ABSTRACT
Low oxygen level is a phenomenon often occurring during the cucumber cultivation period. Genes involved in adaptations to stress can be regulated by non-coding RNA. The aim was the identification of long non-coding RNAs (lncRNAs) involved in the response to long-term waterlogging stress in two cucumber haploid lines, i.e., DH2 (waterlogging tolerant-WL-T) and DH4 (waterlogging sensitive-WL-S). Plants, at the juvenile stage, were waterlogged for 7 days (non-primed, 1xH), and after a 14-day recovery period, plants were stressed again for another 7 days (primed, 2xH). Roots were collected for high-throughput RNA sequencing. Implementation of the bioinformatic pipeline made it possible to determine specific lncRNAs for non-primed and primed plants of both accessions, highlighting differential responses to hypoxia stress. In total, 3738 lncRNA molecules were identified. The highest number (1476) of unique lncRNAs was determined for non-primed WL-S plants. Seventy-one lncRNAs were depicted as potentially being involved in acquiring tolerance to hypoxia in cucumber. Understanding the mechanism of gene regulation under long-term waterlogging by lncRNAs and their interactions with miRNAs provides sufficient information in terms of adaptation to the oxygen deprivation in cucumber. To the best of our knowledge, this is the first report concerning the role of lncRNAs in the regulation of long-term waterlogging tolerance by priming application in cucumber.
Subject(s)
Cucumis sativus/genetics , Gene Expression Regulation, Plant , MicroRNAs/genetics , Oxidative Stress , RNA, Long Noncoding/genetics , Adaptation, Physiological , Cucumis sativus/metabolism , Gene Regulatory Networks , MicroRNAs/metabolism , RNA, Long Noncoding/metabolismABSTRACT
The production of haploid and doubled haploid plants is a biotechnological tool that shortens the breeding process of new cultivars in many species. Doubled haploid plants are homozygous at every locus and they can be utilized as parents to produce F1 hybrids. In this chapter, we describe a protocol for the production of doubled haploid plants in Brassica rapa L. subsp. pekinensis using androgenesis induced by isolated microspore cultures.
Subject(s)
Brassica rapa/growth & development , Brassica rapa/genetics , Plant Breeding/methods , Acclimatization/genetics , Brassica rapa/physiology , Crops, Agricultural/genetics , Crops, Agricultural/growth & development , Crops, Agricultural/physiology , Culture Media/chemistry , DNA, Plant/genetics , Diploidy , Glucose-6-Phosphate Isomerase/genetics , Haploidy , Homozygote , Molecular Biology/methods , Pollen/genetics , Pollen/growth & development , Polymerase Chain Reaction , Regeneration/genetics , Tissue Culture TechniquesABSTRACT
Haploid plants have a gametophytic number of chromosomes (n) in the sporophyte. A doubled haploid (DH) plant results from doubling the chromosome set of a haploid plant, as a consequence a homozygosity plant is produced at every locus (true homozygous plant). DH plants are of great significance in breeding programs for the improvement of plants. Here we describe a protocol for the production of doubled haploid plants in carrot (Daucus carota L.) using parthenogenesis induced by wide pollination.
Subject(s)
Daucus carota/embryology , Haploidy , Ovule/physiology , Parthenogenesis , Plant Breeding/methods , DNA, Plant/genetics , DNA, Plant/isolation & purification , Daucus carota/genetics , Flow Cytometry , Isoenzymes/metabolism , Pollination , Polymerase Chain Reaction , Tissue Culture TechniquesABSTRACT
In this work, we describe an improved protocol for induced parthenogenesis and ovule culture of carrot (Daucus carota L.). The effects of pollination with parsley pollen and/or 2,4-dichlorophenoxyacetic acid (2,4-D) treatment on the stimulation of parthenogenesis were studied using heterozygous donor plants of 30 varieties and breeding populations of carrots. Isolated ovules, cultured in vitro, enlarged and developed embryos or calli. The application of 2,4-D on pollinated flowers stimulated callus development but did not increase the frequency of embryo development from ovules and, thus, was not useful for increasing the frequency of haploid plant recovery. The efficiency of embryo development was accession-dependent and varied from 0 to 24.29%. In optimized conditions, most accessions responded by embryo development exclusively. The highest frequency of embryo development was observed from ovules excised from ovaries 20-22 d after pollination with parsley pollen. Among several media used for ovule culture, 1/2-strength Murashige and Skoog medium with 0.06 µM indole-3-acetic acid (IAA) was the best. It allowed the production of embryos at a similar frequency as on the media supplemented with kinetin, gibberellic acid, putrescine, or thidiazuron, but restricted callus development. Most plants obtained were haploids and diploids derived from parthenogenesis, as evidenced by homozygosity at three independent loci based on isozyme and PCR analyses. In total, considering haploids and embryo-derived homozygous diploids together, 72.6% of regenerated plants were of gametic origin.
ABSTRACT
Ten accessions belonging to the Brassica oleracea subspecies alba and rubra, and to B. oleracea var. sabauda were used in this study. Protoplasts were isolated from leaves and hypocotyls of in vitro grown plants. The influence of selected factors on the yield, viability, and mitotic activity of protoplasts immobilized in calcium alginate layers was investigated. The efficiency of protoplast isolation from hypocotyls was lower (0.7 ± 0.1 × 10(6) ml(-1)) than for protoplasts isolated from leaf mesophyll tissue (2 ± 0.1 × 10(6) ml(-1)). High (70-90%) viabilities of immobilized protoplasts were recorded, independent of the explant sources. The highest proportion of protoplasts undergoing divisions was noted for cv. Reball F1, both from mesophyll (29.8 ± 2.2%) and hypocotyl (17.5 ± 0.3%) tissues. Developed colonies of callus tissue were subjected to regeneration and as a result plants from six accessions were obtained.
