Isolation of cytoplasts from Satsuma mandarin (Citrus unshiu Marc.) and production of alloplasmic hybrid calluses via cytoplast–protoplast fusion

Abstract

Cytoplasm of Satsuma mandarin (Citrus unshiu Marc.) is known to influence seedlessness. Transfer of cytoplasm to a seedy cultivar could possibly lead to the production of seedless citrus fruits. In the present paper cytoplasts were isolated from cell suspension-derived protoplasts of Satsuma mandarin via ultra-centrifugation in a discontinuous gradient. No nucleus could be detected in the cytoplasts by DAPI (4', 6-diamidino-2-phenylindole) staining compared with normal protoplasts. The cytoplasts, with high viability and small size, did not divide during solid embedding culture. Cytoplasts of Satsuma mandarin were electrically fused with embryogenic protoplasts of Murcott tangor (C. reticulata x C. sinensis), which led to regeneration of several cell lines. Flow cytometry (FCM) indicated that the cell lines were diploids. Simple sequence repeats (SSR) and cleaved amplified polymorphism sequence (CAPS) showed that the cell lines got their nuclear DNA from the protoplast parent, whereas the cytoplast parent donated the mtDNA, confirming transfer of mtDNA from Satsuma mandarin into Murcott tangor via cytoplast-protoplast fusion though no polymorphism was detected in chloroplast DNA between the fusion partners. This is the first report on isolation and characterization of cytoplasts, together with cytoplast-protoplast fusion in Citrus, which has a potential for citrus cultivar improvement involving cytoplasm transfer via cytoplast-protoplast fusion.

In vitro induction, regeneration and analysis of autotetraploids derived from protoplasts and callus treated with colchicine in Citrus

“…Flow cytomery (FCM) analysis of ploidy and cell cycle FCM was used to analyze the ploidy of cell lines, embryoids or plantlets as reported before Xu et al 2005Xu et al , 2006. In order to analyze cell cycle, diploid and tetraploid cell lines derived from colchicine-treated 'Meiwa' protoplasts were cultured in SCM and subcultured for three cycles at 12-day intervals before FCM analysis.…”

“…Chromosomes of at least 20 cells with clear metaphases were counted using a compound microscope (Olympus, BH-2) equipped with a · 40 objective. Viability of protoplast was Xu et al (2006). Nearly 300 protoplasts in three observation scopes were counted.…”

Influence of Mitochondria Origin on Fruit Quality in a Citrus Cybrid

“…In citrus, cybrids could be obtained by asymmetric fusion between irradiated protoplast withadestroyednucleusandaniodoacetate-treatedprotoplast (7,8), but most citrus cybrids have been obtained by symmetric hybridization (9)(10)(11). Recently, the production of an alloplasmic hybrid via cytoplast-protoplast fusion has been developed in citrus (12). In the case of somatic hybridization between a mesophyll protoplast and an embryogenic callus protoplast, the cybrid systematically contained mitochondria from the embryogenic callus (13,14).…”

Protoplast Fusion Technology – Somatic Hybridization and Cybridization

“…Cybrids can be produced by the donor-recipient method [33,34] (see Figure 10.2) or by cytoplast-protoplast fusion [35] but can also occur spontaneously from intraspecific, interspecific or intergeneric symmetric hybridization [36]. This is a common phenomenon in some species especially, tobacco and citrus.…”