How to minimize degrading genetics when cloning cannabis

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, Stewart Maxwell explains in a recent Elevated Botanist article.

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. 

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 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.

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.    

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For more information
Elevated Botanist
Stewart Maxwell
stew@elevatedbotanist.com 
www.elevatedbotanist.com 



 


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