Influence of agronomic factors on yield and quality of hemp (Cannabis sativa L.) fibre and implication for an innovative production system

Research and development of an innovative production system for hemp (Cannabis sativa L.) fibre for textile use requires the integration of multidisciplinary knowledge from cultivation technique to realization of end products. Research was carried out to study the effect of the agronomic factors cultivation year (2003–2004), genotype (Futura 75 and Tiborszallasi), plant population (120, 240 and 360 plants m⁻²) and harvesting time (beginning and full flowering) on fibre yield and quality in the whole hemp stem, and in the basal and apical stem portions separately. The study of separate stem portions was done to determine the effect on fibre quality of an innovative harvesting and processing system in which hemp stems are cut in two portions of approximately 1 m at harvest to enable processing on modern flax scutching lines.     

Sowing time and prediction of flowering of different hemp (Cannabis sativa L.) genotypes in southern Europe

Fibre hemp can be grown under a wide range of agro-ecological conditions, but it requires special attention for several physiological features and crop management. A management strategy in order to reduce inputs and thus achieve acceptable yield could be achieved optimizing sowing time. With this respect, the effects of sowing date on hemp biology and yield was studied, using two monoecious and two dioecious genotypes. Field experiments were carried out in two subsequent years (2003-2004) in South of Italy, using drip irrigation system. Sowing time, in the two year period, ranged between March 10th and July 22nd. Optimal sowing time was observed between the end of April and the first three weeks of May; in that range, the dioecious Fibranova yielded the most in terms of aboveground biomass and stem dry yield, followed by Tiborszallasi, while the two monoecious showed the lowest yield. On the contrary, before and after that period, the shorter day length caused an early floral induction that strongly reduced stem and fibre elongation, and thus aboveground dry biomass and consequently stem yields. Based on this study a simulation model focusing on flowering prediction in Mediterranean environment was developed.     

Fatty acid composition and oxidation stability of hemp (Cannabis sativa L.) seed oil extracted by supercritical carbon dioxide

Supercritical carbon dioxide (SC-CO₂) was employed to extract oil from hemp (Cannabis sativa L.) seeds. For ground seeds, the supercritical extraction was carried out at temperatures of 40, 60 and 80 °C and pressures of 300 and 400 bar. Different solvent-ratios were applied. Supercritical CO₂ extractions were compared with a conventional technique, n-hexane in Soxhlet. The extraction yields, fatty acid composition of the oil and oxidation stability were determined. The seed samples used in this work contained 81% PUFAs, of which 59.6% was linoleic acid (ω-6), 3.4% γ-linolenic (ω-3), and 18% α-linolenic (ω-6). The highest oil yield from seeds was 22%, corresponding to 72% recovery, at 300 bar and 40 °C and at 400 bar and 80 °C. The highest oxidation stability corresponding to 2.16 mM Eq Vit E was obtained at 300 bar and 80 °C.     

QTL mapping for crude protein and protein fraction contents in rice (Oryza sativa L.)

Improvement of rice storage proteins is important in rice breeding for high nutritional quality. Seventy-one recombinant inbred lines (RIL) derived from a cross between japonica variety Asominori and indica variety IR24 were used to study the inheritance of crude protein and protein fraction contents in rice. A total of 16 QTL were identified and mapped on eight chromosomes. Several QTL affecting contents of different protein fractions were mapped in the same chromosomal region. In particular, two QTL with a significant contribution were identified to simultaneously affect prolamin and glutelin contents. One QTL denoted as qCP-12 affecting crude protein content (CP) was located in the same region as QTL qGLT-12 affecting glutelin content, in agreement with the positive correlation between glutelin level and protein content. QTL with larger genetic effects were further confirmed using two sets of chromosome segment substitution lines (CSSL), where Asominori and IR24 were used as the recurrent parents. By QTL comparative analysis, two QTL for CP, three for globulin content and one for prolamin content were located in the vicinity of CP QTL previously identified in polished rice. Application of these results in rice breeding is discussed.     

Biorefinery of Cannabis sativa using one- and two-step steam treatments for the production of high quality fibres

Among the sources of lignocellulosic biomass that could be considered as a feedstock for a biorefinery, hemp (Cannabis sativa) is known for the quality and relative abundance of its fibre. In this work, two variations of the Feedstock Impregnation Rapid and Sequential Steam Treatment (FIRSST) process are compared for the production of both fibres and biomolecules (including liquid fuels) as the valorization of the entire carbon content of biomass is needed to reach economical viability. In the first variation, the fibres were isolated via two specific and sequential steam treatments while in the second variation, fibres were isolated using only one steam treatment. Both processes allowed the isolation of a fibre that was comparable to kraft pulp while reducing the amounts of ashes along the process. Compositions of the residual solid fibre at different steps of the treatment were evaluated using ASTM and TAPPI standard methods. Carbohydrates were identified and quantified using HPLC with an anion exchange stationary phase. The qualities of the FIRSST and Kraft pulp produced were compared using standard ATPPC methods. Both pulps showed similar indexes. The two-steps FIRSST process has leaded to direct isolation of cellulose, hemicelluloses and lignin that could be suitable for the production of energy (biofuels) and high value chemicals, with small investments in terms of chemicals and energy.     

A set of near-isogenic lines for blast resistance genes with an Indica-type rainfed lowland elite rice (Oryza sativa L.) genetic background

A set of near-isogenic lines for blast resistance genes was developed by using an Indica-type elite rice variety, IR49830-7-1-2-2, suitable for the rainfed lowland conditions in the tropics, as a genetic background. Initially, we revealed that IR49830-7-1-2-2 harbors five blast resistance genes - Pia, Pib, Pik-s, Pita, and Pi11(t) - by using a differential system involving 19 selected standard blast isolates from the Philippines. Based on this result, we developed nine near-isogenic lines (NILs) targeting eight resistance genes - Pik, Pi7(t), Pi3, Pi5, Pita-2, Piz-5, Pish, and Pi9 - by recurrent backcrossing. The introgression of each resistance gene in the NILs was confirmed by reaction patterns to the blast isolates, allelism tests, and DNA marker analysis. In addition, a genome-wide DNA marker survey revealed that most of the chromosome regions in each NIL were of the IR49830-7-1-2-2 type. The agricultural characteristics of most of the developed NILs were almost the same as those of IR49830-7-1-2-2. Moreover, with one exception, they showed submergence tolerance similar to IR49830-7-1-2-2. The developed NILs could be used as a multiline variety suitable for the rainfed lowland in the tropics.     

Variability in essential oil content and composition of Origanum hirtum L., Origanum onites L., Coridothymus capitatus (L.) and Satureja thymbra L. populations from the Greek island Ikaria

Plants belonging to four “oregano” plant species (Origanum hirtum L., Origanum onites L., Coridothymus capitatus L., and Satureja thymbra L.) were collected during flowering from 33 sites located in the eastern part of the Greek island of Ikaria in the Eastern Aegean during April, May and July 2008. C. capitatus and O. hirtum were mostly observed in higher altitudes, whereas O. onites and S. thymbra in lower ones. The spatial distribution of all species was depicted on a GIS map. All four species exhibited essential oil concentrations higher than those reported in earlier literature, namely O. onites 3-4.3%, S. thymbra 4-6.5%, C. capitatus 3.7-5.6% and O. hirtum 5.5-10.0% (v/w). Carvacrol was the main constituent of the essential oils of all species, followed by γ-terpinene, p-cymene and caryophyllene, while thymol was not detected. All constituents varied remarkably among the four species, with carvacrol exhibiting the lowest variation. Carvacrol content varied between 72.3 and 89.2% in O. onites; 46.5 and 58.0% in S. thymbra; 82.9 and 90.9% in C. capitatus; and 84.4 and 93.8% in O. hirtum. By applying hierarchical cluster analysis on the basis of the essential oil constituents two main groups, divided into four subgroups of the taxa were evident. The first group consisted of O. onites and S. thymbra, while the second one of C. capitatus and O. hirtum. The results are discussed in terms of topography and climatic variation.     

Identification of a GDSL-motif carboxylester hydrolase from rice bran (Oryza sativa L.)

A major esterase (designated OsEST1) showing high activity using 1-naphthyl acetate as a substrate was identified from rice bran and purified approximately 239-fold to near-homogeniety. The purified enzyme migrated as a single polypeptide band on native and SDS-polyacrylamide gels and had a molecular mass of 25 kDa under denaturing conditions. Analysis of its tryptic peptides by MALDI-TOF-MS and subsequent data mining identified a corresponding cDNA OsEST1 consisting of 714 nucleotides and encoding a 238 amino acid protein. Analysis of its primary sequence indicated that OsEST1 is a GDSL-motif carboxylester hydrolase belonging to the SGNH protein subfamily in containing the putative catalytic triad of Ser¹¹, Asp¹⁸⁷, and His¹⁹⁰. OsEST1 showed the highest catalytic activity at approximately pH 8.0–8.5 and at 45 °C with K_m and V_max values for 1-naphthyl acetate of 172 μM and 63.7 μmol/min/mg protein, respectively. However, OsEST1 showed no activity with triacylglycerol. Alignment of the primary sequence of OsEST1 and other rice GDSL-motif esterases/lipases showed that OsEST1 aligns with a specific family of plant SGNH esterases involved in response to dehydration and cuticle formation. These results suggest that OsEST1 is not a lipase but an esterase activity which has some other function in rice, especially during seed development.     

Phloem sugars and amino acids as potential regulators of hordein expression in field grown malting barley (Hordeum vulgare L.)

The commercial quality of malting barley is dependent on the content and composition of grain proteins which are subjected to nitrogen (N) and sulfur (S) control. In nutrient deficient soils, grain protein content is mainly a consequence of the remobilization efficiency. In order to evaluate the effect of N and S soil availability on phloem amino acid and sugar export rates and B- and C-hordein gene expression during grain filling, a factorial combination of N and S fertilization assay was carried out under field conditions. Besides, several carbon (C) and N metabolites were analyzed in leaves and spikes. We observed that, even under soil N and S availabilities that do not limit yield, N and S fertilization induced significant changes in N and C metabolism. N phloem remobilization was promoted by S fertilization independently of N fertilization and C remobilization was also promoted by S fertilization but only in N fertilized plants. The B- and C-hordein gene expression correlated positively with sugar and amino acid exudation rate, respectively. Our results suggest an important role of the export rate of sugars and amino acids in the regulation of grain prolamine expression.     

Identification of deletion mutants with improved performance under water-limited environments in rice (Oryza sativa L.)

Rice (Oryza sativa L.) is a semi-aquatic member of the grass family that is poorly adapted to dry environments and has greater sensitivity to water-deficits than other important cereals in this family. To increase productivity in aerobic or water-limited environments rice must overcome its adaptations to flooded environments. Deletion mutants offer an alternative genetic resource for improving drought tolerance. Almost 3500 IR64 deletion mutants were screened under vegetative and reproductive stage drought stress in the field and evaluated for leaf drying and/or grain yield. Seven novel conditional mutants of rice which showed gain of function through continued growth as drought stress developed compared to the wild type were identified. Mutant recovery rate was 0.1%. Further evaluation of putative drought mutants revealed that their average shoot biomass at maturity and grain yield per plant under stress exceeded those of the wild type by two-fold. Studies under controlled conditions confirmed mutants to have continued growth of both roots and shoots as drought developed compared to the wild type, and a tendency for greater water extraction. We propose that deletions in these mutants have affected a regulator of the highly conservative growth response common to irrigated lowland rice cultivars. Our results suggest that screening deletion mutants for performance under managed drought stress in the field could be a highly effective way to identify valuable genetic resources for improved drought response and aerobic adaptation in rice.     

Genomic regions associated with the degree of red coloration in pericarp of rice (Oryza sativa L.)

The degree of red coloration (DRC) in pericarp of rice depends on the content of flavonoid compounds which have beneficial health effects for humans. In this study, 182 backcross-recombinant inbred lines (BILs) derived from Koshihikari (white pericarp)/Kasalath (red pericarp)//Koshihikari were used to detect the genomic regions associated with DRC through the QTL mapping approach. As a result, a total of four genomic regions were found to associate with DRC on chromosomes 1, 7, 9 and 11, respectively. Interestingly, the two genomic regions having the largest effects corresponded to previously characterized Rc and Rd genes on chromosome 7 and 1, respectively. In addition, two novel genomic regions having minor effects on DRC and located on chromosomes 9 and 11, respectively, are reported here for the first time. These results and the identification of tightly linked molecular markers that flank the genomic regions provide an opportunity for marker-aided improvement of red coloration in pericarp of rice.     

Evaluation of post-emergence herbicides for the control of wild oat (Avena fatua L.) in wheat and barley in Argentina

Wild oat (Avena fatua L.) is the most troublesome weed in cereal crops in Argentina. With the aim of studying the effects of different herbicides, doses, and wild oat growth stage at application on weed control and crop yield, field experiments were conducted in wheat and barley crops during three growing seasons in the south of Buenos Aires Province, Argentina. Treatments were post-emergence applications of new herbicide, pinoxaden + cloquintocet mexyl (5%-1.25%), at doses that ranged from 20 g to 60 g a.i. pinoxaden ha⁻¹, applied at two to three leaves and the beginning of tillering of wild oat. In addition, standard treatments were included and applied at the same wild oat growth stages. Diclofop methyl at 511 g a.i. ha⁻¹ and fenoxaprop-p-ethyl at 55 g a.i. ha⁻¹ were applied in barley. In wheat, diclofop methyl was replaced by clodinafop-propargyl + cloquintocet mexyl (24%-6%) at 36 g a.i. clodinafop-propargyl + 9 g cloquintocet mexyl ha⁻¹ and in 2008/09 wheat experiments, iodosulfuron plus metsulfuron methyl (5%-60%) at 3.75 g a.i. ha⁻¹ + 3 g a.i. ha⁻¹ also was included. In both crops, pinoxaden at 30 g a.i. ha⁻¹ and at higher rates, fenoxaprop-p-ethyl and clodinafop-propargyl gave the best control of wild oat. In 2006/07 wheat crops, treatments applied at tiller initiation provided better control than the early timing averaged across herbicides. However, wheat yield generally was greater with early application. In barley, wild oat control and crop yield were similar regarding time of application. Variations in crop yield were correlated with grain number m⁻² both in wheat and barley, but relationships between both grain number and spikes m⁻² and with grains per spike were identified only in wheat.     

燕麦不同磷效率品种苗期对不同磷源的利用研究

为了探究燕麦不同磷效率品种对有机磷的利用能力,选取磷高效品种燕科1号和磷低效品种MARION为供试材料,在土培盆栽条件下,设置植酸态磷(OP)、无机磷(KP)和不施磷(CK)三个磷处理,研究苗期不同磷处理对燕麦不同磷效率品种生物量、磷累积量、根际土壤酸性磷酸酶活性和土壤有效磷含量的影响。结果表明:(1)OP处理下,燕科1号和MARION的生物量较KP处理显著提高,分别提高70.4%、 9.4%;燕科1号的根冠比显著低于MARION。(2)与CK比较,OP处理可显著提高燕麦地上部、全株磷含量,但比KP处理磷含量低;在OP处理下,燕科1号地上部磷含量比MARION提高15.1%,全株磷含量比MARION降低8.6%。(3)与CK比较,OP处理可以提高燕麦根际土壤有效磷含量,但显著低于KP处理;相同处理下,燕科1号根际土壤有效磷含量均低于MARION。(4)两个燕麦品种在不同磷处理下,燕麦根际土壤酸性磷酸酶活性变化不同,燕科1号根际土壤酸性磷酸酶活性在KP和OP处理比CK分别提高35.2%、26.5%,差异均达到显著水平;MARION在KP和OP处理比CK分别降低4.4%、14.1%,OP与CK之间差异显著。磷高效品种燕科1号利用植酸态有机磷能力高于磷低效品种MARION。     

Radiation use efficiency,N accumulation and biomass production of high-yielding rice in aerobic culture

The concept of aerobic culture is to save water resource while maintaining high productivity in irrigated rice ecosystem. This study compared nitrogen (N) accumulation and radiation use efficiency (RUE) in the biomass production of rice crops in aerobic and flooded cultures. The total water input was 800–1300 mm and 1500–3500 mm in aerobic culture and flooded culture, respectively, and four high-yielding rice cultivars were grown with a high rate of N application (180 kg N ha⁻¹) at two sites (Tokyo and Osaka) in Japan in 2007 and 2008. The aboveground biomass and N accumulation at maturity were significantly higher in aerobic culture (17.2–18.5 t ha⁻¹ and 194–233  kg N ha⁻¹, respectively) than in flooded culture (14.7–15.8 t ha⁻¹ and 142–173 kg N ha⁻¹) except in Tokyo in 2007, where the surface soil moisture content frequently declined. The crop maintained higher N uptake in aerobic culture than in flooded culture, because in aerobic culture there was a higher N accumulation rate in the reproductive stage. RUE in aerobic culture was comparable to, or higher than, that in flooded culture (1.27–1.50 g MJ⁻¹ vs. 1.20–1.37 g MJ⁻¹), except in Tokyo in 2007 (1.30 g MJ⁻¹ vs. 1.37 g MJ⁻¹). These results suggest that higher biomass production in aerobic culture was attributable to greater N accumulation, leading to higher N concentration (N%) than in flooded culture. Cultivar differences in response to water regimes were thought to reflect differences in mainly (1) early vigor and RUE under temporary declines in soil moisture in aerobic culture and (2) the ability to maintain high N% in flooded culture.     

Evaluation of Macroalgae Sulfated Polysaccharides on the Leishmania (L.) amazonensis Promastigote

The sulfated polysaccharides from Solieria filiformis (Sf), Botryocladia occidentalis (Bo), Caulerpa racemosa (Cr) and Gracilaria caudata (Gc) were extracted and extensively purified. These compounds were then subjected to in vitro assays to evaluate the inhibition of these polysaccharides on the growth of Leishmania (L.) amazonensis promastigotes. Under the same assay conditions, only three of the four sulfated polysaccharides were active against L. amazonensis, and the polysaccharide purified from Cr was the most potent (EC₅₀ value: 34.5 μg/mL). The polysaccharides derived from Bo and Sf demonstrated moderate anti-leishmanial activity (EC₅₀ values of 63.7 μg/mL and 137.4 μg/mL). In addition, we also performed in vitro cytotoxic assays toward peritoneal macrophages and J774 macrophages. For the in vitro cytotoxicity assay employing J774 cells, all of the sulfated polysaccharides decreased cell survival, with CC₅₀ values of 27.3 μg/mL, 49.3 μg/mL, 73.2 μg/mL, and 99.8 μg/mL for Bo, Cr, Gc, and Sf, respectively. However, none of the sulfated polysaccharides reduced the cell growth rate of the peritoneal macrophages. These results suggest that macroalgae contain compounds with various chemical properties that can control specific pathogens. According to our results, the assayed sulfated polysaccharides were able to modulate the growth rate and cell survival of Leishmania (L.) amazonensis promastigotes in in vitro assays, and these effects involved the interaction of the sulfated polysaccharides on the cell membrane of the parasites.     

Growth and centelloside production in hydroponically established medicinal plant-Centella asiatica (L.)

Conditions to cultivate medicinally important herb Centella asiatica in hydroponic system are reported here for the first time. Growth kinetics of hydroponically grown plants was monitored over a period of 70 days. The maximum growth and dry matter accumulation (156.3% increment over the initial inoculum weight) in the cultured plants occurred around 42nd day. High Performance Liquid Chromatography (HPLC) analysis of the bioactive centellosides in the crude triterpenoids extract of the harvested leaves showed the presence of 11 mg, 1.7 mg, 36.6 mg and 6.3 mg of madecassoside, asiaticoside, madecassic acid and asiatic acid on per gram dry weight basis, respectively. The results of this study suggest that the cultivation of C. asiatica in hydroponic systems can be an effective platform for the production of clean and good quality C. asiatica herb for the pharmaceutical companies.     

Development of simple and co-dominant PCR markers to genotype puroindoline a and b alleles for grain hardness in bread wheat (Triticum aestivum L.)

Grain hardness is one of the most important quality characteristics of cultivated bread wheat (Triticum aestivum L.). A large deletion in the puroindoline a (Pina) gene or single nucleotide polymorphisms (SNPs) in the puroindoline b (Pinb) gene results in hard grain texture. So far, nine Pina alleles (Pina-D1a–Pina-D1b, Pina-D1k–Pina-D1q) and seventeen Pinb alleles (Pinb-D1a–Pinb-D1g, Pinb-D1p–Pinb-D1ab) have been identified in bread wheat. The major Pina and Pinb alleles identified in hard wheat cultivars are Pina-D1b, Pinb-D1b, Pinb-D1c and Pinb-D1d. In this study, a three-primer PCR system was employed to develop nine co-dominant STS markers for genotyping Pina-D1a and Pina-D1b, whereas temperature-switch (TS) PCR was used to develop six co-dominant SNP markers for genotyping the Pinb-D1a, Pinb-D1b, Pinb-D1c and Pinb-D1d alleles. These STS and TS-PCR markers were used to verify the grain hardness genotype of 100 wheat cultivars. The reliability and genotyping accuracy of TS-PCR markers were confirmed through sequencing of PCR products and a comparison with previously published results. Therefore, STS and TS-PCR markers offer a simple, cost-effective and reliable method for high-throughput genotyping Pina and Pinb alleles to select grain hardness in wheat quality breeding programs and for wheat market classification.     

Weed control and sensitivity of oats (Avena sativa) with various doses of saflufenacil

Saflufenacil is a new herbicide being developed by BASF for broadleaved weed control in maize, soybean and other crops prior to crop emergence. Six field studies were conducted in Ontario, Canada over a three year period (2008-2010) to evaluate the potential of saflufenacil applied pre-emergence (PRE) at various doses for broadleaved weed control in oats. Saflufenacil applied PRE caused minimal visible injury at 1, 2 and 4 weeks after emergence (WAE) in oats. At 4 WAE, the dose of saflufenacil required to provide 95% control of Ambrosia artemisiifolia (common ragweed), Chenopodium album (common lambsquarters), Polygonum convolvulus (wild buckwheat), Polygonum scabrum (green smartweed) and Sinapsis arvensis (wild mustard) was 72 to >100, >100, 74, 58 and >100 g ai ha⁻¹, respectively. Generally, similar saflufenacil dose-response trends were seen at 8 WAE. The doses of saflufenacil required to provide 95% reduction in density and dry weight ranged from 95 to >100 and 42 to >100 g ai ha⁻¹ respectively for A. artemisiifolia, C. album, P. convolvulus, P. scabrum and S. arvensis. Oat yield showed no sensitivity to saflufenacil at the doses evaluated. Based on this study, saflufenacil applied PRE can be safely used in spring planted oats for the control of some troublesome annual broadleaved weeds.     

Genetic analyses of heading date of Japonica rice cultivars from Northeast China

Northeast regions of China (38–55°N latitude) play an important role in Japonica rice planting. Heading dates of 10 Japonica rice cultivars native to the Northeast of China were investigated and their sensitivities to photoperiod and temperature were analyzed. The results showed that these Japonica rice cultivars were insensitive to photoperiod but strongly sensitive to temperature, i.e. a high temperature can markedly shorten the heading date. Genetic analyses were conducted on these 10 cultivars using a set of heading date tester lines. All these Japonica cultivars carried a dominant early heading gene Ef-1, and most of these cultivars carried the photoperiod insensitivity allele e₁, and two types of alleles were presented at the Se-1 locus, including recessive photoperiod insensitivity gene Se-1^e and dominant strong photoperiod-sensitivity (PS) gene Se-1ⁿ. The PS of these cultivars carrying E₁ or Se-1ⁿ can be repressed or weakened by Ef-1 and the recessive allele hd2 they carried. These results provided a reasonable explanation to the adaptability of Japonica rice to the high latitude environment of the Northeast China, and could be useful for breeding new cultivars well adapted to the high latitude regions and expanding the rice cultivation range.     

Differences in leaf yield and indigo precursors production in woad (Isatis tinctoria L.) and Chinese woad (Isatis indigotica Fort.) genotypes

Isatis tinctoria L. (woad) is one of the earliest known sources of indigo in Europe where it was cultivated since the Middle Ages. Isatisindigotica Fort. (Chinese woad), widely distributed in China, had been used from ancient times as indigo-producing plant and medicinal plant. Both species produce indigo precursors indican (indoxyl β-d glucoside) and isatan B (indoxyl ketogluconate) in their leaves. In order to identify new suitable crops for indigo production in Italy, 17 woad lines were studied under field conditions in Central Italy (Pisa, 43°40′N, 10°19′E) from 2001 to 2003. We analyzed the effects of year, genotype, and harvest times together with their reciprocal interactions on leaf yield and indigo precursors production. Woad lines were then compared with seven I. indigotica lines in a field crop experiment set up in 2003. Extraction and quantification of indigo precursors were accomplished by HPLC-ELSD. Isatan B and indican content, as well as equivalent indigo and fresh/dry leaf yield, were compared between species and among genotypes.