The potential of hemp (Cannabis sativa L.) for sustainable fibre production: a crop physiological appraisal

Hemp (Cannabis sativa L.) fibre can be used as a raw material for paper and textile production. A comprehensive research programme in the Netherlands has concluded that fibre hemp is a potentially profitable crop, having the right profile to fit into sustainable farming systems. This paper presents an appraisal of the crop physiological characteristics and the agronomic potential of hemp. Parameter values of basic crop physiological characteristics such as light interception potential, radiation use efficiency and dry matter partitioning coefficients are given. The effect of crop management decisions such as cultivar choice, sowing date, plant density, and harvest date on the value of these parameters is discussed. A simple crop growth model was used to assess the yield potential of hemp for the climate of the Netherlands. Calculations made for a non-stressed late-flowering hemp crop sown on 15 April and harvested on 15 September give a stem dry matter yield of 17.1 t ha^-1. The effects of advancing or delaying sowing or harvest date on stem yield were calculated. Crop physiological characteristics of hemp are compared to those of kenaf (Hibiscus cannabinus L.). Radiation use efficiency and dry matter partitioning coefficients of the two crops are similar. Base temperatures for development and growth are lower in hemp than in kenaf. In a temperate climate with cool springs, canopy establishment will be more rapid in hemp than in kenaf. Hemp seems an excellent candidate to fill the niche for an annual fibre crop in a temperate climate.     

The effect of temperature on leaf appearance and canopy establishment in fibre hemp (Cannabis sativa L.)

The effects of temperature on the development and growth of hemp (Cannabis sativa L.) have never been quantified. Therefore, to establish the effect of temperature on leaf appearance and canopy establishment of fibre hemp under controlled and field conditions, plants were grown in growth chambers at 11 regimes with average temperatures between 10°C and 28°C, and three cultivars were sown in the field in March, April and May in 1990, 1991 and 1992. In the field, thermal time (base 0°C) between sowing and emergence ranged from 68°Cd to 109.5°Cd (average 88.3°Cd). Rates of leaf appearance and stem elongation increased linearly with temperature between 10°C and 28°C. The base temperature for leaf appearance was 5.7°C from the growth chamber experiments and 1°C from the field experiments. In the field, the base temperature for the relationship between light interception by the canopy and thermal time was 2.5°C, and thermal time, calculated at the appropriate base temperature, accounted for about 98% of the variance in the number of leaves and for 98.6% of the variance in the proportion of light intercepted by the canopy. Days from emergence accounted for less of the variance in both parameters than thermal time. Interception of 90% of light was attained on average at 465°Cd (base 0°C) after emergence. It is concluded that thermal time is a simple and accurate tool to describe leaf appearance and light interception in fibre hemp.     

Genotypic variability for callus formation and plant regeneration in rice (Oryza sativa L.)

Summary Sixty rice varieties (Oryza sativa L.), belonging to three subspecies, japonica, indica and javanica (some japonicaXindica hybrids were included), were compared for their capacity for callus growth and plant regeneration. Tissue cultures initiated from mature seeds on Murashige and Skoog's (1962) medium with 2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) were transferred to a medium containing 0.02 mg/l 2,4-D and 10 mg/l kinetin, from which plantlets were regenerated. Large variabilities in callus growth and plant regeneration potentials were revealed among the varieties tested. Most japonica varieties formed a callus that weighed more than 100 mg per seed 30 days after inoculation, and showed a relatively high regenerative potential, whereas indica varieties, japonica-indica hybrids and javanica varieties showed poor callus growth and plant regeneration, although considerable varietal variation was observed in each subspecies. The callus growth potential was not correlated with the plant regeneration potential. Histological observations revealed that the epithelium cells of the scutellum mainly proliferated to form a callus, from which shoot and root primordia were differentiated independently from each other. The shoot primordia developed into plantlets when roots were formed adventitiously. In a few cases, shoots and roots were bilaterally initiated from a single primordium.     

Microgametophytic selection in two alfalfa (Medicago sativa L.) clones

Summary Microgametophytic selection was investigated using two ecologically diverse autotetraploid clones of alfalfa. Several selection pressures (drying, aging, freezing, and high and low temperatures) were applied to microgametophytes at three stages of the life cycle, 1) during microsporogenesis, 2) post-anthesis, and 3) pollen tube growth. Pollen aging produced a progeny population with a greater mean plant size and a lower coefficient of variation than the control progeny. High temperature (29.5 °C) applied both during microsporogenesis and pollen tube growth resulted in progeny populations which were significantly taller and, in one case, had a larger leaf number than the control populations. In contrast, air dried pollen resulted in a progeny population which had significantly smaller character means and larger coefficients of variation than the control population. Also, low temperature (15 °C) during pollen tube growth yielded progeny with reduced branch number and a larger coefficient of variation than the control progeny. In cases where progeny derived from selected microgametophytes were found to differ from the control offspring, corresponding shifts in the reciprocal cross were not observed. For the temperature stress treatments, the lack of reciprocal differences may be related to the different temperature adaptations of the two ecotypes. These results suggest that microgametophytic selection can be effective in shifting the mean of the progeny generation; however, the results obtained will vary depending upon the selection pressure, stage of selection, and the parents used.     

Relationship between growth,nitrogen fixation and assimilation in a legume (Medicago sativa L.)

Summary Symbiotic N₂ fixation, NO ₃ ⁻ assimilation and protein accumulation in the shoots were measured simultaneously in alfalfa (Medicago sativa L.) grown in the field or in pots, in order to study how the balance between the two modes of nitrogen nutrition could be influenced by agronomic factors, such as harvest, mineral nitrogen supply and drought stress. During periods of rapid growth, fixation and assimilation may function simultaneously; they are antagonistic at the beginning and at the end of the growth cycle, when the nitrogen requirement of the plant is lower. When nitrogen nutrition does not limit growth, mineral nitrogen supply favours assimilation at the expense of fixation, but does not modify the amount of nitrogen accumulated, which is adjusted to the growth capacity of the plant. After cutting, nitrate assimilation compensated for the decrease in fixation and supplied the plant with the nitrogen required by the regrowth, the proliferation of which determined the fixation recovery. Drought stress decreased N₂ fixation much more than NO ₃ ⁻ assimilation. The latter made growth recovery possible when water supply conditions became normal again. These results suggested the existence of an optimum level of nitrate assimilation, which differed depending on the age of the plants and allowed both maximum growth and fixing activity.     

Identification of DNA markers linked to the male sex in dioecious hemp (Cannabis sativa L.)

 A 400-bp RAPD marker generated by a primer of random decamer sequence has been found associated with the male sex phenotype in 14 dioecious cultivars and accessions of hemp (Cannabis sativa L.). The primer OPA8 generates a set of bands, most of which polymorphic among all the individual plants tested, and 1 of which, named OPA8₄₀₀, present in all male plants and absent in female plants. A screening of 167 plants belonging to different genotypes for the association of the OPA8₄₀₀ marker with the sex phenotype revealed that only in 3 cases was the 400-bp band was present in plants phenotypically female; on the contrary, in male plants the band was never missing, while in monoecious plants it was never present. Despite this sex-specific association, the sequences corresponding to OPA8₄₀₀ were present in both staminate and carpellate plants, as revealed by Southern blotting and hybridization with the cloned RAPD band. The RAPD marker was sequenced, and specific primers were constructed. These primers generated, on the same genotypes used for RAPD analysis, a SCAR marker 390 bp in length and male-specific. This SCAR is suitable for a precise, early and rapid identification of male plants during breeding programs of dioecious and monoecious hemp. Received: 16 January 1998 / Accepted: 30 April 1998     

In silicio expression analysis of PKS genes isolated from Cannabis sativa L

Cannabinoids, flavonoids, and stilbenoids have been identified in the annual dioecious plant Cannabis sativa L. Of these, the cannabinoids are the best known group of this plant's natural products. Polyketide synthases (PKSs) are responsible for the biosynthesis of diverse secondary metabolites, including flavonoids and stilbenoids. Biosynthetically, the cannabinoids are polyketide substituted with terpenoid moiety. Using an RT-PCR homology search, PKS cDNAs were isolated from cannabis plants. The deduced amino acid sequences showed 51%-73% identity to other CHS/STS type sequences of the PKS family. Further, phylogenetic analysis revealed that these PKS cDNAs grouped with other non-chalcone-producing PKSs. Homology modeling analysis of these cannabis PKSs predicts a 3D overall fold, similar to alfalfa CHS2, with small steric differences on the residues that shape the active site of the cannabis PKSs.     

Heavy metal tolerance and accumulation of Cd, Cr and Ni by Cannabis sativa L.

Experiments in semi-natural conditions were undertaken to assess hemp metal tolerance and its ability to accumulate cadmium, nickel and chromium. Cannabis sativa was grown in two soils, S1 and S2, containing 27, 74, 126 and 82, 115, 139 g g^–1 of Cd, Ni and Cr, respectively. After two months from germination and at ripeness, no significant alteration in plant growth or morphology was detected. On the contrary, a high hemp reactivity to heavy metal stress with an increase in phytochelatin and DNA content was observed during development, suggesting the Cannabis sativa ability to avoid cell damage by activating different molecular mechanisms. Metals were preferentially accumulated in the roots and only partially translocated to the above-ground tissues. The mean shoot Cd content was 14 and 66 g g^–1 for S1 and S2 soil, respectively. Although not negligible concentrations they were about 100 times lower than those calculated for the hyperaccumulator Thlaspi caerulescens. Similarly Ni uptake was limited if compared with that of the Ni-hyperaccumulator Alyssum murale. Chromium uptake was negligible. As expected on the base of the metal concentration detected in ripe plants, no statistically significant variation in soil metal content was detected after one crop of hemp. Nevertheless, a consistent amount (g) of Cd and Ni is expected to be extracted by 1 ha biomass of hemp (about 10 t) per year and along the time a slow restoration of deeper soil portions can be obtained by its wide root system (at least 0,5 m deep). In addition, the possibilities of growing hemp easily in different climates and using its biomass in non-food industries can make heavy metal contaminated soils productive. This means economical advantage along with a better quality of soil.     

Hairy root transformation in alfalfa (Medicago sativa L.)

Summary The widely cultivated forage legume alfalfa (Medicago sativa L.) was transformed with the agropine type Agrobacterium rhizogenes NCPPB 1855. Sterile root and callus cultures were derived from tumorous hairy roots which were easily obtained independent of the plant variety or genotype. Plant regeneration, via somatic embryogenesis, was achieved only when a selected alfalfa line, characterized by high regenerative capability, was utilized. Genetic transformation was confirmed by the presence of agropine and T-DNA. Phenotypic alterations, mainly affecting the root system, were observed in transformed plants. The possibility that T-DNA-induced variations could be useful in the improvement of M. sativa is discussed.Research work was partially supported by Progetto Strategico Agrobiotecnologia C.N.R., Italy     

RFLP mapping of QTLs for yield and related characters in rice (Oryza sativa L.)

Quantitative triat loci (QTLs) for yield and related traits in rice were mapped based on RFLP maps from two indica/indica F₂ populations, Tesanai 2/CB and Waiyin 2/CB. In Tesanai 2/CB, 14 intervals carrying QTLs for eight traits were detected, including 3 for grain weight per plant (GWT), 2 for number of panicles per plant (NP), 2 for number of grains per panicle (NG), 1 for total number of spikelets per panicle (TNS), 1 for spikelet fertility (SF), 3 for 1000-grain weight (TGWT), 1 for spikelet density (SD), and 1 for number of first branches per main panicle. The 3 QTLs for GWT were located on chromosomes 1, 2, and 4, with 1 in each chromosome. The additive effect of the single locus ranged from 2.0 g to 9.1 g. A major gene (np4) for NP was detected on chromosome 4 within the interval of RG143–RG214, about 4cM for RG143, and this locus explained 26.1% of the observed phenotypic variance for NP. The paternal allele of this locus was responsible for reduced panicles per plant (3 panicles per plant). In another population, Waiyin 2/CB, 12 intervals containing QTLs for six of the above-mentioned traits were detected, including 3 for GWT, 2 for each of NP, TNS, TGWT and SD, 1 for SF. Three QTLs for GWT were located on chromosome 1, 4, and 5, respectively. The additive effect of the single locus for GWT ranged from 6.7 g to 8.8 g, while the dominance effect was 1.7–11.5 g. QTL mapping in two populations with a common male parent is compared and discussed.     

Agrobacterium infection of hemp (Cannabis sativa L.): establishment of hairy root cultures

Abstract

Experimental conditions were optimized for hemp, a difficult to transform plant, to be effectively infected with either Ri or Ti plasmid-bearing agrobacteria and to establish stably transformed tissues. Hypocotyl of intact seedlings was the most responsive material and the response depended on both bacterial strain and plant variety. Transformed tissues, hairy roots and tumors, were cultured and stabilized in vitro and showed the characteristic traits of fast and phytohormone-independent growth as well as high incidence of lateral branching and abundance of root hairs in the case of roots. They all contained T-DNA of the corresponding Ri or Ti plasmid as revealed by PCR analysis with specific primers and further hairy roots induced by AR10GUS strain showed normal pattern of β-glucuronidase positive staining. To our knowledge, this represents the first reported protocol for the establishment of Cannabis sativa hairy root cultures.     

Simple dehydration treatment promotes plantlet regeneration of rice (Oryza sativa L.) callus

Summary To increase plantlet regeneration frequency, rice callus was dehydrated in a Petri dish with a single layer of filter paper prior to transfer to the regeneration medium. With a 24 h dehydration treatment, the regeneration frequency was increased to 47 %, while the regeneration frequency of the untreated control was less than 5 %. This relatively simple method provides an alternative method for improving the regeneration frequency of rice callus.     

Fine root demography in alfalfa (Medicago sativa L.)

In perennial forages like alfalfa (Medicago sativa L.), repeated herbage removal may alter root production and mortality which, in turn, could affect deposition of fixed N in soil. Our objective was to determine the extent and patterns of fine-diameter root production and loss during the year of alfalfa stand establishment. The experiment was conducted on a loamy sand soil (Udorthentic Haploboroll) in Minnesota, USA, using horizontally installed minirhizotrons placed directly under the seeded rows at 10, 20, and 40 cm depths in four replicate blocks. We seeded four alfalfa germplasms that differed in N₂ fixation capacity and root system architecture: Agate alfalfa, a winter hardy commercially-available cultivar; Ineffective Agate, which is a non-N₂-fixing near isoline of Agate; a new germplasm that has few fibrous roots and strong tap-rooted traits; and a new germplasm that has many fibrous roots and a strongly branched root system architecture. Video images collected biweekly throughout the initial growing season were processed using C-MAP-ROOTS software.More than one-half of all fine roots in the upper 20 cm were produced during the first 7 weeks of growth. Root production was similar among germplasms, except that the highly fibrous, branch-rooted germplasm produced 29% more fine roots at 20 cm than other germplasms. In all germplasms, about 7% of the fine roots at each depth developed into secondarily thickened roots. By the end of the first growing season, greatest fine root mortality had occurred in the uppermost depth (48%), and least occurred at 40 cm (36%). Survival of contemporaneous root cohorts was not related to soil depth in a simple fashion, although all survivorship curves could be described using only five rates of exponential decline. There was a significant reduction in fine root mortality before the first herbage harvest, followed by a pronounced loss (average 22%) of fine roots at the 10- and 20-cm depths in the 2-week period following herbage removal. Median life spans of these early-season cohorts ranged from 58 to 131 days, based on fitted exponential equations. At all depths, fine roots produced in the 4 weeks before harvest (early- to mid-August) tended to have shorter median life spans than early-season cohorts. Similar patterns of fine root mortality did not occur at the second harvest. Germplasms differed in the pattern, but not the ultimate extent, of fine root mortality. Fine root turnover during the first year of alfalfa establishment in this experiment released an estimated 830 kg C ha^–1 and 60 kg N ha^–1, with no differences due to N₂ fixation capacity or root system architecture.     

Molecular cloning and expression analysis of a monosaccharide transporter gene OsMST4 from rice (Oryza sativa L.)

Two putative glycosyltransferases in Arabidopsis thaliana, designated reduced residual arabinose-1 and -2 (RRA1 and RRA2), are characterized at the molecular level. Both genes are classified in CAZy GT-family-77 and are phylogenetically related to putative glycosyltranferases of Chlamydomonas reinhardtii. The expression pattern of the two genes was analyzed by semi-quantitative RT-PCR using mRNA extracted from various organs of bolting Arabidopsis thaliana plants. In addition, promoter::gusA analysis of transgenic Arabidopsis thaliana containing a fusion between either the RRA-1 or -2 promoter fragment and the gusA reporter gene showed that whereas the RRA1 promoter was primarily active in the apical meristem, the expression pattern of the RRA2 promoter was more diverse but also highly active in the meristematic region. In addition, T-DNA mutant insertion lines of both RRA-1 and -2, were identified and characterized at the molecular and biochemical level. Monosaccharide compositional analyses of cell wall material isolated from the meristematic region showed a ca. 20% reduction in the arabinose content in the insoluble/undigested cell wall residue after enzymatic removal of xyloglucan and pectic polysaccharides. These data indicate that both RRA-1 and -2 play a role in the arabinosylation of cell wall component(s).     

Production of doubled haploid rice plants (Oryza sativa L.) by anther culture

The results of anther culture of F₂ pollen issued from 23 single crosses are presented. A relation between the morphology of the panicle and the microspore stage was established. After cold-pretreatment (8 days at 4°C), the anthers were cultured on the callus-induction medium N6 supplemented with 1 mg l^–1 naphthaleneacetic acid. The calli were transferred to MS plant regeneration medium supplemented with 3 mg l^–1 kinetin + 0.5 mg l^–1 naphthaleneacetic acid. The induction frequency varied from 0.22% to 29% and the regeneration frequency from 0% to 144.4%, dependent upon the crosses used. On average, 27% of the plants obtained were albinos and 59% of the green plants underwent spontaneous chromosome doubling. Thirtynine doubled haploid lines were evaluated and multiplied in the field. Lines with an excellent behaviour in upland culture conditions were selected from two crosses.     

Ammonium nutrition increases photosynthesis rate under water stress at early development stage of rice (Oryza sativa L.)

An hydroponic experiment with a simulated water stress induced by PEG (6000) was conducted in a greenhouse to study the effects of nitrate (NO₃ ⁻), ammonium (NH₄ ⁺) and the mixture of NO₃ ⁻ and NH₄ ⁺, on water stress tolerance of rice seedlings. Rice (Shanyou 63) was grown under non- or simulated water stress condition (10% (w/v) PEG, MW6000) with the 3 different N forms during 4 weeks. Under non-stressed condition no difference was observed among the N treatments. Under simulated water stress, seedlings grown on N-NO₃ ⁻ were stunted. Addition of PEG did not affect rice seedling growth in the treatment of only NH₄ ⁺ supply but slightly inhibited the rice seedling growth in the treatment of mixed supply of NO₃ ⁻ and NH₄ ⁺. Simulated water stress, when only N-NH₄ ⁺ was present, did not affect leaf area and photosynthesis rate, however, both parameters decreased significantly in the NO₃ ⁻ containing solutions. Under water stress, Rubisco content in newly expanded leaves significantly increased in the sole NH₄ ⁺ supplied plants as compared to that in plants of the other two N treatments. Under water stress, the ratio of carboxylation efficiency to Rubisco content was, respectively, decreased by 13 and 23% in NH₄ ⁺ and NO₃ ⁻ treatments, respectively. It is concluded that, water stress influenced the Rubisco activity than stomatal limitation, and this effects could be regulated by N forms. Responsible Editor: Herbert Johannes Kronzucker. Shiwei Guo and Gui Chen contributed equally to this paper.     

Selection of higher regenerative callus and change in isozyme pattern in rice (Oryza sativa L.)

Summary Calli were initiated from seedling roots in rice (Oryza sativa L. var. Tadukan) and subcultured at 45-day intervals on MS medium supplemented with 2 mg/l 2,4-D. Sectors of callus which differentiated shoot meristems (green spots) under the same 2,4-D concentration were selected from the calli subcultured 90 days after initiation. The selection was continued for about 2 years. Responses to 2,4-D between original and selected lines differed considerably, although differentiation was not generally seen in rice callus in the presence of 2 mg/l 2,4-D. After 180 days, calli of the selected line differentiated into numerous shoot-bud primordia and grew out new callus tissues under 2 mg/l 2,4-D concentration; the frequency of the differentiation exceeded 90%. On the other hand, no calli of non-selected line differentiated into shootbuds under 2 mg/l 2,4-D, and the frequency of the shootbud was only about 50% under lower 2,4-D concentration (0.1 mg/l). The pattern and activity of peroxidase isozyme varied markedly between calli of the selected and non-selected lines. First, two strong peroxidase bands which show fast mobility and one intermediate peroxidase band with slow mobility were detected only in the calli of selected line. Secondly, changes in band pattern of proteins separated by SDS-PAGE were observed. In the calli of selected line, there was a loss of the polypeptide bands with molecular weight of 24 and 42 K in the selected calli, but they were clearly present in the unselected line. The appearance of new peroxidase isozyme bands and loss of polypeptide bands, change in response to auxin and increased ability for shoot bud differentiation are closely correlated to each other.