Haploid Embryogenesis in Isolated Microspore Culture of Carrots (Daucus carota L.).

Natalia Shmykova,Tatiana Vjurtts,Arthur Domblides,Elena Domblides

doi:10.3390/life11010020

Natalia Shmykova, Tatiana Vjurtts + Show 2 more

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https://doi.org/10.3390/life11010020

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Journal: Life	Publication Date: Dec 31, 2020
Citations: 11	License type: CC BY 4.0

Affiliation: Federal Scientific Center for Vegetable Growing

Abstract

The process of embryogenesis in isolated microspore culture was studied in eight carrot accessions of different origin. The ½NLN-13 medium supplemented with 0.2 mg/L 2,4D and 0.2mg/L kinetin was used to induce embryogenesis. The temperature treatment was performed at 5–6 °C for three days, followed by cultivation at 25 °C in darkness. As was shown, the first embryogenesis was only observed in microspores at the late vacuolated stage when the nucleus moved from the center to one pole following the long cell axis. Depending on the nucleus position, the microspore can divide into two equal or two different sized cells. Following divisions occurred either in one of these cells or in two. However, microspores that divided into two unequal cells were morphologically different form bi-cellular pollen grain. Embryogenic divisions in bi-cellular pollen grains were not observed. First divisions began by the third day of cultivation, and continued until the globular embryoid stage that was well-seen after the fourth week of cultivation. The already-formed embryoids can develop the secondary embryoids on their surface. Depending on the genotype, up to 1000 secondary embryoids can be produced from one embryoid in the liquid MSm medium supplemented with 0.1 mg/L of kinetin for regeneration. All carrot accessions studied were split into three groups: responsive genotypes, weakly responsive genotypes, and reluctant genotypes. The highest yield was 53 initial embryoids per a 6 cm diameter petri dish. Thus, the Nantskaya 4 cultivar totally produced 256 initial embryoids, out of which 94 developed into green plantlets and 162 into albino plantlets, whereas 97 initial embryoids with 45 albino plantlets formed from them were obtained from Chantenay cultivar.

Highlights

In world practice, despite the fact that carrots (Daucus carota L.) are an economically important vegetable crop, doubled haploid plants (DH-plants) in isolated microspore culture in vitro were first obtained only at the end of the first decade of the twenty-first century
The first studies began in the eighties in Denmark and Russia, in the nineties in Japan, which showed the possibility of obtaining homozygous plants in the anther culture [1,2,3]
It was found that the induction of embryogenesis is possible only in carrot microspores, which are at a late stage of vacuolization during the movement of the nucleus from the center of the cell to the pole along the length of the cell axis

Summary

Introduction

Despite the fact that carrots (Daucus carota L.) are an economically important vegetable crop, doubled haploid plants (DH-plants) in isolated microspore culture in vitro were first obtained only at the end of the first decade of the twenty-first century. The first studies began in the eighties in Denmark and Russia, in the nineties in Japan, which showed the possibility of obtaining homozygous plants in the anther culture [1,2,3]. The yield of doubled haploid plants in the carrot anther culture was low. The anther culture has a big drawback, since embryoids or callus can develop from microspores, and from somatic tissues of the anther [7], which complicates their use in breeding. Tangible results in isolated microspore culture in vitro of carrots were obtained much later

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