Paolo Ranalli

Hemp as a raw material for industrial applications

SummaryDeveloped countries need to promote alternatives to crops produced in excess (such as cereals), and cultivation with limited environmental impact. Hemp (Cannabis sativa L.) is a potentially profitable crop, fitting into sustainable farming systems. Interest in “new” fibre crops is in fact increasing, to find alternatives to a high-input crop such as cotton, or to relieve the pressure of the paper industry on remaining natural forests. Besides, innovative applications are provided by hemp in renewable raw materials (oils from seeds and essential oils or secondary metabolites from inflorescences for food, cosmetic or pharmaceutical industry; straw and hurds for the building industry and for energy production). The development of more specialized hemp plants, needs a better understanding and control of metabolism. Since the renewed interest for this crop is increasing all over the world, it is important to update the knowledge on this crop, to understand whether it is a really sustainable, alternative and economically rewarding crop, and what contribution plant breeding, agronomical practice, and processing techniques are making, or could further make, to improve the relevant traits. The symposium “Hemp: perspectives for advanced utilization”, held in Bologna (Italy) on March 2004, was an opportunity to update the research carried out in different fields.

The sexual differentiation of Cannabis sativa L.: A morphological and molecular study

SummaryCannabis sativa L. is a dioecious species with sexual dimorphism occurring in a late stage of plant development. Sex is determined by heteromorphic chromosomes (X and Y): male is the heterogametic sex (XY) and female is the homogametic one (XX). The sexual phenotype of Cannabis often shows some flexibility leading to the differentiation of hermaphrodite flowers or bisexual inflorescences (monoecious phenotype). Sex is considered an important trait for hemp genetic improvement; therefore, the study of the mechanism of sexual differentiation is of paramount interest in hemp research. A morphological and molecular study of Cannabis sativa sexual differentiation has been carried out in the Italian dioecious cultivar Fibranova.Microscopic analysis of male and female apices revealed that their reproductive commitment may occur as soon as the leaves of the fourth node emerge; the genetic expression of male and female apices at this stage has been compared by cDNA-AFLP. A rapid method for the early sex discrimination has been developed, based on the PCR amplification of a male-specific SCAR marker directly from a tissue fragment.Five of the several cDNA-AFLP polymorphic fragments identified have been confirmed to be differentially expressed in male and female apices at the fourth node. Cloning and sequencing revealed that they belong to nine different mRNAs that were all induced in the female apices at this stage. Four out of them showed a high degree of similarity with known sequences: a putative permease, a SMT3-like protein, a putative kinesin and a RAC-GTP binding protein.

Current status and future scenarios of hemp breeding

SummaryHemp is a multi-use crop, able to provide fiber, cellulose, seeds and seed oil, cannabinoid, and biomass. Integrating many agroindustrial chains, Cannabis is considered a crop model in which insights into specific metabolic pathways and biosynthetic processes are valuable for improvement of the plant for all sets of industrial derivatives. In this review the hemp breeding status is elucidated and many aspects are focused: (i) recovering, maintenance and characterization of genetic resources; (ii) widening of germplasm and genetic variability; (iii) marker-assisted selection and development of breeding programs; (iv) sexual differentiation; (v) monitoring of THC content. Modern hemp varieties for fiber and other specific end uses have been developed and new varieties are entering production. The scenario for the breeding advances in hemp relies on basic and applied research which provides insights to identify a strategy for the design of modified plants with enhanced performance. This is accounted by the dissection of traits into components and the modification of single steps of the related metabolic pathways. These advances are provided by genomic techniques and are able: (i) to identify key genes encoding enzymes and regulatory factors participating in cannabinoid, fiber and oil biosynthesis; (ii) to identify the mode of regulation of these genes; (iii) to characterize the function of the selected genes through higher, lower or specific expression incited by specific promoters. The identification of molecular markers for specific traits, gathered in a saturated linkage map, will have a remarkable impact on hemp breeding. The advances in basic and applied research make it possible to design methods for the identification of superior parents and cross combinations and the development of selection schemes that rely on less labour-intensive and time-consuming methods.

Qualitative and Quantitative Aspects of the Inheritance of Chemical Phenotype in Cannabis

Abstract Four crosses were made between Cannabis plants with “pure” CBD and THC chemotypes. The F1 plants obtained were self-fertilised to produce F2s. Chemotypical distributions were analysed by gas-chromatography. A segregation analysis of the different F2 progenies obtained showed that chemotype, estimated as CBD/THC ratio, behaves as a qualitative character, and a model for a single locus B, with two co-dominant alleles, B D and B T is described. The CBD/THC ratios in the F1 offsprings were found to be significantly different in the heterozygous plants from the different pedigrees. The amount of CBD plus THC in the same pedigrees was also described. Heterosis was found to be a common feature, but not a general one, of cannabinoid accumulation in the F1s. Distribution of the values of cannabinoid content in classes was found to be normal. RAPD markers linked to the segregating chemotypes (“pure” CBD and “pure” THC) were identified by bulk segregant analysis, and the degree of linkage of these markers with the chemotype was described.

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.

The Applications of Molecular Markers in Genetics and Breeding of Hemp

Abstract Molecular markers were employed to the characterization and analysis of hemp genetic structure by using RAPD technique. The results are presented about the statistical treatment of the molecular data. In addition, markers tightly linked to the male sex and their applications are discussed, and a short protocol for direct amplification of such markers from hemp tissue is provided. A molecular map of hemp, including a number of RAPD markers obtained from a progeny of a cross between a female Carmagnola plant and a monoecious accession is also presented. Finally, the state of the art of sex genetics in hemp and the possibilities of developing molecular markers linked to different hemp chemotypes are discussed.

Hemp in Italy

Abstract Traditionally, hemp is a plant very well suited to Italian pedo-climatic conditions. The textile fibre produced in the past was of the finest quality due to integrated selected local varieties, good agrotechnology and experienced retting techniques. Attempts to reintro-duce this crop in Italy rely upon updating the chain of fibre production and its processing that lead to the textile and its derivatives. This article discusses a new research project designed to study production and utilization of the plant.

Effects of gamma-ray treatment on Cannabis sativa pollen viability

The viability and thein vitro germination capability of hemp pollen (cv. Carmagnola) were studied. Viability tests were based on the microscopic observation of the fluorescence of loaded fluorescein diacetate (FDA), while, for germinability tests, five different media were tested. The effects of irradiation with γ-rays on pollen viability and germination and on seed set were also studied, at three different irradiation doses (20, 60 and 100krad). The results show that in one of the media tested, about 85–90% of the pollen grains are viable and able to germinate in control samples, and that while viability measured by FDA test is not affected by increased γ-ray doses, the pollenin vitro germinability drops to about one-half of the controls at the maximum γ-ray dose employed, 100krad. Seed set of hemp plants pollinated with the irradiated pollen dropped to less than 1% of that of plants pollinated by untreated pollen for the higher dose used. The different media suitable forin vitro germination of hemp pollen, and the observed lack of correspondence between viability and germination capacity tests are discussed.

An International Symposium on the Most Advanced Applications that Can Be Foreseen for Hemp

Abstract A report of the recent symposium on “Hemp: perspectives for advanced utilisation,” held in Bologna (Italy) on March 5-6, 2004, and organised by the Istituto Sperimentale per le Colture Industriali, under the sponsorship of the Italian Ministry of Agriculture and Forestry, is presented. The symposium was organised into an Introduction and four sessions: Agronomy; Fibre and Seed; Cannabinoids and Endo-cannabinoids; and Germplasm and Marker Assisted Selection. Twenty-two speakers were present, from nine different countries. The main topics covered during the symposium are reported.