Identification of Polyploidy of in vitro Anther-derived Shoots of Asparagus officinalis L. by Flow Cytometric Analysis and Measurement of Stomatal Length

Abstract

Ploidy levels of in vitro shoots produced through anther culture of asparagus were determined by flow cytometry (FCM) analysis instead of counting the chromosome number of root tips that were difficult to regenerate. Of the 110 anther-derived shoots, there were 83 diploids, 21 tetraploids, 3 octaploids, and 3 mixoploids, but no haploids. When flower buds of different length were used as starting material for anther culture, the richest variation in polyploidy originated from short flower buds (1.0-1.5 mm). This result suggested that polyploids might develop by the fusion of nuclei during pollen-mother cell division before the tetrad stage. We used scanning electron microscopy (SEM) to measure the stomatal length of in vitro and field-grown shoots in order to clarify the differences among polyploids determined by FCM analysis. When the developmental stage of tissue was taken into account, we found statistically significant differences in stomatal length among all polyploids (diploid, tetraploid, and octaploid) for both stems and phyllodes (P < 0.001). Stomata on stems were longer than those on phyllodes, and mature tissue had longer stomata than immature tissue. Morphological observations with SEM found that immature tissue had guard cells set below epidermal cells, and were poorly developed. Field-grown diploids had much shorter stomatal length than in vitro diploids. Male plants grown in the field had significantly longer stomata than female plants in both their immature stems and mature phyllodes (P < 0.001). These results indicated that stomatal length can be a reliable ploidy index by removing any other influential factors such as sex, environment and developmental stage.

Genomic aspects of research involving polyploid plants

“…The counting of chloroplast number in the epidermal guard cells of stomata can be used as an indirect ploidy indicator for the rapid screening of polyploids (Ewald et al 2009;Ho et al 1990). Measurement of the stomatal diameter with scanning electron microscopy has also been used to clarify the differences among polyploids (Shiga et al 2009). Besnard et al (2008) used flow cytometry to estimate genome content and six highly variable nuclear microsatellites to assess the presence of multiple alleles at co-dominant loci in Olea europaea.…”

Mitotic chromosome doubling of plant tissues in vitro

“…), Pyrus communis(Sun et al 2009), Pyrus pyrifolia(Kadota and Niimi 2002), Vitis vinifera(Yang et al 2006), Musa(Hamill et al 1992), Morus alba(Chakraborti et al 1998), Asparagus(Shiga et al 2009), Pennisetum(Campos et al 2009), Zantedeschia…”

Production of “super-males” of asparagus by anther culture and its detection with SSR-ESTs

“…Furthermore, different ploidy levels in the plantlets regenerated from the same anther were detected. The regeneration of tetraploid and octoploid plantlets from diploid microspores, as well as the mix of ploidy levels, can be possible due to the high endoreduplication rate occurring in the callus culture in A. officinalis (Kunitake et al 1998;Raimondi et al 2001;Pontaroli and Camadro 2005;Regalado et al 2015b), also described in the anther culture of A. officinalis Wolyn and Feng 1993;Shiga et al 2009). Other important result obtained in this ploidy analysis is the increase of the spear diameter of the plantlets associated with ploidy level (Fig.…”