Cannabidiol (CBD) is now one of the most discussed and craved chemicals on the market. The three famous letters drew in 6.4 million Google searches in the United States alone during April 2019 – outpacing other leading wellbeing-related search terms, such as acupuncture, meditation, and veganism. And this curiosity isn’t idle; US sales of cannabis- and hemp-derived CBD products are expected to surge to $20 billion by 2024.

With such values at stake, cannabis cultivators aren’t taking any chances with their CBD contents. Since the passage of the 2018 Farm Bill last December, the total number of US acres used for cultivating hemp has increased by 328 percent.

But is there a better way of boosting CBD supplies other than just growing more hemp? With the top tools of genetic science now at the cannabis cultivator’s disposal, there just might be.





Galvanizing green genes

“We have several assays designed for multiple different markers across the cannabis genome,” Anthony Torres explained. As a research scientist for Steep Hill, one of the first commercial medical cannabis labs in the US, Torres spends his time carrying out biochemical and genetic tests on multiple cannabis cultivars to determine their cannabinoid contents. And through such studies, it hasn’t escaped Torres’ notice which of cannabis’ green genes are vital to CBD production.

“For a couple of CBD markers, we’ve refined the assays to identify individuals that will produce CBDA at a ratio greater than 20:1,” Torres said during Analytical Cannabis’ recent online symposia.

CBDA, or cannabidiolic acid, is the chemical precursor to CBD. While not as notable as its successor, the compound was been shown to be a thousand times more powerful than CBD in binding to a specific serotonin receptor linked to anti-nausea and anti-anxiety effects.

Working with a cannabis cultivator in Tennessee, Torres and his team at Steep Hill tested and bred generations of hemp to produce a new, high-CBDA variety.

“When we looked at the F1 (first generation) genotypes, we identified the classes that matched the parents and used the chemical phenotype information to identify which diverse individuals would produce high CBDA,” he said.

A mixture of standard genetic tests and some unique innovations, Torres’ genotyping process relies on fluorescent markers and equipment to carry out polymerase chain reactions (PCRs) – a typical method of replicating DNA.

“For a CBD marker, a specific segment of the CBD gene will create lots of copies of this CBD marker using PCR,” Torres explained. “The [PCR] process will raise the temperature to 95 degrees, [after which] the double stranded DNA will come apart and mix with the fluorescent dye that is emitting light to the detector of the instrument. As the nucleotides reanneal, you’ll observe a differential DNA sequence. So if there’s any kind of diversity, you’ll observe a unique melting signature.”

It may sound complex – and it is – but Torres’ test is routine enough to be easily replicated for any grower looking to find and boost their CBD genes, as Torres’ study on the Tennessee hemp demonstrated.

“We identified the blue group individuals as high ratio CBD-producing genotypes that had the capacity of producing ratios of CBD 20:1,” he told Analytical Cannabis. “[These] were crossed with red group individuals and we observed a diversity of different genotypes that were all high CBD-producing.”

“So through chemical phenotyping and chemical tracking, we helped the breeder identify some key F1 individuals that will be carried out through production and used to produce high CBD individuals.”





The birds and the bees – and the CBD boost

Traditionally, when cannabis cultivation was a more clandestine process, growers didn’t have access to such genetic aids. If a cultivator wanted to encourage a particular characteristic in their crop, they’d have to taste, smell, and smoke the plant just to know what attributes it had in the first place.

But whether in 1979 or 2019, the process of selectively breeding cannabis is still the same: both a male and female plant have to be identified. Fortunately, genetic tests can help on that front, too.

“The female flowers are what are commercially sought after,” Torres explained. “And within the last decade or so breeders have been utilizing chemical potency analysis to track traits as well. And they can use those services from Steep Hill.”

As the bud-producing sex, feminized plants are coveted for their THC and CBD, while pollen-producing males are often regarded as worthless.So by clearly identifying CBD-producing females, growers can pair them with masculinized flowers to create a second generation, highly potent in CBD.

“Since its inception, we’ve tested nearly 10,000 individuals for sex genotypes,” Torres added. “Our assay targets a very specific DNA element that we’ve identified in true male cannabis that possess a Y chromosome.”

“We’re fully aware that the sex expression of cannabis is very diverse,” he clarified. “But there are true, genetically-diverged male individuals.”

As another PCR-based method, Steep Hill’s sex determination assay functions a lot like its cannabinoid test.

“We track a fluorescent probe of DNA through a PCR, and as the copies of DNA accumulate and cross a threshold, we’ll observe a fluorescent signal,” Torres said.

Although it’s not suited to every cultivation strategy – outdoor growing notoriously results in natural pollen distribution – Steep Hill’s sexing assay could help countless cultivators curate an ideal CBD-rich crop. And twinned with the company’s cannabinoid-identifying assays, the test has the potential to cement genetic screening as a key tool in a cannabis cultivator’s arsenal.

“There’s more genetic diversity in cannabis than there is in humans,” Torres added. “And the potential for breeding definitely could be maximized through biochemical and genetic applications.”





This article originally appeared in Analytical Cannabis' Advances in Cannabis Cultivation Science ebook in December 2019.

