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Stem cells, Meristems and Tissue Culture - A Cannabis Perspective​

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Whether exposure comes via news articles covering the use of the controversial embryonic stem cells, or the extraction and reuse of adult stem cells (for example in treating sporting injuries), an increasing majority of the general public has an awareness of stem cells –  not only what they are, but the potential therapeutic outputs they can achieve. From regenerating lost or deformed body parts, to curing cancers and many more diseases, stem cells arguably hold the most promise of any single disciple within the life sciences. Consequently, most of the knowledge the public have of stem cells comes from cells of mammalian origin. In the last 10 years, there has been a growing number of people, including professional athletes who have had their own ‘adult’ stem cells extracted and re-administered in problems areas, which then go on to alleviate the issues therein (Ahmad et al 2012, Han et al 2019). Moreover, many private hospitals will offer expectant parents the chance to freeze afterbirth for the future use on the child should any issues arise in later life which embryonic stems cells could remedy (placenta and associated tissue contains a ripe source of embryonic stem cells).

Personalised medicine is predicted to be the way of the future

As impressive as this is, and not undermining the importance of the individual, these solutions are limited to individuals – much like the use of genome sequencing for treating disease, this is a branch of medicine often referred to as personalised medicine. However, what has been almost entirely overlooked in the public domain – and has the potential to have a greater impact on the planetary ecosystem – is a class of stem cells which, among their primary benefits, could potentially solve world hunger issues, provide unlimited biofuels, and provide oxygen to a slowly suffocating planet. These stem cells are plant stem cells.

We may note with interest that animals and plants had already separated on the evolutionary timescale before they developed from single-celled organisms into the more complex multicellular organisms (Heidstra et al 2014). This means that the emergence of stems cells in both kingdoms is not due to divergence of a common stem cell ancestor, but instead the product of independent evolution resulting in a similar end product – a phenomenon known as convergent evolution. However, there remain fundamental differences between stems cells in animals’ verses those found in plants.

Plant cell under microscope (low magnification)

Two words that often arise when discussing stem cells which relate to the potential of the stem cell type:

1) Totipotent stem cell – A cell capable of differentiating into all cell types, and can give rise to the entire organism

2) Pluripotent stem cell – A cell capable of differentiating into almost any other cell type in the organism – but cannot generate a new organism

To simplify – totipotent stem cells can produce pluripotent cells, but pluripotent cells cannot produce totipotent stem cells.

The regenerative power of plants

The strategy of plants is somewhat unique when compared to most animals, as they can maintain a consistent pool of post-embryonic stem cells. It is this distinctive attribute which enables grass to regenerate after grazing, trees to live for thousands of years, and allows humans to clone plants via regeneration of cuttings from a mother plant and the establishment of a totipotent cell mass from almost any plant tissue called a callus – the starting point to producing plant tissue culture (Vasil and Hildebrandt 1965(1) & Vasil and Hildebrandt 1965(2)). Plants have three main centres of constant stem cell production known as the meristem. The two primary meristems are the growth expansion points and are positioned at the shoot apex – shoot apical meristem, the apical root – root apical meristem and a secondary meristem known as the cambium, usually positioned between the xylem and phloem (the plant’s internal transport system for water, nutrients, and mineral movement) (Gaillochet and Lohmann 2015).

The potential for almost any tissue of a plant to be treated to induce callus formation gives rise to a wealth of possibilities and positive solutions, but the output of this can also cause some problems. Although important to address the potential issues, this article refers readers to look at the issues of monocrops and a lack of genetic diversity therein as one of the main issues – i.e., a lack of resistance to either biotic or abiotic stress can wipe out entire cultivars if not managed well. Otherwise, the main considerations around tissue culture generation are the specialised training/personnel, infrastructure requirements and time frames involved in for example the genetic clean up. This is all assuming well-established protocols are in place and easily transferable between cultivars – by no means a given. In fact, arguably the biggest problem with cannabis tissue culture is the lack of well-proven protocols that are useful to both drug-type and hemp-type genotypes. In many published articles to date researchers have used hemp-type cultivars which are not like-for-like when attempting tissue culture on high THC cultivars – A comprehensive review of this can be found in a recent 2021 publication – The Past, Present and Future of Cannabis sativa Tissue Culture by Adrian Monthony et al.

Plant Tissue Culture

growing seeds and microscope

Clonal propagation via cuttings from a mother plant and the generation of tissue culture both require starting tissue from an established mother plant. In more refined protocols (rice, for example), calli can be generated by isolating the embryo from the seed (Lee et al 2002, Hoque et al 2007). Tissue culture generally requires the use of plant growth regulators which can come in various forms. It has been shown in the model species Arabidopsis thaliana that the shoot apical meristem can be activated by light and metabolic signals via a hormone called cytokinin (Pfeiffer et al 2016). This type of discovery is not yet applicable to large scale propagation, but gives an idea of the different ways stem cell production can be induced. In general for cannabis tissue culture, there are up to five main stages, and four main influential inputs to consider (Monthony et al 2021).

Stages:

  1. Stock plant: selection and maintenance
  2. Multiplication (this step can be omitted)
  3. Pre-transplant treatments
  4. Acclimation
  5. Planting

Input factors:

Explant: Cultivar, genetic clean up, tissue type use – the specifics pertaining to any one cultivar need to be optimised. This may include consideration of factors such as tissue type and age of plant used to generate callus formation.

Basal media: Nutrients, additives, pH – here are various recipes for making media for plant growth, this would include both different ingredients and variation of concentration to reach the optimal base media. This is something which can take a while to optimised, especially in a new species with so many variables involved.

Plant Growth regulators: Phytohormones – the correct combination and concentration may be cultivar specific, or may require different isoforms of the hormone depending on the tissue type used. This, again, is something which requires a good amount of tweaking in order to fully optimise. It also usually involves different ratios at different stages of development.

Environmental factors: Light, temperature, humidity – these are always factors involved in plant growth, but fine tuning is required for establishing a tissue culture optimised environment. This is another example of factors that may require changing over different stage of development.

Conclusions

The discussion on cannabis tissue culture will be further explored in a follow-up article. There is a need to have open dialogues about this technology as it has the potential to revolutionise the cannabis industry as it has done for other species, especially in the research capacity. It is very hard to keep all variables under control when experimenting and one thing that tissue culture can help with is to the lessen the variability with the plants themselves, one callus can give rise to hundreds of genetically identical plants which can be kept in fairly small spaces and maintained over time.

Plant stem cells show extremely high levels of plasticity, and over the course of fifty years, plant scientists have refined various protocols to induce totipotency from almost all tissue types. It makes sense that stem cell research in plants has so much promise when we consider they are sessile organisms (anchored to one place) which can live for thousands of years, can be almost completely destroyed but fully regenerate, and they can be asexually propagated often, with minimal fuss and effort*.

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*As a child, I remember once on a long walk with my grandfather, we found a beautiful large rose bush with a mottled patterned flower. My grandfather pulled out his pocket-knife and cut a pre-budded branch off as he told me he would plant it when we got home. I remember being completely dismissive of this and thinking that it would never grow. My grandfather simply stuck it cut-end down into a pot of soil when we got home, and within a few weeks it had clearly started growing. Eventually, it produced a large bush from which he created a beautiful archway, the centre point to this garden , and although I didn’t know at the time, this was an early lesson in the power of plant stem cells.