Cotyledon Leaf Research Articles

Transgenic Cucumis sativus Expressing the Hepatitis B Surface Antigen

A DNA fragment encoding the hepatitis B virus surface antigen was amplified from a positive blood (hepatitis B) sample and introduced into the pET 32c prokaryotic expression vector. The gene encoding the HBV surface protein antigen was introduced into pCAMBIA 3300, and immobilized into Agrobacterium tumefaciens strain LBA4404. Cotyledonary leaf sections of Cucumis sativus (cucumber) cv ‘Swarnamukhi’ were cocultivated with Agrobacterium harboring the binary vector pCAMBIA 3300 carrying the HBV surface antigen gene driven by the CaMV35S promoter and the herbicide resistance gene phosphinothricin. Putative transformed shoots were induced on a Murashige and Skoog (MS) medium containing phosphinothricin, and these were then rooted on MS basal medium supplemented with 1 mg/L Indole 3-butyric acid (IBA). Integration of the T-DNA into in putative transgenic plants was confirmed by PCR and Southern blot analyses. RT-PCR and Northern blot analyses were conducted to determine RNA expression. Levels of expression in transgenic plants were confirmed by Western blot analysis, and quantification of the protein was determined by enzyme linked immuno assay (ELISA). Molecular mass of the recombinant protein was measured by Matrix-Assisted Laser Desorption Ionization-Time-of-Flight (MALDI-TOF) Mass Spectrometry.

Shoot regeneration from cotyledonary leaf explants of Jatropha curcas: a biodiesel plant

A simple, high frequency, and reproducible method for plant regeneration through direct organogenesis from cotyledonary leaf explants of Jatropha curcas was developed using Murashige and Skoog (MS) medium supplemented with different concentrations of thidiazuron (TDZ) or 6-benzyl aminopurine (BAP). Medium containing TDZ has greater influence on regeneration as compared to BAP. The induced shoot buds were transferred to MS medium containing 10 mu M kinetin (Kn), 4.5 mu M BAP, and 5.5 mu M a-naphthaleneacetic acid (NAA) for shoot proliferation. The proliferated shoots could be elongated on MS medium supplemented with different concentrations and combinations of BAP, indole-3-acetic acid (IAA), NAA, and indole-3-butyric acid (IBA). MS medium with 2.25 mu M BAP and 8.5 mu M IAA was found to be the best combination for shoot elongation. However, significant differences in plant regeneration and shoot elongation were observed among the genotypes studied. Rooting was achieved when the basal cut end of elongated shoots were dipped in half strength MS liquid medium containing different concentrations and combinations of IBA, IAA, and NAA for 4 days, followed by transfer to growth regulators free half strength MS medium supplemented 0.25 mg l(-1) activated charcoal. Elongated shoot treated with 15 mu M IBA, 5.7 mu M IAA, and 11 mu M NAA resulted in highest percent rooting. The rooted plants could be established in soil with more than 90% survival rate. The method developed may be useful in improvement of J. curcas through genetic modification.

Age and orientation of the cotyledonary leaf explants determine the efficiency of de novo plant regeneration and Agrobacterium tumefaciens-mediated transformation in Jatropha curcas L.

Effects of age and orientation of the explant on callus induction and de novo shoot regeneration from cotyledonary leaf segments of Jatropha curcas were studied. The callus induction and shoot regeneration capacity of cotyledonary leaf segments were found significantly related to the age of the explants and their orientation in culture medium. The youngest explant, derived from the cotyledonary leaf of germinated seed induced the highest regeneration response as compared to one- and two-week-old explants. A gradient response with age of the explant was observed in percentage of callus induction, shoot regeneration from callus and the number of shoots per regenerating callus. The explants cultured with their abaxial side in medium showed significantly higher regeneration response. The youngest explant was found to be most amenable to Agrobacterium-mediated transformation as compared to older explants. The fact that callus induced from the edges of the explant followed by de novo shoot induction, and strong transient gus expression observed in the edges of the explant are significant for routine Agrobacterium-mediated transformation and generation of stable transgenic plants in J. curcas.

Effect of explant age, hormones on somatic embryogenesis and production of multiple shoot from cotyledonary leaf explants of Solanum trilobatum L.

The present study examines the effect of explant age and various concentrations of kinetin and BAP on somatic embryogenesis and organogenesis in Solanum trilobatum L. MS medium fortified with 11.1 iM BAP + 13.95 iM KN produced highest frequency of embryogenesis (97.3%) and average number of multiple shoots (48.4 ± 1.33) from cotyledonary leaf explants at two opened leaves stage. The presence of BAP and/or KN was critical in increasing embryos and multiple shoots per explant with its increasing concentrations which was also supported from significant positive corrilation (p observed that embryogenic and organogenic response was significantly affected by explant type in all concentrations of BAP and KN alone as well as in combinations (P on half strength MS medium supplemented with 7.35 and 9.8 iM IBA respectively. The rooted plantlets were well accomplished with a survival frequency of 86.5 ± 5%. Moreover, there were no phenotypic differences observed between the in vitro regenerated and in vivo plantlets.

Pathological and biochemical changes in Brassica juncea (mustard) infected with Albugo candida (white rust)

Components of disease reaction, including incubation period, pustule types, inoculum production and disease index (DI); and contents of protein, phenols, soluble sugars and reducing and non‐reducing sugars were investigated in cotyledonary and true leaves of six genotypes of Brassica juncea: Varuna, Kranti, EC‐399296, EC‐399299, EC‐399313 and EC‐399301, inoculated with Albugo candida. Cotyledonary leaves were examined 14 days after inoculation (d.a.i.), whereas true leaves were scored 14 and 21 d.a.i. Disease indices were assessed on a 0% (resistant) to 100% (susceptible) scale. DIs at the cotyledonary leaf stage in the above six genotypes were 67, 65, 32, 31, 31 and 38%, respectively, whereas at the true‐leaf stage they were 21, 28, 12, 17, 9 and 4%, respectively at 14 d.a.i., and 35, 45, 17, 19, 20 and 6%, respectively at 21 d.a.i. Protein contents were highest in the genotypes with the highest DIs, such as Varuna at the cotyledonary leaf stage and Kranti at the true‐leaf stage, and lowest in the genotypes with the lowest DIs, such as EC‐399299 at the cotyledonary stage and EC‐399301 at the true‐leaf stage. Total phenols, total sugars, reducing sugars and non‐reducing sugars were generally negatively correlated with DI, but were not always consistent, particularly when differences in DI were small. The results indicated that factors conditioning the response of host genotypes to A. candida may differ or operate in different ways at different growth stages.

Uzun Yapraklı Üçgülün (Trifolium pannonicum ssp. elongatum) Hipokotil ve Yaprak Sapı Eksplantından in vitro Çoğaltılması

The study reports effects of various concentrations of Kinetin and α- Naftalin asetik asit (NAA) (kinetin and 0.1+ 0.1, 0.2, 0.4 NAA mg/l) on shoot regeneration (percentage, number and length of shoot) of Trifolium pannonicum ssp. elongatum from hypocotyl and petiole of cotyledon leaf. The maximum (91.7%) shoot regeneration frequency (25%) was recorded on hypocotyl explant on Murashige and Skoog ( MS) medium containing 1 mg/l Kinetin + 0.4 mg/l NAA; whereas, minimum shoot regeneration frequency (25%) was in medium containing 1 mg/l Kinetin + 0.1 NAA respectively, in both hypcotyl and petiole of cotyledon leaf explant. The highest number of 11.2±1.3 shoots per explant was recorded on MS medium containing 1 mg/l Kinetin+0.4 mg/l NAA and the lowest number of 2.5±0.6 was in medium containing 1 mg/l Kinetin+0.1 mg/l NAA respectively. The longest shoots (3.5±0.5 cm) were recorded on MS medium containing 1 mg/l kinetin+ 0,1 mg/L NAA, and the smallest shoots (1.5±0.2 cm) were recorded on MS medium containing 1 mg/l kinetin + 0.4 NAA. Maximum frequency of 95% roots was recorded on MS medium containing 1 mg/l IBA.

Plasmopara halstedii

<i>Plasmopara halstedii</i>

Agrobacterium rhizogenes mediated transformation of Scutellaria baicalensis and production of flavonoids in hairy roots

Using different explants of in vitro seed grown Scutellaria baicalensis Georgi plantlets, hairy roots were induced following inoculation of Agrobacterium rhizogenes strains A4GUS, R1000 LBA 9402 and ATCC11325. The A4GUS proved to be more competent than other strains and the highest transformation rates were observed in cotyledonary leaf explant (42.6 %). The transformed roots appeared after 15–20 d of incubation on hormone free Murashige and Skoog medium. Growth of hairy roots was assessed on the basis of total root elongation, lateral root density and biomass accumulation. Maximum growth rate was recorded in root:medium ratio 1:100 (m/v). Hairy root lines were further established in Gamborg B5 medium and the biomass increase was maximum from 15 to 30 d. PCR, Southern hybridization and RT-PCR confirmed integration and expression of left and right termini-linked Ri T-DNA fragment of the Ri plasmid from A4GUS into the genome of Scutellaria baicalensis hairy roots. GUS assay was also performed for further integration and expression. All the clones showed higher growth rate them non-transformed root and accumulated considerable amounts of the root-specific flavonoids. Baicalin content was 14.1–30.0 % of dry root mass which was significantly higher then that of control field grown roots (18 %). The wogonin content varies from 0.08 to 0.18 % among the hairy root clones which was also higher than in non-transformed roots (0.07 %).

Effects of Growth Regulators and Explant-Type on Agrobacterium-Mediated Transformation in Brinjal (Solanum melongena L.) cv. Manjarigota

Effects of growth regulators and type of explants on transformation and in vitro morphogenetic responses of brinjal cv. Manjarigota were studied. Both hypocotyl and cotyledonary explants showed marked influence on in vitro morphogenetic responses after Agrobacterium co-cultivation. Hypocotyl explants showed callus initiation and regeneration responses earlier than cotyledonary leaves. Hypocotyl explants were found to be better than cotyledonary leaf explants in regenerating shoots after Agrobacterium co-cultivation. There was delay and reduction in both callus and regeneration responses in Agrobacterium co-cultivated explants. Hypocotyl explants showed the highest regeneration response on MS medium containing 2 μM BAP and 0.05 μM NAA while cotyledonary leaves did not show regeneration response after Agrobacterium co-cultivation. However, they showed green buds on MS medium containing 10 μM BAP and 1 μM NAA, which could not differentiate into shoots. Overall, hypocotyl explants were found better in regenerating shoots after Agrobacterium co-cultivation.

Truffle volatiles inhibit growth and induce an oxidative burst in Arabidopsis thaliana

The function of fungal volatiles in fungal-plant interactions is poorly understood. The aim here was to address this lack of knowledge, focusing on truffles, ectomycorrhizal fungi that are highly appreciated for their aroma. The effect of volatiles released by truffles was tested on Arabidopsis thaliana in a closed chamber bioassay. The volatiles produced by Tuber melanosporum, Tuber indicum and Tuber borchii fruiting bodies inhibited A. thaliana in terms of root length and cotyledon leaf size, and in some cases induced a bleaching of the seedlings, thus indicating toxicity. Ten synthetic volatiles were tested in a similar way. The strongest inhibitory effect was observed with C(8) molecules such as 1-octen-3-ol, an alcohol with a typical 'fungal smell'. Two of these C(8) compounds were further tested to investigate their mechanism of action. 1-Octen-3-ol and trans-2-octenal induced an oxidative burst (hydrogen peroxide, H(2)O(2)) in the A. thaliana leaves as well as a strong increase in the activities of three reactive oxygen species (ROS)-scavenging enzymes. These results demonstrate that fungal volatiles inhibit the development of A. thaliana and modify its oxidative metabolism. Even though limited to laboratory observations, these results indicate the presence of a hitherto unknown function of fungal volatiles as molecules that mediate fungal-plant interactions.

Adventitious bud regeneration from leaf and cotyledon explants of Chinese hawthorn (Crataegus pinnatifida Bge. var. major N.E.Br.)

Hawthorn (Crataegus spp.) is an important plant with a long history as an ornamental and a source of medicine. A protocol is outlined for adventitious bud regeneration from leaf and cotyledon explants of Chinese hawthorn (C. pinnatifida Bge. var. major N.E.Br.). Adventitious buds were induced on both the leaves of sprouting winter buds and the leaves of in vitro plants, but the percentage of bud regeneration from leaves of in vitro plants was very low—less than 6%. On N6 medium supplemented with 31.08 μM BA and 9.67 μM NAA, the percentages of bud regeneration from leaves of sprouting winter buds of cultivars “Liaohong” and “Qiujinxing” were 31.4% and 17.6%, respectively. The regeneration abilities of three kinds of cotyledon explants, immature cotyledon, mature cotyledon, and cotyledon leaf, were compared. The percentage of bud regeneration from cotyledon leaves was higher. On MS media supplemented with 4.44 μM BA and 4.54–9.08 μM TDZ, the percentages of bud regeneration from cotyledon leaves of cultivars “Qiujinxing” and “Xiajinxing” were 27.7 ± 7.8% and 20.1 ± 4.7%, respectively, and the numbers of buds per explant were 5.9 ± 1.6 and 3.2 ± 0.7, respectively. On B5 medium supplemented with 2.22 μM BA, 2.32 μM Kn, and 0.57 μM IAA, adventitious buds grew quickly and 80–100% of buds developed into shoots. The shoots rooted successfully with the two-step rooting method. Ninety days after transplantation, more than 80% plants were survived. This system of adventitious bud regeneration from leaf and cotyledon explants could be useful for the genetic transformation and polyploidization of Chinese hawthorn.

A Complex Mode of Glucose Signaling

In Arabidopsis , hexokinase 1 (HXK1) acts as a glucose sensor to regulate gene expression and plant growth, a role distinct from its function in glycolysis; however, the mechanisms whereby HXK1 mediates glucose signaling have been unclear. After showing that a portion of Arabidopsis HXK was present in the nucleus, Cho et al . used proteomic and binary-interaction screens to identify two proteins--vacuolar H + -ATPase B1 (VHA-B1) and the 19 S regulatory particle of proteasome subunit (RPT5B)--as nuclear-specific HXK1-interacting partners that formed a complex with HXK1. Genetic analysis revealed that vha-B1 and rpt5b loss-of-function mutants resembled the HXK1 gin2 ( glucose-insensitive2 ) mutant: All three mutants were insensitive to repression of cotyledon expansion, chlorophyll accumulation, and leaf and root development in response to high-glucose conditions and showed growth retardation compared with wild-type plants under low-light, low-nutrient conditions. The gin2 -like phenotypes of the vha-B1 and rpt5b mutants did not depend on loss of HXK1 expression, HXK1 protein abundance, or reduction in glucose or fructose phosphorylation. Like gin2 , the vha-B1 and rpt5b mutants were insensitive to glucose-mediated repression of chlorophyll a/b binding protein ( CAB ) and carbonic anhydrase ( CAA ) gene expression. Chromatin immunoprecipitation analysis showed that the HXK1 complex bound to the CAB2 promoter; this binding was reduced but not abolished in the vha-B1 and rpt5b mutants. Thus, these three proteins appear to form a nucleus-specific complex that is critical to glucose-mediated regulation of gene transcription. Y.-H. Cho, S.-D. Yoo, J. Sheen, Regulatory functions of nuclear hexokinase1 complex in glucose signaling. Cell 127 , 579-589 (2006). [PubMed]

In vitro propagation of Albizia odoratissima L.F. (Benth.) from cotyledonary node and leaf nodal explants

Cotyledonary node and leaf nodal explants excised from 14-d-old in vitro-grown seedlings of Albizia odoratissima were cultured on a Murashige and Skoog (MS) medium with different concentrations of 6-benzylaminopurine (BAP), N6-(2-isopentenyl) adenine (2-iP) and kinetin, used either solely or in combinations. The highest frequency for shoot regeneration (82.5%), the maximum number of shoots per explant (6.9), and the maximum shoot length (2.55 cm) were obtained from cotyledonary node explants cultured on a MS medium containing 10 μM BAP and 10 μM 2-iP with 30 gl−1 sucrose. Successful rooting was achieved by placing the microshoots on MS medium with 25 μM indole-3-butyric acid (IBA) for 24h first, then transferring to the same medium without IBA. Of the various substrates tested, vermiculite was best for plant acclimatization, in which 75% of plants survived.

The Effect of Repeated Short-term Flooding on Mycorrhizal Survival in Snap Bean Roots

Since arbuscular–mycorrhizal (AM) fungi are aerobic, symbiosis was not considered significant under flooded conditions. However, AM colonization of wetland plants is now believed more common than previously thought. In the humid tropics, storms that result in standing water for 24 hours or less are common. Short-term floods, especially on sandy soils, may leach banded fertilizer, reducing uptake efficiency. Crops planted in flood prone areas are not normally enhanced with mycorrhizal mixes. However, mycorrhizal associations tolerant to wet conditions may improve nutrient uptake as plants recover from short-term flooding. Greenhouse studies were initiated to determine the effects of frequent short-term floods (two to four events) on mycorrhizal colonization and subsequent development in snap bean (Phaseolus vulgaris Phaseolus vulgaris L.) plants. Flooding produced no obvious long-term physical effects on plant shoots. In the first study, flooding did not affect survival of colonies established before the first flood event. Percent root colonization in flooded vs. nonflooded treatments was not significantly different at either 31 or 50 days after planting (DAP). As root length increased there was a concomitant increase in colonization so that percent colonization remained approximately the same in both flooded and nonflooded treatments. In the second study, three weekly floods beginning 13 DAP (cotyledon leaf open only) did not inhibit initial mycorrhizal colonization. Mycorrhizal associations should form with snap bean under conditions subject to short-term flooding. Additional research is needed to determine the efficacy of different mycorrhizal mixes under short-term flooded conditions in the field.

Effects of different drying rates on the physiological quality of Coffea canephora Pierre seeds

Desiccation tolerance in seeds depends on the species, development stage and drying conditions, especially the water removal rate. Coffea seeds are considered of intermediate performance, because they tolerate relative dehydration compared to orthodox seeds and are sensitive to low temperatures. The objective of this study was to verify the effect of different drying rates on the viability and storability of Coffea canephora seeds. A complete randomized experimental design was used, in a factorial 3 x 5 x 2 design, with three drying rates (fast, intermediate and slow), five final mean water contents after drying (51, 42, 33, 22 and 15 %) and two storage temperatures (10 and 20°C). The germination and seed vigor assessments, using radicle protrusion, cotyledon leaf opening, seedling emergence and emergence speed index, were performed shortly after drying and after two and four months storage. It was observed that with reduction in the water content there was reduction in the germination values and seed vigor, for all the drying rates. The greatest reductions in physiological quality occurred when the seeds were dried quickly and the best results were obtained at the intermediate drying rate. There was an effect of drying rate and storage temperature on the physiological quality of the seeds, and lower germination and vigor values were observed in seeds with lower water content stored at 20°C. C. canephora seeds were tolerant to desiccation down to 15 % water content and can be stored for four months at 10°C. A temperature of 20ºC can be used to store C. canephora seeds, as long as the water content is not reduced to values below 22 % water content.

Histological and inheritance studies of partial resistance in the Brassica napus–Albugo candida host‐pathogen interaction

AbstractTraditional and doubled haploid (DH) genotypes of oilseed Brassica spp. resistant, partially resistant, moderately susceptible, and susceptible to Albugo candida were compared for phenotypic development of host‐pathogen interaction and histology of host‐pathogen interaction. The partially resistant genotype showed pinhead‐size pustules, mainly on the upper surface of cotyledonary leaves. Relatively less mycelium was observed in the partially resistant genotype compared with the susceptible genotype. In resistant B. napus genotypes, there was neither pustule development nor any mycelial growth. In the moderately susceptible genotype, the pustules were similar to those in the partially resistant genotype in being of pinhead‐size and occasionally coalescing. However, ample mycelial growth in the mesophyll tissue in the moderately susceptible genotype was similar to that in the susceptible control B. rapa cv. ‘Torch’. The susceptible genotype B. rapa cv. ‘Torch’ also showed large coalescing pustules. In the non‐host B. juncea cv. ‘Commercial Brown’, no pustules were formed although some mycelial growth was observed beneath the epidermal cell layer and in the mesophyll cell layer of the cotyledonary leaf tissue. For inheritance studies, two partially resistant B. napus genotypes were crossed with a resistant B. napus genotype. Various generations viz., F1, F1(reciprocal), F2, and DHs produced from the crosses were inoculated with a zoospore suspension of race 7v of A. candida. The partially resistant phenotype appeared to be controlled by a single recessive gene designated as wpr with variable expression. The simple inheritance of partial resistance has implications for disease resistance breeding against white rust, as this type of resistance can be easily incorporated into elite breeding lines through conventional and DH breeding methods.

EVALUATION OF SELECTED IN-FURROW AND SEED APPLIED INSECTICIDES, WITH AND WITHOUT AN ADDITIONAL FOLIAR BROADCAST APPLICATION AT COYTLEDON OR FIRST TRUE LEAF STAGE, FOR CONTROL OF THRIPS, 2004

Selected insecticides applied onto seed or into the seed furrow at planting, both with and without an additional broadcast treatment at cotyledon (BC at cotyl) or first true leaf stage (BC at 1 tl), were evaluated for control of thrips in cotton. ‘Delta Pine 432 RR’ cotton was planted 28 Apr at the Virginia Tech Tidewater Agric. Res. & Ext. Ctr., Suffolk, VA, using 36-inch row spacing. In-furrow (IF) treatments were applied into the seed furrow at the time of planting using tractor-mounted inverted jars with lid holes calibrated to deliver exact amounts via gravity. Cruiser 5FS and Gaucho 480 seed treatments were applied commercially. The foliar treatments were applied with a CO2 pressurized backpack sprayer as a broadcast at 14.3 gpa and 18 psi through 8002VS nozzles spaced 18 inches apart on the spray boom. Foliar applications were made on 11 May at the cotyledon stage or on 17 May at the first sign of true leaf emergence after the cotyledon stage. A RCB design was used with 4 replicates; plots were 4 rows by 40 ft. Thrips injury to plants was determined by visually rating injury using a 0 to 5 scale, where 0 = no thrips induced plant injury, 1 = 10% injured leaves, no bud injury; 2 = 25% injured leaves, no bud injury; 3 = 75% injured leaves, and 0 to 25% buds injured; 4 = 90% injured leaves, and greater than 25% buds injured; and 5 = dead plants. Yield was determined by harvesting the 2 center rows of each plot (80 row ft) using a commercial 2-row John Deere cotton picker. Sub-samples were ginned to determine lint versus seed and trash weight (41% lint, 59% seed and trash). Data were analyzed using ANOVA and means were separated using LSD (P ≤ 0.10).

Developing propagation techniques for conservation of Heracleum candicans – an endangered medicinal plant of the Himalayan region

SummaryHeracleum candicans, an endangered medicinal plant of commercial value, is subject to heavy exploitation in the wild. This paper presents findings on the effect of the growing medium (or substrate) on seedling performance, propagation from rhizome cuttings and in vitro propagation protocols. Seedlings were obtained from 14 d prechilled seeds. Seedling growth was best in a mixture of sand and soil (1:1 by volume). Cuttings taken from the terminal growing part of rhizomes resulted in 67% rooting, following treatment with 100 mg l–1 indole-3-butyric acid (IBA). Cotyledonary leaf explants responded better (29 shoots/explant) than cotyledonary node explants for organogenesis using MS medium supplemented with 10 μM 6-benzyladenine. Of the harvested shoots 74 % rooted in MS medium supplemented with 4 μM IBA. Rooted shoots transferred to 120 g sterile soil, sand and peat mixture (1:1:1 by volume) showed 70% survival ex vitro.

Callus induction and plantlet regeneration in Withania somnifera (L.) dunal

Callus induction was observed from hypocotyl, root, and cotyledonary leaf segments, grown on Murashige and Skoog (MS) medium supplemented with various concentrations and combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin (KN). Maximum callusing (100%) was obtained from root and cotyledonary leaf segments grown on MS medium supplemented with a combination of 2 mg l−1 (9.1 μM) 2,4-D and 0.2 mg l−1 (0.9 μM) KN. The calluses, when subcultured in the same medium, showed profuse callusing. However, these calluses remained recalcitrant to regenerate regardless of the quality and combinations of plant growth regulators in the nutrient pool. When hypocotyl segments were used as explants, callus induction was noticed in 91% of cultures which showed shoot regeneration on MS medium supplemented with 2 mg l−1 2,4-D and 0.2 mg l−1 KN. These shoots were transferred to fresh medium containing various concentrations and combinations of 6-benzyladenine (BA) and N6-(2-isopentenyl)adenosine (2-iP). Maximum shoot multiplication was observed after 60 d of the second subculture on MS medium containing 2 mg l−1 (8.9 μM) BA. These shoots were rooted best (87%) on MS medium containing 2 mg l−1 (9.9 μM) indole-3-butyric acid (IBA). The plantlets were transferred to the field after acclimatization and showed 60% survival.

First Report of Blackleg Disease Caused by Leptosphaeria maculans on Canola in Brazil.

Canola (Brassica napus L.) is a relatively new crop in Brazil, having been grown there for approximately 8 years. In 2000, leaf lesions and stem cankers were observed in cvs. Hyola 420 and Hyola 401 in farmers' fields in the state of Rio Grande do Sul. Cankered stems were received at the University of Manitoba, Canada, from Rio Grande do Sul for disease identification. Small pieces of the stem were cut from the cankered area, and standard protocol was followed to surface sterilize the stem pieces. Stem pieces were plated on V8 agar medium and incubated under light for 12 days. Typical fungal colonies with concentric rings containing pycnidia formed on the V8 agar. The colony characteristics were typical of the blackleg pathogen, Leptosphaeria maculans (Desmaz.) Ces. & De Not. (anamorph = Phoma lingam) (Tode:Fr.) Desmaz.). Blackleg is an economically important and serious disease in many parts of the world including Australia, Canada, the United States, and Europe. L. maculans strains can be characterized in four pathogenicity groups (PG1 through PG4) based on differential testing procedures giving interaction phenotype (IP) reactions (2). Two weeks after plating on V8 media, plates were flooded with sterile distilled water, and pycnidiospores were harvested. Flats of multipots filled with Metro Mix were seeded with three cultivars (Westar, Glacier, and Quinta). One-week-old cotyledons from the three cultivars were inoculated with pycnidiospore suspensions (2 × 107 pycnidiospores per ml) of seven Brazilian isolates, numbered 7, 8, 9, 11, 15, 16, and 18, respectively. Each cotyledon leaf, punctured in the center with a needle, was inoculated with a 10-μl droplet of the inoculum. Disease evaluations were made 11 days after inoculation using a 0 to 9 rating scale (1). This screening was repeated three times from February 2001 to October 2001. After the second repeat, the isolates from Rio Grande do Sul were passed through the highly susceptible canola cv. Westar. Results from all four trials were consistent, and yielded one PG1 isolate (No. 7) and six PG3 isolates. PG1 is classified as a nonaggressive strain, whereas PG3 isolates are classified as aggressive. PG3 isolates would have an IP reaction of 7 to 9, 7 to 9, and 3 to 6 on cvs. Westar, Glacier, and Quinta, respectively. PG2 is the most commonly found aggressive strain in the Canadian prairies. PG3 is predominantly found in Australia, the United Kingdom, and the United States. To our knowledge, this is the first report of blackleg disease caused by L. maculans on canola in Brazil. Differential testing fulfilled Koch's postulates and determined the PG groups found in Brazil (PG1 and PG3).