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 (La& 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 f 1.33 pg DNAhucleus 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 DNAhucleus up to as much as 35 pg DNA/nucleus can be tested using mouse hepatocytes as a standard while retaining an optimal samplektandard ratio. o 1993 ~i l e y -~i s s ,
Inc.Key terms: Diploid, haploid, plant tissue, standardsThe potential of biotechnology to facilitate the establishment and to increase the productivity of forests through the use of tissue culture, cell selection, and genetic engineering is vast. However, it is important to confirm the genetic stability of selected genotypes multiplied through tissue culture. Although chromosome counts reveal changes in ploidy status, this method is limited since only actively dividing cells can be analyzed (root tips or other meristematic tissue). Further, significant changes in the amount of DNA may not be revealed (for review see 19).One of the most commonly used methods of measuring nuclear DNA contents involves the cytophotometric analysis of nuclei subjected to the Feulgen reaction (21). Despite the fact that the Feulgen reaction is considered to be one of the most specific in histochemistry, it is subject to some drawbacks (7). Flow cytometry offers the possibility of rapid, accurate DNA analysis without these limitations. Several studies have shown that flow cytometric analysis techniques are applicable to plant systems for the determination of ploidy levels (e.g., 13,27,28).Despite the fact that it is possible to release intact nuclei both from mechanical chopping of fresh tissue (15) and protoplasts lysis (6), the vast majority of studies on plant tissue, and in fact all reports on coniferous species, use protoplast lysis. However, a protoplast population might not be truly representative of the starting tissue (15) and, depending upon the tissue, protoplast yield might not be sufficient from individual samples to test differences in nuclear DNA content between them.For accurate and repeatable determinations of DNA content, the choice of referenc...