Raman spectroscopy sorts male hemp plants from females
Cannabis sativa (courtesy Wikipedia)

It turns out that growers of medicinal cannabis and those catering for a more recreational market love females but hate males. Gender bias is certainly in the news today but horticulturalists have long known that female hemp plants have higher levels of pharmacologically active compounds than male plants. The difference in yield between the sexes is so large that it can make cultivation uneconomic.

There are three main pharmacologically active chemical compounds in hemp (Cannabis sativa) plants. Cannabidiol (CBD) and cannabigerol (CBG) are the main cannabis compounds of pharmaceutical interest, while hemp plants which contain more than 0.3% tetrahydrocannabinol (THC) are frequently classified as marijuana. Collectively, this family of compounds are known as cannabinoids.

Male cannabis plants produce male flowers and female plants produce female flowers with higher levels of cannabinoids. Flowers are relatively easy to tell apart but the plants need to be identified as male or female before flowering so that males can be removed and preferably not cultivated at all. It is therefore important to pharmacology research and therapeutics development that plants are identified before flowering. Genetic tests exist but cannot give an immediate result in the field.

Researchers at Texas A&M University have developed an immediate, non-invasive method of determining the sex of hemp plants with a success rate of up to 94%. Professor Kurouski’s team used a handheld Raman spectrometer to measure not the cannabinoids themselves but the pigments that give plants their colour. A commercially available Raman spectrometer (shown below) with a 830 nm wavelength near infrared laser was used to analyse the pigments in the plant leaves.

Agilent Resolve handheld Raman spectrometer (courtesy of Agilent Technologies Inc.)

Raman spectroscopy is a powerful chemical analysis technique that records a ‘chemical fingerprint’ of the vibrations of bonds in molecules. When a laser is focused onto a substance, almost all over the laser light is reflected or scattered with exactly the same colour as the laser. A tiny fraction, less than a millionth of the incident light, interacts with vibrations of molecules in the substance. As a result the light loses energy and becomes red-shifted. A Raman spectrometer spreads out the red-shifted light to reveal an array of slightly different coloured spots. Each one of these light peaks corresponds to a specific molecular vibration and forms the Raman spectrum or ‘fingerprint’ of the substance.

Averaged Raman spectra of female (red) and male (green) hemp leaves (*normalised to the 1440 cm-1 peak) (courtesy Texas A&M University and Springer-Verlag)

Averaged Raman spectra from male and female plant leaves showed small but consistent differences. Female plant leaves gave higher intensities of peaks associated with plant pigments and other biomolecules. Plant pigments typically include chlorophyll, lutein, lycopene, beta carotene, cryptoxanthin and zeaxanthin.

In order to investigate which plant pigment could be contributing to the differences between the male and female Raman spectra, the Texas A&M team took leaf samples from the same plants and analysed them by high pressure liquid chromatography (HPLC). HPLC comparison showed that female plant leaves generally had higher levels of pigments and had clearly higher levels of the pigment lutein. Differences in the Raman spectra of male and female hemp leaves were therefore associated with the plant carotenoid lutein.

In order to make a predictive model they used partial least squares discriminant analysis (PLS-DA), based on two components of the second derivative of the Raman spectra. When the male and female results were analysed based on the two components, it was found that around 90% of young plant leaves could be correctly identified as male or female and 94% of mature plant leaves could be correctly classified.

Professor Dmitry Kurouski and his group have therefore developed a new method of quickly determining the sex of young cannabis hemp plants in the field or greenhouse. The Raman method has the potential to improve the efficiency of medicinal cannabis production and aid research into new pharmacologically active substances.

You can find the full paper recently published by Springer-Verlag here.

News from Dmitry’s group at Texas A&M can be found on his website.

Related research on Raman analysis of hemp leaves can be downloaded from this Open Access Source.

Related research on using Raman analysis to measure plant disease can be found here.

If you want to try your Python skills at PLS-DA there is a useful practical introduction here.