CMA Banding Pattern of Chromosomes Is Useful for the Identification of Chromosome Doubling in Haploid Citrus

Abstract

A variant plant showing long internodes arose from a haploid plant obtained from clementine (Citrus clementina). Flow cytometric analysis and chromosome count revealed that it was a mixoploid with both haploid (2n = x = 9) and diploid (2n = 2x = 18) cells. The average length of the guard cells of the variant was almost the same as that of the haploid plant. This suggested that the epidermis of the variant consisted of haploid cells and that other leaf tissues consisted of diploid cells. Since in citrus the first germ layer forms the epidermis and the second and third layers form all the leaf tissues except for the epidermis, the variant was considered to be a ploidy periclinal chimera. When chromosomes of the young leaves were stained with chromomycin A 3 (CMA), they could be classified into four types based on the number and the position of the CMA-positive bands. The chromosome configuration of the variant differed from that of the original diploid clementine. The number of chromosomes of each type in the diploid cells of the variant was twice the number of those in the haploid plant. These results demonstrated that the diploid cells of the variant were doubled haploid cells, and that CMA analysis of chromosomes is useful for the identification of doubled haploids in citrus.

Recovery and characterization of a Citrus clementinaHort. ex Tan. 'Clemenules' haploid plant selected to establish the reference whole Citrus genome sequence

“…Duplication of the chromosome stock of nucellar cell-producing embryos is frequent in apomictic diploid plants of citrus [ 60 ]. However, it is rare in somatic vegetative tissue of diploid plants [ 61 ] although Yamamoto and Tominaga [ 62 ] obtained a haploid-diploid periclinal chimera from a haploid plant recovered in a cross between clementine and a triploid hybrid. This double haploid line is still young and, therefore has not been characterized yet from the reproductive point of view.…”

Haploids in flowering plants: origins and exploitation

“…It has been known for many years that contrary to initial expectations, the plants derived from the various in vitro techniques are not all haploid and that a range of ploidies, including ploidy chimaeras (Yamamoto and Tominaga, 2004), can be generated. A good example of this is provided by an analysis of microspore‐derived plants regenerated from cultured anthers of Datura innoxia .…”

Production of a Doubled Haploid from a Haploid Pummelo Using Colchicine Treatment of Axillary Shoot Buds

“…In previous studies (Germana and Chiancone, 2003;Germana et al, 1994Germana et al, , 2000, isozyme, RAPD, and microsatellite analysis were used for the identifi cation of doubled haploids in Citrus. Recently, a combination of enzymatic maceration and fl uorescent staining, such as CMA and/or 4´-6-diamidino-2-phenyl-indole (DAPI), has been used for identifying Citrus chromosomes and has been applied for biotechnological studies such as genome analysis, somatic hybridization, and ploidy manipulation (Befu et al, 2000;Guerra, 1993;Miranda et al, 1997aMiranda et al, , 1997bYahata et al, 2005b;Yamamoto and Tominaga, 2004;Yang et al, 2002). By CMA staining of the chromosomes, Yamamoto and Tominaga (2004) demonstrated that diploid cells in a haploid-diploid periclinal chimera were doubled haploid cells arisen from a haploid plant obtained from clementine.…”

“…Recently, a combination of enzymatic maceration and fl uorescent staining, such as CMA and/or 4´-6-diamidino-2-phenyl-indole (DAPI), has been used for identifying Citrus chromosomes and has been applied for biotechnological studies such as genome analysis, somatic hybridization, and ploidy manipulation (Befu et al, 2000;Guerra, 1993;Miranda et al, 1997aMiranda et al, , 1997bYahata et al, 2005b;Yamamoto and Tominaga, 2004;Yang et al, 2002). By CMA staining of the chromosomes, Yamamoto and Tominaga (2004) demonstrated that diploid cells in a haploid-diploid periclinal chimera were doubled haploid cells arisen from a haploid plant obtained from clementine. In the present study, we could also easily identify that the diploid strain was the complete doubled haploid of a haploid from ʻBanpeiyuʼ pummelo by using CMA staining.…”