Citrus Tissue Culture Practice Makes Perfect

I recently posted an article that outlined some challenges and opportunities related to tissue culture (TC) production of citrus scions. This issue was brought to the forefront as a result of overnight interest in the UF/IFAS 7-6-27 mandarin hybrid (FAST TRACK Suite III). Grower demand for budwood quickly exceeded supply and nurseries began to look to TC as a potential solution. While the wheels are in motion to address the regulatory aspects of producing scions through TC, labs and nurseries are being licensed and they are beginning the process of establishing material, developing lab procedures, and taking orders.

Citrus tissue culture sample

Photo courtesy of Agri-Starts

It is important that citrus nurseries and growers understand the fundamentals of the TC process, the opportunities, and any risks that may be present. DPI is working hard to protect industry interests without unnecessarily hindering variety introductions through technological advancements and the use of proven laboratory procedures.

Advertisement

I reached out to TC laboratories and nurseries to better understand the process and how this may be a useful tool to restore acreage and help achieve critical mass of new varieties. This is a technical issue. The details and specific procedures are important. However, to avoid breach of confidentiality and competitive advantage of the companies involved, we will top-line the issue. Hopefully, those most interested will elect to seek more information from their nursery partners and follow the issue in future research and trade publications.

Why consider TC?

Top Articles
Healthy Soils at Center of New $5 Million Initiative for California Citrus Growers

Mathew Konrad of Citrific puts it like this: “At its very core, plant tissue culture is a collection of techniques used to maintain or grow plants under sterile conditions on a nutrient culture medium of known composition (think synthetic soil). That being said, the only difference between tissue culture and a greenhouse cutting operation is that tissue culture is done in a sterile environment, which allows for faster growth; the ability to use smaller size material; more potent and larger database of synthetic chemicals; and more control that leads to a higher and faster success rate than one might get in a cuttings operation.”

Process Overview

David Lawson of Agri-Starts explains there are two traditional means of regenerating plants from TC. Indirect Organogenesis: “The plant is taken through a callus stage (undifferentiated cells, the cells have not chosen an organ to develop into) and then, by adding select hormones, you tell those cells to regenerate into shoots or embryos. This is one method where you get single cell regeneration. Typically, it has been shown this type of method increases the chances for off types or variations. However, it is still widely used as the percent of off types is typically so low that the risk does not outweigh the benefits. The best example of this is coffee. The majority of coffee trees worldwide are propagated this way. They generate embryonic callus the same as breeders do when they are doing transformations. These turn into plant embryos and eventually an entire plant.”

Direct Shoot Organogenesis: “New plants are regenerated from existing organs. Cells do not go through a callus stage. This process is considered the safer route to avoid mutation. It still encompasses regeneration from single cells of plants as long as they do not enter a callus stage. This typically takes higher concentrations of hormones to accomplish. Most cautious labs do not increase hormone concentrations to high enough levels for mutations to happen. Instead the propagators increase hormone levels just enough to break apical dominance without having to mechanically prune the plant. By doing this you can promote lateral growth from existing meristems (buds) much more rapidly than conventional methods ex vivo (in a greenhouse).”

Click on the following pages to read the Q&A with our plant nursery insiders.

There has been a lot of talk about higher risk of mutations when scions are produced through TC. Is this true?

The type and quantity of hormone used can impact the likelihood of variation. Conventional wisdom seems to support that any risk through TC is more procedural than the science/technology. The technology has been used in many crops and ornamentals for decades.

Citrus tissue culture lab

Photo courtesy of Agri-Starts

It also should be mentioned that at least one lab, Citrific, is working on TC procedures for citrus scion material that would not use hormones. Konrad states their approach in this way: “Many consider the only way to multiply is through a cytokinin called BA or BAP.  These are hormones that force a single bud to reproduce multiple times. There is absolutely nothing wrong with these hormones, and its common place to use them. There is, however, uncertainty whether or not these hormones can cause genetic mutations in scions that may slightly alter a true to type scion, thus over time, every multiplication is getting farther from true to types.

“Citrific is unsure on this topic and does not want the liability of finding out in the coming years that through using a cytokinin we altered the scion the farmer got. We instead are taking a more ‘organic’ approach using nothing more than LEDs, robotics, and the standard Murashige and Skoog medium at 4.43 grams per liter with 30 grams per liter sucrose (that’s right, that’s our secret sauce). We take the buds, sterilize them, dissect them, and place them on this medium. Once new shoots grow, we dismember the new shoots from the old mother material. We get the new shoots to root using something every cuttings house uses: IBA, NAA, or IAA. Once the shoots root, we have a micro-tree with four to five micro-buds on it. We then use robotics to dissect these micro buds at an efficient speed, leaving only the stumps with roots. In four weeks’ time, the stumps will grow back with four to five more buds and the process starts all over again and we can harvest more buds, all done without any cytokinins. This truly would be an inefficient way of doing things but, due to some technologies we use, it has become a more efficient way than forcing one bud to push multiple shoots.”

Agromillora, an international TC micropropagator, also was consulted on this issue. They have 10 years of experience with micropropagation of citrus rootstocks. Agromillora is interested and willing to help develop protocols but stressed that “the researchers and experimental work should show that TC production of citrus scions is reliable or how risky it could be.”

Phillip Rucks of Phillip Rucks Citrus Nursery, says, “TC propagation of scion varieties must be carefully considered and executed with caution using the most conservative lab methods. This is necessary to prevent natural occurring mutations.”

Are certain varieties or plants more likely to incur variation through TC regeneration than others?

Lawson continues, “Variations can occur in any plant in culture. It would be a bold statement to say that something is more susceptible to variation than something else. It is biology we are dealing with and not everything can be predicted. It heavily depends on how the plants are treated. Does the propagator push the plant as hard as possible to generate the most plants in a short time frame or do they give it just enough to make it worthwhile so that they can sustain the cultures over a longer duration of time?”

Many believe it is important to limit the number of generations away from the mother culture in order to minimize risk of variation. Do all agree?

Of course, nothing is unanimous in the citrus industry. Some labs have successfully maintained multiple genera and cultivars in culture for more than 30 years. Though this has not been done in citrus, they believe it is disciplined adherence to established procedures that yields stability. These labs would object to any requirement that mother material be replaced in an annual or other arbitrarily assigned basis. They do not see any correlation between the amount of time that a plant is in culture and the likelihood of mutation. Furthermore, they fear a short turnover requirement for mother material will negate any multiplication advantage achievable through TC. Plants must acclimate to culture. Until they acclimate, they might only multiply two-fold each month. However, once acclimated to culture, they might multiple five-fold each month. It is only after acclimatization that TC makes economic sense.

This raises the issue of cost vs conventional methods of increase and propagation. Until recently, TC was viewed as a more costly intermediary step. The increased cost was justified by the opportunity to accelerate commercial scale production of emerging varieties. Citrific comments, “Seed, even for things like Swingle (rootstock) or Valencia (scion) will never be able to touch the cost we will be able to achieve once efficiency and mass TC production starts. Yes, it will be a few years, but we are taking the playbook straight from the reforestation industry. Putting the cost of liners well below the cost any nursery could even produce a liner themselves by seed.”

Are TC labs planning to produce complete trees or just plant material?

Some laboratories are planning to produce complete trees. These labs also are producing TC rootstock liners and will ultimately graft the TC scion to the liner. Other labs are focused entirely on providing scion plantlets to other nurseries as a budwood source. Finally, there will be some scion plantlets going to nurseries for repotting as a tree on its own root.

Rucks sums up the issues nicely: “In the past, scion variety TC propagation has been difficult to economically justify compared to nursery conventional scion production practices. Despite the costs, TC is a viable option when diversifying scion production in the presence of HLB and to meet the challenges presented by more frequent variety introductions.”

3