JARS v44n3 - Tissue Culture Troubles

Tissue Culture Troubles
Anna J. Knuttel
East Windsor, Connecticut

Reprinted with permission from American Nurseryman, December 1989.

Plants have been commercially micropropagated for over 20 years. The first items produced this way were tropical foliage plants. They were followed by herbaceous perennials and, finally, woody ornamentals.
Knuttel Nursery Inc., East Windsor, Connecticut, has been a wholesale grower of rhododendron, azalea and kalmia for 15 years. Thus we have considerable experience growing these genera. We initially started purchasing tissue culture plants because they give more depth to our production program. Using this material, we can quickly build up an inventory of plants that are in demand. This is a particular advantage if a cultivar is difficult to propagate by conventional methods, or if we experience a crop failure.
To be knowledgeable about our product and its cultural needs, we test new cultivars before we release them for sale. The rapid multiplication process inherent in tissue culture allows us to grow a representative number of plants in a short time. During this period, we evaluate hardiness, vigor, efficiency of production and marketability. If a plant shows promise in these areas, we may purchase large numbers of tissue culture plants to facilitate full-scale production.
We thus consider tissue culture plants essential to our operation. We buy 45 to 50 different cultivars - a total of 45,000 plants a year - from five commercial labs. Most of the plants have been normal, and equal, if not superior, to our conventionally propagated plants.
However, we are very concerned about some serious problems that now seem to be arising with certain aspects of micropropagation. We have observed plant variability, a tendency toward irregular growth (especially with small-leaved rhododendrons), lab-to-lab inconsistencies, and mislabeling. Growers and micro-propagators need to recognize these problems and work together to solve them.
The most costly problem we have encountered is the variability of some plants from some sources. This variability may be genetic, epigenetic or both. We have observed several types of variation, including changes in flower color or form; variegation in foliage; distortion of leaf shape; and dwarfed growth habit. Here are a few examples:
Two years ago, we purchased 175 'Crimson Tide' deciduous azaleas from a tissue culture lab. We wanted to test this cultivar, which had been shown to us as a large-trussed, double red flower. This spring, the plants flowered in a range of colors from light pink to salmon to dark red. Blossom shape also ran the gamut: single, double, frilled, plain, complete and incomplete. We have been forced to stop producing 'Crimson Tide'.
In 1986, we purchased 2,500 Rhododendron catawbiense 'Album' as rooted cuttings from tissue culture. Twenty percent of this crop exhibits white-and-green foliar variegation. Although we are growing these plants on in the hope that the variegation will eventually disappear, two years of production time indicates that this probably will not occur.
Three years ago, due to increased demand and favorable initial trials, we purchased 5,000 Rhododendron 'Molly Fordham' as micropropagated rooted cuttings. Seventy-five percent of these plants have shown excessive brooming that varies not only from plant to plant but also from branch to branch.
The plants' appearance is dramatically altered, and we are unable to sell them as 'Molly Fordham'. Losing 3,750 plants - at an average sales price of $10 - is a considerable blow. Five hundred conventionally propagated stem cuttings given the same cultural conditions showed no variability and have been sold.
In 1987, we purchased 2,000 Rhododendron 'Aglo'. Of these plants, 1,570 show abnormal foliage and branching. The rest appear true to form. Again, at a sales price of $10 each, our losses greatly outweigh any benefit we had hoped to receive from this crop.
The tendency toward leaf distortion, compacting and irregular growth is most apparent on small-leaved rhododendron cultivars. I should note that we occasionally observe this phenomenon on conventionally propagated small-leaved varieties. In our experience, tissue culture exacerbates this tendency - and the proportion of such irregularity varies from lab to lab.
An identical cultivar purchased from several sources may vary in growth habit and rate because the suppliers use differing production techniques. For example, last year we purchased 1,250 Rhododendron 'Black Satin' from two laboratories. The plants from one lab were true to form and vigorous; they performed well. However, the plants from the second lab showed severe stem contortion and compression, as well as deformed foliage. Their growth rate was substantially slower than normal. Our experience indicates that these plants will continue to exhibit abnormal growth and are highly susceptible to disease.
This variation from lab to lab may be imperceptible at first, revealing itself only after several years of production. We once purchased R. 'Henry's Red' from two sources. The initial growth in both blocks appeared true to form. However, during the second year of growth, the foliage was light green in the first block and dark green in the second.
Because we top dress with a slow-release fertilizer, we initially assumed that we'd skipped the first block. However, investigation showed that it had been fertilized at the proper rate. Repeated applications, followed by a liquid feed and chelated iron, did nothing to "green up" the plants. We monitored the soil fertility and pH to assure that the plants had every opportunity for proper nutritional uptake - but to no avail. (The flowers from this deficient block also bloomed in double and polypetaloid forms, although the true 'Henry's Red' has a single flower.)
Another tissue culture concern is mislabeling. Always worrisome with conventional propagation, this problem can manifest itself more seriously in tissue culture. Due to the increased productivity inherent in micropropagation, large numbers of incorrectly labeled plants can enter the trade. Compounding this problem is the fact that tissue culture plantlets do not always resemble the parent plant. In addition, different cultivars of microcuttings are virtually indistinguishable. A grower may invest several years of costly production time before discovering a labeling mistake. And, if the grower is unfamiliar with the original cultivar, he may never be aware that he is growing and selling a misidentified plant.
For instance, in 1988, after observing Rhododendron 'April Rose' in Dr. Gustav A.L. Mehlquist's test garden, we decided this outstanding new hybrid would be a valuable addition to our specialized line. We purchased 500 rooted tissue culture plants from the only source that had released 'April Rose' at the time.
Because our greenhouses were filled, we subcontracted another nursery to grow these plants in a manner similar to ours. The subcontractor accepted the shipment of plants and grew them to 4 inches. As with all new or unfamiliar varieties, we and the subcontractor were particularly careful to avoid varietal mix-ups.
When we received these plants from the subcontractor, we put them into our production schedule and potted them. We took 3,000 cuttings from them last fall.
We routinely test our plants for trueness to name before releasing them. When forced, five randomly selected (yet representative) plants proved to be Rhododendron 'P.J.M.' In this case, a labeling error has cost us not only the initial purchase price but also the subcontractor's fees and our own production and propagation expenses.
Such financial losses are indeed serious. But it is equally important to consider the possible loss of reputation for the hybridizer. By releasing mislabeled plants, a careless lab can destroy a minimum of seven to 10 years of hybridization work - more usually, a lifetime's worth - in less than one year.
Labeling mistakes are generally inadvertent. Thus it is essential that both the grower and the lab are knowledgeable, and that they test the cultivars they are growing. Most tissue culture labs do guarantee their cultivars to be true-to-name, and it is important that they honor this guarantee. To preserve the integrity of the industry, they must also notify everyone who has purchased the mislabeled plants.
As promising as micropropagation appears, growers must remember that it remains a relatively new technology (especially for woody plants) that is still undergoing evolution. At this point, the grower is usually saddled with the financial burden of disposing of abnormal plants, as well as the ethical dilemma of possibly marketing plants that are not true-to-name. It is vital that growers purchase plants only from reputable, established labs that participate in associations where technical and cultural problems are discussed and information is exchanged.
Growers should also ask labs if they grow the products they sell beyond the liner stage. We have found that the labs that do this are most aware of the critical need for uniformity and quality control.
Growers and labs must communicate freely. The grower can alert the lab to potential problems before they escalate, and the lab can give the grower information that may facilitate proper culture of its stock.
Reputable suppliers are responsive to input from their customers. (Most of our nursery's bad experiences have involved tissue culture labs that defensively said they didn't have product problems or, when informed that problems existed, chose to ignore them.) In addition, growers must understand that not every production problem stems from the stock's origin as micropropagated plants. For instance, in January 1986, we purchased 500 micropropagated Kalmia latifolia 'Carousel'. We grew these plants to liner size in the greenhouse. In May, we potted them into 1-gallon pots and placed them in our normal container production cycle.
The following spring, our losses of 'Carousel' exceeded 50 percent. Most of the plants had rotted at the base. We initially thought the problem was associated with the fact that they were micropropagated. However, after looking more closely at the crop, we realized we had potted the plants too deep. They had suffocated.
The moral of the story is that growers must carefully examine their own cultural practices. Only then can they work with their suppliers to determine the true source of problems.
After purchasing 225,000 plantlets over the past five years, we still feel that tissue culture is fundamental to our production program. However, we are careful to choose only those labs that are committed to quality and to preserving the integrity of the nursery industry.

Anna J. Knuttel is general manager of Knuttel Nursery Inc., East Windsor, CT.