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.

Diploid and haploid embryogenesis in Larixleptolepis, L. decidua, and their reciprocal hybrids

Diploid and haploid embryogenesis was induced in two Larix species (L. decidua and L. leptolepis) and their reciprocal hybrids. Diploid embryogenic tissue was initiated in immature zygotic embryos isolated with the micropylar half of the megagametophyte left attached. These were placed either on modified LM or MSG medium supplemented with the growth regulators 2,4-D and 6-benzyladenine. MSG medium was solidified with either gellan gum or agar. There was no appreciable difference in response between the two. Haploid embryogenesis was induced in isolated megagametophytes placed on modified LM medium supplemented with 2,4-D and 6-benzyladenine. Diploid embryogenic tissue was subcultured on medium with growth regulators, but haploid embryogenic tissue grew well on medium without growth regulators. There were few morphological differences between the diploid and haploid embryogenic tissue. In all species and hybrids, haploid cultures contained more coenocytic long cells. Binucleate cells were most common, but tetranucleate and octanucleate cells were also present. Haploid cultures showed poorer organization than the diploid ones, with only a few cultures having well-developed embryoids. Haploid tissue originated from expanded cells of the megagametophyte. Diploid tissue originated from the suspensor region of the zygotic embryo; it proliferated from isolated clusters of meristematic cells in early embryoids. Diploid and haploid cultures differed not only from the outset, but also in the mature embryoids they produced.