The spontaneous diploidization rates in oilseed rape (Brassica napus L.) via in vitro androgenesis are too low for practical applications. In contrast, artificial doubling of chromosomes of the microspore has proven to be more successful and allows homozygous plants to be obtained in a short time. Here, we present the efficiency of diploidization of B. napus haploids using three different chromosome doubling methods.
Using the in vitro approach in microspores, the rate of chromosome doubling in 24 populations of androgenic plants ranged from 15.8% to 94.0%. An alternative in vivo method for the induction of chromosome doubling involves colchicine treatment of young haploid plants, and this yielded doubling rates ranging from 47.5% to 86.4% in 10 different plant populations. Another in vivo method of chromosome doubling is colchicine treatment of the excised young axillary shoots of haploid plants at the early flowering stage. The high efficiency of this method was confirmed in haploid plant populations from 11 genetically distinct donors in which the frequency of occurrence of diploids ranged from 53.3% to 100%. However, in this case, the time required for seed formation from doubled haploids increased by about 3–5 months. The availability of several methods of chromosome doubling at various stages of the androgenic process – from isolated microspores through to young plants and flowering plants – allows seeds to be obtained from nearly every selected individual haploid.
It has long been observed that toxic heavy metals at different concentrations can induce root hair development in plants. In oilseed rape we studied ethylene levels and root hair initiation under Cd2+ stress. Growth of the primary root was inhibited but close to root tips the development of subapical root hairs was significantly stimulated by Cd2+ at 30 μM. Versus the control, the distance between the root tip and the root hair zone and the length of the epidermal cell in the elongation zone were significantly reduced by Cd2+ at the same concentration. Exogenous application of Cd2+ and 1-aminocyclopropane-1-carboxylate (ACC) to roots had similar effects on subapical root hair development. Hair density increase and hair elongation in the presence of Cd2+ were reduced by the ethylene inhibitors CoCl2 at 15 μM and aminooxyacetic acid (AOA) at 10 μM. Exposing roots to Cd2+ caused a rapid increase in superoxide radical (O2 ·-) production in the root hair differentiation zone, and at the tips of emerging and newly formed root hairs. Cd2+-induced O2 ·- production at the growing hair tips was blocked in the presence of AOA. Our findings suggest that Cd2+-induced ethylene signaling may act upstream of O2 ·-. Cd2+ promotion of O2 ·- production may operate through an ethylene signaling pathway, and O2 ·- itself may stimulate root hair elongation.
Isozyme, RAPD and AFLP markers were evaluated and compared for their ability to determine genetic similarity in a set of 18 parental lines of winter oilseed rape F<sub>1</sub> hybrids developed using CMS ogura. Five isozyme systems, 64 RAPD starters and 23 EcoRI+3/MseI+3 AFLP primer combinations generated 597 polymorphic markers. These polymorphic fragments were chosen for estimation of genetic similarity. Of the total number of polymorphic products, polymorphic zymograms constituted only 3.0% of the markers, 57 RAPD primers 37.7%, and 23 AFLP primer combinations 59.3%. The size of RAPD polymorphic products ranged from 564 to 2100 bp. On average there were four amplified bands per primer, with 61.0% polymorphism. The AFLP polymorphic fragments ranged from 72 to 1352 bp in size. AFLP assays generated 15 bands per primer pair on average and detected roughly four times more bands than with RAPD analysis. The genetic similarity coefficients based on all marker data range from 0.52 to 0.84. The correlation of genetic similarities based on RAPD and AFLP markers was 0.58. Estimated genetic similarity based on isozyme data was not correlated with genetic similarity derived from the two DNA-based markers. The dendrogram constructed with the three types of markers taken together grouped all the winter oilseed rape parental lines into several similar clusters. The genomic distribution and frequency of the RAPD and AFLP markers can serve well as estimators of genetic similarity between parental lines of F<sub>1</sub> CMS ogura hybrids
We investigated direct and indirect formation of somatic embryogenesis in Brassica oleracea var. botrytis (cauliflower), a very important vegetable crop worldwide. Direct somatic embryogenesis, which is rather rare, was achieved in culture of 2-week-old hypocotyl explants of Brassica oleracea var. botrytis on MS medium supplemented with 1.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5; 1.0; and 1.5 mg/l kinetin. Initial induction of embryogenic callus was achieved on MS supplemented with very low concentrations of 2,4-D (0.05 mg/l and 0.1 mg/l). Indirect somatic embryogenesis from leaf sections was obtained on MS supplemented with 0.05 or 0.1 mg/l 2,4-D. We examined various stages of somatic embryos (globular, heart, torpedo, cotyledonary). More embryos per explant were produced through the indirect pathway (23-25) than through the direct pathway (14-19). The number of embryos produced was high. There is a potential for recurrent, repeated or secondary somatic embryogenesis, possibly an unlimited source for mass propagation and ideal for synthetic seed production in this species. Plant regeneration was achieved on half-strength MS medium without any hormones.
To keep genetic diversity, flowering plants have developed a self-incompatibility system, which can prevent self-pollination.
It has been reported that calcium concentration in pistil papilla cells was increased after self-pollination
in transformed self-incompatible Arabidopsis thaliana. In this study, we found that CML27 changed its expression
level for both mRNA and protein when compared to transcriptome and proteome. At the same time, CML27 was
expressed in the anther and pistil at a high level and reached up to 5-fold up-regulated expression in the pistil
at 1 h post-pollination when compared to 0 min. In order to find out potential proteins that may interact with
BoCML27, BoCML27 was expressed in and isolated from E. coli. After its co-incubation with Brassica oleracea
pistil proteins, the products were separated on SDS-PAGE gels. We found a specific band at the position between
130–180 kDa. Through LC-MS-MS (Q-TOF) analysis, eight proteins were identified from the band. The proteins
include 26S proteasome non-ATPase regulatory (26S), Phospholipase D, alpha 2 (PLDα2) involved in Ca2+ binding
and Coatomer subunit alpha-2-like (Coatomer) involved in vesicle mediated transport. All of these identified
proteins provide new insights for the self-incompatibility response in B. oleracea, specific for increasing Ca2+
concentration in pistil papilla cells.