Evaluation and verification of resistance to Meloidogyne hapla Chitwood in a Cannabis germplasm collection

A collection of Cannabis accessions was evaluated for resistance to the root-knot nematode Meloidogyne hapla Chitwood in a seedling test. After inoculation with a larval suspension, significant variation was found for the number of galls and egg masses on the seedling roots. These parameters were considered as estimates for nematode infection and larval multiplication, respectively. A subset of the tested accessions was grown on a naturally infested arable field to study the relation between the test results and host characteristics in the field. The ranking order of accessions for the number of galls in the seedling test agreed well with that for the number of root galls on juvenile plants in the field. Therefore the test provides a useful indication for nematode infection. The number of egg masses in the seedling test and the population density of Meloidogyne measured in post-treatment soil samples of the field trial, showed a comparable ranking of accessions. Differences between accessions in the field were however smaller and not statistically significant.     

The CPRO Cannabis germplasm collection

Summary A collection of more than a 150 Cannabis accessions is being established as a source for evaluation and breeding experiments. Origin of the accessions and maintenance of the collection are described.     

The inheritance of chemical phenotype in Cannabis sativa L. (II): Cannabigerol predominant plants

This paper aims to clarify the genetic mechanism that is responsible for the accumulation of cannabigerol (CBG) in certain phenotypes of Cannabis sativa L. CBG is the direct precursor of the cannabinoids CBD, THC and CBC. Plants strongly predominant in CBG have been found in different fibre hemp accessions. Inbred offspring derived from one such individual were crossed with true breeding THC predominant- and CBD predominant plants, respectively. The segregations in the cross progenies indicate that CBG accumulation is due to the homozygous presence of a minimally functional allele, tentatively called B₀, at the single locus B that normally controls the conversion of CBG into THC (allele B_T) and/or CBD (allele B_D). The fact that CBG accumulating plants have so far been found in European fibre hemp populations that are generally composed of B_D/B_D plants, and the observation that the here investigated B₀ allele possesses a residual ability to convert small amounts of CBG into CBD, make it plausible that this B₀ is a mutation of normally functional B_D. Therefore, B₀ is considered as a member of the B_D allelic series encoding a CBD synthase isoform with greatly weakened substrate affinity and/or catalytic capacity.     

Occurrence and frequency of putatively Y chromosome linked DNA markers in Cannabis sativa L.

DNA from female and male hemp (Cannabis sativa L.) plants belonging to nine different varieties were screened with180 RAPD primers in a search for sex-associated DNA markers. About 1500bands were produced in total, nine primers were found yielding one or two DNA bands amplified in all nine male DNA bulks and absent in all female DNA bulks. These putatively male-associated markers were then scored in three different F1progenies, deriving from a cross between a common male parent and three different female plants. The sex of the progeny was accurately scored on the basis of the floral phenotype, and the presence of the nine male-associated markers was verified by RAPD analysis. In all three progenies examined, all the male plants showed the DNA markers previously identified by bulk segregant analysis (BSA) on the hemp varieties, while all the female plants lacked them. The fact that the association between these markers and the staminate phenotype is found when examining male plants of distantly related varieties, and that such linkage is never broken when different progenies are examined, strongly supports the hypothesis that the markers found are physically located on the Y chromosome, in a region excluded from recombination during meiosis. Another marker was shown to be present in the male parent, in all the male plants of each progeny, and in 50% of the female progenies, while it was absent in the female parent; the possible occurrence of markers deriving from multiple amplification sites of the genome is discussed. This revised version was published online in August 2006 with corrections to the Cover Date.     

Time course of cannabinoid accumulation and chemotype development during the growth of <Emphasis Type="Italic">Cannabis sativa</Emphasis> L

The time course of cannabinoid accumulation in the leaves of individual plants of three Cannabis accessions was determined by gas-chromatographic analysis in greenhouse-grown plants. The total amounts and the concentration ratios of CBD, THC and CBG were determined; two accessions (an experimental hybrid, (21R × 15R) × NL, and plants from a seized seed lot) were found chemotypically uniform, with all plants belonging to chemotpe II (mixed) and I (high THC) respectively. The Carmagnola accession showed chemotypic heterogeneity, with a majority of plants belonging to chemotype III. The CBD/THC and CBG/CBD ratios were shown to be largely constant in the leaves, since 28 and until 103 days after sowing, and consistent with the ratios determined on mature inflorescences. CBD and THC maximum amounts in the leaves showed a peak in the leaves around 80 days from sowing, and were shown to be simultaneous during the growth period, irrespective of the chemotypes. Callus cultures were obtained from all the five different chemotypes (I, II, III, IV, V), and GC analyses were performed. Independently of the type and amount of cannabinoids in the mother plants, it was confirmed that callus cultures of Cannabis were not able to produce detectable amounts of any cannabinoids.     

Survey of safflower (Carthamus tinctorius L.) germplasm for variants in fatty acid composition and other seed characters

Summary Two hundred safflower accessions, originated in 37 countries, and multiplied in two environments, were evaluated for fatty acid composition of the seed oil and other seed characters. Overall mean values of stearic and palmitic acids were similar in both environments but differed for seed weight and oil content. Oleic and linoleic acids showed also similar overall mean content in both environments but some entries with intermediate contents of these acids displayed significant variation among environments. Oleic and linoleic acids showed a tremendous range of variation, from 3.1 to 90.60% and from 3.9 to 88.8%, respectively. The ranges of variation observed for stearic, oleic and linoleic acids indicate that all the reccessive genes, already discovered, controlling high content of these acids, st, ol and li, are present in the collection. Moreover, the upper values of oleic, ten points higher than the published values for the high oleic genotype olol, suggest than other genes controlling such levels may be present.     

Hempseed as a nutritional resource: An overview

Summary The seed of Cannabis sativa L. has been an important source of nutrition for thousands of years in Old World cultures. Non-drug varieties of Cannabis, commonly referred to as hemp, have not been studied extensively for their nutritional potential in recent years, nor has hempseed been utilized to any great extent by the industrial processes and food markets that have developed during the 20th century. Technically a nut, hempseed typically contains over 30% oil and about 25% protein, with considerable amounts of dietary fiber, vitamins and minerals. Hempseed oil is over 80% in polyunsaturated fatty acids (PUFAs), and is an exceptionally rich source of the two essential fatty acids (EFAs) linoleic acid (18:2 omega-6) and alpha-linolenic acid (18:3 omega-3). The omega-6 to omega-3 ratio (n6/n3) in hempseed oil is normally between 2:1 and 3:1, which is considered to be optimal for human health. In addition, the biological metabolites of the two EFAs, gamma-linolenic acid (18:3 omega-6; GLA) and stearidonic acid (18:4 omega-3; SDA), are also present in hempseed oil. The two main proteins in hempseed are edestin and albumin. Both of these high-quality storage proteins are easily digested and contain nutritionally significant amounts of all essential amino acids. In addition, hempseed has exceptionally high levels of the amino acid arginine. Hempseed has been used to treat various disorders for thousands of years in traditional oriental medicine. Recent clinical trials have identified hempseed oil as a functional food, and animal feeding studies demonstrate the long-standing utility of hempseed as an important food resource.     

Oil content, tocopherol composition and fatty acid patterns of the seeds of 51 Cannabis sativa L. genotypes

The oil content, the tocopherol composition, the plastochromanol-8 (P-8) content and the fatty acid composition (19 fatty acids) of the seed of 51 hemp (Cannabis sativa L.) genotypes were studied in the 2000 and 2001 seasons. The oil content of the hemp seed ranged from 26.25% (w/w) to 37.50%. Analysis of variance revealed significant effects of genotype, year and of the interaction (genotype × year) on the oil content. The oil contents of the 51 genotypes in 2000 and 2001 were correlated (r = 0.37**) and averaged 33.19 ± 1.45% in 2000 and 31.21 ± 0.96% in 2001. The -tocopherol, -tocopherol, -tocopherol, P-8- and -tocopherol contents of the 51 genotypes averaged 21.68 ± 3.19, 1.82 ± 0.49, 1.20 ± 0.40, 0.18 ± 0.07 and 0.16 ± 0.04 mg 100g^–1 of seeds, respectively (2000 and 2001 data pooled). Hierarchical clustering of the fatty acid data did not group the hemp genotypes according to their geographic origin. The -linolenic acid yield of hemp (3–30 kg ha^–1) was similar to the -linolenic acid yield of plant species that are currently used as sources of -linolenic acid (borage (19–30 kg ha^–1), evening primrose (7–30 kg ha^–1)). The linoleic acid yield of hemp (129–326 kg ha^–1) was similar to flax (102–250 kg ha^–1), but less than in sunflower (868–1320 kg ha^–1). Significant positive correlations were detected between some fatty acids and some tocopherols. Even though the average content of P-8 in hemp seeds was only 1/120^th of the average -tocopherol content, P-8 content was more closely correlated with the unsaturated fatty acid content than -tocopherol or any other tocopherol fraction. The average broad-sense heritabilities of the oil content, the antioxidants (tocopherols and P-8) and the fatty acids were 0.53, 0.14 and 0.23, respectively. The genotypes Fibrimon 56, P57, Juso 31, GB29, Beniko, P60, FxT, Félina 34, Ramo and GB18 were capable of producing the largest amounts of high quality hemp oil.     

Somaclonal variation in Lotus corniculatus L. in relation to plant breeding purposes

Summary Seventy-two plants regenerated from leaf-derived calli of a single plant of Lotus corniculatus have been evaluated for several morphological and agronomical traits. The analysis of selfed and polycross progenies of the regenerants indicates that the variation among regenerants was, at least in part, of genetic origin. Most of the mutations induced by tissue culture were recessive and were detected only after sexual propagation. Although in vitro culture had a depressive effect for most of the traits, the selfed progenies of 2 regenerants displayed higher values for leaflet width and seed yield than the selfed progeny of the initial plant. However the somaclonal variation did not increase the variation for any trait with respect to the variation of the donor cultivar of the initial plant.     

The inheritance of chemical phenotype in <Emphasis Type="Italic">Cannabis sativa</Emphasis> L. (III): variation in cannabichromene proportion

The mechanism that controls the proportion of cannabichromene (CBC), a potential pharmaceutical, in the cannabinoid fraction of Cannabis sativa L. is explored. As with tetrahydrocannabinol (THC) and cannabidiol (CBD), CBC is an enzymatic conversion product of the precursor cannabigerol (CBG). CBC is reported to dominate the cannabinoid fraction of juveniles and to decline with maturation. This ontogeny was confirmed in inbred lines with different mature chemotypes. A consistent CBC presence was found in early leaves from a diverse clone collection, suggesting that CBC synthase is encoded by a fixed locus. Morphological variants possessing a ‘prolonged juvenile chemotype’ (PJC), a substantial proportion of CBC persisting up to maturity, are presented. PJC is associated with a reduced presence of floral bracts, bracteoles, and capitate-stalked trichomes. Genetic factors causing these features were independent of the allelic chemotype locus B that was previously postulated and regulates THC and CBD synthesis and CBG accumulation. In contrast to previously described Cannabis chemotypes, the cannabinoid composition of PJCs showed plasticity in that reduced light levels increased the CBC proportion. The ability of PJC plants to enable the production of pharmaceutical raw material with high CBC purity is demonstrated.     

The combined effect of plant stature and maturity on the response of wheat and triticale accessions to Septoria tritici

Summary The relationships between percent pycnidia coverage on the four uppermost leaves (PCD), plant height (PHT) and days to heading (HED) were evaluated for 21,000 wheat and triticale accessions tested in artificially inoculated (with fixed combination of S. tritici isolates) field nurseries over 8 trial years. A general Linear Model procedure (GLM) estimated Septoria severity using two correlative models: model 133-1 Year and model II–PCD=b₁PHT+b₂HED+C. The regression coefficients for PHT and HED in the two models were –0.54 and –0.40, respectively, with a R²=0.80^** and R²=0.29^** for model I and model II, respectively. The predicted cultivar best fitted to the model would be characterized as a semidwarf (PHT=115 cm) with an early-moderate maturity (HED=95 days to heading). The estimated mean percent pycnidial coverage for the two models over the 8 trial years was 40.8%. The performance of a group of 38 cultivars replicated yearly during the 8 trial years was assessed relative to model I. The deviation of each cultivar from the model was calculated using two functions: a) Sum Relative Serial Deviation (SRSD) and b) Total Relative deviation (TRD), in addition to Standard errors (SE). The proposed analytical protocol enabled identification of cultivars which expressed consistent yearly deviation (from the model) in host response combined with low-moderate mean pycnidial coverage (±30%). Such cultivars may possess a more stable type of genetic protection against the adverse effects of septoria tritici blotch.     

Copper localization in Cannabis sativa L. grown in a copper-rich solution

Summary With the aim to examine its potential as a renewable resource to decontaminate polluted soils, electron microscopy combined with X-ray microanalysis was used to investigate the localization of copper in Cannabis sativa grown in hydroponic copper-rich culture. Cu was found to accumulate preferentially in the upper leaf epidermal cells; it was also detected in spiculae and in abaxial trichomes too. Primary bast fibres seem to be not involved in copper accumulation.     

Variation in the crude protein content of field beans (Vicia faba L.) in relation to the possible improvement of the protein content of the crop

Summary A survey of the protein content of various field bean varieties indicates a range of variation extending over sixteen units of crude protein from 22–38%. Analysis of single plants from within three WPBS varieties Dylan. Danas and Dacre indicates that a similar range exists within commercial varieties. Examination of the progenies of high and low protein single plant selections together with a preliminary estimation of heritability suggest that protein content is highly heritable. No significant correlations were found between protein content and seed yield.     

Artificial neural networks as a tool for plant identification: a case study on Vietnamese tea accessions

Seventeen tea accessions belonging to Chinese (Camellia sinensis), Assamic (C. sinensis var. assamica), and Shan tea (C. sinensis var. pubilimba) groups, which are either commercially planted or new promising tea germplasm, were morphologically described at Phu Tho province (Viet Nam) and assessed for their diversity. Fourteen phyllometric parameters were qualitatively and quantitatively investigated using digital image analysis. The accessions were then discriminated by a dedicated artificial neural network for univocal plant identification and a hierarchical cluster analysis was performed in order to build a dendrogram reporting the relationships among them. Results proved the diversity of investigated tea morphotypes from Phu Tho province based on a morphological screening. More, the artificial neural network was able to perform a correct identification for almost all the accessions using simple dedicated instruments.     

Effectiveness of selection for plant characters of barley in simulated segregating rows

Summary Single plants of 12 barley (Hordeum vulgare L. and H. distichum L.) cultivars were randomized to form simulated segregating (mixed) rows. There were 16 mixed rows 15 cm apart, each row containing 24 plants 5 cm apart. In addition, every third row was a control row of one of the cultivars, Bonanza. Each plant was harvested separately.The cultivars differed significantly for all seven characters measured. These seven characters fell into 3 performance groups. Group I characters, seeds/head, 1000 kernel weight, days to ripe and height, showed a higher variance in the mixed rows than in the control rows, had high average heritabilities, and were consistent in performance from one row to another when measured by correlation amongst the cultivars. In Group II, yield/plant and seeds/plant, variance, heritabilities and performance consistency were all low or non-significant. Group III, heads/plant, fell between Groups I and II. These results suggested that single plants with the desired genotype could be successfully selected for characters in Group I only.The frequency with which the most desirable genotype would actually have been chosen with 12.5% selection intensity generally confirmed the above conclusions but indicated that the degree of differences among genotypes was also important in selecting the most desirable genotypes.Contribution No. NRG 77–9 from the Beaverlodge Research Station.     

Predicting the chemical composition of fibre and core fraction of hemp (Cannabis sativa L.)

Summary Fibre formation in hemp (Cannabis sativa L.) is a dynamic process. In order to follow this process, the chemical composition of the hemp stem was studied during plant development using the acid and neutral detergent fibre and lignin methods. Additionally, near infra-red spectroscopy was carried out. To predict the chemical composition of the stem samples partial least square (PLS) analysis was carried out. The developed PLS models can predict the level of %ADF and %NDF in the hemp bast and core samples. For %ADL, the models are only suited for either core or bast tissues and can not be used for both tissues simultaneously.     

Evaluation of Brassica genotypes for resistance to shatter. II. Variation in siliqua strengh within and between accessions

Summary Within-plant variation in strength of the siliquae, studied in seven Brassica accessions, appears to be mostly associated with variation in size, rather than the position of the siliquae in the infructescence. Position and strength did not show correlated variation in three accessions where the position of the siliqua in the infructescence appeared to significantly influence its strength (adjusted for the effect of size). There was significant variation in strength between plants within accessions. Considering the above results, variation between siliquae from the same accession could be reduced by sampling siliquae of average size as represented by length. When comparing accessions, a few siliquae from many plants would provide a more representative sample than many siliquae from few plants. Use of strength measures adjusted for the size of the siliquae (bending moment/length² and energy/length) would enhance the accuracy of estimation of inherent shatter-resistance. A minimum of three to six tests per accession appeared to be necessary for satisfactory evaluation of shatter-resistance.     

Screening fruit tree genetic resources in Belgium for disease resistance and other desirable characters

Summary The wide diversity of old fruit-tree cultivars originating or introduced into Belgium during the 18 ^th and 19 ^th centuries was collected as far as feasible over the last fifteen years at the State Plant Pathology Station in Gembloux. Out of the 2400 accessions now collected, one quarter was recovered from old public collections, and three quarters came from farms or gardens. The initial intention was to screen the material for disease resistance and other characters of agronomic interest with a view to using the best cultivars as breeding parents. However, as the collection developed, genetic resources conservation also became an objectiveper se. The collection presently contains 1150 apple, 850 pear and 300 plum accessions, and smaller numbers of other fruit species. Each accession is evaluated in an experimental orchard for at least ten years. In view of the growing public interest in old fruit-tree cultivars, the Plant Pathology Station has for several years been releasing to the nursery trade the better cultivars emerging from the evaluation, namely nine apple and four plum cultivars, and one peach cultivar. The principal features of the apple cultivars are presented in this paper. Since 1988, old apple and plum cultivars have been being used at the Station as parents in a breeding programme, with both controlled and open pollination. In some instances, old apple cultivars have also been crossed with a modern parent carrying the Vf gene for scab resistance. The preliminary observations on some of these seedlings are presented.     

Reaction of triticale,wheat and rye accessions to graminaceous Fusarium spp. infection at the seedling and adult plant growth stages

Summary Pathogenicity of 20 isolates of 12 Fusarium species recovered from triticale seed against seedlings of 14 varieties of winter cereals (triticale, wheat, and rye) was tested. The most pathogenic inoculum was a mixture of isolates (a composite isolate) of all the species. The following species were individually the most pathogenic: F. avenaceum, F. culmorum, F. sambucinum var. coeruleum, and F. graminearum. Winter triticale was more resistant to seedling blight than rye but more susceptible than wheat.Also reactions of 31 winter and 12 spring varieties of cereals to head inoculation with a composite isolate of 4 Fusarium spp. (F. avenaceum, F. culmorum, F. graminearum, and F. sambucinum var. coeruleum) was studied. In comparison to other cereals of similar type winter and spring wheat appeared to be the most susceptible while winter rye reaction was comparable to winter triticale. Spring and winter triticale varieties responded to head infection intermediately.There was no significant correlation between seedling and head reactions to infection with Fusarium spp. for winter rye and triticale. For winter wheat a negative trend was found. The above findings imply that screening of cereals at the seedling stage can not be used to predict the resistance to head blight. Nevertheless, resistance at the stage is highly desirable to prevent excessive damage of the crops due to the seedling blight incited by Fusarium spp..     

Optimisation of breeding for agronomic traits in fibre hemp (Cannabis sativa L.) by study of parent-offspring relationships

Summary In the framework of a comprehensive Dutch research project the feasibility of hemp cultivation for paper pulp production is being investigated. This project also includes a hemp breeding programme which was initiated at CPRO-DLO in 1990. Hemp breeding is primarily aimed at improving bast fibre production since bast fibre is qualitatively superior to woody core fibre for paper pulp production. The progress of the ongoing breeding programme is hampered by the lack of knowledge on the inheritance of agronomic traits.The following traits were examined on 252 parental plants in 1991 and subsequently on their progenies in 1992: fibre content, plant height, date of flowering, stem diameter and cannabinoid contents. For each trait heritabilities were estimated and direct and indirect effects of artificial selection were studied.The heritability of bast fibre content was high and mass selection proved to be an efficient method causing no undesirable influence on other characters. Characters not directly related to bast fibre yield such as date of flowering, plant height and stem diameter were shown to have disadvantages as selection criteria for the improvement of bast fibre yield.The cannabinoids THC and CBD were studied, as the acceptance of hemp cultivation requires a low level. The content of THC, the psychoactive component can be successfully reduced by mass selection, but it is not certain that mass selection is the most efficient method.