Improved doubled haploid production protocol for <i>Brassica napus</i> using microspore colchicine treatment <i>in vitro</i> and ploidy determination by flow cytometry

Weber, S.; ÜNker, F.; Friedt, W.

doi:10.1111/j.1439-0523.2005.01114.x

Cited by 53 publications

(38 citation statements)

References 11 publications

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“…The results are consistent with previous reports on the positive effect of colchicine on embryo development (Zaki & Dickinson 1995;Zhou et al 2002), direct plant regeneration (Weber et al 2005) and improvement of embryo germination by trifluralin (Eikenberry 1994). The lower embryo frequency in the untreated control in comparison with in vitro colchicine-treated variants described by Zhou et al (2002) in spring rape was not proved.…”

Section: Discussionsupporting

confidence: 83%

“…Similar results were reported by Weber et al (2005) in spring Canadian cultivars. They further revealed that spontaneous diploidization showed a large variation depending on the genotype.…”

Section: Discussionsupporting

confidence: 80%

“…It was revealed later that colchicine added directly to microspore cultures improved embryogenesis and increased the diploidization rate up to 80-95% (Chen et al 1994;Möllers et al 1994;Weber et al 2005). Several microtubule depolymerising herbicides were also proved to be efficient for in vitro chromosome doubling of microspores.…”

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confidence: 98%

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Chromosome doubling effects of selected antimitotic agents in Brassica napus microspore culture

Klíma¹,

Vyvadilová²,

Kučera³

2008

CZECH J GENET PLANT

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Effects of microspore culture treatment with antimitotic agents colchicine, trifluralin and oryzalin on the frequency of embryo formation, embryo development, plant regeneration and diploidization rate in three F 1 hybrids of winter rapeseed cultivars were compared. The ploidy level analysis of 1709 flowering microspore-derived plants showed that in vitro applications of all antimitotic drugs increased the rate of doubled haploid (DH) plants significantly. The mean rate of DH plants from the trifluralin treatment was 85.7%, from colchicine 74.1% and 66.5% in the case of oryzalin, while only 42.3% in the untreated control variant whereas in vivo additional application of colchicine at the plantlet stage did not significantly increase the mean rate of DH plants (55.6%). Although there were no significant differences in diploidization efficiency between the in vitro applications of particular antimitotic agents, trifluralin showed to be the most suitable because of its positive effect on embryo development and conversion into whole plants. In addition, the diploidization rate was sufficient and stable in all genotypes tested. The results indicate that the trifluralin treatment of microspore cultures could provide efficient chromosome doubling for the production of doubled haploid lines from winter oilseed rape breeding materials.

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Section: Discussionsupporting

confidence: 83%

“…Similar results were reported by Weber et al (2005) in spring Canadian cultivars. They further revealed that spontaneous diploidization showed a large variation depending on the genotype.…”

Section: Discussionsupporting

confidence: 80%

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Chromosome doubling effects of selected antimitotic agents in Brassica napus microspore culture

Klíma¹,

Vyvadilová²,

Kučera³

2008

CZECH J GENET PLANT

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“…Indeed, it is thought that the most flowering plant species have one or several rounds of polyploidisation in their ancestry (1,2). This adaptability to ploidy change has led to a number of plant research disciplines that analyze ploidy changes, including taxonomy [ploidy series (3)], plant development [endopolyploidy (4)], and plant breeding/genetics [doubled haploidy (5)(6)(7)(8)]. …”

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confidence: 99%

An efficient high‐throughput flow cytometric method for estimating DNA ploidy level in plants

Cousin¹,

Heel

Cowling

et al. 2009

Cytometry Pt A

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We present an efficient high-throughput flow cytometric method that builds on previously published methods and permits rapid ploidy discrimination in plants. By using Brassica napus L. microspore-derived plants as an example, we describe how 192 leaf tissue samples may be processed and analyzed comfortably by one operator in 6 h from tissue sampling to ploidy determination. The technique involves placing young leaf samples in two 96-well racks, using a bead-beating procedure to release nuclei into a lysis solution, filtering the samples on 96-well filter plates, staining with propidium iodide, and then rapidly estimating DNA ploidy using a plate loader on a BD FACSCanto II flow cytometer. Throughout the sample preparation process, multichannel pipetting allows faster and less error-prone sample handling. In two 96-well plates of samples, the histogram peaks of DNA content from flow cytometry were wellresolved in 189 of 192 samples tested (98.4%), with CV values ranging from 2.98% to 6.20% with an average CV of 4.35% (SD 5 0.68%). This new method is useful in doubled haploid plant breeding programs where early discrimination of haploid and doubled haploid (i.e., diploid) plantlets can confer significantly improved operational efficiencies. We discuss how this method could be further refined including adapting the method to robotic sample processing. Flow cytometry (FCM) is the predominant method for measuring nuclear DNA content (9). FCM involves staining cells with a DNA-specific fluorescent dye and separating the cells into single file within the liquid core stream of a flow chamber, in which they are intercepted by a high-intensity light source or laser focused on a small region known as the observation point. The laser excites the fluorescent dye that is bound to the DNA from which light scatters and fluorescence emissions are measured (10). FCM of plant cells is made difficult by structural irregularities (e.g., interlinked cells of irregular shape and rigid cell walls) typical of plant tissues. Furthermore, plant cells are frequently larger than the orifices in the flow chamber (50-100 lm diameter) of most flow cytometers (11-13). These problems associated with large and irregular cells may be overcome by isolating the nuclei that are smaller and more regularly shaped (13). In early protocols, nuclear suspensions were prepared from plant single-cell suspensions (protoplasts) using enzymes to digest cell wall materials.

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“…During the development of DH lines in rapeseed, diploization rates are increased by a treatment with colchicine, a natural alkaloid extracted mainly from the plant Colchicum autumnale, which depolymerises microtubules and disrupts regular mitotic cell division. Möllers et al 1994;Zhao et al 1996aZhao et al , 1996bHansen and Andersen 1996;Zhou et al 2002aZhou et al , 2002bWeber et al 2005;Seguí-Simarro and Nuez 2008). As discussed by Möllers and Iqbal (2009), the most efficient protocol for the colchicine treatment is a treatment of the freshly isolated microspores during the first days of cultivation, which will lead to entirely diploid plants with full fertility and diploidization rates of up to 90 %.…”

Section: Introductionmentioning

confidence: 99%

Identification and evaluation of intervarietal substitution lines of rapeseed (Brassica napus L.) with donor segments affecting the diploidization rate of isolated microspores

et al. 2016

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In Brassica species microspore derived doubled haploid lines are an important tool in breeding and research. A limiting step in the production of doubled haploid lines is the diploidization of the microspores. Strong differences have been observed in diploidization rates between different genotypes but little is know about the genetic control of these differences. To identify genomic regions that carry genetic factors controlling the diploidization rate of isolated microspores of rapeseed, marker segregations were compared between segregating populations of diploid and haploid microspore derived embryos and a BC 1 from a cross between 'Express 617' and 'RS239'. After map construction ten intervarietal substitution lines from the same cross were selected with donor segments covering five genomic regions that showed a pattern of skewed marker segregations across the three populations indicative of the segregation of genes controlling the diploidization rates. The diploidization rates of microspores of the ten lines ranged from 23.9 to 58.7 % while the recurrent parent 'Express 617' showed a rate of 52.5 %. For three lines the diploidization rates were significantly lower (P = 0.05) than the rate of 'Express 617'. By comparing donor segments between the significant and the non-significant lines, seven genomic regions that cover just between 4.17 and 6.16 % of the rapeseed genome were identified that may contain genetic factors controlling diploidization rates in rapeseed. In addition, one marker was found that has a high probability to be linked to such a factor. The significant lines represent an ideal material for further in depth studies of this trait.

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Improved doubled haploid production protocol for Brassica napus using microspore colchicine treatment in vitro and ploidy determination by flow cytometry

Cited by 53 publications

References 11 publications

Chromosome doubling effects of selected antimitotic agents in Brassica napus microspore culture

Chromosome doubling effects of selected antimitotic agents in Brassica napus microspore culture

An efficient high‐throughput flow cytometric method for estimating DNA ploidy level in plants

Identification and evaluation of intervarietal substitution lines of rapeseed (Brassica napus L.) with donor segments affecting the diploidization rate of isolated microspores

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