Microstructural Characterization of Prefabricated Hempcrete Blocks

Sustainable building materials have been developed to reduce the polluting emissions and the exploitation of natural resources of the building sector. Among these materials, an outstanding category is that of nature-based solutions which are produced recovering waste or by-products of agricultural cultivations and using them as vegetal aggregates to replace the traditional ones. This paper focusses on hempcrete which is produced mixing the by-product of industrial hemp cultivation (i.e., shives) and lime to obtain a sustainable, breathable and insulating material. The strength of hempcrete develops through carbonation of the binder that, leading to the formation of calcium or magnesium carbonates and mineralization of shives, determines the microstructure and hence most of the characteristic properties of the material. The aim of this research is to investigate how carbonation influences the microstructure of hempcrete when different recipes are used for blocks production. This study consists in the characterization of the material through techniques such as XRD (X-ray Diffractometry), SEM (Scanning Electron Microscopy) and TG-DTG (thermogravimetric analyses). Moreover, the evolution of carbonation is studied analyzing samples at different maturation times. The investigation of the carbonation reaction degree is also crucial to evaluate the environmental performances of the material because it allows the quantification of the carbon dioxide uptake. Also, periodic characterization allows to assess the durability of hempcrete and to select the best formulation according to the designed application and the corresponding service conditions.

The Effect of TIBA and NPA on Shoot Regeneration of Cannabis sativa L. Epicotyl Explants

Industrial hemp (Cannabis sativa L., family Cannabaceae) is a multi-purpose crop, used in the production of food, nutraceuticals, cosmetics and medicines. Therefore, development of new varieties with specific chemical profiles is necessary. In vitro culture methods could be complementary to conventional breeding and a useful tool for large-scale propagation. Strong apical dominance is considered as one of the factors contributing to the recalcitrance of industrial hemp in shoot proliferation. In this study, we tested the polar transport inhibitors N-1-naphtylphtalamic acid (NPA) and 2,3,5-triiodobenzoic acid (TIBA) to enhance shoot regeneration as the result of suppression of apical dominance and to develop in vitro protocols for Diana, Finola and Fedora 17 cultivars. Shoot tips derived from epicotyls were cultivated on Murashige and Skoog medium (MS) supplemented with meta-topolin (mT) and NPA, and also thidiazuron (TDZ) with a combination of TIBA and NPA. The results showed that the combination of TDZ with NPA (1–5 mg L−1) and TDZ with TIBA (0.5–2.5 mg L−1) increased the response of explants and the multiplication rate, but the effect was genotype-dependent and malformations were observed. To optimize the developed protocol, a two-step procedure with shortened time of exposure to inhibitors and reduced concentrations of them was applied. Shoots were rooted on media containing indole-3-butyric acid (IBA) and then successfully acclimatized. The obtained results will be useful in micropropagation of recalcitrant industrial hemp varieties.

Cytotoxic Potential of Industrial Hemp Extracts

Industrial hemp (Cannabis sativa L.) is a source of fibers, oil and valuable secondary metabolites. Regarding phenolic compounds, it has to be noted that the plant biosynthesizes molecules with various pharmacological benefits. The aim of this study was to prove the cytotoxic potential of hemp polar and non-polar fractions against two cancer cell lines (BT-20 and U87). The study revealed the potential antitumor activity of industrial hemp selective fractions but a correlation between polyphenols content and the cytotoxic effect could not be established.

Industrial Hemp (Cannabis sativa L.) Varieties and Seed Pre-Treatments Affect Seed Germination and Early Growth of Seedlings

Seed germination and seedling growth are two essential early determinants of subsequent crop yield and quality. A high germination percentage of industrial hemp (Cannabis sativa L.) seed is required to import into Australia. The viability of hemp seed can decline rapidly depending on storage and other factors; hence, the quality of imported seed is not always reliable. Here, we aimed to investigate germination and early seedling growth responses of 14 industrial hemp varieties after being imported from various countries. Germination trials were conducted with 100 seeds of 14 varieties using a soil-less Petri dish assay and a compost growth medium under glasshouse conditions. We also assessed the effect of seed pre-treatments such as gibberellic acid (500 and 1000 mg·L−1), chlorine dioxide (500 and 1000 mg·L−1) and cold temperature (4 °C for 72 h) using 300 seeds of each of the three selected varieties in compost growth medium. Hemp varieties imported from China had higher germination and better seedling growth indices than those imported from Europe. All seed pre-treatments were associated with a decreasing trend in germination, but a positive effect on early growth responses was observed. Our findings indicate that the hemp variety Han FNQ performed better than many other varieties did regarding seed germination and seedling growth. Hemp seeds sanitising with 500 mg·L−1 of chlorine dioxide might improve the germination and early growth of seedlings.

Selection and validation of reference genes for normalization of qRT-PCR data to study the cannabinoid pathway genes in industrial hemp

There has been significant interest in researching the pharmaceutical applications of Industrial hemp since its legalization three years ago. The crop is mostly dioecious and known for its production of phytocannabinoids, flavonoids, and terpenes. Although many scientific reports have showed gene expression analysis of hemp through OMICs approaches, unreliable reference genes for normalization of qRT-PCR data make it difficult to validate the OMICs data. Four software packages: geNorm, NormFinder, BestKeeper, and RefFinder were used to evaluate the differential gene expression patterns of 13 candidate reference genes under osmotic, heavy metal, hormonal, and UV stresses. EF-1α ranked as the most stable reference gene across all stresses, TUB was the most stable under osmotic stress, and TATA was the most stable under both heavy metal stress and hormonal stimuli. The expression patterns of two cannabinoid pathway genes, AAE1 and CBDAS, were used to validate the reliability of the selected reference genes. This work provides useful information for gene expression characterization in hemp and future research in the synthesis, transport, and accumulation of secondary metabolites.

Whole-Genome Resequencing of Wild and Cultivated Cannabis Reveals the Genetic Structure and Adaptive Selection of Important Traits

Background: Cannabis is an important industrial crop, whose bast fiber, seed, flowers and leaves are widely used by humans，especially cannabinoids extracted from plants as medicine is a hot spot in recent years. China is one of the origins of cannabis, where it has been cultivated and utilized for more than 6000 years, with the largest planting area of industrial hemp at present. China is rich in cannabis germplasm resources covering different latitudes (23 to 51°N) and is one of the few countries with wild cannabis resources. However, the genetic structure of Chinese cannabis populations and the adaptive selection of important traits remain unclear.Results: We identified the main morphological and physiological characteristics of wild cannabis and defined the genetic structure and relationships among wild and cultivated Chinese cannabis accessions and foreign representatives. This suggested that wild resources in Xinjiang have played an important role in the process of cannabis domestication. Adaptive selection analysis revealed that cultivated cannabis has undergone selective evolution or adaptation in flowering, growth and stress tolerance, and many functional genes were identified. Flowering characteristics analysis implied that wild cannabis is native to high latitudes and possesses the typical characteristic of early flowering, while cultivated cannabis has undergone a process of adaptive evolution to adjust to natural photoperiod conditions in different latitudes through regulation of FT-like expression.Conclusion: This study clarifies the genetic structure of Chinese cannabis and provides insight into adaptive selection and breeding in cannabis.

First Report of Fusarium falciforme (FSSC 3+4) Causing Rot of Industrial Hemp (Cannabis sativa) in California

Industrial hemp (Cannabis sativa) is a newly legal crop in California that is grown for cannabidiol oil, fiber and seed. In August 2019, whole plant decline and root rot were observed affecting <5% of plants in two industrial fields in Fresno County, CA. Symptoms included chlorotic, collapsed foliage, stem vascular discoloration, and root rot with abundant mycelial growth. Stem and root segments (1-2 cm) from three to five diseased plants were agitated in 0.1% tween-20 and soaked in 70% ethanol for 30 s and 1% NaOCl for 2 min. After incubating for 5 to 7 days on 1:10 potato dextrose agar (PDA) amended with tetracycline, Fusarium selective medium (FSM), and PARP (pimaricin + ampicillin + rifampicin + pentachloronitrobenzene [PCNB] agar) medium, white to pale cream aerial mycelium emerged from tissue of all plants on PDA and FSM but not PARP. Isolates cultured on 0.1% potassium chloride agar formed heads of microconidia on long monophialides consistent with the Fusarium solani species complex (FSSC) (Leslie and Summerell 2008). To obtain pure cultures of two isolates (CS529 and CS530), a single-hyphal tip was excised and grown on PDA. DNA was extracted from actively growing mycelium (PrepMan Ultra kit). The translation elongation factor gene (EF-1α) was amplified via PCR using EF1/EF2 primers (O’Donnell et al. 1998). Sequences of the two isolates were identical and deposited under accession number MW892973 in GenBank. The 599 bp sequence was 99.33% identical to FSSC 3 + 4 (Fusarium falciforme) accessions FD_01443_EF-1a based on FUSARIUM-ID BLAST analysis. To evaluate pathogenicity, stems of hemp plants (cv. ‘Berry Blossom’; n=8 plants per isolate) were wounded by penetrating the epidermis in an area about 0.5-cm square by 1-mm deep and 8-inches above the soil line. A 0.5 cm-diameter plug of 7-day old F. falciforme-colonized PDA was placed against the wound. Inoculation sites were loosely wrapped with parafilm for 2 days. A negative control consisted of a sterile PDA plug (n=3). Treatments were arranged in a completely randomized design in a greenhouse. The experiment was conducted once, due to regulatory restrictions at campus facilities. At 61 days post-inoculation, external stem lesions were significantly larger in diameter (P < 0.05; Tukey’s HSD) in plants inoculated with CS529 (8 ± 1 mm) compared to the control (2 ± 0 mm), and larger but not significant for CS530 (6 ± 1 mm). Internal stem lesions (i.e., rot in stele) were observed in plants inoculated with CS529 (9 ± 3 mm); stem rot was very minor in plants treated with CS530 (1 ± 1 mm) and nonexistent for control plants. No other disease symptoms were observed. F. falciforme was isolated from stems of CS529- and C530-inoculated plants. Sequences of re-isolates matched 100% with accession MW892973. These results suggest that F. falciforme causes rot in hemp in California. These studies specifically confirm stem rot abilities; field observations of root rot indicate root rotting abilities, but further tests are needed for confirmation. This is the first report of F. falciforme causing disease in industrial hemp. FSSC was described as causing foot rot in hemp in Italy (Sorrentino et al. 2019), but these isolates belonged to phylogenetic species 5 (F. solani) not F. falciforme. In addition, F. falciforme was reported as causing root rot in hydroponically grown cannabis (Punja and Rodriguez 2018). These studies provide the foundation for development of management tools for hemp disease.