Laima S. Kott

Parental effects in Pieris rapae in response to variation in food quality: adaptive plasticity across generations?

1. Herbivores using seasonal resources must cope with variation in the quality of their host plants. The effects of variation in protein concentration of artificial diet and glucosinolate concentration in canola, Brassica napus, on Pieris rapae parental and progeny growth were investigated.

Detection of molecular markers associated with linolenic and erucic acid levels in spring rapeseed (Brassica napus L.)

Undesirable characteristic of rapeseed oil is a relatively high level of linolenic acid (18:3), which is easily oxidized leading to rancidity and a shortened shelf life of the oil. Previous attempts to reduce linolenic acid levels in rapeseed oil through breeding have been impaired by complex genetics and strong environmental sensitivity of this trait. Therefore, our objective was to develop molecular markers for low linolenic acid that could facilitate the breeding of low linolenic rapeseed. Bulked segregant analysis was employed to identify two RAPD markers associated with 18:3 in a doubled haploid population segregating for linolenic and erucic acid levels. Based on analysis of individual DH lines, the markers RM350 and RM574, representing two independent loci, accounted for a total of 39% of the genetic variability in this population. This marker RM350 alone accounted for 25% genetic variation for this trait with no evidence of recombination. Significant interlocus interaction found between the markers RM350 and RM574 suggested that epistasis was involved in the genetic control of 18:3 level in this population. Another marker designated as RM322, which was independent of the other two, was found significantly associated with the erucic acid level and oil content. RAPD markers identified in this study should be a useful tool for the early detection of low linolenic, or low or high erucic acid genotypes in rapeseed breeding programs based on doubled haploids.

Desiccation of microspore derived embryos of oilseed rape (Brassica napus L.)

SummaryMicrospore-derived embryos from Brassica napus L. were dried to less than 15% moisture and stored dry for a minimum of 7 days. Successful plant regeneration was observed when embryos at the cotyledonary stage of development were treated with 50 uM ABA for 7 days prior to desiccation. Solid agar or liquid medium gave similar results. The rate of drying of embryos after ABA pretreatment had only minor effects on embryo survival, but for untreated embryos, slow drying gave a small degree of survival. These results are very comparable to those with alfalfa somatic embryos, suggesting that the ABA treatment of cotyledonary stage embryos may be broadly used as a pretreatment for inducing the expression of desiccation tolerance in plant embryos.

Embryogenesis following cryopreservation in isolated microspores of rapeseed (Brassica napus L.)

A simple procedure is described for cryopreservation of isolated microspores of rapeseed in liquid nitrogen without loss of embryogenic capacity (i.e. embryogenes is can still be induced following freezing). Microspores frozen in Lichters (1982) medium with 13% sucrose produced ca. 10% of the embryos yielded by an unfrozen control. Microspores frozen in Lichters medium with 13% sucrose, and supplemented with 0.5 M glycerol and 0.5 M DMSO produced no embryos. Regeneration of embryos obtained from frozen microspores yielded 88% diploid and 12% haploid plants, while embryos from unfrozen controls produced 7% diploids and 93% haploids. The potential to increase the efficiency of the rapeseed haploidy system using cryopreservation is discussed in light of these results.

Protection against LPS-induced cartilage inflammation and degradation provided by a biological extract of Mentha spicata

BackgroundA variety of mint [Mentha spicata] has been bred which over-expresses Rosmarinic acid (RA) by approximately 20-fold. RA has demonstrated significant anti-inflammatory activity in vitro and in small rodents; thus it was hypothesized that this plant would demonstrate significant anti-inflammatory activity in vitro. The objectives of this study were: a) to develop an in vitro extraction procedure which mimics digestion and hepatic metabolism, b) to compare anti-inflammatory properties of High-Rosmarinic-Acid Mentha spicata (HRAM) with wild-type control M. spicata (CM), and c) to quantify the relative contributions of RA and three of its hepatic metabolites [ferulic acid (FA), caffeic acid (CA), coumaric acid (CO)] to anti-inflammatory activity of HRAM.MethodsHRAM and CM were incubated in simulated gastric and intestinal fluid, liver microsomes (from male rat) and NADPH. Concentrations of RA, CA, CO, and FA in simulated digest of HRAM (HRAMsim) and CM (CMsim) were determined (HPLC) and compared with concentrations in aqueous extracts of HRAM and CM. Cartilage explants (porcine) were cultured with LPS (0 or 3 μg/mL) and test article [HRAMsim (0, 8, 40, 80, 240, or 400 μg/mL), or CMsim (0, 1, 5 or 10 mg/mL), or RA (0.640 μg/mL), or CA (0.384 μg/mL), or CO (0.057 μg/mL) or FA (0.038 μg/mL)] for 96 h. Media samples were analyzed for prostaglandin E2 (PGE2), interleukin 1β (IL-1), glycosaminoglycan (GAG), nitric oxide (NO) and cell viability (differential live-dead cell staining).ResultsRA concentration of HRAMsim and CMsim was 49.3 and 0.4 μg/mL, respectively. CA, FA and CO were identified in HRAMsim but not in aqueous extract of HRAM. HRAMsim (≥ 8 μg/mL) inhibited LPS-induced PGE2 and NO; HRAMsim (≥ 80 μg/mL) inhibited LPS-induced GAG release. RA inhibited LPS-induced GAG release. No anti-inflammatory or chondroprotective effects of RA metabolites on cartilage explants were identified.ConclusionsOur biological extraction procedure produces a substance which is similar in composition to post-hepatic products. HRAMsim is an effective inhibitor of LPS-induced inflammation in cartilage explants, and effects are primarily independent of RA. Further research is needed to identify bioactive phytochemical(s) in HRAMsim.

Induction of desiccation tolerance in microspore-derived embryos of Brassica napus L. by thermal stress

Abstract The use of dry microspore derived embryos as cloning materials greatly facilitates long term storage and transportability. The effects of heat and cold pretreatments on microspore-derived embryos of Brassica napus L. were investigated in an attempt to induce desiccation tolerance in these embryos before drying to about 12% moisture. Heat treatment was optimal between 30 and 33°C for shoot survival and plant regeneration from dry embryos. Induction of desiccation tolerance also was achieved with 2 day exposure to 2°C; however, its effectiveness was relatively less than heat shock treatment. Cold stratification at 4°C for 10 days enhances germination of embryos whetther they were previously dried or not. Following desiccation, plant regeneration frequencies varied considerably among 4 winter lines tested, which may have been associated with genotype factors that influence synchronized embryo development and maturation. The results presented here demonstrate that thermal stress is capable of expressing an enhanced desiccation tolerance in microspore derived embryos of Brassica napus L. and this response is related to genotype, stress temperature, age of the embryos and stratification.

Development of haploid cell lines from immature barley,Hordeum vulgare, embryos.

Callus and suspension cell lines were derived from haploid barley embryos produced by the Bulbosum method. Embryos 1 to 2 mm long callused on medium containing a low concentration of 2,4-dichlorophenoxyacetic acid (2,4-D). Fast-growing nodular, beige callus (Type 1), slow-growing, light brown, watery callus (Type 2) and a dense, light yellow, nodular callus (Type 3) were recovered. Type 3 callus was embryogenic and was produced on embryos 1 to 2 mm in length. Although callus cultures gradually became polyploid, a small proportion of haploid cells was retained and the majority of regenerated plantlets were haploid. The organogenic potential of long-term (Type 1) callus cultures was generally low and decreased with time. Attempts to inducedenovo shoot formation in Type 1 cultures were not successful.

Environmental Factors Affecting the Accumulation of Rosmarinic Acid in Spearmint (Mentha spicata L.) and Peppermint (Mentha piperita L.)

Four spearmint, and two peppermint clonal lines, selected for enhanced rosmarinic acid content (50-120 mg g -1 rosmarinic acid DW), where up to 80% of the antioxidant activity was correlated to rosmarinic acid content, were examined to determine the effects of environmental and physiological conditions on the accumulation of rosmarinic acid in leaf tissues. Exposure to a short photoperiod of 12 hours in comparison to 16 hours reduced rosmarinic acid accumulation in two mint lines, but no significant difference was found between photoperiods of 14 and 16 hours. The physiological age of the plant strongly influenced the accumulation of rosmarinic acid with the highest levels recorded in the vegetative state, and a significant reduction in the concentration of rosmarinic acid in the leaves in both the bud initiation and flowering stages in the mint lines. Cold stress, impacted over a six week period had no effect on rosmarinic acid production. A field study of the commercial chemotype 700B indicated that soil type plays an essential role in the accumulation of rosmarinic acid in the leaf tissue, probably due to retention of moisture which favours rosmarinic acid production. For producers and extractors, taking these factors into account would significantly increase rosmarinic acid accumulation in commercially high rosmarinic acid mint and increase the quality control of plant extracts for the natural products industry.

Oral rosmarinic acid-enhanced Mentha spicata modulates synovial fluid biomarkers of inflammation in horses challenged with intra-articular LPS

A biological extract of high-rosmarinic acid mint (HRAM) has previously demonstrated inhibitory effects on lipopolysaccharide (LPS)-induced prostaglandin E(2) (PGE(2)), nitric oxide (NO) and glycosaminoglycan (GAG) release in vitro. This study was undertaken to determine whether HRAM added to feed produces similar effects in horses challenged with intra-articular LPS. Eight horses received HRAM (0 or 28.1 ± 1.3 g/day; n = 4 per group) in their feed for 24 days in a blinded manner. On day 21, all horses received an intra-articular injection of LPS (0.3 ng) into their left or right intercarpal joint. Synovial fluid (SF) samples were taken on postinjection day (PID)-21 (i.e. prior to commencement of supplementation), PID0, PID0.25, PID0.5, PID1 and PID3 and analysed for PGE(2), GAG, NO, protein and total nucleated cells counts. Blood biochemistry and haematology screens were conducted at PID-21, PID0, PID1 and PID3. There was a significant reduction in LPS-induced PGE(2) and GAG in SF in horses supplemented with HRAM compared with controls and a tendency to increase complement recognition protein accumulation in synovial fluid of HRAM horses. Plasma from HRAM horses had reduced total white blood cells, segmented neutrophils (compared with baseline concentrations) and lymphocytes (compared with controls), and increased SF nucleated cell count (compared with baseline concentrations and controls). It is concluded that HRAM offered as part of the feed alter biomarkers of inflammation in SF of LPS-challenged horses. Larger studies that seek to clarify effects of HRAM on synovial fluid cell counts and possible role of HRAM-induced interference with complement signalling are warranted.

Reduction of saturated fats by mutagenesis and heat selection in Brassica napus L

The reduction of saturated fats in canola oil has recently been promoted as a goal for breeders for commercial and human health benefits. Currently, saturated fatty acids in Canadian produced canola oil are above the 7% level, and the objective of this study was to generate canola lines with reduced major saturates (palmitic and stearic), by several percent. Mutant embryos generated from direct ultraviolet radiation mutagenesis of microspores in vitro were subjected to heat during the maturation stage. Heat artificially elevated the saturate levels in developing mutant embryos, allowing efficient identification of those with reduced saturates within the expanded range using HPLC fatty acid analysis of the embryo cotyledons. Mutagenesis produced embryos with fatty acids altered in both directions. Major saturate levels in the cotyledons of heat-treated mutant embryos ranged from 3.3 to 16.4% (heated control ca. 6–9%) and 1.3–10% (heated control ca. 2–4%) for palmitic and stearic fatty acids, respectively. Doubled haploid seed derived from embryos grown at normal temperatures confirmed the reduction of major saturates. HPLC fatty acid analysis of DH seed identified saturate levels ranging from 3.9 to 6.5% (control ca. 5.5%) and 0.9–2.7% (control ca. 1.7%) for palmitic and stearic fatty acids, respectively. Various doubled haploids were identified with major saturate levels below 5.5%. Concomitant positive changes in the unsaturated fatty acids (18:1, 18:2, 18:3) among the mutant lines are also discussed.