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

Nuclear DNA content variation in seeds from 22 half‐sib families of jack pine (Pinus banksiana, Pinaceae)

“…In order to estimate peak channel numbers for haploid and diploid tissue, nuclei isolated from megagametophyte tissue excised from dry seeds were passed on the same settings. A previous study (Wyman, Tremblay, and Laliberté, 1996) has shown that the majority of nuclei of gametophytic tissue from the dry seeds were in the 1C state. From this it was established that nuclei falling on the channel approximately twice this value contained DNA levels equivalent to the 2C state.…”

“…Thus, in order to compare embryos at the same physiological stage of germination the greening of embryos was taken into account. When we compared the percentage nuclei in the ''normal'' 2C-4C cell cycle between superior and inferior families, we noted that inferior families had significantly more nuclei ''blocked'' in the 2C state, although families begin imbibition with the same proportion of cells in the 2C state (Wyman, Tremblay, and Laliberté, 1996). After 144 h of imbibition this ''unblocking'' of the nuclei is greater in the families classified as superior.…”

“…Twenty embryos from each family were analyzed. Nuclei extractions were carried out following a modification of the procedure of Wyman et al (1993). Briefly, 0.5 mL of cold (4ЊC) chopping buffer of the following composition: 0.44 mol/L sucrose, 2.5% (w/v) Ficoll 400 (Sigma), 25 mmol/L Tris-HCl (hydroxymethyl aminomethane) pH 7.6, 10 mmol/L MgCl 2 , 10 mmol/L ␤-mercaptoethanol, and 30% (v/v) glycerol (Chiatante et al, 1990) was added to the embryo tissue in an approximate ratio of 3:1 (volume buffer: volume plant tissue).…”

“…Three separate extractions of ten embryos or megagametophytes per family were carried out on seeds that had undergone no imbibition. Absolute DNA values were determined with rat hepatocytes as a standard (Wyman et al, 1993).…”

“…The proportion of jack pine embryos that had undergone greening at the time of sampling was related to the per- centage of seeds with cracked seed coat. It has been established that the distribution of nuclei in the various C classes became increasingly more marked between green and white embryos as imbibition period augmented and were significantly different after 144 h (Wyman, Tremblay, and Laliberté, 1996). Thus, in order to compare embryos at the same physiological stage of germination the greening of embryos was taken into account.…”