Plant Tissue Culture

Opportunities present themselves, problems arise, and solutions are found. The story of cannabis plant  tissue culture follows that same cadence.  But what exactly is plant tissue culture? How was it developed? Why is it crucial to the rapidly expanding cannabis industry? The early adopters of this biotechnology have seen the countless benefits related to crop performance, pathogen management, and mitigation of operational risks through genetic banking. The use of plant  tissue culture technologies will continue to proliferate as the cannabis industry enters the most competitive time it has seen since the inception of commercial production. 

Plant  tissue culture is a broad term that refers to the in vitro growth of plant cells under defined physical and chemical conditions. It can be applied to a range of technologies and biological processes performed in vitro. The in vitro growth of plant cells can originate from a singular cell, a piece of leaf or stem tissue, or a seed placed in growth media. Some of the more common plant  tissue culture technologies are listed below: 

• Anther Culture
• Callus Culture
• Meristem Culture
• Organ Culture  
• Organogenesis
• Protoplast Culture
• Somatic Embryogenesis
• Seed Culture
• Suspension Culture

These technologies categorize a series of different specializations and techniques used to research and produce clean, individual plantlets and are often used in combination with one another depending on the crop or product. To produce our clean-stock cannabis plantlets, Segra utilizes organ culture, meristem culture, and organogenesis. To understand the relevance of these specific processes in modern agricultural and horticultural practices, we must first understand how and why these technologies were developed. Click here to watch our webinar “The ABC’s of PTC.”

The idea of studying and growing plant cells in vitro was proposed by the German scientist Gottlieb Haberlandt in the early 1900’s^[3]. Haberlandt studied the properties and interrelationships of individual cells within a multicellular organism and proposed that researchers could successfully propagate artificial embryos from individual vegetative plant cells^[4]. Today, this process is referred to as “plant tissue culture micropropagation.” 

From the inception of Haberlandt’s theory and throughout the early to mid 1900’s, further development in new plant tissue culture emerged.  Augmentation of existing processes continued. Refinement of plant tissue culture media and discoveries of how various growth regulators work to influence the performance and development of plant cells occurred. These discoveries led to an understanding of the balance between auxin and cytokinin to influence the morphological structure of plants. A high ratio of auxin to cytokinin favours rooting, the opposite favours shoot development, and an intermediate mix of both favours the growth of wound parenchyma tissue (callus tissue)^[5]. With the ability to direct the growth and development of a single vegetative plant cell into a fully developed multicellular plantlet equipped with leaves, shoots, and roots, scientists had the opportunity to begin growing plantlets at scale under sterile conditions. The cut flower industry demonstrated the significance of plant  tissue culture in the 1960’s when they developed protocols to initiate meristem shoot tips in tissue to produce clean orchids, a discovery that scientists embraced as a solution for many other crops. 

The ability to distribute clean plants while leveraging logistical efficiencies attracted commercial agriculture companies. Today, plant tissue culture is used to produce a plethora of crops at scale, including potatoes, bananas, eggplants, pineapple, strawberries, blueberries, raspberries, and more^[6].  

With plant tissue culture, however, there is no facile solution for every plant species. Individualized agar growth media formulas and protocols need to be tailored not only to cannabis generally, but to each specific cultivar within the cannabis genome. When developing plant tissue culture protocols for a species, researchers must  develop plant tissue culture growth agar formulas and handling techniques for each specific cultivar as all have nuanced in vitro performance requirements. This presented a very time-consuming challenge for cannabis growth in plant tissue culture due to the inherent genetic diversity in the industry. In other commercially produced crops around the world such as strawberries or raspberries, approximately 5-10 main production cultivars are utilized globally. When approaching plant tissue culture for cannabis, thousands of varieties currently exist and consumer demand requires a constant flow of new cultivars. As a result, extensive research is required to successfully operate a cannabis plant tissue culture facility with the capabilities and expertise to tailor operating procedures for every new cultivar required for mass production. Segra has pioneered related research in the cannabis tissue culture and nursery space over the past four years.

The vast genetic variance existent in the cannabis genome causes another challenge that cannabis plant  tissue culture providers face: soma-clonal variation. Soma-clonal variation is a genetic variation observed within progeny created from somatic cells regenerated in vitro. Following the successful initiation of a cultivar in tissue culture, the cell’s DNA risk mutation into a distinct variant from original donor material. With cannabis higher than average rates of soma-clonal variation can be attributed to the broad genome and genetic instability that exists in the cannabis industry. It has been observed that some cultivars are more prone than others to soma-clonal variation, causing challenges when tailoring plant tissue culture protocols for specific cannabis cultivars. All cannabis plant tissue culture providers face these challenges, and it will take years of further research and development to fully overcome them. 

As a recognised leader in the cannabis tissue culture industry, Segra has studied these issues and developed unique proprietary protocols. These fall into three of the main technological categories of plant tissue culture: organ culture, meristem culture, and organogenesis. At the forefront of our protocols is pathogen identification and DNA fingerprinting using Variable Nucleotide Tandem Repeat (VNTR) technology. It’s an essential step in determining the correct cultivar ID and the proper plant tissue culture techniques to use on certain plant material, depending on the existing pathogen load. For instance, some pathogens can be removed using techniques that fall under the organ culture category. These techniques include a process called shoot-tip tissue culture, where anti-viral and sterilization agents are applied to multiple growth nodes of a cannabis plant to remove existent pathogens. Shoot-tip tissue culture, when performed properly, is often a fast and efficient way to clean and initiate plant material in tissue culture. 

For pathogens that cannot be removed using shoot-tip culture techniques, Segra employees meristem culture. This method isolates meristematic cells not yet vascularized within the plant and initiates them in the appropriate growth media. Meristematic plant tissue culture is the gold standard for effectively removing pathogens and producing clean plantlets with augmented production performance; however, this method can take significantly longer than shoot-tip plant tissue culture techniques to return a clean plantlet to a receiving cultivator ranging from 4-8 months for shoot-tip plant tissue culture and 8-12 months for meristem culture. Segra uses this method for every cultivar in its genetic library and the process is generally performed simultaneously with shoot-tip culture depending on initial pathogenic load and our client’s operational timeline for clean plantlet provisioning. 

Following the successful removal of pathogens, Segra then applies organogenesis techniques to rapidly multiply the clean population and return scaled plant tissue culture plantlets. Organogenesis is the practice of taking in vitro nodal cuttings from existing shoots within a cultivar population and multiplying them to larger inventories.