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

Abstract

In the present paper attempts were made to induce chromosome doubling of 'Meiwa' kumquat (Fortunella crassifolia) protoplasts and 'Frost' navel orange (Citrus sinensis Osbeck) embryogenic callus via colchicine treatment. Colchicine decreased protoplast viability, delayed protoplast division and inhibited callus growth, indicating presence of toxicity to cells. Cell lines established from 'Meiwa' protoplasts treated with 0.01 and 0.1% colchicine for 8, 16 and 24 h at each concentration showed different responses when they were cultured on embryoid-induction medium. Flow cytometry (FCM) demonstrated that tetraploids were detected in cell lines and embryoids from all of the treatments, with the highest frequency being 19.23%. As for 'Frost', tetraploid cells were only detected when the callus was treated with 0.1% colchicine for either 4 or 8 days, from which plantlets were regenerated. FCM and chromosome counting confirmed them as true tetraploids. The diploid cells were more active in mitotic division during a 12-day culture and smaller in size than their tetraploid counterpart. Potential applications of the novel tetraploid germplasms obtained through in vitro chromosome doubling to citrus cultivar improvement are discussed.

Tetraploidization in citrus rootstocks: effect of genetic constitution and environment in chromosome duplication

“…These are characterized as being 'spontaneous polyploids', or autopolyploid, for being genetically identical to the mother plant. This result is in accordance to those obtained by Zeng et al (2006), Allario et al (2011) and Hussain et al (2011). In this study, the use of microsatellite markers was efficient in identifying the molecular profile of the tetraploid plants.…”

“…However, plants with a different number of chromosomes, such as triploids (2n=3x=27) and tetraploids (2n=4x=36), are of natural occurrence, originated by both sexual and somatic ways. Polyploids of sexual origin can be obtained through the fusion of an unreduced gamete with a normal haploid or of two unreduced gametes; polyploids of somatic origin, or 'spontaneous polyploids', can appear through natural processes of mutation through the duplication of chromosomes in nucellar cells and the lack of division during mitosis, originating tetraploid plants (Cameron and Frost 1968, Lee 1988, Zeng et al 2006). …”

Induction of tetraploid hardy rubber tree, <i>Eucommia ulmoides</i>, and phenotypic differences from diploid

“…When seeds were treated in 0.01% colchicine for 24 h, about half of the seeds germinated, but only five individuals were tetraploid. Colchicine causes serious cellular toxicity to plants, so higher concentrations and longer treatment times could lead to a lower survival rate, despite effective induction of polyploids (Zeng et al 2006). For Acacia crassicarpa (Fabaceae), the effective concentration was 0.001-0.020% (Harbard et al 2012).…”

Tetraploid production through zygotic chromosome doubling in <i>Populus</i>

“…In plants, tetraploids can be induced by treating seeds, shoots, somatic embryos, calli and protoplasts with chemical mutagenic agents (Särkilahti and Valanne 1990;Kadota and Niimi 2002;Zeng et al 2006;Ewald et al 2009;Dhooghe et al 2010;Kanchanapoom and Koarapatchaikul 2012). In Populus, Mattila (1961) treated seeds of P. tremula × P. tremuloides with 0.1% colchicine solution for 1 h before sowing, resulting in some tetraploid seedlings.…”