Stephen R. Bowley

Desiccation tolerance of alfalfa (Medicago sativa L.) somatic embroys. Influence of abscisic acid, stress pretreatments and drying rates

Abstract Desiccation tolerance was induced in alfalfa (Medicago sativa L.) somatic embryos by exogenous application of abscisic acid (ABA). Subsequently, embroys were dried to 10–15% moisture and stored for at least 3 weeks in the dry state. To estimate embryo survival, dried somatic embryos were imhibed on moist paper with no moist filter paper with no added nutrients. Under the appropriate treatment conditions, 65% of the somatic embryos survived and germinated in a manner analagous to true seed. The rate of drying affected the survival of the somatic embryos under some but not all treatment conditions. A slow drying rate of 1.2 g H2O · g−1 · day−1 over 6 days, generally gave higher and more consistent embryo survival, compared to fast (6.9 g H2 g H2O · g−1 · day−1) drying over 1 day. Tolerance of desiccation was also induced in these somatic embryos by exposure to sub-lethal levels of low temperature, water, nutrient or heat stress prior to desiccation. However, some of these stress pretreatments had other deleterious effects on embryo maturation and seedling vigor after imbibition. Treatment of embryos with the triazole, uniconazole, also induced desiccation tolerance in these somatic embryos, but other undesirable side effects on seedling growth were observed. The only stress pretreatment that was comparable to exogenous application of ABA, as a means of inducing desiccation tolerance without detrimental effects on other characteristics, was heat shock.

Artificial seeds of alfalfa (Medicago sativa L.) induction of desiccation tolerance in somatic embryos

SummaryThe use of somatic embryos from cell culture systems in the clonal propagation of plants would be greatly facilitated if the somatic embryos could be dried and stored in a dormant state similar to true seeds. A cell culture system was developed for alfalfa (Medicago sativa L.) line RL34 which gave high yields of somatic embryos in an approximately synchronized pattern. These somatic embryos were treated with abscisic acid (ABA) at the cotyledonary stage of development to induce desiccation tolerance. With no visual preselection, approximately 60% of the dried embryos converted into plants upon reimbibition. When high quality embryos were selected prior to drying, 90 to 100% conversion rates were observed. The timing of the application of ABA in terms of embryo development was critical with an optimum being at cotyledonary stage spanning approximately 4 days; thus, synchronized embryo development is required for optimal expression in bulk samples. The vigor of the seedlings from dried somatic embryos was greater than those from embryos which had not been dried, but remained substantially lower than those from true seeds.

Carbohydrate composition and freezing tolerance of canes and buds in Vitis vinifera

Summary In grapevines, buds and canes have different strategies of freezing tolerance - buds avoid freezing by supercooling, whereas canes tolerate extracellular freezing. To further understand the relationship between freezing tolerance and carbohydrate composition of these tissues in Vitis vinifera, analyses were made of starch, glucose, fructose, sucrose and raffinose of field-grown vines during natural acclimation and deacclimation over two years, on artificially deacclimated field material, and on daylength induced dormant growthroom material. Statistics on correlation and path coefficients were calculated to detect relationships, and a was set at 0.05. During the acclimation and deacclimation of grapevines in the field, the level of most carbohydrates was correlated with freezing tolerance in both buds and canes. Starch levels were negatively correlated with hardiness whereas most soluble sugars were positively correlated. Artificial deacclimation with heat treatment supported these relationships between LTSo and carbohydrates. Dormancy induction by short day treatment did not increase freezing tolerance but did increase raffinose in buds. Statistical path analysis highlighted the importance of starch and fructose in freezing tolerance of grapevines, and indicated that the different soluble sugars may have different roles in buds and canes. This statistical method holds great potential, particularly for identifying transformation targets from data on metabolites.

Active Oxygen and Freezing Tolerance in Transgenic Plants

Winterhardiness is a complex trait involving tolerances to freezing, water deprivation, ice-encasement (severe anoxia), flooding (milder anoxia) and disease. The combination and severity of these stresses that crops must tolerate varies from environment to environment and from year to year. Different crops, even in the same environment, experience different stresses because of their growth habit. For example, a winter annual crop like wheat (Triticum aestivum L.) grows close to the ground and is covered by snow, whereas a woody fruit crop grows above the snow and is not insulated against cold air temperatures. In northern climates, management of our major crops is based on the avoidance of winter injury. For example, a summer annual is grown in these areas instead of a winter annual, i. e. spring wheat is grown instead of winter wheat. Also, production practices for perennial forage crops, such as alfalfa (Medicago sativa L.), include planting before critical seeding dates, harvesting before critical fall harvest dates, leaving shoot growth to hold snow that will insulate the plants over-winter, and grading to improve surface drainage.

Manipulating freezing tolerance in transgenic plants

Winterhardiness is a composite of tolerances to freezing, desiccation, ice-encasement, flooding and diseases. From one point of view, winterhardiness may not be easily manipulated by genetic engineering technology because many different genes are involved in the tolerance of these diverse stresses. However, these various stresses have similarities. They promote formation of activated forms of oxygen, promote membrane lipid and protein degradation, cause similar biophysical changes in membrane structure, and culminate with increased leakage of cytoplasmic solutes and loss of cellular membrane functions. These similarities led to the hypothesis that winter injury might be reduced in crop plants if their tolerance of oxidative stress was increased.Towards that objective we created transgenic alfalfa (Medicago sativa L.) plants that overexpress either Mn-SOD or Fe-SOD cDNA (provided by Dirk Inzé, Universiteit Gent). Petiole explants were transformed using Agrobacterium tumefaciens and plants were regenerated by somatic embryogenesis. The primary transgenic plants were screened using PCR (polymerase chain reaction), Southern hybridization and native PAGE for SOD activity. Greenhouse and laboratory studies showed a minimal difference in stress tolerance between the primary transgenic and non-transgenic plants. In the first field trial, four primary transgenic plants expressing two forms of the Mn-SOD cDNA had greater survival after two winters than the non-transgenic RA3. Similar results were obtained in a second field trial, comparing 18 independent transformants with Mn-SOD targeted to the mitochondria, 11 independent transformants with Mn-SOD targeted to the chloroplast and 39 independent transformants with Fe-SOD targeted to the chloroplast, expressed in three different non-transgenic plants. The transgenic plants averaged over 25% higher survival than the non-transgenic controls after one winter. There was no effect of subcellular targeting or SOD type on field survival, but there was variation among independent transformants containing the same SOD construct. Activated oxygen therefore appears to be one of the possible causes of winter injury, and it should be possible to reduce winter injury in transgenic plants by constitutive overexpression of SOD.

Responses of Dry Beans to Flumioxazin

There is little information on the sensitivity of dry beans to flumioxazin. Tolerance of eight cultivars of dry beans representing four market classes (black, cranberry, kidney, and white beans) to preplant incorporated (PPI) and preemergence (PRE) applications of flumioxazin at the rate of 52.5, 70, and 140 g ai/ha were studied in three field experiments in Ontario in 2002 and 2003. There were no differences (P < 0.05) between two cultivars within a market class in their responses to flumioxazin. However, the four market classes differed in their responses to flumioxazin. Black and white beans were more sensitive to the PRE application of flumioxazin than cranberry and kidney beans. Flumioxazin applied PRE at 140 g/ha caused as much as 34% visual injury and reduced plant height by 23 to 28%, shoot dry weight by 35 to 39%, and yield by 20 to 30% in black and white bean market classes. Flumioxazin-applied PPI did not injure any market class. On the basis of this research, there is an acceptable margin of crop safety in these black and white bean cultivars only when flumioxazin is applied PPI. The two cranberry and kidney bean cultivars were tolerant to all rates of flumioxazin applied both PPI and PRE. Nomenclature: Flumioxazin; black bean, cranberry bean, kidney bean, navy bean, white bean, Phaseolus vulgaris L. Additional index words: Dry beans, herbicide tolerance, preemergence herbicides, preplant incorporated herbicides. Abbreviations: DAE, days after emergence; OM, organic matter; PPI, preplant incorporated; PRE, preemergence.

Isolation and characterization of class A4 heat shock transcription factor from alfalfa

Plant heat shock transcription factors (HSFs) regulate transcription of heat shock (HS) genes. In Arabidopsis thaliana, 21 HSFs have been classified into groups A-C. Members of class A act as typical transcriptional activators, whereas B HSFs function as coactivators or repressors depending on promoter context. The function of class C HSFs is still unclear. Here, we present the isolation and characterization of the first HSF from alfalfa (Medicago sativa L.) and designate it MsHSFA4 based on amino acid sequence analysis. The MsHSFA4 gene was determined to be single copy and was detected at two separate genetic loci in the tetraploid Medicago sativa. Overexpression of MsHSFA4 in tobacco mesophyll protoplasts resulted in weak transcriptional activity, similar to that exhibited by Arabidopsis AtHSFA4a. The MsHSFA4 proximal promoter contains three putative HSE elements, and the gene itself is activated both by heat and cold stress.

Development of locomotion over inclined surfaces in laying hens

The purpose of the present study was to evaluate locomotor strategies during development in domestic chickens (Gallus gallus domesticus); we were motivated, in part, by current efforts to improve the design of housing systems for laying hens which aim to reduce injury and over-exertion. Using four strains of laying hens (Lohmann Brown, Lohmann LSL lite, Dekalb White and Hyline Brown) throughout this longitudinal study, we investigated their locomotor style and climbing capacity in relation to the degree (0 to 70°) of incline, age (2 to 36 weeks) and the surface substrate (sandpaper or wire grid). Chicks and adult fowl performed only walking behavior to climb inclines ⩽40° and performed a combination of wing-assisted incline running (WAIR) or aerial ascent on steeper inclines. Fewer birds used their wings to aid their hind limbs when climbing 50° inclines on wire grid surface compared with sandpaper. The steepness of angle achieved during WAIR and the tendency to fly instead of using WAIR increased with increasing age and experience. White-feathered strains performed more wing-associated locomotor behavior compared with brown-feathered strains. A subset of birds was never able to climb incline angles >40° even when using WAIR. Therefore, we suggest that inclines of up to 40° should be provided for hens in three-dimensional housing systems, which are easily negotiated (without wing use) by chicks and adult fowl.

Tolerance of Processing Tomato to Thifensulfuron-Methyl1

Limited information exists on the tolerance of processing tomato to postemergence (POST) application of thifensulfuron-methyl. The tolerance of 13 processing tomato varieties, ‘CC337’, ‘H9144’, ‘H9314’, ‘H9478’, ‘H9492’, ‘H9553’, ‘H9909’, ‘N1069’, ‘N1082’, ‘N1480E’, ‘N1480L’, ‘N1522’, and ‘PETO696’, to POST applications of thifensulfuron-methyl at the maximum use rate (6 g ai/ha) and twice the maximum use rate (12 g/ha) for soybean was evaluated at two Ontario locations in 2001 and 2002. At 7 days after treatment (DAT), thifensulfuron applied POST caused 0.2 to 1% visible injury to CC337, H9144, N1082, N1522, and PETO696 at the high rate. H9553, H9909, N1069, and N1480E were the most sensitive to POST thifensulfuron-methyl, with visible injury ranging from 1 to 6% at the high rate. There was no visible injury to H9314, H9478, H9492, or N1480L at either application rate of thifensulfuron-methyl. By 28 DAT, no visible injury was noted to any variety, except for H9909, N1069, and N1480L, which showed minimal (<2%) visible injury. There were no adverse effects on shoot dry weight and marketable yield for any variety at either rate. Although thifensulfuron-methyl applied POST caused minimal and transient injury to the varieties tested, more tolerance trials with other fresh and processing tomato varieties are required to confirm these initial results. Nomenclature: Thifensulfuron-methyl; tomato, Lycopersicon esculentum. Additional index words: Crop tolerance, tomato injury, postemergence herbicides, yield, processing tomato. Abbreviations: DAT, days after treatment; OM, organic matter; POST, postemergence.