, 8 µM propyzamide and 20 µM cytochalasin B at 25°C in the dark on a rotary shaker (30 rpm) to isolate micronucleated protoplasts (Fig. 2E).…”

“…4. Isolation of somatic microprotoplasts 22 Continuous iso-osmotic gradients of PERCOLL were prepared by the addition of 0.5 M sorbitol to PER-COLL followed by ultra-centrifugation (40,000 rpm, 200,000 g in the center of tube) for 30 min at 4°C in a Hitachi ultra-centrifuge 70P-72 using a 6×13 mL swingout rotor RPS40T. The top layer (45 mm from the top) was removed from the preformed gradient, and 5 mL of the protoplast suspension maintained on ice was layered on the top followed by ultra-centrifugation (40,000 rpm for 1.5 h).…”

“…5. Enrichment and characterization of somatic microprotoplasts 22 In order to enrich microprotoplasts containing one or a few chromosomes, the gathered suspensions were diluted with a 0.5 M sorbitol solution and then sequentially filtered through nylon sieves with decreasing pore sizes (50, 20 and 10 µm). The sequential filtration resulted in a population containing predominantly smaller microprotoplasts (Fig.…”