Clone Cannabis Forever and Minimize the Drift

Most of us rely on cannabis clones to populate our cultivation facilities. 

Some of the most successful cannabis brands have been built on the quality reputation of a single cultivar.

But what happens when genetic assets become impaired?

When success is dependent on the phenotypic stability of your germplasms, it is worthwhile to understand why genetic expression might drift over generations, how to minimize this occurrence, and what the options are to rescue degraded genetics.

The cultivation of exemplary cannabis begins with the propagation of healthy, vigorous, and phenotypically consistent plants.

Asexual reproduction might not sound like much fun, but it confers distinct benefits when cultivating cannabis plants for intensive cropping systems.

Due to its outcrossing nature, and decades of clandestine hybridization, many cannabis populations are highly variable when grown from seed.

Phenotypic consistency for horticultural traits like vigor and pest resistance matters for all crops. 

For the cultivation of cannabis flowers with distinctive biochemical profiles, a higher order of phenotypic consistency is required.

Biosynthesis

The desirable features of cannabis flowers are the by-product of complex interactions between various compounds including cannabinoids, terpenoids, flavonoids and esters. 

The synthesis potential of these various biochemicals is determined by the independent inheritance of many different genes and their expression within the production environment. 

This horizontal trait inheritance increases the likelihood of variability in the biochemical features of populations grown from seed. These aesthetic distinctions are often evident even among closely related plants. 

Plasticity

Plants are sessile organisms.  They are rooted in place, and vulnerable to many biotic and abiotic pressures. 

Over hundreds of millions of years, plants have developed a comprehensive plasticity, or ability to change physical form in response to environmental pressure. 

This plasticity extends to cellular biology and enables the incredible regenerative capacity of plants.

Plants have developed pathways to regenerate tissue to close wounds and replace lost organs. 

This feature has important implications for both natural and cultivated asexual reproduction systems.

Asexual Reproduction

Some plants have evolved asexual reproduction as a norm. 

The earth’s largest organism is Pando, a quaking aspen colony that consists of 40,000 individual trunks over a 100-acre site.  This single organism originated as a seed, tens of thousands of years ago, and has sustained its presence through repeated clonal regeneration from a common root system.

Many modern horticultural crops are exclusively reliant on asexual propagation due to genetic variability, sterility, and other physiological traits. Asexual propagation is highly dependent on hormone regulation for cell division and differentiation.

Hormone Regulation

Hormones are signaling compounds that direct plant development based on environmental and genetic stimuli. Without hormone regulation, plants would consist of masses of undifferentiated cells. All plant cells can synthesize, store, and exchange these simple bio chemicals. 

Hormones move within plant cells, between cells through diffusion, and translocate to other plant parts through vascular tissue. In the case of ethylene, a gaseous hormone involved with reproduction and ripening, signals can be transmitted to remote parts of a plant, or to other nearby organisms, through the air. The classes of hormones most commonly applied to promote root and shoot formation are auxins and cytokinins.

Auxins

Auxins are an important class of phytohormone that are involved with cell differentiation, root formation, and phototropism among many other aspects of plant physiology. 

Auxins are primarily synthesized in apical shoots and transported to roots, where cell division and root development is promoted. The synthetic auxin most applied to soft tissue cuttings for promotion of adventitious rooting is indole-3-butyric acid (IBA).  Auxins naturally occurring in solutions such as willow water, and aloe vera have also been used for vegetative propagation of cuttings.

Cytokinins

Cytokinins are another class of hormones with potent implications for plant organization and structure.  Cytokinins generally favor shoot growth, and promote cell division.  Cytokinins are synthesized in root cells and transported to shoots through the xylem and synthesized in shoots and transported to roots through phloem tissues.

Cytokinins also contribute to many root functions including nutrient signaling and uptake.  Plant growth regulators including Thidiazuron (TDZ) are commonly used for cannabis micropropagation due to their cytokinin activity.

A simplified model of auxin-cytokinin interactions is demonstrated by tissue culture practices.  When plants are cultured in sterile media, addition of synthetic auxins promotes roots, while addition of cytokinins promotes shoots.  When both hormones are present and balanced, both root and shoot development occur. 

Cloning Cannabis

Plants maintain a store of undifferentiated stem cells throughout their lifetimes. 

These cells are pluripotent and can transform into many other types of plant cell. 

Undifferentiated cells are maintained in the cambium layer which is a thin layer of vascular tissue present in all plant stems.

This feature enables plants to be reproduced via vegetative propagation.

Most Cannabis crops currently cultivated in Controlled Environmental Agriculture (CEA) facilities are cloned, or vegetatively propagated.

Cannabis is easy to root from soft tissue cuttings when climate parameters can be managed.

Love Your Moms

Many legacy growers have reported the maintenance of clone consistency following decades of continual reproduction from generations of mother plants. Almost universally, this phenotype stability has been attributed to the continual renewal of mother stock under ideal growing conditions.

When plants have been cloned continuously for production in protected horticulture, they may change phenotypic expression to suit their conditions. This shift might benefit the plant, but changes in phenotype are usually unwelcome in prized cultivars.

Cultivating mother plants outdoors, in optimal and diverse (living soil) environments can provide an opportunity for a seasonal reset of some epigenetic traits. The health of mother plants should always be maintained at an optimal level. If you want to understand how well run a cultivation facility is, check out the mother room.

Cannabis cuttings should be taken from actively growing shoots of healthy plants. Cuttings are typically removed under low to medium light conditions. When cuttings are removed from plants experiencing high photosynthetic rates, the leaf stomata are open for gas exchange, and cuttings will quickly lose turgidity. 

At the time of removal from the donor, cuttings are trimmed and then immediately immersed in water or a rooting solution to maintain water transport through the shoot.

Rooting Solution

Rooting solutions commonly include plant tonics such as B1, growth factors present in seaweed and other botanicals, and bioprotectants to limit fungal pathogen establishment.

The cloning process usually involves humidity, temperature, and stagnant air conditions which favor fungal pathogen establishment. 

Unless cloning directly into a suppressive soil, bioprotectants should be considered as a drench for rooting media.

Several organisms, including Bacillus subtilis, Streptomyces lydicus, and Tricoderma harzianum have shown efficacy in suppression of fungal pathogens, and are available in branded formulations.

Rooting Media

Cuttings can be successfully rooted in various media provided that appropriate cultural conditions are maintained. A small format (plug, puck, or cube) is typically used for rooting.

The air–water ratio within the media is critical to the successful establishment of cuttings. Initially, high saturation percentages are required to establish water transport through cuttings, and gradual drying of the media will encourage oxygen access and promote adventitious rooting.

Media is prepared using a dibble to receive clones. As each cutting is removed from solution, the lower portion of the stem is excised 3 to 4” from the growth tip using sharp, clean scissors. This cut is executed at an acute angle to promote water uptake by xylem and expose undifferentiated cells within phloem tissues for root initiation. Dipping the cutting in rooting formulations containing synthetic auxins prior to seating can decrease the time required for formation of root primordia.

Adventitious rooting

Once firmly seated in media, trays of clones are placed in an environment that will enable the cuttings to retain turgidity until roots form. This climate typically entails 75 to 90% relative humidity, with a temperature of 72 to 80 degrees Fahrenheit.

Lighting should be low intensity (150–250 µmol) to limit photosynthesis and attendant water transport demands. These climate parameters can be maintained within propagation areas using humidifiers, energy curtains, and other climate modifiers. Adventitious rooting will occur within 6 to 12 days for most cultivars.

Plant Tissue Culture

Plant tissue culture includes a diversity of techniques to maintain and multiply germplasms within sterile in vitro (in glass) laboratory conditions.

This is enabled by development of variety specific protocols, including formulation of nutrient culture media which contain a composition of plant growth regulators to initiate the desired tissue formation.

Significant research, and trial and error are required to develop cultivar specific tissue culture protocols.

Micropropagation                                         

Tissue culture techniques that are intended to produce large numbers of plants for commercial production are referred to as micropropagation.  Micropropagation systems consist of several cultural stages which require diverse protocols, and media formulations optimized to enable the successful production of plantlets.

Plants are cultivated in vessels, within multi-tier culture chambers which accommodate large numbers of plants within a small space.

Although some tissue culture process can be used to eradicate pathogens and viruses from plant materials, it is beneficial to start with healthy stock material.  Stock plants should be as pathogen and virus free as possible with testing and analysis applied to select the cleanest specimens for reproduction.

Induction

After removal, plant tissue is surface sterilized and inducted onto an aseptic media in a clean room designed to minimize contamination. 

Sterility in the transfer environment is maintained by hygiene, equipment sanitation, air management and filtration, and the experience of skilled laboratory technicians.  Despite all possible precaution, a percentage of newly cultured vessels will become contaminated during transfer.

Plant tissue will often undergo a period of sporadic development following induction due to the radical shift from vegetative growth to existence within a sealed jar.  Carbon, which would normally be acquired from the heliosphere during photosynthesis, is instead delivered through the nutrient media.  The vessel headspace is humid, and gas makeup within it can become co2 depleted, and ethylene rich during tissue development.

Multiplication

When plantlets have been inducted, and well established in vitro, they can then be multiplied. 

Multiplication includes division of established cultures into multiple vessels containing a nutrient media optimized for this sub culture stage. Stem tissue is commonly used for multiplication.

Depending on the volume of tissue inducted, and the number of plants desired for production, multiple rounds of multiplication may be necessary.

When plant tissues have been multiplied, the plantlets are again transferred to a vessel and nutrient media formulation to optimize shoot and root development. 

When plantlets have developed organs suitable to enable their transition to a horticultural growing environment they are moved to the acclimation stage.

Acclimation

At this stage most of the plants basic life functions including transpiration, photosynthesis, symbiosis, and reproduction have been transformed to enable existence within a test tube.  It is due to an incredible plasticity, and evolved response to adversity that plant cells are capable of this adaptation.

Transitioning an in vitro plantlet to a horticultural climate rife with biotic stressors requires a measured and gradual acclimation period.  Plants do not typically develop a cuticle layer on leaves in vitro, and stomata may be underformed due to low light levels, and lack of photosynthesis under culture conditions. 

Acclimation protocols begin prior to removing plantlets from their rooting vessels and can continue for several weeks as plantlets are transitioned to cultivation.  Plants can be acclimated within climate chambers including domes or plastic bags which are placed over pots containing juvenile plants. 

Phenotypic Drift

Asexual propagation of plants has the potential to enable indefinite storage, and multiplication of exceptional cultivars.  In practice, phenotypic stability following many generations of asexual propagation can be difficult to achieve, and a gradual degradation of plant quality is often reported. There are several factors that can contribute to this phenomenon of phenotypic drift.

Over many generations of production, contaminants can accumulate within plant tissues.  Endophytic pathogens, intracellular bacteria, viruses, and viroids can all proliferate within plants to the detriment of phenotypic expression. Diagnosis of viral pathogens requires laboratory testing.

Epigenetic shifts

DNA is resilient and germline mutations to plants are rarely caused by propagation practices.  Epigenetic changes are more common and linked to shifts in phenotypic expression in many plant species. 

Epigenetics encompasses a range of modifiers by which genetic expression can be altered based on environmental inputs. 

DNA methylation is a common agent regulating epigenetic expression in plants. This chemical modification of a DNA base by a methyl group can turn on or quiet gene expression in response to environmental and developmental stimuli. Plant DNA methylation changes during many developmental processes including sexual reproduction.

With repeated cloning of stock plants grown in sub-optimal horticulture conditions, epigenetic changes can accrue to the detriment of plant performance.

Somaclonal variation

In vitro plant culture can give rise to phenotypic and molecular changes in plant regenerates due to the stress associated with continuous sub-culturing.

These changes are known collectively as somaclonal variation and affect traits across the spectrum of plant morphology.  Changes in DNA methylation patterns are common in plant tissue culture microenvironments and can give rise to epigenetic variants with heritable changes in phenotype.

The propensity of plantlets to undergo changes in DNA methylation patterns while in vitro also presents opportunities to rescue cultivars that have degraded due to phenotypic drift, and contamination. The rescue of degraded cultivars is usually enabled from the culture of stem cells from the shoot apical meristem.  This small cluster of cells is excised using microscopy and proliferated in vitro.

Meristem culture

Meristem tissue has the lowest pathogen load in the plant due to the brand-new nature of meristem cells. 

Additional treatment with heat and chemicals can eliminate most viruses, viroids and other contaminants from the tissue. 

Plantlets that have been regenerated through shoot apical meristem culture can sometimes regain phenotypic currency due to changes in DNA methylation patterns, and the elimination of viruses and other contaminants.

For the rehabilitation and long-term storage of plant tissue, meristem tissue culture has great utility.

Conclusion

Asexual propagation provides opportunities for the indefinite maintenance and proliferation of genetic information. 

Optimized tissue culture protocols can enable long-term storage of valuable cultivars within a compact footprint and the unlimited multiplication for production.

Each crop rotation presents the opportunity for an exceptional production cycle. The propagation and establishment of superior plants is the first step in this pursuit of excellence.

To ensure an ongoing consistency in plant phenotype, mother stock must be maintained in optimal health, tested for viruses, and sometimes renewed through meristem culture and optimal growing conditions.