PROCEDURE
INTRODUCTION
The process to be covered in this paper is a particular area of the general operation known as Plant Tissue Culture. This process has been referred to as 'Meristem Culture', 'Meristem Tip Culture', 'Shoot Tip Culture', etc, depending on the type and size of the tissue used for culturing . In this paper the term 'Meristem Tip Culture' will be employed as the term best describing the tissue being treated.
Description of this operation is possible only through use of and reliance on certain botanical processes. It will not be an exercise in Botany although certain botanical terms will occasionally be used. In fact, a discussion such as this would not be possible without resorting to botanical terms on occasion. Where such terms are necessary they are explained in a way that the reader, even with a limited knowledge of Botany, will be able to understand and follow the procedure. In order to be as explicit as possible it will be necessary at times to make general statements. The statements may not necessarily be completely true for all specific details. If the reader notices any such statements he is requested to consider the reason and bear along
Why is Meristem Tip Culture Important?
In an article written as a special publication for the Ameican Dahlia Society in 1959(1) Mr. Roland A Milner Jr. summarized much of what was known about dahlia virus disease to that time and drew several conclusions regarding the extent, damage and general effect of various virus diseases in dahlia, and stated an urgent need for research into the control and identification of such diseases. The opening paragraph of the article states "Virus diseases are the chief limiting factors in dahlia production. All other diseases and pests, indeed all other problems concerning dahlia growing are insignificant in comparison." Other widely publicized and quoted reports(2,3,4) arrive at substantially the same conclusions.
In the same opening paragraph Mildner states, "moreover the situation is confounded by the refusal of growers, both professional and amateur to admit that the problem exists".
In the years since that report more and more growers are admitting the presence of the problem, and many have expressed concern. A great many have become aware the Meristem Tip Culture process offers a possible solution but appear to be unfamiliar with that process. This paper will describe in detail a program conducted under routine home environmental conditions that has successfully recovered symptomless plants of dahlia from stock which had previously expressed very pronounced symptoms of virus infection.
History of Plant Tissue Culture
The entire idea may be said to have started in 1902 with a German scientist named Haberlandt who attempted to culture Chlorophyll-containing cells and demonstrate the totopotentiality of cells. (totopotentility means that every cell of an organism has the potential of every other cells or total potential).
To say it another way, each cell of the organism contains factors which determine the final form of the entire organism. (These factors are known today as Genes) Although Dr. Haberlandt's success was not enthusiastically received, he did manage to initiate a new method of plant propagation which has become known as 'plant tissue culture'.
In 1946 another Scientist by the name of Ball(5) obtained complete plants of Lupine through the culture of shoot tips. About this same time several people were studying the distribution of virus proteins in the interior of plants. They found that the presence of virus proteins decreased rapidly when going from the adult leaves to the younger growth. The virus in the terminal buds was so feeble that even with very precise tests its presence could not be determined. Analyzing this thought two French Research Scientists, Dr.Georges Morel and Dr. Claude Martin concluded that it might be possible to recover plants from virus infected stock through the technique used by Ball.
For three years they experimented along that line. We can be thankful that one of the plants chosen for the experiment was dahlia. The results of their experiments were published in a paper presented to the Academie des Sciences in November 1952(6) Unfortunately dahlia has never been a truly commercial flower. Drs Morel and Martin were forced to abandon their dahlia research in favor of crops of a commercial nature, such as carnation, asparagus, strawberries, potatoes etc.
In the years since 1952 there has been an increasing interest in Plant Tissue Culture for propagating many types of plants. Meristem Tip Culture has been primarily used to increase stock of desired plants which do not reproduce true through sexual means. Other applications include the genetic improvement of crops, the production of pharmaceutical products, preservation of germ plasm and more recently the recovery of disease free plant stock..
The title of the paper describing the experiments of Drs. Morel and Martin when translated to English was "Cure of Dahlias Affected by a Virus Disease Through Culture of Apical Meristems" In a strict sense the dahlias were not cured but rather virus free dahlia cultivars were recovered from a variety known to be totally infected with one or more virus diseases. They succeeded in recovering a small number of plants of the variety Reve Rose (Pink Dream) from dahlia mosaic. As reported, the plants "developed normally showing none of the symptoms of dahlia mosaic. On two occasions serological tests were carried out on the plants; both tests gave negative results."
In 1955, F. O. Holmes(7) reported on his success in the elimination of Tomato Spotted Wilt virus (TSWV) from dahlas by propagation of small tip cuttings. In 1962. Michael Hollings(8) reported he had successfully recovered dahlias from Cucumber Mosaic Virus (CMV) by growing the plants under heat for four weeks then taking small tip cuttings. There is no known record of the recovery of dahlias from any other type of virus.
It is difficult to determine exactly how many virus diseases affect dahlias. There have been a dozen or more virus diseases reported; Dahlia Mosaic, Yellow Ringspot, Dahlia Oakleaf, Tomato Spotted Wilt, Cucumber Mosaic , Dahlia Stunt Disease, Dahlia Leaf Curl, Dahlia Leaf Roll, and Aster Yellows, to name the most commonly listed ones. Brierley and Smith writing in 1950(9) stated that Tomato spotted Wilt induced most of the ringspot and oakleaf patterns in the United States. Dr. John Grainger(10) attributed Oakleaf to a strain of Cucumber Mosaic virus. Consequently there appears to be only a few different viruses which affect dahlias and manifest themselves differently in different varieties of dahlias.
At the present time most authorities agree that there are three or possibly four distinct virus diseases which affect dahlia. Dr. Lawson(4) identified four viruses which he had isolated from different varieties of dahlias: Dahlia Mosaic virus (DMV), Cucumber Mosaic Virus (CMV) , Tomato Spotted Wilt Virus (TSMV) . His method of identification was to compare virus isolates with type viruses. The virus isolates were obtained from various varieties of dahlia; the type viruses are those which have been positively identified through laboratory procedures and maintained as laboratory stock. Features and reactions of two isolates compared favorably with the same features and reactions of CMV and TSWV. No type viruses for DMV was available so his comparisons were made against descriptions in available literature on the subject. The comparison seemed sufficiently accurate to establish DMV as a third definite pathogen. The fourth pathogen was less definite. He summed it up as "a ringspot virus possessing host range and physical properties similar to those of Tomato Ringspot Virus" Other authorities seem to lean to the conclusion that TRV is a mutant or variant of TSWV.

AUTHOR'S NOTE: ( In the five years preceding 1998 several other viruses have been reported as affecting dahlia. They are not included here as definite viruses since no information on method of determining their presence or identification has been published).

From the results of experiments with these diseases on other plants it appears highly likely that the basic method used by Dr. Morel and Dr. Martin, can be readily used for recovery of plants afflicted by DMV. Furthermore, since it appears from the Holmes and Hollings operations that TSWV and CWV respectively can be overcome by relatively large cuttings, it certainly seems likely that any process which will accomplish recovery of stock from plants infected with DMV will also eliminate the other pathogens, if present. Since Meristem Tip Culture has been used successfully to recover healthy plants from varieties infected with DMV and presumably any other known type of virus present in the specimens used, it appears that MTC offers a definite promise of recovering healthy dahlia plants under any conditions of virus infection.
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