Abstract
Flow cytometry is widely applied in determination of nuclear DNA content and ploidy level in many organisms. However, flow cytometers suffer from their intrinsic inability to tolerate large particles that associate with the isolated nuclei. A plant nuclei suspension can often contain a high level of crystalline calcium oxalate that blocks the fluidics system of the flow cytometer. First, we designed a cotton column, and added PVP-40 to the buffer to remove phenolic impurities and cytoplasmic compounds from plant nuclei, making the suspension suitable for flow cytometry. This simple and highly efficient protocol enables isolation of intact nuclei from plant tissues containing high levels of polysaccharides, calcium oxalate crystals, and other metabolites. Our protocol resulted in the isolation of intact nuclei from mature orchid leaves and flowers. This method can be used on recalcitrant tissues and is particularly effective on plants containing calcium oxalate crystals. It was also found that endoreduplication occurred during orchid leaf and flower development.Endoreduplication, a process to amplify nuclear DNA without cell division, is widespread in plants. We analyzed the ploidy levels during orchid flower development and proposed an improved model to describe the relationship between endoreduplication and cell growth. Our model combined a logistic growth model with an endoreduplication model. We found that using the Fermi function to describe the transition rates from one C value to next higher C value significantly improved simulation of changes in growth and endoreduplication. The growth rate, endoreduplication transition rates, and total cell number of each C level at different developmental stages were computed. Our results indicated that cells with higher C values had lower transition rates and less potential for further endoreduplication, and the time that endoreduplication stopped occurred at the same time flower fresh weight stopped increasing. In addition, average cell fresh weight was positively correlated to average C value, suggesting that endoreduplication is a contributing factor to cell growth.Temperature is the primary climatic factor affecting flower growth rate. A work used a new approach to quantify revealing environmental effect using an endoreduplication dynamic model and system identification techniques. Our study is much different from the previous studies for analyzing endoreduplication process determining the temperature effects on endoreduplication and cell division during flower development. The growth rates and endoreduplication transition rates decreased at lower temperature, but were compensated for by a longer period of growth. Therefore, the total cell number, floral structure and polyploidy pattern were not significantly affected. Our results indicated that systemic endoreduplication is intrinsically controlled by a differentiation program, and affected by environmental influences such as temperature changes. The relationship between the average C value and the average fresh weight is positive correlation and that can be effected by growth temperature, indicating that the final size of a cell is linked to its DNA content and the ploidy levels are regulated by environmental signals. This is clearly illustrated by the observation that the same cell with the same DNA content can reach different sizes depending on the environment. Endoreduplication cycle may determine nuclear size and be an important factor controlling cell size and organ size in plant.