Tag Archives: clone

Heavy Metals Testing: Methods, Strategies & Sampling

By Charles Deibel
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Editor’s Note: The following is based on research and studies performed in their Santa Cruz Lab, with contributions from Mikhail Gadomski, Lab Manager, Ryan Maus Technical Services Analyst, Laurie Post, Director of Food Safety & Compliance, and Charles Deibel, President Deibel Cannabis Labs.


Heavy metals are common environmental contaminants resulting from human industrial activities such as mining operations, industrial waste, automotive emissions, coal fired power plants and farm/house hold water run-off. They affect the water and soil, and become concentrated in plants, animals, pesticides and the sediments used to make fertilizers. They can also be present in low quality glass or plastic packaging materials that can leach into the final cannabis product upon contact. The inputs used by cultivators that can be contaminated with heavy metals include fertilizers, growing media, air, water and even the clone/plant itself.

The four heavy metals tested in the cannabis industry are lead, arsenic, mercury and cadmium. The California Bureau of Cannabis Control (BCC) mandates heavy metals testing for all three categories of cannabis products (inhalable cannabis, inhalable cannabis products and other cannabis and cannabis products) starting December 31, 2018. On an ongoing basis, we recommend cultivators test for the regulated heavy metals in R&D samples any time there are changes in a growing process including changes to growing media, cannabis strains, a water system or source, packaging materials and fertilizers or pesticides. Cultivators should test the soil, nutrient medium, water and any new clones or plants for heavy metals. Pre-qualifying a new packaging material supplier or a water source prior to use is a proactive approach that could bypass issues with finished product.

Testing Strategies

The best approach to heavy metal detection is the use of an instrument called an Inductively Coupled Plasma Mass Spectrometry (ICP-MS). There are many other instruments that can test for heavy metals, but in order to achieve the very low detection limits imposed by most states including California, the detector must be the ICP-MS. Prior to detection using ICP-MS, cannabis and cannabis related products go through a sample preparation stage consisting of some form of digestion to completely break down the complex matrix and extract the heavy metals for analysis. This two-step process is relatively fast and can be done in a single day, however, the instruments used to perform the digestion are usually the limiting step as the digesters run in a batch of 8-16 samples over a 2-hour period.

Only trace amounts of heavy metals are allowed by California’s BCC in cannabis and cannabis products. A highly sensitive detection system finds these trace amounts and also allows troubleshooting when a product is found to be out of specification.

For example, during the course of testing, we have seen lead levels exceed the BCC’s allowable limit of 0.5 ppm in resin from plastic vape cartridges. An investigation determined that the plastic used to make the vape cartridge was the source of the excessive lead levels. Even if a concentrate passes the limits at the time of sampling, the concern is that over time, the lead leached from the plastic into the resin, increasing the concentration of heavy metals to unsafe levels.

Getting a Representative Sample

The ability to detect trace levels of heavy metals is based on the sample size and how well the sample represents the entire batch. The current California recommended amount of sample is 1 gram of product per batch.  Batch sizes can vary but cannot be larger than 50 pounds of flower. There is no upper limit to the batch sizes for other inhalable cannabis products (Category II).

It is entirely likely that two different 1 gram samples of flower can have two different results for heavy metals because of how small a sample is collected compared to an entire batch. In addition, has the entire plant evenly collected and concentrated the heavy metals into every square inch of it’s leaves? No, probably not. In fact, preliminary research in leafy greens shows that heavy metals are not evenly distributed in a plant. Results from soil testing can also be inconsistent due to clumping or granularity. Heavy metals are not equally distributed within a lot of soil and the one small sample that is taken may not represent the entire batch. That is why it is imperative to take a “random” sample by collecting several smaller samples from different areas of the entire batch, combining them, and taking a 1 g sample from this composite for analysis.


References

California Cannabis CPA. 12/18/2018.  “What to Know About California’s Cannabis Testing Requirements”. https://www.californiacannabiscpa.com/blog/what-to-know-about-californias-cannabis-testing-requirements. Accessed January 10, 2019.

Citterio, S., A. Santagostino, P. Fumagalli, N. Prato, P. Ranalli and S. Sgorbati. 2003.  Heavy metal tolerance and accumulation of Cd, Cr and Ni by Cannabis sativa L.. Plant and Soil 256: 243–252.

Handwerk, B. 2015.  “Modern Marijuana Is Often Laced With Heavy Metals and Fungus.” Smithsonian.com. https://www.smithsonianmag.com/science-nature/modern-marijuana-more-potent-often-laced-heavy-metals-and-fungus-180954696/

Linger, P.  J. Mussig, H. Fischer, J. Kobert. 2002.  Industrial hemp (Cannabis sativa L.) growing on heavy metal contaminated soil: fibre quality and phytoremediation potential. Ind. Crops Prod. 11, 73–84.

McPartland, J. and K. J McKernan. 2017.  “Contaminants of Concern in Cannabis: Microbes, Heavy Metals and Pesticides”.  In: S. Chandra et al. (Eds.) Cannabis sativa L. – Botany and Biotechnology.  Springer International Publishing AG. P. 466-467.  https://www.researchgate.net/publication/318020615_Contaminants_of_Concern_in_Cannabis_Microbes_Heavy_Metals_and_Pesticides.  Accessed January 10, 2019.

Sidhu, G.P.S.  2016.  Heavy metal toxicity in soils: sources, remediation technologies and challenges.   Adv Plants AgricRes. 5(1):445‒446.

Applications for Tissue Culture in Cannabis Growing: Part 1

By Aaron G. Biros
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Dr. Hope Jones, chief scientific officer of C4 Laboratories, believes there are a number of opportunities for cannabis growers to scale their cultivation up with micropropagation. In her presentation at the CannaGrow conference recently, Dr. Jones discussed the applications and advantages of tissue culture techniques in cannabis growing.

Dr. Hope Jones, chief scientific officer at C4 Labs

Dr. Jones’ work in large-scale plant production led her to the University of Arizona Controlled Environment Agriculture Center (CEAC) where she worked to propagate a particularly difficult plant to grow- a native orchid species- using tissue culture techniques. With that experience in tissue culture, hydroponics and controlled environments, she took a position at the Kennedy Space Center working for NASA where she developed technologies and protocols to grow crops for space missions. “I started with strawberry TC [tissue culture], because of the shelf life & weight compared with potted plants, plus you can’t really ‘water’ plants in space- at least not in the traditional way,” says Dr. Jones. “Strawberries pack a lot of antioxidants. Foods high in antioxidants, I argued, could boost internal protection of astronauts from high levels of cosmic radiation that they are exposed to in space.” That research led to a focus on cancer biology and a Ph.D. in molecular & cellular biology and plant sciences, culminating in her introduction to the cannabis industry and now with C4 Labs in Arizona.

Working with tissue culture since 2003, Dr. Jones is familiar with this technology that is fairly new to cannabis, but has been around for decades now and is widely used in the horticulture industry today. For example, Phytelligence is an agricultural biotechnology company using genetic analysis and tissue culture to help food crop growers increase speed to harvest, screen for diseases, store genetic material and secure intellectual property. “Big horticulture does this very well,” says Dr. Jones. “There are many companies generating millions of clones per year.” The Department of Plant Sciences Pomology Program at the Davis campus of the University of California uses tissue culture with the Foundation Plant Services (FPS) to eliminate viruses and pathogens, while breeding unique cultivars of strawberries.

A large tissue culture facility run in the Sacramento area that produces millions of nut and fruit trees clones a year.

First, let’s define some terms. Tissue culture is a propagation tool where the cultivator would grow tissue or cells outside of the plant itself, commonly referred to as micropropagation. “Micropropagation produces new plants via the cloning of plant tissue samples on a very small scale, and I mean very small,” says Dr. Jones. “While the tissue used in micropropagation is small, the scale of production can be huge.” Micropropagation allows a cultivator to grow a clone from just a leaf, bud, root segment or even just a few cells collected from a mother plant, according to Dr. Jones.

The science behind growing plants from just a few cells relies on a characteristic of plant cells called totipotency. “Totipotency refers to a cell’s ability to divide and differentiate, eventually regenerating a whole new organism,” says Dr. Jones. “Plant cells are unique in that fully differentiated, specialized cells can be induced to dedifferentiate, reverting back to a ‘stem cell’-like state, capable of developing into any cell type.”

Cannabis growers already utilize the properties of totipotency in cloning, according to Dr. Jones. “When cloning from a mother plant, stem cuttings are taken from the mother, dipped into rooting hormone and two to five days later healthy roots show up,” says Dr. Jones. “That stem tissue dedifferentiates and specializes into new root cells. In this case, we humans helped the process of totipotency and dedifferentiation along using a rooting hormone to ‘steer’ the type of growth needed.” Dr. Jones is helping cannabis growers use tissue culture as a new way to generate clones, instead of or in addition to using mother plants.

With cannabis micropropagation, the same principles still apply, just on a much smaller scale and with greater precision. “In this case, very small tissue samples (called explants) are sterilized and placed into specialized media vessels containing food, nutrients, and hormones,” says Dr. Jones. “Just like with cuttings, the hormones in the TC media induce specific types of growth over time, helping to steer explant growth to form all the organs necessary to regenerate a whole new plant.”

Having existed for decades, but still so new to cannabis, tissue culture is an effective propagation tool for advanced breeders or growers looking to scale up. In the next part of this series, we will discuss some of issues with mother plants and advantages of tissue culture to consider. In Part 2 we will delve into topics like sterility, genetic reboot, viral infection and pathogen protection.

An Introduction to Cannabis Genetics, Part III

By Dr. CJ Schwartz
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Polyploidy in Cannabis

Polyploidy is defined as containing more than two homologous sets of chromosomes. Most species are diploid (all animals) and chromosomal duplications are usually lethal, even partial duplications have devastating effects (Down’s syndrome). Plants are unique as in being able to somewhat “tolerate” chromosomal duplications. We often observe hybrid vigor in the F1, while the progeny of the F1 (F2) will produce mostly sickly or dead plants, as the chromosomes are unable to cleanly segregate.

polyploidy
Polyploids are generated when chromosomes fail to separate (non-disjunction) during pollen and egg generation. The chromosomes normally exist in pairs, thus having only one, or three, interferes in pairing in subsequent generations.

Chromosomal duplications, either one chromosome or the whole genome, happen frequently in nature, and actually serves as a mechanism for evolution. However the vast majority (>99.99%) results in lethality.

Thus there is polyploidy in Cannabis, and a few examples are supported by scientific evidence. The initial hybrid may show superior phenotypes and can be propagated through cloning, but there may be little potential for successful breeding with these plants.

Epigenetics and Phenotypic Consistency in Clones

One mechanism of turning off genes is by the DNA becoming physically inaccessible due to a structure resembling a ball. In addition, making molecules similar to DNA (RNA) that prevents expression of a gene can turn off certain genes. Both mechanisms are generally termed epigenetics.

These mice are all genetically identical yet they manifest different phenotypes for fur color.
These mice are genetically identical, yet their coat color phenotype is variable. Something above or beyond (epi) the gene (genetic) is controlling the phenotype.

Epigenetic regulation is often dependent on concentrations of certain proteins. Through the repeated process of cloning, it is possible that some of these proteins may be diluted, due to so many total cell divisions and epigenetic control of gene expression can be attenuated and results in phenotypic variability.

Sexual reproduction, and possibly tissue culture propagation, may re-establish complete epigenetic gene regulation, however the science is lacking. Epigenetic gene regulation is one of the hottest scientific topics and is being heavily investigated in many species including humans.

Hermaphrodites and Sex Determination

Cannabis is an extremely interesting genus (species?) for researching sex determination. Plants are usually either monoecious (both male and female organs on a single plant), or dioecious, separate sexes. Sex determination has evolved many times in many species. Comparing the mechanisms of sex determination in different organisms provides valuable opportunities to contrast and compare, thereby developing techniques to control sex determinations.

The sex organs on a Cannabis plant identified.
The sex organs on a Cannabis plant identified.

Cannabis is considered a male if it contains a Y-chromosome. Females have two X chromosomes. Even though female Cannabis plants do not have the “male” chromosome, they are capable of producing viable pollen (hermaphrodite) that is the source of feminized seeds. Therefore, the genes required to make pollen are NOT on the Y-chromosome, but are located throughout the remainder of the Cannabis genome. However, DNA based tests are available to identify Male Associated Sequence (MAS) that can be used as a test for the Y-chromosome in seedlings/plants.

Natural hermaphrodites may have resulted from Polyploidization (XXXY), or spontaneous hermaphrodites could be a result of epigenetic effects, which may be sensitive to the environment and specific chemical treatments.

Feminized seeds will still have genes segregating, thus they are not genetically identical. This shouldn’t lead to a necessary decrease in health, but could. A clone does not have this problem.

The other issue is that “inbreeding depression” is a common biological phenomenon, where if you are too inbred, it is bad…like humans. Feminized seeds are truly inbred. Each generation will decrease Heterozygosity, but some seeds (lines) may be unhealthy and thus are not ideal plants for a grower.

GMO– The Future of Cannabis?

Is there GMO (genetically modified organism) Cannabis? Probably, but it is likely in a lab somewhere…deep underground! Companies will make GMO Cannabis. One huge advantage to doing so is that you create patentable material…it is unique and it has been created.

The definition of a GMO is…well, undefined. New techniques exist whereby a single nucleotide can be changed out of 820 million and no “foreign” DNA remains in the plant. If this nucleotide change already exists in the Cannabis gene pool, it could happen naturally and may not be considered a GMO. This debate will continue for years or decades.

Proponents of GMO plants cite the substantial increase in productivity and yield, which is supported by science. What remains to be determined, and is being studied, are the long-term effects on the environment, ecosystem and individual species, in both plants and animals. Science-based opponent arguments follow the logic that each species has evolved within itself a homeostasis and messing with its genes can cause drastic changes in how this GMO acts in the environment/ecosystem (Frankenstein effect). Similarly, introducing an altered organism into a balanced ecosystem can lead to drastic changes in the dynamics of the species occupying those ecological niches. As in most things in life, it is not black and white; what is required is a solid understanding of the risks of each GMO, and for science to prove or disprove the benefits and risks of GMO crops.