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

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

Optimizing Different Medium Component Concentration and Temperature Stress Pretreatment for Gynogenesis Induction in Unpollinated Ovule Culture of Sugar Beet (<i>Beta vulgaris</i> L.)

The great economic importance of sugar beet determines the ongoing biotechnological studies conducted worldwide to improve the technology of obtaining doubled haploids (DHs) using the method of unpollinated ovule culture in vitro. To improve the induction of gynogenesis, we tested the effect of thidiazuron (TDZ), temperature bud pretreatment, different concentrations of sucrose, and culturing on liquid or solid medium. Three genotypes were tested in this study. The use of TDZ at a concentration of 0.4 mg/L in solid IMB (induction medium for Beta vulgaris) induction nutrient medium with 3 g/L phytagel, 50 g/L sucrose, 200 mg/L ampicillin and cultivation at 28◦C in the dark produced up to 16.7% induced ovules. The liquid nutrient medium of the same composition induced up to 8% ovules. Increasing TDZ concentration to 0.8 mg/L resulted in reduction or total inhibition of gynogenesis, depending on the genotype. Reducing the sucrose concentration to 20 g/L or increasing it to 80 g/L was not effective. In all three genotypes, the absence of temperature pretreatment of buds (5–6 °C) showed the best results. The plant regeneration with MS nutrient medium of 20 g/L sucrose, 3 g/L phytagel, 1 mg/L 6-benzylaminopurine (BAP) and 0.1 mg/L gibberellic acid (GA3) resulted in up to seven shoots from one induced ovule in the most responsive genotype. We showed by flow cytometry, chromosome counting and chloroplast number assessment that all regenerant plants were haploid (2n = x = 9)

Embryogenesis induction of carrot (Daucus carota L.) in isolated microspore culture

Haploid technologies are used to create homozygous lines for accelerated breeding. We aimed to optimize the technology for using the isolated microspore culture in vitro to obtain doubled haploids of the carrot (Daucus carota L.). We studied two carrot varieties with different responsiveness to embryogenesis, Altajskaya lakomka and Breeding line 17. Carrot microspores were isolated from buds and cultivated in liquid nutrient media supplemented with an antibiotic and activated carbon in vitro. They were exposed to different thermal treatments. The experiment showed the benefits of combining cold pre-treatment of buds (5°C for 1 day) with heat shock of isolated microspores in vitro (32°C for 2 days). The induction of embryogenesis on the NLN-13 medium was twice as high as on the MSm-13 medium. The use of 1% activated carbon in 0.5% agarose increased the yield of embryoids by more than 1.5 times. 100 mg/L of ampicillin was found to be the most efficient concentration. After 30 days of cultivation under optimized conditions, the yield was 161.3 and 44.0 embryoids per Petri dish for the cultivar Altajskaya lakomka and Breeding line 17, respectively. The induction of carrot embryogenesis is determined by the type and duration of thermal stress, the composition of the nutrient medium, the use of activated carbon as a sorbent, the addition of β-lactam antibiotics, and the type of explant exposed to thermal treatment. Our technology enabled us to obtain homozygous doubled haploid lines of carrots during a year, and these lines were included in the breeding process to create F1 hybrids