Efficient Methods for Evaluation on Ploidy Level of Cucurbita pepo L. Regenerant Plants Obtained in Unpollinated Ovule Culture In Vitro

Abstract

An important stage in doubled haploid (DH) production is to evaluate and to differentiate the ploidy level of regenerant plants at least two–three times during the technology. Therefore, rapid and reliable methods are necessary for particular species taken into the technology. In this study, Cucurbita pepo regenerants obtained through unpollinated ovule culture in vitro were evaluated including three different methods: direct chromosome counting in apical meristems, flow cytometry of the cell nucleus, and estimation of morphological parameters of the abaxial epidermis. Methods were optimized for each of three evaluations, and main criteria were determined for ploidy level differentiation. As a result, four ploidy levels, namely, 2n, 3n, 4n, and 8n, were defined among regenerant plants adapted to ex vitro conditions, while true haploids were only found among plants that remained in the in vitro culture. In total, 32.35%, 26.47%, 33.82%, 4.41%, and 2.94% of regenerant plants of courgette and patisson were diploid, triploid, tetraploid, octaploid, and aneuploid, respectively. According to results of flow cytometry of the cell nucleus, two cytotypes in diploid samples with DNA content of 2C = 1.07 ± 0.03 pg for courgette belonging to subsp. pepo and 2C = 0.95 ± 0.03 pg for patisson samples belonging to subsp. ovifera were revealed. The images of metaphase chromosomes of haploid, triploid, and tetraploid C. pepo specimens obtained using the propion–lacmoid chromosome staining method were presented for the first time. Parameters of abaxial epidermis in diploid samples of courgette and patisson grown in open-field and greenhouse conditions were described and compared. It was shown that the most robust parameter not depending on external factors was the number of chloroplasts in stomatal guard cells, which contained 9.41 to 11.31, 14.84 to 16.3, and up to 17.58 chloroplasts in diploid, triploid, and tetraploid samples, respectively. The application of several methods for estimation enables avoiding the misidentification of ploidy levels in adapted regenerant plants produced with the use of DH technology.

Spontaneous and Chemically Induced Genome Doubling and Polyploidization in Vegetable Crops

“…Since the dye stoichiometrically binds DNA, it allows for the differences in the DNA content between the standard and the analyzed sample to be distinguished (Figure 1G-G″, Figure 2C-F). Although rapid processing samples have made flow cytometry the most efficient approach for determining the ploidy, its use in many laboratories is still limited due to the high cost of equipment and challenges in mastering quality sample preparation and instrument management [165,166].…”

“…The root tips or developing flower buds are fixed, macerated with enzymes, and stained with DNA dyes for chromosome visualization. Direct chromosome counting is a very reliable method for ploidy determination, but this method is extremely time-consuming and technically challenging [166,167].…”

“…The smaller the number of stomata, the larger the stomata, and the larger the number of chloroplasts in stomatal guard cells, the more they are associated with higher ploidy (Figure 1H-H″). This method is widely used since it is fast and inexpensive, but it can be applied only to ex vitro plants raised under the same conditions [166,167].…”