Factors affecting DH plants in vitro production from microspores of European radish.

E V Kozar,E A Domblides,A V Soldatenko

doi:10.18699/vj20.592

Abstract

Over the recent years the market demand for scaling up the production of European radish (Raphanus sativus L.) varieties and hybrids for open and protected production, varying in ripeness group, root shape and color, has drastically increased. Therefore, the expansion of genetic diversity and acceleration of the selection process are important. Doubled haploid technology considerably curtails the time required for creation of homozygous constant parental cell lines when in vitro microspore culture is used as the most promising method. For the first time, we were able to realize the full production cycle of DH plants of European radish by in vitro microspore culture up to inclusion of the produced material into the selection process. We have selected: preferable flower bud size, heat shock parameters, induction and regeneration media. It was revealed that linear length on the flower buds with the best possible stage of microspore development is genotype-specific: the flower bud length 2.8–3.3 mm is optimal for accessions of Rhodes and 3.7–4.2 mm is optimal for accessions of Teplichny Gribovsky. Heat shock at 32 °C for 48 hours is the most suitable for most genotypes. For the first time Murashige and Skoog based culture medium has been used for embryogenesis induction, and a major dependence of embryogenesis induction on the genotype × medium interaction was found. At regeneration and tiller stage it is advisable to add 1 mg/mL of benzylaminopurine and 0.1 mg/L of gibberellic acid to the medium, and rotting of micro-sprouts is performed with the use of hormone-free medium. Analysis of the produced regenerant plants by chromosome count and cell nucleus flow cytometry showed that 69 % of plants have a diploid chromosome set, 9 % have a haploid chromosome set, and 22 % have mixoploids and aneuploids chromosome sets. The seed progeny from doubled haploids and mixoploids were obtained by self-pollination, where all R1 plants had a doubled set of chromosomes. This study launches the development of an efficient method of radish doubled haploid production to be used in the selection process.

Highlights

In modern crop breeding, the priority is to create F1 hybrids that differ from the cultivars in high yield and evenness of plants in terms of ripening and quality of productive organs
To identify the significance of the qualitative composition of isolated microspores for the induction of embryogenesis, we evaluated the yield of embryoids in an in vitro microspore culture, isolating microspores from buds of various sizes using the example of the Teplichny Gribovsky variety (Fig. 1)
It has been shown that for the European radish the linear size of buds that contain the maximum concentration of microspores at the optimal stage of development for embryogenesis is genotype-specific

Summary

Introduction

The priority is to create F1 hybrids that differ from the cultivars in high yield and evenness of plants in terms of ripening and quality of productive organs. Technologies for double haploid production (DH technologies) are currently widely used to accelerate breeding (Dunwell, 2010), which makes it possible to accelerate the selection process by at least 3–4 years (Ferrie, Möllers, 2011). The main methods for obtaining haploids and their classification are considered in a number of reviews (Maluszynski et al, 2003; Dunwell, 2010; Asif, 2013). By the centenary year in 2020 from the establishment of Federal Scientific Vegetable Centre (VNIISSOK) hybrids in major vegetable crops such as head cabbage, broccoli, sweet pepper, winter squash and others have been developed with the use of doubled haploids (Domblides al., 2017)

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