Imagine stepping into your workplace, greeted by raucous, screaming animal calls. Imagine looking around and above you, wondering what in the world could be making so much noise. And finally, imagine your delight when you discover that the calls are coming from a group of wild cockatiels perched high up in the tree branches ahead. Well, during one of my visits to the Lyon Arboretum, I didn't have to imagine. I was lucky enough to observe the three cockatiels flying from tree to tree, arguing amongst each other-- or were they playing?

Such rare moments serve to refresh me during the long week, and make up part of the allure of the Arboretum. Each week I look forward to the surprises that may be in store for me. During the month of March, I've learned about so much, ranging from the technical to the aesthetic. Perhaps what has made the learning easier are the people whom I work with, all so open and helpful. Through them I've been taught the recipes for the different plantlet growth media, the procedures for transplantation and seed preparation, and the important points of the Endangered Species Act, especially as it relates to plants.

As in microbiology lab at KCC, broth and gel media are used to grow the organisms in the Arboretum micropropagation lab. This did not come as surprise to me. What did surprise me, however, was the discovery that there are many, many different kinds of growth media, each serving a different purpose. The choice of which media to use with each plant species depends on two things: the species' needs, and the lab's needs. Some species grow better on one type of media over any other. Some media promote rooting, some encourage shoot formation, while still others inhibit growth. Thus the media can be used to manipulate the plantlets into a desired pattern of growth.

Each media type is identified by an ID number, used by micropropagation labs around the nation. One of the most commonly used media is M0103. It is a general growth medium, which can support most any species of plantlet. Just to illustrate the complexity of the different medias, here is M0103's recipe:

2 g. Activated Charcoal

50 mL MS Macro Stock (M0005)

5 mL MS Micro Stock (M0006)

5 mL MS Iron Stock (M0007)

5 mL MS Vitamin Stock (M0038)

10 mL Myo-inositol Stock (M0072)

20 g C12H22O11

as needed to maintain pH 5.7-5.8, add NaOH or HCl

also add de-ionized water to bring up to volume

All of the Stock ingredients mentioned above have their recipes as well. For example, MS Macro Stock is made of (per liter) 16.5 grams ammonium nitrate, 4.4 grams calcium chloride dihydrate, 3.7 grams magnesium sulfate, 19.0 grams potassium nitrate, and 1.7 grams potassium phosphate. Considering that this is just one small part of the entire M0103 recipe, it becomes apparent that plant growth media are very complex indeed.

As mentioned earlier, the media can be used either as broth or gel, depending on the presence or absence of phytogel, the solidifying agent of choice among plant micropropagation labs. Phytogel is derived from the sap of the gummen tree, and, in this case, works better than agar.

I obtained this information on the day that I helped to make the M0103 media. We used a 2000 mL plastic graduated cylinder, an electronic pH meter, and a magnetic stirrer. The actual mixing of the ingredients was straightforward and quick, but the hard part was getting the pH to stay at 5.7. We spent two hours to add just the right amount of HCl to achieve a stable pH! Pretty frustrating.

Seed preparation, on the other hand, is not nearly as tedious. Seeds may be donated to the Arboretum by plant lovers, or they may be collected from their natural setting by Dr. Greg Koob, who runs the micropropagation lab. The important thing to remember is to treat all incoming seeds as if they were contaminated with microorganisms. If they are still embedded within fruit, they are extracted before planting, removing as much superfluous flesh as possible. Decreasing the amount of organic material around the seed decreases the possible sites for microorganism growth. The seeds are then rinsed and soaked in bleach solution before they are placed in media test tubes.

So far, I've worked with the seeds of two different species: the Silversword, and Sicyon, a relative of the cucumber. The lab had never cultivated Silversword seeds before, so I was excited to be given the chance to work with them. We prepared them in the following way:

Immerse seeds in 10% bleach for fifteen minutes, stirring vigorously with magnetic stirrer. Transfer to 5% bleach and stir for ten minutes; repeat 5% rinse. Plant into test tubes (do not rinse off the 5% bleach) The bleach helps to kill off microbial growth. Different seeds are rinsed to different extents, depending on the condition they are received. Generally, the seed with more exposure to possible contamination gets longer and stronger rinses.

For the Sicyon, we used only one rinse in 10% bleach, because we extracted the seeds directly from the fruit. They really hadn't had much time to accumulate many microorganisms. The Silversword seeds, on the other hand, had been extracted from the plant and flown over from Maui. The long period of time between formation in the plant and their arrival at the Arboretum, as well as possible excessive handling qualified the seeds for the three bleach rinses.

In addition to the rinses, sometimes seeds are placed in liquid media and then the whole container of seeds is placed on a platform shaker, which continuously stirs up the seeds. This treatment aerates the seeds and promotes multiple root formation.

Over the course of March I learned many of the technical aspects of saving endangered plant species. But I also learned about the Endangered Species Act. Greg gave me a set of papers to read-- the Act itself, in its entirety, and the Act as it pertains to plants. Some important points: under the ESA, destruction of endangered species is illegal, whether direct or indirect. Taking endangered plants from their native habitat to cultivate on your own is illegal, unless your actions promote the well-being of the species. Even so, a permit is required. With the support of the ESA, the Lyon Arboretum struggles on behalf of those few individuals left on these Hawaiian islands. Through the skills that I have learned over the past two months, I can join the struggle. I am so happy to be a part of the fight.

MOVE ON TO JOANNE'S LAST ENTRY

RETURN TO THE MICROBIOLOGY SERVICE LEARNING HOME PAGE