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Germplasm selection – from seed culture to tissue culture

In this article, Dr Gary Yates, chief scientific officer at PharmaSeeds, discusses the pros and cons of types of germplasm selection.

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Germplasm selection – from seed culture to tissue culture
Home » News » Production » Germplasm selection – from seed culture to tissue culture

Much has been written about the advantages and disadvantages of using different genetic starting material to start a grow. Whether it is seed, cloned cut, tissue culture derived or otherwise, it stands to reason there are pros and cons to all options. 

Currently there are four options for germplasm selection: Seed, Clone from seeded mother, Tissue culture plantlet, Clone from tissue culture derived mother, see table 1 for overview of the differences.

Germplasm selection – from seed culture to tissue culture

Table 1. The various forms in which a commercial facility can receive germplasm for planting.

Seed – sexual propagation

With seed, you have the highest possibility of genetic uniqueness – even within a specified lot. This is due to the fact that each seed produced is a result of individual pollen and ovule combing their DNA and producing a unique embryo, hence why it is called sexual propagation.

Cannabis is a highly adaptive species and as such is more likely to mutate and adapt, due to this, stable and uniform cannabis seed lots are not easy to achieve, but there are also ways to reduce the genetic variation further by inbreeding, selfing and backcrossing. Driving the genetics this way will often reduce the vigour over time, and a trade-off between uniformity and vigour will undoubtedly manifest at some point.

Read more from PharmaSeeds: The parallels between biology and cannabis production

Seeds are great for phenohunting as this genetic variation can be used to find high-performing plants, and they are also good for certain outdoor cultivation sites, especially for extraction. Otherwise, seeds are easy to transport and store, and they provide a good stable source for your future stock replenishment.

Autoflowering seeds are a great way to produce crops in glasshouses and outdoors, where day length is too long for photoperiod plants. The early versions of autoflowering genetics often came with flaws such as the loss of yield – however, modern-day autoflowering genetics are fast catching up with their photoperiod cousins, and some breeders claim their autoflowering lines are as good as their photoperiod lines. Stable and uniform seed lots do exist, and they can be generated from nearly any generation, but they will nearly always have a proportion of variability.

Growing from seeds will require designated space; germination, plantlet establishment, vegetation and flowering all require variations on conditions and if a facility is staggering their grow cycles, they will need appropriate separation of growth spaces to operate optimally.

Rough timeline based on seven week flowering:

Week 1 germinate

Week 2 seedling establishment/hardening begins

Week 3 vegetative growth

Week 5-7 flowering growth begins

Week 12-14 harvest

Clone from seeded mother – asexual propagation

Cloning is a way to ensure there is no genotypic variation in the genetic starting material, and therefore unlike seeds, no combining of DNA from pollen and ovule takes place, hence asexual propagation. Taking a part of a maturing plant and turning it into an entirely new plant means that there is no developmental opportunity to recombine the DNA, and the genotype should be fixed in place.

Read more from PharmaSeeds: Improving cannabis yield 

When highly consistent cannabinoid levels are to be produced in flowers, using a cloning system is usually best, whether that be inhouse nursery or externally provided. However, as this requires around 25 to 30 per cent of a commercial facility’s floorspace, additional labour, and time requirements for selection and propagation, having an inhouse cloning room/nursery is not always the most efficient way forward. Having clones delivered into the facility ensures maximum use of space for flowering.

Although clones can be more expensive than seeds, it does take the plants beyond the first two fragile parts of life, and if handled correctly should not pose much of a challenge in terms of success rate, especially in well-seasoned hands.

Individual differences can still be observed from cut clones, due to the plant’s ability to react to changes in its micro-environment. There is no doubt they are genetically identical, however, this does not necessarily mean they will express the same genes at the same point in time. Without well-managed plant touching techniques and consistency in the grow area, gene expression may vary from clone to clone, thus creating variability between individuals.

Rough timeline based on seven week flowering:

Week 1 receive clone begin vegetative growth

Week 3-5 flowering growth begins

Week 10-12 harvest

Tissue culture plantlet – Asexual propagation

The use of tissue culture to produce plantlets is not new, nor is it unique to cannabis – many commercially available fruits, cut flowers and vegetables are derived from tissue culture. Utilising the power of the plant’s meristems – specialised stem cells – it is possible to revert mature tissue into producing a fresh stem cell mass which can be used to generate a new genetically identical clone/plantlet. Another way to achieve a tissue culture starter is through the extraction and propagation of the embryo from within the seed.

One thing that is often overlooked when comparing tissue culture plantlets to those derived from cuttings, is the fact that tissue culture plantlets regrow an entire organism from the stem cells up, creating newly synthesised and fresh tissue as it develops – whereas cutting-based clones are essentially the age of the mother plant, and start life as branches which are forced into becoming stocks and producing roots. This may have an implication in how long it is possible to go from mother to clone to mother to clone before experiencing phenotypic drift due to natural aging, stress priming, build-up of pathogens and build-up of somatic mutations.

Tissue culture clones can be individually propagated from a lab-based substrate to a more grower-friendly substrate; however this involves a greater financial expense. This method will not improve the individual variability, as stated above, there is still a microenvironment to manage, and fluctuation will result in some plants reacting differently compared to other individuals generated from the same cell mass. Depending on the stage of life when received, tissue culture plantlets may need specialised handling, something not all facilities can accommodate.

Rough timeline based on seven week flowering:

Week 1 receive plantlet begin vegetative growth (very dependent on the age/development of plantlet)

Week 3-5 flowering growth begins

Week 10-12 harvest

Clone from tissue culture derived mother – asexual propagation

Tissue culture is best optimised for mother plant production. It is possible to create a clean, pathogen-free mother plant from which, still under sterile conditions, cuts can be taken and kept in a pathogen free environment before reaching the grow space. This method will reduce the need for specialised handling, but still provide a very clean and healthy plantlet. Whether a facility has the ability to create tissue culture mother plants in house, or the facility receives it as provided by an approved nursey, the same rules apply as above. Well-managed cultivation styles will have a greater impact on the uniformity than letting genetically identical plants grow as they are. Cloning from a tissue culture mother plant brings the a lot of the advantages of the tissue culture method, without the high cost of actual tissue culture plantlets.

Rough timeline based on seven week flowering:

Week 1 receive clone begin vegetative growth

Week 3-5 flowering growth begins

Week 10-12 harvest

So, what’s the best…

As with most things, utilisation of all the above can be greater than the sum of the parts. It should be noted that this choice – seeds, over cut clones, over tissue culture – will be hugely influenced by the desired end product, the persons handling the plants, the budget, and of course the growing environment – however, in the scenario of producing high-quality flower for the medical market for example, and not having a tissue culture lab on site, here is how to progress:

Usually selected from good seed stock, a phenohunt is used to find individual plants with exceptional performance or output. Once this is identified, the plant is placed into a tissue culture lab for genetic clean up, rendering it pest and pathogen-free. Once this cultivar is ready, mother stock plants will be grown up from tissue culture and from here, still in sterile conditions, cloned cuttings are nursed, hardened, and ready for vegetative growth. Most facilities offering this will provide a level of testing of the plantlet lots for pathogens before sending and this is one way to ensure no diseases are sent with the plants.

Although it is very much a case of horses for courses in terms of germplasm choice, combining all three systems is a way of utilising the advantages of each, and minimising the drawbacks of any one system. It is also true that there are many ways to cultivate cannabis and that this is just a part of the story. The best advice for those unsure of which options to pursue, is to allow those dedicated to the genetic selection process to lead the way.

Setting the benchmark

One such company setting the benchmark in this field is PharmaSeeds, which is reimagining the genetic selection process for the industry on a global scale. By combining horticultural and agricultural learnings with cutting edge data and scientific innovations, PharmaSeeds is able to offer the industry an entirely holistic approach to genetic selection, yield enhancement and cannabinoid optimisation. Through its proprietary database, PhytoLog, PharmaSeeds tracks and records multiple critical data points and performance indicators based on a diverse range of climactic and environmental parameters. Having access to this level of tailored agronomic intelligence increases cultivators’ likelihood of a successful crop – moreover, one that precisely matches the specific requirements of its off-takers and customers. This process includes the inclusion or elimination of certain germplasm types with specialist training provided on the topic by PharmaSeeds.

Once product requirements, budget and environment suitability are determined it may leave only one option for germplasm, however, by most accounts tissue culture will grow in popularity as the industry progresses. Due to the strictness of some of the legislators, tissue culture provides, at least theoretically, best practises for cleanliness, and this lack of pathogens makes it both easier to pass quarantine at borders and lessens the risk to the cultivation floor. 

If, after the above considerations, all three options are still viable, select a good cultivar – preferably derived from tissue culture – and use clean, cloned cuttings for filling the production floor. For expert support and advice on germplasm selection, please contact PharmaSeeds [email protected]

Gary Yates
Chief scientific officer

PharmaSeeds

Production

New research could help cultivators control THC and CBD levels in crops

A team of researchers has used firefly genes to understand cannabis biology.

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New research could help cultivators control THC and CBD levels in crops
Home » News » Production » Germplasm selection – from seed culture to tissue culture

A better understanding of how cannabis produces THC means scientists could selectively knock out the enzyme that synthesises THC using genome editing techniques such as CRISPR. This would produce plants with lower levels of, or no levels of, THC.

With strict regulations surrounding the levels of CBD and THC in cultivated cannabis, controlling these levels is vital to prevent destruction of crops and lost licences, for example. 

Cannabinoids are produced by trichomes, the small, spikey and sticky protrusions on the surface of cannabis flowers, however, scientists know very little about how cannabinoid biosynthesis is controlled.

To discover the underlying molecular mechanisms behind trichrome development and cannabinoid synthesis, Yi Ma, research assistant professor, and Gerry Berkowitz, professor in UConn’s College of Agriculture, Health and Natural Resources received funding through the National Research Initiative from the US Department of Agriculture.

The research has been published in the journal Plants.

Read more: Cannabis cultivation – the artificial lighting minefield

Cannabinoid biosynthesis

Berkowitz and Ma, and former graduate students Samuel Haiden and Peter Apicella, have discovered transcription factors responsible for trichome initiation and cannabinoid biosynthesis. 

Transcription factors are molecules that determine if a piece of an organism’s DNA will be transcribed into RNA, and thus expressed. In this case, the transcription factors cause epidermal cells on the flowers to morph into trichomes. 

With this new grant, the researchers will continue to explore how these transcription factors play a role in trichome development during flower maturation.

Berkowitz and Ma will clone the promoters – the part of DNA that transcription factors bind to – of interest, and will then put the promoters into the cells of a model plant along with a copy of the gene that makes fireflies light up, known as firefly luciferase; the luciferase is fused to the cannabis promoter so if the promoter is activated by a signal, the luciferase reporter will generate light. 

Berkowitz commented: “It’s a nifty way to evaluate signals that orchestrate cannabinoid synthesis and trichome development.”

The researchers will load the cloned promoters and luciferase into a plasmid. Plasmids are circular DNA molecules that can replicate independently of the chromosomes. This allows the scientists to express the genes of interest even though they aren’t part of the plant’s genomic DNA. They will deliver these plasmids into the plant leaves or protoplasts, plant cells without the cell wall.

When the promoter controlling luciferase expression comes into contact with the transcription factors responsible for trichome development (or triggered by other signals such as plant hormones), the luciferase ‘reporter’ will produce light. 

Ma and Berkowitz will use an instrument called a luminometer, which measures how much light comes from the sample. This will tell the researchers if the promoter regions they are looking at are controlled by transcription factors responsible for increasing trichome development or modulating genes that code for cannabinoid biosynthetic enzymes. They can also learn if the promoters respond to hormonal signals.

In prior work underlying the rationale for this experimental approach, Ma and Berkowitz along with graduate student Peter Apicella found that the enzyme that makes THC in cannabis trichomes may not be the critical limiting step regulating THC production, but rather the generation of the precursor for THC (and CBD) production and the transporter-facilitated shuttling of the precursor to the extracellular bulb might be key determinants in developing cannabis strains with high THC or CBD.

Most cannabis farmers grow hemp, a variety of cannabis with naturally lower THC levels than marijuana. Currently, most hemp varieties that have high CBD levels also contain unacceptably high levels of THC. This is likely because the hemp plants still make the enzyme that produces THC. If the plant contains over 0.3% THC, it is considered federally illegal and, in many cases, must be destroyed. 

The researchers said: “We envision that the fundamental knowledge obtained can be translated into novel genetic tools and strategies to improve the cannabinoid profile, aid hemp farmers with the common problem of overproducing THC, and benefit human health.”

This knowledge could lead to the production of cannabis plants that produce more of a desired cannabinoid, making it more valuable and profitable.

As well as sharing these findings with cannabis scientists, industry, and growers, the researchers will incorporate this new knowledge into UConn courses on cannabis horticulture.

This grant will also support the training of undergraduates interested in cannabis research, providing them with the skills to enter the workforce.

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Planning application submitted for Isle of Man cannabis facility

The facility will include a science and innovation centre.

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Planning application submitted for Isle of Man cannabis facility
SIRC CGI Isle of Man Eye Level (Peel NRE)
Home » News » Production » Germplasm selection – from seed culture to tissue culture

Plans to kickstart the medical cannabis industry on the Isle of Man have progressed with the submission of a formal planning application for a cultivation facility on the island.

Earlier this year, Peel NRE proposed to establish a multi-million-pound science innovation and research centre (SIRC), sustainable energy park and medical cannabis facility on the Isle of Man.

The regeneration and clean energy specialist has now applied for planning consent for its proposals for the 72-acre site in between the A6 and A5 on New Castletown Road. 

The plan, which represents an investment of over £150m from Peel NRE, would create around 250 jobs across a range of skills from botany and technology to security and exports. More than 178,000 sqft of cannabis cultivation space will be created with around 102,000 sqft for research and development.

Read more: Isle of Man opens for business to the medical cannabis industry

Managing director of Peel NRE, part of Peel L&P, Myles Kitcher, said: “This is a game-changing opportunity for the Isle of Man to get ahead in a new and exciting industry that will bring many benefits to the Island and its people and we also hope that it will encourage more renewable projects in the area.

“As expected, our proposals have attracted a lot of attention and, we are pleased to say, significant support from the community during the extensive public consultation. This includes positivity around the emerging industry on the Island, the proposed campus-style development and new educational and career opportunities.

“We will continue to work with the community over the coming weeks and months as the project progresses and will be holding an industry event later in the year for businesses interested in finding out more about available opportunities.”

Read more: Proposal for medical cannabis facility put forward on Isle of Man

The proposals (22/00678/B) follow a change in the legislation of cannabis production and exportation on the island. The Isle of Man Government announced in June last year that opening the island up could make it “a world-leading exporter” of cannabis, and establish it as a global destination for science and technological excellence that will contribute to cutting-edge research into cannabinoids for pharmaceutical uses.

It will also set a gold standard for the burgeoning cannabis industry across the world and unlock partnerships in the public and private sectors.

Additionally, the scheme will feature a solar farm to power the site which would be the Island’s only grid-scale renewable project, contributing to the Isle of Man Government’s ambitions on climate change.

SIRC Isle of Man CGI – A5 Looking East (Peel NRE)

Peel NRE launched a public consultation to help shape the plans before the application was submitted to the Department of Environment, Food and Agriculture later this year, and has stated that it has had positive feedback from the Isle of Man community. 

Isle of Man Minister for Treasury, Dr Alex Allinson MHK, said: “The development of the Island’s medicinal cannabis sector is a key part of the Government’s ambitions to diversify the economy as well as creating a vibrant new sector to attract more investment and skills to the Isle of Man. 

“We are looking to create a world-class infrastructure and continue to welcome interest in developing this sector further.”

Speaking to Cannabis Wealth at the time of the proposal’s announcement, Laurence Skelly, President of Tynwald and Minister for Enterprise, commented: “Diversifying the economy is a significant part of the Isle of Man Government’s Island Plan and the development of Medicinal Cannabis for export is one of the key sectors to bring forward this diversification.

“The Island’s Medicinal Cannabis export proposition is to develop high-quality products and attract new investment through utilising the Island’s stellar reputation as a well-regulated jurisdiction.”

Planning application submitted for Isle of Man cannabis facility

SIRC CGI Isle of Man Aerial view (Peel NRE)

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MoU to establish medical cannabis production facility

The site will be based in Madrid, Spain.

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MoU to establish medical cannabis production facility
Home » News » Production » Germplasm selection – from seed culture to tissue culture

Kanabo has signed a Memorandum of Understanding (MoU)  for an indoor medical cannabis cultivation project in Spain.

The facility will enable 4,000kg per annum indoor cultivation and processing of cannabis. The facility, which is to be established in Madrid, Spain, will focus exclusively on medical cannabis.

Kanabo Group has formed an Israeli subsidiary company, Kanabo Agritec Ltd. (Agritec), which Kanabo Research Ltd, a wholly owned subsidiary of Kanabo Group, holds a 40 per cent shareholding, together with certain additional control rights over the strategic direction of the subsidiary.

Kanabo Agritec will enter into agreements with customers to offer consulting advice and support on cultivation, processing and production of medical cannabis products to organisations and entrepreneurs entering or already active in the cannabis market.

Read more: Kanabo Group extends GP Service contract with major UK retailer

CEO of Kanabo Group, Avihu Tamir, commented: “We are truly excited by today’s announcement. Agritec provides Kanabo Group plc an opportunity to realise the value of our extensive, existing intellectual capital in medical Cannabis cultivation and production, providing near-term consulting revenues.  

“Secondly, it provides us a way to diversify, secure and quality control our supply of medical-grade Cannabis that will always meet our exacting standards, avoiding any wasted margin from product that does not meet the grade.  

“In short, the new venture provides all the benefits and value of owning the cultivation supply chain, without the Capex and Opex requirement of building a cultivation operation ourselves.  Furthermore, Kanabo consumers will be guaranteed a consistent, highest quality supply of medical Cannabis products, at all times.”

Read more: Kanabo launches online CBD store for UK customers

CEO of Agritec, Ophir Shimshi, commented: “We are excited to combine Agritec’s team experience with Kanabo’s extensive R&D knowledge and expertise, to bring this powerful, full-service consultancy offering to market.  

“Agritec’s clients will benefit from Kanabo’s immensely valuable, tried, tested and proven playbook.  

“Our range of consulting services deliver every strategic and operational consideration required by enterprises and entrepreneurs who wish to take advantage of the immense growth opportunities this market has to offer.”

Kanabo has stated that Agritec provides the company with complementary near-term revenue opportunities and will offer Kanabo improved security of cannabis supply through a diverse range of suppliers, who will all adopt Kanabo’s high-quality manufacturing standards without Kanabo having to fund or become directly involved in cannabis cultivation.

Agritec customers will benefit from Kanabo’s services including: procurement, design and build of commercial scale, medical cannabis cultivation facilities; high-quality genetics and standard operating procedures for optimal medical cannabis cultivation; post-harvest services including offtake agreements with Kanabo Group; design and draft reporting and documentation filing services with the local authorities; and, local training and ongoing consulting services.

As per the terms of the MoU, Agritec may elect to receive up to 20 per cent ownership of the project based on the achievement of agreed milestones.

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