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MERISTEM TIP CULTURE

MERISTEM TIP CULTURE
by Gerald G. Weland

In the summer of 1974 the author conducted a program of Meristem Tip Culture for the purpose of developing a procedure to recover healthy stock from virus infected dahlias in a home environment,. A second similar program was conducted during the summer of 1975 using the same procedure. This article is to document the procedure developed during the conduct of those programs and supply it to the general dahlia public. A step-by-step procedure has been written in report form and made available to several persons who have expressed an interest in the program and the resulting procedure. That detailed procedure is still available to anyone desiring a copy. The following article, although complete in operational details, is hereby edited and condensed for this publication. The original article was first published in the Dahlia Reporter in the fall 1975 edition.

FOREWORD

Sometime in the mid 1960s the author attended a seminar conducted by Dr. Georges Morel at which he reported on a program of plant tissue culture that he and his partner Dr. Claude Martin had conducted. He reported that their experiments were connected with a program of Meristem Tip Culture of several types of plants including dahlias. For the next several years the author began reading, researching and gathering information about the subject. During the summer of 1974 he was fortunate to be able to attend a workshop program in plant tissue culture at the University of California at Riverside. This article summarizes that experience, the results of two small programs of Meristem Tip Culture which was a fallout of that course, and some conclusions about the future of this process in its application to dahlias.

INTRODUCTION

There are many branches of plant tissue culture. In the workshop course at the University of California. Each student is encouraged to follow their particular branch of interest as well as to work with plant varieties of their choice. The authors choices were to work with dahlias in using the culture process which utilizes the meristem tips of axial shoots. The reason for these choices was primarily to attempt to recover healthy stock from dahlia plants infected with virus.
In 1952 Doctors George Morel and Claude Martin of France showed that certain plants no matter how badly affected with virus diseases, could be recovered and again propagated in a healthy state. Their experiments showed that virus is not equally distributed into all parts of a growing plant, and that the rapidly growing shoot tips have a very good chance of being free of virus pathogens. Fortunately, some of those early experiments were with dahlia. Unfortunately, due to lack of a substantial commercial market for dahlias they were forced to give up that program in favor of crops with a larger commercial demand. There has since been a few small programs with dahlias but none sufficient to recover any large amount of plants or to refine to any large extent, the techniques proposed by Morel and Martin. The first dahlia recovered was Reve Rose (Pink Dream) and altogether they succeeded in recovering a total of 15 varieties. The system used for recovering healthy plants from virus infected stock was that of Meristem Tip culture (MTC). By this process a very tiny piece of growing plant issue is removed under extremely sanitary conditons, placed in a culture tube on a nutrient medium and grown under artificial conditions until large enough and mature enough to be grown satisfactorily in the open. Meristem Tip Culture takes advantage of one of the natural developments of plant growth. There are three basic ways in which plants grow; cell division, in which cells are formed; enlargement where existing cells increase in size; and cell differentiation where cells take on the shape and orientation which determines the form and structure of the plant organs. Meristem Tip Culture is concerned with the first of these, i.e. cell division.
In many areas of a plant there are small regions where cell division is nuch more rapid than in others. These areas of rapid cell division are referred to as meristematic regions or simply meristems. Any of the several meristems could be used for culturing but the most practical for dahlias seems to be the one near the tip of the growing shoots. This meristem is not extreme tip of the growing shoot but is located a few rows of cells behind or back of the tip. The meristem is exposed by removing as many leaves as practicable from the growing shoot. The growing tip then appears rounded, shiny and semi-transparent. When the visible leaves have been removed and the shoot tip placed under a fairly strong microscope several protruberances may be seen about the rounded tip. These are leaves in the process of being formed (an example of cell differentiation) and are called primordial leaves. As many of these are removed as possible without damaging the rounded shoot tip. [IMAGE] This tip, very tiny (smaller than a period) , is then cut from the shoot and placed in a culture tube containing the nutrient solution. The culture tubes are then capped and placed in an environment with controlled heat and artificial light to facilitate their development into a plant. All this must be done under extremely sanitary conditions to prevent contamination of the cuttings or tube by fungus spores.
As can be noted from this description the cutting used for propagation (called a propagule) includes not only the meristem but also the few rows of cells between it and the tip, some small amount of tissue below or sub-jacent to the meristem and probably the first two primordial leaves. For this reason the process is properly know as Meristem Tip Culture and not simply Meristem Culture as so commonly used in the past. The amount of sub-jacent tissue used , and the number of primordial leaves retained are a matter of compromise. The smaller the amount of tissue used the better the chance of obtaining a virus-free propagule. On the other hand, the smaller the propagule, the less likely to survive in culture. .
Although not a recent development, MTC is only recently becoming popular with plant breeders and propagators. There are several uses for MTC: genetic improvement of crops; production of pharmaceutical products; preservation of germ plasm; the rapid increase of such flower crop as Carnations, Gerberas, Orchids, Poinsettias, etc; and most recently as a means of recovery of healthy stock from plants infected with virus.
Most dahlia growers are aware that many of the older varities (and newer varieties too for that matter) are quite thoroughly infected with various virus diseases . A method of propagating which would assure that a dahia root or plant for which the growers pay their hard-earned money, was free of those debilitating viruses should be a boon to dahlia circles. This is the promise of MTC and seems definitely within reach.
The basic technique for recovering virus free stock has been developed. It has been applied successfully to many varieties of commercial crops such as potatoes, asparagus, strawberries and tobacco, to name a few. There have been very few programs or efforts designed especially for application to dahlias although it was one of the very first plants to be recovered through MTC..
Dr. Morel and Dr.Martin experienced and reported difficulties in working with dahlias. One such difficulty is that the propagules failed to make roots in the culture tubes. This necessitated grafting the propagules onto a young seedling plant. Since the propagules grown in culture are quite etiolated (spindly) they are generally very delicate, a high mortality rate was experienced. Several hundred cultures were required to get even a very few plants. Additional research and experimentation will be required to perfect the grafting procedure. Recent developments in plant tissue culture techniques has led to enhanced root formation on plants other than dahlias. Through use of those techniques the author has been able to establish a procedure leading to a substantial increase in root formation in his cultures. That procedure will be detailed in the following procedure.
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