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How are varieties stabilized?

The breeding of cannabis has become a central topic of conversation in the industry's hobbyist circles in recent years. With the increase in knowledge, people have started to be more interested in the backgrounds and development work of the varieties. Some industry professionals organize courses and workshops related to breeding. Interest in the development of varieties is probably here to stay. One important part of the breeding work of varieties is stabilization. Stabilizing the characteristics of varieties may require precise selection work spanning several generations. In this article, we will get to know the most commonly used tools and models used by breeders.

Phenotypic variation causes individual differences in plants

The word phenotype means the appearance of an individual and it generally refers to the plant's anatomical characteristics, physiological aspects such as the length of flowering times and chemical composition - i.e. also to the terps and cannabinoids expressed by the plant, which produce the plants' psychoactive effects. The manifestation of the phenotype is regulated not only by genetic factors but also by the growth environment. These factors together make each cannabis plant unique.

Most cannabis breeders strive to create varieties that can be promised to produce, for example, a certain kind of aroma or a large amount of yield, and which do not cause additional problems with, for example, too wide a phenotypic variation. For this reason, it is important for the breeder to get to know the inheritance of crossbred individuals and the degrees of same or different inheritance.

Homozygotism and heterozygotism – the keys to genetic stabilization

Hemp is a diploid species that inherits half of its chromosomes from the female plant that produced the seeds and half from the plant that acted as a pollinator. The sets of chromosomes originating from both parents complement each other and the genes find their counterparts, i.e. alleles. The same gene can have several alleles and their dominant and recessive forms. The whole formed by these determines the genotype of the plant, i.e. the genetic basis for the phenotypic expression.

The phenotype formed by recessive alleles occurs only if the recessive gene form is inherited from both parents. A good example of this phenomenon is the inheritance of the autoflower trait: Crossing a photoperiodic plant (e.g. OG Kush) with an autoflower variety (such as Lowryder) does not produce autoflower offspring in the first generation, but the line must be crossed further.

If the alleles of the gene are different, this is called heterozygosis, same inheritance homozygosis. When the genotype is heterozygous, only the dominant alleles determine the appearance of the individual.

Punnett squares can be used to illustrate the inheritance of alleles.

Punnett squares can be used to illustrate the inheritance of alleles.

The enhanced growth ability of hybrids results from heterozygotism

A cross made between two genetically different and internally identical strains is called a hybrid. Hybrids are referred to as F1 crosses – this reading tells you the generation of the lineage.

Usually only crosses between pure strains are called F1 crosses, but among cannabis breeders this name is also often used for polyhybrids (crosses between several hybrid varieties).

The offspring of the F1 generation resulting from hybridization have enhanced growth ability. This phenomenon is called hybrid vigor (heterosis).

In the mosaic-like new inheritance, the characteristics of the two strains complement each other and the recessive alleles do not affect the growth characteristics.

Inbreeding helps to strengthen the desired characteristics

Some desired characteristics (such as autoflowering tendency) do not yet appear in the first generation and, for example, there may be great variation in terpene profiles, so in many cases it is essential to create inbred lines (IBL) from crosses with the selected characteristic in mind.

The degree of inbreeding is a mathematical value that tells the probability that an individual will inherit certain characteristics from their parents. When you get to the eighth generation (F8), you can talk about true breeding. Pure inheritance refers to strains that are more than 90% identical, in which case their phenotypic variation is very little. It is generally thought that in this case the position is stabilized.

Individuals of the F1 generation also often resemble each other very homogeneously, because only dominant genes are expressed in individuals. However, with the next generation, recessive expressions start to appear and the phenotypic variation is more abundant. Even with the F2 generation, it can be observed that the vigor of the cross is disappearing. The decline in cross-breeding vigor is also the steepest when moving to the F3 generation, so plant breeders have to make the selection of breeding individuals at this stage very carefully. Also, the phenotypic similarity of the individuals of the F3 generation depends entirely on the F2 selections.

When trying to stabilize the variety through inbreeding, it is essential to work systematically and keep an eye on the desired characteristics. The breeder's most important tool is his selection criteria. Large populations are an essential asset, as they enable mapping of the range of phenotypic traits and offer greater opportunities for selection.

Most breeders also keep their champion individuals as cuttings for very long periods of time, which makes it possible to work on complex elite lines and strengthen traits by backcrossing.

Skunk #1 is the best-known example of a modern cannabis strain with fully stabilized properties and pure genetics. David Watson, also known as "Skunkman Sam", grew over ten generations and tens of thousands of plant individuals during the selection work of a hybrid strain that contained Mexican, Colombian and Afghan heritage. The cuttings chosen by Watson are still the credit individuals of the breeding stables of many European seed banks. Skunk's pure genetics show up in a very predictable way in the crosses that include it, and its growth characteristics can be considered stellar.

Blueberry Muffin is a slightly more modern example. The Humboldt Seed Company has spent several years refining this variety, which is known for its fast flowering and excellently delicious blueberry aroma. The seeds found in our selection are a cross between two separate F8 strains. When the strains are genetically different, but composed of the same hereditary factors and established for the same characteristics, this kind of line crossing can restore the vigor of the cross to the strain, which is inbred. Such an advanced method aims to reverse inbreeding depression, which normally impairs growth performance in inbred lines.

Feminization and backcrossing as means of stabilization

Sometimes cannabis breeders find an exceptional individual whose best qualities they want to capture as seeds. In such a case, the only options are feminization and traditional (male-line) backcrossing: The first of these is really popular today, but backcrossing is used especially when you want to create a collection of the strain that also contains male offspring and is fully reproductive.

When producing feminized seeds, the plant's normal hormonal activity during the flowering period is chemically prevented, so that the female plant begins to develop male flowers. Becoming a hermaphrodite is also a stress reaction for the plant, but if carried out correctly, the feminization treatment should not cause the plant any stress. The pollen produced by such a plant has an XX sex chromosome set instead of the XY chromosome set produced by a male plant, so any flowers fertilized by it produce only female plant seeds.

The pollen produced by the female plant can be used for either cross-pollination or self-pollination. If the plant self-pollinates, the inbreeding coefficient increases faster than with conventional cross-pollination. "Self-fatted" S1 seeds are more than 70% of the same heritage. The majority of feminized seeds are S1 seeds.

Backcrossing means using a hybrid offspring as a pollen-producing plant and crossing it to its own parent. The seeds born from this again contain a little more of the genetic material of the parent plant. With the fourth backcross (BX4), the degree of same-heritability is more than 90 percent - that is, it can be considered that the variety stabilizes twice as fast as when conventional crosses between siblings are made.

It is also reasoned that when using male seeds for breeding, it would be an advantage that in hemp plants the Y chromosome is larger than the X chromosome and it transfers more genetic information to the offspring.

Among other things, Cinderella 99, which rose to legendary fame in the early 2000s, is a backcross to a special offspring of Jack Herer (and an unknown pollinator). Our selection includes a feminized version of Cinderella 99 from its original producer (Cinderella XX) as well as a lot of interesting Cindy crosses.

Chemotypal differences create variations in cannabinoid levels

The plant phenotype also refers to the appearance of the plant and the secondary metabolic products it expresses, such as terpenes and cannabinoids. These components are part of the so-called chemotype. Two outwardly similar plants can produce different metabolic products – and also different effects.

Cannabinoid concentrations vary between individuals. This is due to the codominance and variation in inherited alleles of the genes for THC-producing THCA synthase and CBD-producing CBDA synthase.

The growth environment and conditions also have a decisive effect on the phenotype and chemotype. For example, from OG Kush, which originated in 1991, several different cuttings are available that are genetically identical but phenotypically different. This is due to the very long age of the cuttings and changes in the internal structure of the plant, which occur when the plant adapts to a new growing environment. It is possible that two different cuttings of the same plant also transfer different epigenetic factors to their offspring.

The plant produces its main aroma substances with the help of terpene synthases. The expression of these depends on genetic factors, but the microbiome of the growing environment has also been found to have a minor effect on the occurrence of terpenes. In a stabilized (i.e. broadly homogenous) strain, certain terpenes are on the surface of all plant individuals.

In our next article, we will take a closer look at how these flavoring substances originate and how their presence is also related to the generation of cannabinoids found in plants - and how they are possibly responsible for part of the effect of cannabis.

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