Establishment of a cell line derived from embryos of the potato tuber mothPhthorimaea operculella (Zeller)

Abstract

A cell line from the main insect pest of potatoes in tropical and subtropical areas, Phthorimaea operculella (Zeller), was obtained from embryoculture. These cells were cultured in Grace's modified medium. The cell line, designated ORS-Pop-93, had a heterogeneous population consisting of spherical and spindle cells with great capacity to adhere and a doubling time of 40 h. They were subcultured for more than 60 passages. Their polypeptidic profile was different from profiles of other lepidopteran cell lines. The cell line supports the multiplication of the Autographa californica nuclear polyhedrosis virus.

Multiplication of a granulosis virus isolated from the potato tuber moth in a new established cell line of Phthorimaea operculella

“…After the 5 'h passage, only rounded cells of varied sizes were observed. The morphology of the ORS-Pop-95 cell line is similar to the cell lines previously established at 27 ° C 03, 15,20). Increasing the temperature to 27 ° C does not affect the morphology of the eells established at 1W C. Winstanley and Crook (29) observed that a cell line established from embryos of Cydia pomonella at 19 ° C changed morphology when euhivated at 27 ° C. Thus, they deeided to select different cultures to obtain more homogeneous cell lines.…”

“…The ORS-Pop-93 cell line, consisting of heterogeneous cell types (15) and five other cell lines with homogeneous cell types, obtained by selecting (Pop2, Pop3 and Pop4) or cloning (cllA and cl2B) from embryonic cells of P. operculella (13), were cultivated in modified Grocers medium (14) containing 10% fetal bovine serum (FBS) at 27 ° C. The cells were routinely subcultured every 5 to 7 d and seeded with 1 to 2 × 10 ° cells in 25-cm 2 flasks.…”

“…Derivation of new cell lines cultivated at 19 ° C. Eggs from several layings of laboratory reared insects were used for primary cultures as previously described (15). The fragments and cells obtained from embryos were seeded in Nunc tissue culture flasks (25 cm 2) with modified Grace's medium containing 20% FBS and incubated at 19 ° C. During the first 6 mo., 1 ml of fresh medium was added every month.…”

“…Polypeptidic profiles were determined after electrophoresis in 12% polyacrylamide gels with the method of Laemmli (12). Cellular extracts were prepared with a technique of freezing-thawing (15). Electrophoresis was performed with a Biorad Miniprotean It dual slab cell, and the polypeptide profile of all the established cell lines were compared.…”

“…Initial results were negative for GV replication (15), but more recently viral DNA multiplication was obtained in cloned cell lines of PTM derived from our first established cell line (ORS-Pop-93) which was maintained at 27 ° C (13), Because no complete virions or granules were obtained from the PTM GV infections in these cell lines or clones, new cell cultures were initiated and systematically tested against the PTM GV.…”