Use of mouse hepatocytes for the flow cytometric determination of DNA levels of nuclei extracted from fresh tissue of hybrid larch (Larix x eurolepis Henry).

Abstract

A rapid and reliable method is presented to release intact nuclei from small amounts (100 mg) of fresh plant tissue. Further, an accurate and readily accessible new standard is proposed. Both techniques have potential application for many plant systems. The system chosen as a standard (inbred mouse strain Balb/C or B6/AF1 hepatocyte nuclei) contains both diploid and polyploid cells. This system was applied in the flow cytometric determination of absolute nuclear DNA values of female gametophytes and in vitro propagated shoots of hybrid larch (Larix x eurolepis Henry). The amount of DNA in 2C nuclei of in vitro grown larch is 32.48 +/- 4.04 or 31.97 +/- 6.14 pg/nucleus, respectively, when calculated using the mouse hepatocyte 4C or 8C nuclear peak as a reference standard. The amount of DNA in female gametophyte nuclei is 17.47 +/- 1.33 pg DNA/nucleus when these haploid larch nuclei were analyzed with trout red blood cell nuclei as the standard. When hepatocyte 4C nuclei were used as a standard, the absolute value of DNA per haploid larch nucleus was estimated as 16.8 +/- 0.53 pg. Plant tissue with as little as 4-6 pg DNA/nucleus up to as much as 35 pg DNA/nucleus can be tested using mouse hepatocytes as a standard while retaining an optimal sample/standard ratio.