Cannabis is a hot topic in many respects today. The cannabis industry is among the fastest growing sectors in our economy. According to an article from Forbes Magazine in December 2016 [1], the industry’s projected growth could be as large as 700% by 2020, a figure that only captures the cannabidiol (CBD) side of the market, regarding a non-intoxicating cannabinoid extract that is projected to generate $2.1 billion dollars in consumer sales. In February 2017, another Forbes article cited a report from New Frontier Data that stated that by 2020, the cannabis industry alone will create a quarter of a million jobs surpassing the manufacturing industry. [2]
The framework that made the cannabis industry what it is today can be attributed largely to the persistent home and outdoor cannabis breeding techniques of passionate independent farmers. One thing is for certain — this current species of plant has come lightyears from what humans experienced 5,000 years ago. Due to the Cannabis genus’s inherent promiscuity and competency for heterosis, farmers utilizing traditional breeding techniques were able to propagate this plant generation after generation. The flowering species of Cannabis sativa L. has not been part of any major breeding programs in modern times mainly due to federally imposed Schedule I drug classifications associated with this plant. Due to the cannabis’s legal status, cropping methods and breeding techniques have, for the most part, remained unsubstantiated or improperly quantified, with some published propagation/breeding data [3], but mostly unsubstantiated rumors and claims that lack peer-review. New, credible knowledge on cannabis breeding is, therefore, needed. Recently, efforts to centralize this knowledge and change the status quo through scientific research have begun to emerge with literature being published in higher volumes. [4]
The objective of this paper is to further contribute to cannabis’ zeitgeist by presenting peer-reviewed techniques which can be applied in common situations for cannabis breeding. With respect to breeding techniques which have established the bar for cannabis in the United States, the propagation techniques and breeding methods presented will be applicable to an in-home breeding program, working with feminized (self-pollinated) and nearly homogenous germplasm. Some of the techniques covered are for the context of stabilizing a personal line and developing other unique cannabis chemovars in home gardens.
Why Cannabis?
As a species, humans have cultivated cannabis for longer than we have been writing our history. [5] From ancient breast cancer patients who utilized cannabis to alleviate their pain to people getting high recreationally, our relationship with Cannabis sativa L is as old as the concept of writing. [5] The evidence suggests cannabis’ role in societies around the world has been multifaceted and important, and therefore, still relevant in modern horticultural practices.
With many states now adopting cannabis as a viable medicinal crop, research on every level pertaining to this species is needed now more than ever, especially chemotyping for licensing and sequencing the cannabis genome for specific medical prescriptions. In order to facilitate research into clinical safety and effectiveness, the American Medical Association has recently called for the rescheduling of cannabis’ legal status from Schedule I to Schedule II (Hoffman et al. 2010). [6]
What is Cannabis?
The Cannabis genus is dioecious and generally separated into two recognized species C. sativa and C. indica. [7] Cannabis is diploid with 20 chromosomes that naturally outcross, and the plant has high heteroic capabilities. With copious interspecies crosses, this promiscuous plant has no breeding barriers, a trait which makes itself very easy to diversify genetically (refer to illustration). [8] C. sativa achieves a higher canopy than the other species bearing long thin leaves, while C. indica is shorter in stature, bearing broader leaves. [9]
Cannabis is predominantly farmed for the euphoric/pain relieving effects of delta-9- tetrahydrocannabinol (THC) and secondary metabolites such as terpenoids. [10-11] Ubiquitous in nature, terpenoids can be found in anything that grows that has a smell or taste. Terpenoids may affect the competence of THC receptors in the brain by disturbing the annular lipids surrounding them and increasing the fluidity of the neuronal membranes, intensifying the effects of the psychoactive cannabinoids. [12]
What Does Cannabis Want?
Prior to implementing a cannabis population, becoming familiar with the nutritional requirements is good practice. Improving the quality of cannabis through proper nutrient management, cropping methods, and simple breeding techniques starts with knowing the different nitrogen-phosphorus-kalium (potassium) ratios for various cultivars in their individual life-stages. This is vitally important to the plant’s quality of life and will affect all aspects of plant material use after harvest. Flowering ornamentals and aromatic and stimulating crops tend to favor nitrogen for the flowering process. The other macronutrients, phosphorus and kalium (potassium), support the alternate key plant functions needed for competent flowering such as node development, stem strength, and efficient photosynthate translocation. Micronutrients Cl (chlorine), Mg (magnesium), Mn (manganese), S (sulfur) contribute greatly to correlations in taste and smell. [13]
Implementation
Selecting genetics that perform properly in your greenhouse is important. Having a plant that produces a flower after 5 ft. of vertical growth while utilizing a 5’ x 5’x 6’ greenhouse would not be suitable as the overall morphology (dimensions) of selected plants would conflict with greenhouse/ growing space. Selecting a hybrid that reaches a mature height of 4 ft but branches horizontally, however, may be better suited for that particular garden exploiting the footprint from the artificial light most efficiently. When the objective of your cannabis farm is solely yield, starting with the right genetics or clones from a reputable source will usually achieve the best results. If you are phenotype “hunting”, starting with as much genetic variation within populations of propagated seeds will ensure successful F2 generations by maintaining a diverse gene pool. Most reputable seed banks will provide a synopsis of expected growth times from seed to flower along with terpene profiles of inflorescences, as well as progenitor origins.
In a clean area designated for your cannabis in the clone/seedling phase, begin the selection of the best phenotype based on how it performs in the “sea of green” method. This involves rooting clones in a densely oriented pattern which promotes uniform growth. As cuttings mature, the individual plants that grow most vigorously and display the highest competency for rooting should be advanced to the next phase of propagation. [14-15]
Cannabis is an extremely photosensitive plant and, in most cases, a “short-day” plant. [16] This refers to when the length of daylight is equal to or less than the critical photoperiod, which is the length of light necessary to begin the flowering phase, after which the light measuring phytochrome (Z) is transmitted via the constant (C) to trigger the flowering time gene Florigen (FT). [17] During the juvenile phase, maintaining 18-to-20-hour photoperiods is best. Once stems have hardened, and terminal axillary growth has given way to increased branching, the vegetative stage has successfully begun.
The Vegetative Stage
Recently, a published study with the objective to increase publicly accessible, peer-reviewed, applicable, cannabis propagation knowledge included identifying the optimal fertilization rate for cannabis in the vegetative state. [18] The experiment studied container-grown plants with an organic coir-based growing medium placed in a growth chamber. Utilizing an organic liquid fertilizer with a N-P-K ratio of 4.0N-1.3P-1.7K and administered in 389 mL/L produced yields 1.8 times higher while possessing increased concentrations of mono- and sesquiterpenoids. This study also provided the ratio of N-P-K at which THC synthesis is boosted. While certain terpenoids decreased in abundance, the study recommends that fertilizer should be administered at a rate of 418 mL L to bolster THC synthesis.
One of two objectives can be achieved while in vegetative phase just by augmenting the N-P-K ratio. One objective is to enhance inflorescence weight while the other enhances terpene diversity. These researchers aim to produce more of these “standardized recipes” available for the public to grow high-yield, THC- and terpenoid-rich cannabis and also plan to apply similar techniques to quantify similar data for the flowering phase.
The Flowering Phase
The flowering phase requirements of N-P-K will differ from the vegetative stage. The actual time to competency in each phase will differ between plant varieties but can be generally divided into four timeframes.
These include the pre-flower phase, when the sex of the plant is showing towards the end of a competent vegetative state, the early-flowering stage, which occurs after the FT gene has been triggered by adjusting the photoperiod to 12/12 lasting about three weeks; the peak flower phase that can be seen after inflorescences have set on the stem and their floral structure is mature, lasting usually from week 3 to 7 (this phase sees the most floral development); and the last late-flowering phase, 7 to 14 days before harvest, signaling the plan to terminate.
Over the course of an 8-to-9-week flowering period, the N-P-K ratio should not be standardized but rather, increased weekly by increments that support these growth patterns and fluctuating nutrient requirements. Not implementing fixed N-P-K ratios while in this developmental process minimizes the “plateau” effect, similar in context to bodybuilding, where physical gains are not achieved due muscular insensitivity to a repetitive regimen. This same logic can be said for a standardized N-P-K ratio used for irrigation during the flowering phase.
The photoperiod could be the input that, when not understood properly, can be the difference in pinnacle inflorescences or an expensive waste of effort. No other facet of cannabis growing affects hormone production more than the solar cycles. Inconsistency in photoperiods or interruptions in sleep schedule will trigger unwanted stresses such as asexual seeding or apomixis specifically though agamospermy. [17]
Towards the end of a successful flowering phase, shortening the photoperiod weekly by increments of one hour until ten-hour photoperiods are reached helps hasten the ripening process. During this time the styles will take on an amber color. You can assess the glandular trichomes with a 30x microscope for an opaque, cloudy and/or amber color for further confirmation of ripeness and end of flowering phase.
Cannabis Breeding
In the development of a new cultivar, locating the live landraces is involved. The parents of all interspecies crosses of cannabis are pure C. sativa or C. indica. To truly breed “new” varieties of cannabis, the progeny developed from hybridizing landrace plants will be “true-breeding” having the ability to produce consistent F1 populations with unique phenotypic characteristics. Even though an individual plant may express highly dominant sativa or indica characteristics, it can still transmit undesirable alleles even if propagated from a “stable” (predictable and homozygous) germplasm since a stable line is not true-breeding, and therefore, chance transmission of deleterious alleles is possible.
The odds of developing a phenotypically consistent F1 from a stable line are much higher than from a heterozygous population. This is due to the heritability factor in the cannabis species. In a broad sense: H2 = V sub G / V sub G + V sub E, where H represents the heritability, V sub G represents variance due to genotype and V sub E represents variance due to the environment. This equation considers actual genetic heritability versus environmentally induced factors. The possibility that a specific trait is transmissible drops precipitously when only expressed in an individual plant amongst a population. If trying to propagate from this individual, the cloning technique is most suitable. Taking clones allows for the breeder to replicate the desirable trait(s) in a later harvest. If the phenotypic trait is desirable, establishing a mother plant (cannabis in perpetual vegetative growth) for cloneable material is recommended.
To bring a desirable phenotype to seed, inducing apomixis in one of two methods can be used to limit the amount of variability in the F1 population. One technique involves selecting a single candidate and transforming her into a male pollinator by deliberately disturbing the critical photoperiod and causing a hermaphroditic change. In a wild setting, this survival mechanism engages in the event of a shortened flowering time or unsuccessful fertilization. This allows the cannabis plant to produce a progeny regardless of the situation given the chance to have a competent vegetative phase. This technique exploits this mechanism in a controlled manner to create a germplasm. After selecting an individual plant to induce apomixis, this individual can be used to pollinate a single female to produce a progeny that will express favorable traits in a narrower variability within that phenotype.
Alternatively, the transformed pollinator can be used to pollinate the whole population en masse (given traits expressed across the population are desirable and within an acceptable amount of variation) to maintain desirable alleles in the germplasm while simultaneously maintaining predictable drift of heritable traits. This will allow a sundry gene pool limiting the chances of breeding depressions. [19] that can occur in cannabis especially when working from a small population even though the species is naturally outcrossing.
Parents closely related increase the chances of transmissible deleterious alleles. Antagonizing this mechanism by propagating from populations with shallow gene pools is dangerous. Given this situation, undiversified germplasms may see signs of crops deteriorating rapidly into expressing recessive alleles ergo undesirable traits. [20] The diagram below illustrates how this can happen over just a few generations.
Figure: This illustration shows how, after a few filial generations, a deleterious allele can have the commanding genetic influence.
The genetic configuration of the cannabis species allows for protogynous or protandrous mutations, whichever better suits the individual in that time and space. Dichogamy or sequential hermaphroditical mutations occur when overly exploited by stress-inducing techniques for germplasm and contribute to breeding depressions. Circumvent this in later generations by exogenous applications of silver thiosulfate solution [21], colloidal silver, or gibberellic acid to mature female plants. This inhibits female hormones allowing production of male flowers producing feminized seeds.
Due to the extreme photosensitivity of this plant, if producing a progeny from a suitable pollinator is not an option, then preservation of genetics through revegetation is possible, a process in which competent flowering material is forced back to a vegetative phase. This survival mechanism ensures as many flowering phases as possible, triggered by vernalization and photoperiodism reactions occurring in the plant. [22] By altering the photoperiod back to a vegetative cycle and reversing the growth phase, a mother plant can be established to provide a perpetual source of clonally propagated crops.
Alternative Compounds
Secondary metabolites found in cannabis include terpenoids. Terpenoids are lipophilic compounds that penetrate membrane barriers and are formed via the cytosolic mevalonate (MEV) and methylerythritol phosphate (MEP) pathways. [23] The MEV and MEP pathways are the regulators of the different substrate pools available for terpene synthases. [24] The major class of terpenoids are monoterpenoids, such as alpha-pinene and limonene, which produce effects representing their namesakes (e.g., pine trees and lemon/lime).
These isoprenes can be differentiated into a few subclasses of which, the most prevalent are sesquiterpenoids. Highly volatile, these molecules are organoleptically interpreted in ranges from mangoes (ß-myrcene) to cloves (caryophyllene) and hints of chamomile (alpha-bisabolol) with lavender (linalool). [25] These secondary metabolites contribute greatly to the growth pattern of cannabis via allelopathy, an ability which allows this plant to be successful amongst competitors in a wild polyculture.
Terpenes in wild settings serve as an attractant to pollinators and as an antagonist to harmful insects or diseases, while catalyzing reactions that protect the plant from environmental stresses. They also provide a chemical building block for all other cannabinoid synthesis. [26] These pathways are located in the cytoplasm and plastids of photosynthetic, chlorophyll-containing cells most prevalent in the leaves; therefore, isoprenoids are diversified into various terpenoids in the leaves of cannabis. Maintaining optimum health of your plant may foster copious and diverse substrate pools from which these isoprenes can synthesize into cannabinoids. These molecules work synergistically with each other enhancing the effects of THC in what’s known as an “entourage effect.” [9] Terpenes are thought to modulate the uptake of THC by sequestering and perturbing receptor sites.
Bouquets of different monoterpenes and sesquiterpenes are important components of cannabis resin as they define some of the unique organoleptic properties. These may also influence medicinal qualities of different varieties. [22] Terpene enhancers are formulated using natural sources of isoprenes that can be isolated from other plants and applied exogenously to the roots in an aqueous solution. The micronutrients that give way to these syntheses P, K, S, Mn, Cu, and B all contribute to the isoprene precursors of monoterpenoids, sesquiterpenoids, and other supporting cannabinoids. [13,26]
Critical photoperiods vary for cannabis depending on growth phase. Controlling the light spectrum will also provide the plant a chance to standardize the cannabidiol content. [18] Failure to provide the adequate photoperiods can lead to a lack thereof, or underdeveloped flowers in cannabis. Starting from seed and ending with harvest (excluding curing time) will require a three-to-four-month investment of time. Developing cropping methods that exploit the natural morphology of each cannabis plant can contribute an enhanced quality and yield.
Harvest
Flushing, a process done in the late flowering phase typically consists of irrigating with plain pH balanced (7.0) water for 7-14 days to rid any residual salted nutrients in the vascular system. Cannabis is typically ready for harvesting when the hairs (styles) have all taken on an amber color and when the trichomes’ glandular heads are cloudy or amber in appearance. Another sign is the chlorosis (yellowing) and abscising (shedding) of the large fan leaves. Do not feed the plants and keep them in complete darkness for 2-3 days prior to harvesting; this allows a lot of moisture to evaporate.
The most common harvesting technique involves cutting plants from rootstalk, removing all leaves attached to a petiole (leaf stem attached to main stem), and hanging to dry in a well-ventilated dark place preferably with a dehumidifier for 5-10 days. When dry to the touch, remove inflorescences from stems and continue the drying process in a secured container over at least a two-week period. This type of aging allows terpenes to balance much like wine or whiskey.
Cannabis has many biological tools which, when exploited properly, contribute to successful small-scale breeding while implementing traditional techniques. Some advanced methods can be useful on a small-scale as well, Companies like Medicinal Genomics offer affordable partial panel quantitative trait locus tests to anyone who seeks to understand their germplasm genetically. They utilize single nucleotide polymorphisms (SNPs) to identify major linked genes and whether they are heritable this kind of test can be what sets your chemovar apart from other breeders.
Photocycle, nutrition, growing media, pest control, and environment are the foundations on which this whole system remains functional, but having an intimate knowledge of the genome unlocking potential or creating genetic potential is the influence a breeder has on the species.
References
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