Oaks from Test Tubes: An Introduction to Oak Micropropagation

 

by Bryan R. Denig, Miles Schwartz Sax, Nina L. Bassuk

 

Putative hybrid Quercus muehlenbergii × virginiana
Putative hybrid Q. macrocarpa × turbinella

Our Interest in Cloning Oaks

One research focus of the Urban Horticulture Institute (UHI) at Cornell University involves the selection, evaluation, and propagation of superior plants for urban environments. Oaks are especially of interest, as many Quercus can tolerate the various stresses associated with growing in urban landscapes. Hoping to combine the best characteristics from multiple species, we have created a number of interesting hybrids which we have been propagating and evaluating for about 10 years. The ultimate goal of this long-term project is to introduce these superior hybrid oak selections into the nursery trade as named cultivars.

A reliable asexual propagation method for oaks is crucial for meeting our goals, and the UHI has been pursuing such a method for almost 20 years. We have been successful in utilizing a modified stool bed layering technique, which is described in detail in International Oaks No. 27 (pp.99-106), but the yield is too low for commercial applications. To overcome this challenge, tissue culture techniques are currently being developed as a means of rapid multiplication of clonally propagated oaks.

What is plant tissue culture?

Plant tissue culture refers to various techniques used to grow plant cells, tissues, organs or whole plants in vitro (“in glass”) on a nutrient medium. This is done aseptically, meaning that the containers are free from microorganisms, insects, etc. These containers, usually test tubes or small glass jars, essentially act as tiny greenhouses, maintaining ideal humidity levels. They are kept in a growth room that is controlled for temperature, photoperiod, and light intensity. The media that the plants grow on are carefully created to provide the right mixtures of macro- and micro-nutrients, vitamins, and plant hormones. All these factors add up to an extremely controlled environment, one that is designed to provide ideal growing conditions.

The techniques associated with plant tissue culture can be used for many diverse purposes. Our interest in plant tissue culture at the UHI is for the purpose of asexually propagating (cloning) our hybrid oaks. This type of plant tissue culture is often referred to as micropropagation. Micropropagation takes advantage of a remarkable ability inherent in plant cells. Even just a single cell is capable of multiplying and differentiating into an entire plant.

When grown in vitro, plants adjust their growth based on the media compositions. Media manipulation will produce varied effects, such as root or shoot development, and typically the plants stay small-statured. This is important, as it allows them to be grown in these confined spaces. Because of the reduced size, these  micropropagated plants are often referred to as “plantlets.” Once planted outside of the laboratory, the plantlet will once again produce normal sized leaves and reassume its normal features.

Plant tissue culture may seem like a new technology, but it has been utilized on a commercial scale longer than one might expect. Since the 1950s it has been a component of the orchid industry.  Woody plants have not been commercially micropropagated as long as orchids, but today in the United States there are wholesale laboratories producing a variety of trees and shrubs. Birches, apples, maples, lilacs, and flowering cherries are just a few examples of the woody plants currently available from commercial micropropagation companies. We

A coppiced stock plant with shoots appropriate for use in tissue culture (Putative hybrid Quercus montana × geminata)

are unaware of any company currently offering micropropagated oaks, but a handful of researchers around the world have developed plant tissue culture protocols that have proven successful with a small number of Quercus species.

How we micropropagate oaks

In traditional propagation, one seed or one cutting usually results in one plant. Using micropropagation, one small piece of stem can theoretically produce an infinite number of plants. The process of micropropagation is often broken down into five stages, and what follows is a summary of the stages as it relates to our work with oaks.  

STAGE 0: Stock Plant Selection and Preparation

There are a number of factors that influence successful initiation of plants into tissue culture. Not every shoot can be taken into tissue culture. Juvenile shoots have the highest affinity for establishment in culture, but success has also been achieved using epicormic buds forced from mature trees. For the most part, our cultures have come from the stock plants we used for our stool bed propagation procedure. Because they are cut back each year, these stock plants grow new stems from the ground that exhibit juvenile characteristics. We collect the shoots early in the spring, when they are about half the thickness of a pencil, and between 5 and 10 cm in length.

STAGE I: Establishment of Aseptic Cultures

After collecting suitable shoots, we cut them into small pieces (a few cm in length), that possess at least one bud. In order to remove fungi, bacteria, etc., they undergo a soaking in ethanol, followed by a soaking in a

Buds starting to swell on the shoot  New shoot growth that is ready for stage II

dilute bleach solution. Sterilization of everything except for the oak shoot can prove difficult at times. After disinfestation, the shoot is placed into a test tube, and is stuck into a medium that contains plant hormones that induce shoot production. Once shoots are produced (which may take a few months), the cultures are ready to be taken into the multiplication stage.

STAGE II: Multiplication

Once new shoots develop, they are cut up, and all leaves and apical buds are removed. The shoots are then placed horizontally on fresh medium in jars. This is done to encourage the lateral buds to develop into shoots. This procedure, along with manipulation of plant hormones in the media, allow the oaks to grow continuously throughout the year. During the multiplication phase, the plantlets are transferred to fresh medium every two weeks. After six weeks, there is enough shoot growth for them to be divided. The shoots are cut into pieces as previously described, and the multiplication cycle is repeated. This stage can go on indefinitely.

Shoots at the start of a multiplication cycle... After a few weeks growth... After 6 weeks, the newly grown shoots are ready to repeat the multiplication cycle or go into stage III

 

Some shoots ready for rooting Shoots at the start of stage III, rooting

STAGE III: In Vitro Rooting

When it is desired to root the oak plantlets, they are taken out of the multiplication cycle and go into the rooting stage. After a complete multiplication cycle, rather than dividing the plantlets, shoots of sufficient length are selected for rooting. The lower leaves are removed, and these shoots are then placed into a medium that contains a rooting hormone. After a week, they are transferred to another medium that has no hormones. It is here that the plantlets 

A rooted plantlet ready for potting up

develop roots, which usually takes about a month.

STAGE IV: Transplanting and Acclimatization (Hardening Off)

When sufficient roots have developed, the oak plantlets can be taken out of the medium and potted up. Much like a cutting or seedling, they will have to be carefully cared for until they become acclimatized to the world ex vitro. Right now our research is focused on stages 0, I, and II, which we believe to be the most difficult. Our experience with stages III and IV is  limited at  this time, but we hope to do more with them in the future. There is still much work for us to do to in order to develop a reliable method for micropropagating oaks!

 

 

All photos © Bryan Denig