RobLaffin said:I'd be very interested to hear your thoughts on the work JG is doing on unusual ploidys as set forth here:
http://www.heavenlygardens.com...
Jamie is using a flow cytometer to measure the amount of DNA in cells from various daylily plants. From those estimates he calculates the ploidy of the plant.
Flow cytometry (FCM) is a scientific method and so I would examine his methods, protocols, analyses, conclusions using scientific standards. He has not provided the details of those or indeed enough details.
The characteristics described for Heavenly New Frontiers, described as a pentaploid, are unusual for a pentaploid. Pentaploids, like triploids, have unbalanced chromosome counts and will have abnormal chromosome alignment. They are usually infertile. They are not always completely sterile and some may have some fertility, for example as Arisumi found with the triploid daylily 'Garnet Robe'. However, it would be very unusual for a pentaploid to be as fertile as Heavenly New Frontiers (described as extremely pod and pollen fertile).
Using a flow cytometer to determine the amount of DNA is an accepted technique. However, there are many possible problems that make the next step of determining the ploidy difficult and many precautions that one should take. I will quote several passages from a book on flow cytometry. FCM is the abbreviation for flow cytometry.
"FCM has many advantages over other methods; however, these benefits are realized only with awareness of potential pitfalls and technical guidelines specific to measuring ploidy."
"It must be emphasized that conventional chromosome counting should follow any suspicion of chromosomal heterogeneity inferred from FCM data in
order to elucidate its nature. "
"A fundamental assumption in using FCM to assign ploidy is that increments of DNA content correspond in a predictable way to increments in chromosome
number. Errors in interpretation can occur when this assumption fails..."
"Moreover, errors (ploidy underestimation or overestimation) may sometimes be introduced even when comparing different cytotypes of the same species, unless FCM measures are combined with chromosome counts"
"To ensure that these advantages of FCM are realized in a consistent way, efforts have been made in recent years to promote universal guidelines for the measurement of nuclear DNA content in plants (Bennett et al. 2000a; Dolezˇel and Bartosˇ 2005). These include: (i) the use of histograms obtained after the analysis of >5000 nuclei and with DNA peaks with coefficients of variation (CVs)<3%, (ii) tissue preparation methods (i.e. chopping) and tissue types (i.e. fresh leaves) that will yield these values, (iii) replication of individuals across three different days, (iv) stains that are not basepair specific (e.g. propidium iodide, PI), and (v) internal plant standards that are as close as possible to the study species in DNA content, without overlap (Dolezˇel and Bartosˇ 2005; Johnston et al. 1999; Marie and Brown 1993; Chapter 4). These recommendations have been driven by the requirements of genome size studies and, at their most basic level, are intended to ensure that such estimates are accurate (i.e. reflect the true DNA content) and precise (i.e. have a variance low enough to allow for the resolution of small differences in absolute DNA content)."
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This is from a published paper, it requests that the term "ploidy" be used only when chromosomes have been counted and that "DNA ploidy" be used when the amount of DNA has been estimated - basically because DNA ploidy may not provide a correct estimate of the number of chromosomes or ploidy,
"There is an ever-increasing amount of cytogenetic data in plant sciences obtained by cytometric techniques, particularly flow cytometry. However, as these methods determine nuclear DNA amount irrespective of the number of chromosomes, a discrepancy between cytometric and karyological data may occur. To avoid potential bias, we appeal for consistency, distinguishing between the terms "ploidy / aneuploidy" referring to chromosome numbers and "DNA ploidy / DNA aneuploidy" to nuclear DNA content."
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The daylily ploidy characteristics need to be confirmed with valid observations of chromosome counts.
