(WWIN!MlIWNHNHNVHIHIIVHIHHHIIWHIHHI THESIS 5’95?” -..._ W 1 fl ‘ ... ._ d i, 113A.“ FRY . Michigan Sue: to, This is to certify that the thesis entitled The Isolation, Fractionation and Uptake of Plant Chromosomes presented by Robert James Griesbach has been accepted towards fulfillment of the requirements for doctoratedegree in Genetics KW /\ Major professor Date ll/l/QO 0-7639 t.» A——— :1 all s1»: ,5~L " ‘OL‘ KIRK» t ' no ._ {V‘s-XVI”? , ; ' Tan—.7“ .! .‘l.. ‘ uvcnwc r mt): 25¢ per day per icon RETURNING LIBRARY MATERIALS: -——-—-——_____—_ Place in book return to remove charge from circulation records THE ISOLATION, FRACTIONATION AND UPTAKE OF PLANT CHROMOSOMES By Robert James Griesbach A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Program in Genetics 1980 ABSTRACT THE ISOLATION, FRACTIONATION AND UPTAKE or PLANT CHROMOSOMES By Robert James Griesbach Plant chromosomes can be efficiently isolated from several different tissues--root tips, microspores and suspen- sion cultures. All these tissues have two characteristics in common. They have a high mitotic index and are easily converted into protoplasts. The procedure for isolating chromosomes involves exposing protoplasts to a buffer which ruptures the cell membrane and inhibits the activity of nucleases and proteases. The procedure is efficient with yields of over 50 percent of the available chromosomes. Sucrose gradients then allow the chromosomes to partially fractionated into several different size classes. Chromosome uptake can be measured by fluorescence microscopy. Isolated chromosomes which are stained with 4'6-diamidino-2-phenthukfle fluoresce green. These chromosomes are easily seen when taken up by mesophyll protoplasts which fluoresce red due to their chlorophyll. About 1 percent uptake is obtained when a chromosome/protoplast suspension is incubated for 20 minutes in 35 percent polyethylene glycol. ACKNOWLEDGMENTS I wish to thank Dr. Peter Carlson for his guidance and for allowing me complete freedom to pursue other areas of my own interest. I am also grateful to Brenda Floyd, Dr. Russell Malmberg and Dr. James Asher. I am indebted to my parents for their support, as well as to my Aunt, Ann Stoegbauer, for her initial help. Finally, I would like to thank Klehm's Nursery for their financial aid. ii TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES LIST OF ABBREVIATIONS INTRODUCTION . MATERIALS Cell Cultures Root Tips Microspores METHODS Chromosome Isolation Chromatin Isolation . Histone Isolation‘. Histone Electrophoresis Chromosome Fractionation rRNA Isolation RNA Iodination DNA Isolation . . . DNA-RNA Hybridization . Chromosome Uptake . RESULTS AND DISCUSSION . Chromosome Isolation Chromosome Fractionation Chromosome Uptake . BIBLIOGRAPHY . iii vi 13 13 13 13 15 15 18 19 19 20 20 21 21 21 22 23 23 38 47 59 Table Table Table Table Table LIST OF TABLES Several of the More Common, Mammalian Chromosome Isolation Buffers Several Methods of Fractionating Isolated, Mammalian Chromosomes Via Differential Sedimentation through Sucrose Gradients List of the Major Isolation Buffers Tried . Chromosome Yields for Various Species Ratio of the Optical Densities at 260nm and 280nm for Isolated Chromosomes . . . . . . . iv 11 16 3O 31 Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure LIST OF FIGURES Procedures for Isolating Chromosomes Isolated, Mitotic Chromosomes Containing Multiple Constric- tions Collection of Isolated, Meiotic Chromosomes Histone Gels Mitotic, Tobacco Chromosomes after Exposure to Various Buffers Differentially Condensed, Mitotic Onion Chromosomes Daylily Chromosome Fraction- ation . Hybridization of DNA from Fractionated Chromosomes with ZSI-RNA Chromosome Uptake . Generalized Procedure for PEG- Mediated Uptake . Conditions for the Optimal Uptake of Chromosomes by Proto- plasts . . . . . . . . . 25 27 29 35 41 43 46 50 52 54 TRIS CAPES MES HEPES EDTA EGTA IAA 2, 4—D DTT sos BIS TEMED 2XSSC DAPI PEG LIST OF ABBREVIATIONS TRIS-(Hydroxymethyl)Aminomethane Cyclohexylaminopropanesulfonic Acid 2(N-Morholino)ethanesulfonic Acid N-Z-Hydroxyethyl piperazine-N-Z ethanesulfonic Acid Ethylenediaminetetraacetic Acid Ethyleneglycol-Bis-(B Aminoethyl Ether) NlNl-Tetraacetic Acid Indoleacetic Acid 2,4-Dichlorophenoxyacetic Acid Dithiothreitol Sodium Dodecyl Sulfate NlNl-Methylene-Bis-Acrylamide NlNlNllNl-Tetramethylethylenediamine 3.0M NaCl + 0.3M Sodium Citrate 4'6'Diamidino-Z-Phenylindole Polyethylene Glycol vi INTRODUCTION The improvement of horticultural and agronomic plants depends upon selecting better producing varieties and also on increasing the amount of genetic variability that is available, so that there is a broad base from which to choose new desired types. Plant breeders have for a long time been improving most of the economic plant species. They have already efficiently utilized a large quantity of the available genetic variability, thus increasing the dif- ficulty in gaining major, genetic improvements in many traits. This situation has caused many breeders to turn to various non-conventional methods of breeding, such as somatic hybridization and in vitrg mutagenesis, in order to obtain new sources of desirable genetic variability. During the last 25 years, the techniques for the gene— tic manipulation of plant somatic cells have been developing. Methods are now available for the production of haploids (Kasha, 1979) and the regeneration of whole plants from a wide variety of tissue cultured cells (Pierik, 1979). Addi- tionally, procedures for the isolation, fusion and regener- ation of whole plants from protoplasts are available for a 'more limited range of species (Butenko, 1979). The ability to regenerate plants from tissue cultured cells or protoplasts is paramount in the application of the newer techniques of genetic engineering to plant breeding, for the breeder generally requires more than one sexual cycle beyond the initial engineering for further selection. During the mid-1970's it was often considered that somatic cell genetics would solve many of the breeder's problems for it could theo- retically allow an unlimited gene pool (Heyn, gt 31., 1974). The use of in vitrg cell selection and somatic hybridization would allow the geneticist an Opportunity to create or intro- duce new sources of variability. This variability could then be funnelled into a classical, practical, plant breeding pro- gram. Some of the problems in applying somatic cell genetics to plant breeding are now being recognized (Carlson, 1980). One of the major problems stems from a lack of well charac- terized genetic systems in which mutants can be rapidly selected. Perhaps the most severe limitation is an insuf- ficient amount of biochemical and genetic characterization of many horticultural and agronomic traits. For example, many economic traits like heterosis and yield are now only abstractly and statistically defined. These traits need to be broken down into their individual biochemical components and genetically analyzed. Without a more precise character- ization these traits cannot be analyzed in 11559. Another problem which is also very serious concerns the tissue speci- ficity of many of the horticultural and agronomic traits. Many of these traits like leaf angle are exclusively expressed at the whole plant level; thus it is impossible to do 13 31333 selection. Ever since the original report of fusing plant proto- plasts to create a somatic cell hybrid (Carlson, 33 31., 1972), there has been a great deal of interest in using this method to increase genetic variability and to create new genetic combinations. It is now theoretically possible to fuse any two somatic cells; they need not belong to the same species, family or kingdom (Butenko, 1979). There are, how- ever, several problems in applying somatic hybridization to plant breeding. The most severe difficulty lies in selecting and regenerating the hybrid cells from the parental cells. Even if one is able to select and regenerate the hybrids, there are several reasons why such plants may not be of much use to the breeder. First, most of the hybrids which have been regenerated from cell fusions can also be produced via sexual means (Vasil, 33 31., 1979). Second, if the parents are only distantly related, then the hybrid progeny will be developmentally unstable. The resulting somatic hybrids will have many morphological abnormalities which will lead to low fertility and lack of vigor (Melchers, 33 31., 1978; Gleba and Hoffman, 1980). Third, whole genomes, instead of individual desirable genes, are transferred. This means that several subsequent, sexual generations are required before the undesirable genes are eliminated from the hybrid; however, in many somatic hybrids advanced sexual generations are impossible. Finally, in many of the wide hybrids the chromosomes of one parental type can be preferentially eliminated (Kao, 1977). The problems inherent in somatic hybridization make DNA- mediated transformation an attractive alternative; however, there has never been reported an example of a stable, long- term, DNA-mediated transformation in higher plants. All the reported data on successful transformation in higher plants are very weak and in some cases the appropriate controls are even absent (Lurquin, 1977; Kleinshoff and Behki, 1977). It is fairly well established that foreign DNA can be taken up by plant cells and even expressed for a very brief period (Lurquin, 1977; Kleinshoff and Behki, 1977). Almost all the DNA, however, is eventually degrated (Slavik and Widholm, 1978). Another problem in DNA-mediated transformation stems from the lack of specific genetic markers and selection systems. This makes it almost impossible to confirm trans- formation, for there are vitually no markers to transform. A new approach, chromosome-mediated transformation, might be able to overcome many of the difficulties associated with applying somatic cell genetics to plant breeding. It offers the geneticist a unique means for introducing into a genome small amounts of foreign information without potentially affecting developmental processes, relative vigor, fertility or gene balance. The first, physiologically active chromo- somes were isolated from cultured mammalian cells (Chorazy, t al., 1963). Since then consider- 3; 31., 1963; Summers, able progress has been made in isolating mammalian chromosanes. Figure 1 outlines the procedure generally used. Cultured cells are first exposed to an agent which holds the cells in mitotic metaphase. Colchicine and its derivatives are the most widely used agents. Once sufficient quantities of mito- tic cells are obtained, the cells are placed in a hypotonic solution and swollen. This process helps disperse the meta- phase chromosomes. The cells are then lysed by adding a suitable buffer and the cellular debris removed via differ- ential centrifugation. Finally, the chromosomes are centri- fuged down. The most critical step in the isolation procedure is the composition of the lysis buffer (Hanson, 1973). Table 1 lists the chemicals found in several of the more successful buffers used in isolating mammalian chromosomes. The selec- tion of a specific buffer depends upon how the chromosomes will be subsequently used after isolation. Certain proper— ties are required of the chromosomes if they are to be used in transformation studies. One, the chromosomes must con- tain high molecular weight DNA which is unnicked or modified. Two, the acidic and basic proteins must be unmodified and correctly associated with the DNA. Finally, the number of contaminating interphase nuclei and chromatin should be kept to a minimum. The method of cell lysis affects chromosome morphology. For example, prolonged incubation in hypotonic solutions prior to lysis or lysis in hypotonic solutions can lead to irreversible chromosome expansion or disintegration mmEomoBouno fiBoo Gama mflunoo fiBov mama mammoa owauopoazs m stounu mmmm Hmmmdn mwmxa mum qutfio muwmamuoua Sowz mam uooaaoo moamuco o>HumowHo .Ham3 HHoo mam mcwowSUHou ou osmmwu omomxo .H Aowaa isomnmamou a wnmneflmzv mHHou “swam moEOmoEouno GBOU swam .o mwhnov CBOu swam .m oaoooa UHEuouomms m amnounu mHHoo mama .o hummus mnmsa cam .m mHHoo onu HHoBm kHHmUHCOuomhn .N wHEooHoo ou mHHoo ponduadouoSmmHu omoaxo .H Amkaa .comcmmw,waaoo cmaflmeeaz .< moEOmoEousu wawumaomH pom mousvoooum .H madman Table 1. Several of the More Common, Mammalian Chromosome Isolation Buffers, Sommers, 33 31., 1963 0.5mM MgCl2 0.5mM CaCl2 0.5M sucrose unbuffered Mendelsohn, 33 31., 1968 1mM MgCl2 1mM CaCl2 0.1M sucrose 0.1M sodium acetate pH 3.0 Blumenthal, st el-. Maio & Schildkraut, 1967 1mM CaCl2 1mM MgCl2 1mM ZnCl2 0.02M Tris (pH 7.0) 1% Triton X-100 Stubblefield, 33 31., 1978 1mM CaCl2 1M hexylene glycol 1mM CAPS or HEPES pH 10.5 or 6.5 1979 15mM Tris (pH 7.2) 0.2mM spermine 0.5mM spermidine 2mM EDTA 0.5mM EGTA 14mM mercaptoethanol 0.5M hexylene glycol 0.34M sucrose 0.1% digitonin 80mM KCl 20mM NaCl (Mendelsohn, 33 31., 1968; Bak and Zeuthen, 1977). The use of non-ionic detergents should also be avoided; because they can cause nuclei to rupture, as well as, increase the chromo- somes' sensitivity to shearing forces (Blumenthal, 33 31., 1979). In lysing the cells, the use of mechanical means, such as passage through a hypodermic needle, prove to be the least disruptive of chromosome morphology (Hanson, 1973). Chromosomes with a high molecular weight DNA component (6.5 x 107 daltons) were isolated by Wray (Wray, 1973). He found that a basic lysis buffer (pH around 10) could reduce most of the nuclease activity without some of the harmful side-effects found when using low pH or metal chelators to reduce the enzyme activity. Low pH can reduce nuclease degra- dation; however, it can also lead to depurination of the DNA, as well as, remove histones and other basic, chromosomal pro- teins (Lewin, 1980). A high pH may prevent DNA degradation; however, it does not preserve the protein integrity of the chromosome. Up to 25 percent of the acidic chromosomal proteins can be removed by high pH (Hearst and Botchan, 1970). Thus, basic and acidic pHs can preserve the integrity of the DNA; but they destroy the chromosomal protein composition. As a consequence, chromosomes must be isolated at near physi- ological pH if one wishes to preserve the protein composition. It is possible at neutral pH to reduce the degradative effects nucleases have on DNA. For example, DNA which is bound to protein is protected from nuclease attack. The greater the quantity of protein bound, the less likely nucleases can bind and digest the DNA (Lewin, 1980). By adding protein stabilizers, such as DTT and hexylene glycol, it is possible to help preserve the protein composition of the chromosomes (Wray, 1973). Metal chelators like EDTA can also restrict nuclease activity by binding Mg++ and Mn++ cations. These cations are the cofactors which are needed for nuclease activity (Lewin, 1980). Agents like colchicine, cold temperature, high ionic strength and polyamines can also reduce nuclease binding by helping to condense the chro- mosomes (Lewin, 1980). Chromosomes with highest molecular weight DNA (2 x 108 daltons) were isolated from neutral buf- fers containing polyamines, chelators and protein stabilizers (Blumenthal, 33 31., 1979). Isolated, mammalian chromosomes have been fractionated based upon their mass (Huberman and Attardi, 1967), size (Hanson, 1973), density (Stubblefield and Wray, 1973) and electrical charge (Landel, 33 31., 1972). In order to sepa- rate or fractionate chromosomes based upon their charge or density, the protein/DNA composition of the chromosomes must be selectively modified prior to fractionation. For example, unmodified HeLa cell chromosomes all band at the same den- sity of 1.31 g/ml (Huberman and Attardi, 1967) and all have the same pK of about 4.0 (Landel, 33 31., 1972). If, how- ever, the chromosomes are treated with trypsin before frac- tionation, it is possible to selectively change the density and the electrical charge of the individual chromosomes (Stubblefield and Wray, 1973). One very serious problem 10 with these methods of fractionation is that they permenantly alter the composition of the chromosomes. Although separa- tion based upon size via selective filtration does not damage the chromosomes' composition, it does have severe limita- tions. The major problem lies in the limited availability of filters which do not absorb chromosomes and which have a suf- ficient range of uniform pore sizes. As a result, selective filtration is only of use in very crude separations or in selecting chromosomes of unusually large or small size. The most widely used technique for fractionating unmodified chromosomes is based upon their density or differential sedi- mentation through sucrose gradients. In this method, an unfractionated chromosome mixture is layered onto a preformed sucrose gradient and centrifuged for a brief period of time. The exact centrifugal force and sucrose concentrations depend upon the species (Table 2). It has only been very recent that studies in isolating higher plant chromosomes have begun. Plant chromosomes have now been isolated from a mixture of tissues from various species (Malmberg and Griesbach, 1980). Figure 1 shows the procedure used to isolate plant chromosomes. From this pre- liminary study several things were noted. First, the use of techniques developed to separate mammalian chromosomes will not work with plant tissues since plant chromosomes respond differently to these extraction procedures. Second, in order to isolate chromosomes from a given tissue, protoplasts with a fairly high mitotic index ( ou ousmomxm Moumm mmEOmoEouno ooomnoe owuouflz .m muswfim 35 36 it to supercoil. Likewise, the KCl and NaCl are needed to ob- tain the correct ionic strength which maintains chromosome con- densation. DTT is added to preserve protein structure, as well as, help reduce nuclease and protease activity. Hexylene glycol, besides rupturing membranes and disaggregating chromo- somes, also helps maintain protein structure. The sucrose is added to keep the interphase nuclei intact. Tris is needed to maintain the pH at near physiological conditions. One unique aspect of isolating mitotic chromosomes is seen in Figure 6. It appears that isolated chromosomes can become quite sticky. In this case, the chromosomes have become associated at both the primary and secondary constrictions. This secondary association is quite strong, for the physical strength needed to break the association shears the chromosomes at their centromere or primary constriction. In some prepara- tions up to 10 percent of the chromosomes can be linked in such a way. Unlike mammalian systems, meiotic chromosomes can be iso- lated from higher plants (Figure 3). It is even possible to isolate quadrivalents in which the nucleolus is still attached to the nucleolar organizer (Figure 3 I). Once again, the yield depends upon several factors. The most important factor is the stage of meiosis. From prOphase I to anaphase II, microspores were easily converted into protoplasts. After anaphase II, it was not possible tx>convert the tetrads into protoplasts. This was probably due to the build up of callose and the beginning of the exine formation. These compounds 37 have 0 1—3 linkages instead of the p 1-4 linkages that the standard cell wall digestive enzymes degrade. Eventhough protoplasts can be isolated from.most stages of meiosis, some stages produce more fragile protoplasts. For example, pro- phase I and metaphase I protoplasts are extremely weak and can easily be ruptured in the centrifugation process. Extreme care must be taken when handling these cells. The other stages produce protoplasts which are less fragile but not as resilient as somatically derived protoplasts. Meiotic chromosomes are also much more fragile than their mitotic counterparts. A quick passage through a hypodermic needle can completely destroy all but the most highly condensed, metaphase I univalents. In terms of the general biochemical and structural analy- sis of chromosomes, plant systems potentially offer more than mammalian systems, for meiotic chromosomes have so far only been isolated from plants. Much information can be gained from a comparison of meiotic and mitotic chromosomes. For example, the way in which histones bind to meiotic chromo- somes seems to be different than the way they bind to mitotic chromosomes. In chromatin, all 4 histones are non-covalently linked together into 2 tetramere- (H2a-H2b)2 and (H3-H4)2 (Lewin, 1980). These tetrameres can be disassociated by heating at 100°C in the presence of SDS. A two minute incu- bation is sufficient to break the mitotic, histone complex (Figurefi ); while a longer exposure is required to disassoci- ate the meiotic complex. Another minute of heating is needed 38 before the meiotic histone complex separates. This suggests that in meiotic chromosomes the histones are somehow more tightly attached to each other. There are many other struc- tural and biochemical differences between meiotic and mitotic chromosomes which can also be studied at the isolated chromo- some level. For example, one very important question concerns the difference between meiotic and mitotic chromosome conden— sation. Meiotic metaphase I chromosomes are generally about ten times more compacted than mitotic metaphase chromosomes. Additionally, meiotic metaphase I chromosomes have a banded appearance and lack primary and secondary constrictions. Besides comparing the two types of chromosomes, meiotic chromosomes in themself have a great potential for research. Using isolated chromosomes, it will be much easier to study the underlying biochemistry of meiosis. It will also be simplier to look at synapsis formation and crossing over at a 13 vitro level, rather than at the typical 13 vivo level. Chromosome Fractionation The techniques for chromosome fractionation are based upon differential sedimentation through sucrose gradients. The more uniformly condensed the chromosomes, the more effi- cient is fractionation. One problem in isolating uniformly condensed chromosmes is in the colchicine pretreatment. Excessive colchicine is required to initially condense the chromosomes, as well as, increase the mitotic index. The colchicine also helps disrupt the spindle, thereby separating 39 the chromosomes. The long colchicine treatment, however, has one serious drawback, differential condensation. The cells in which mitosis began at the start of the colchicine treatment will contain chromosomes which have been exposed to the chemical for 18 hours. These chromosomes will be highly condensed at the end of the incubation. Other cells which start mitosis at the end of the colchicine treatment will contain chromosomes which have only been exposed to the chemical for a short period of time and will be less con- densed. An example will illustrate this phenomenon. 13 3133, onion chromosomes are all about the same size. Figure 6 shows several isolated onion chromosomes in which there is up to a ten fold difference in chromosome condensation. This causes some very serious problems in chromosome fractionation, for a given chromosome will be present in varying degrees of condensation in an unfractionated chromosome mixture. When this mixture is sedimented through sucrose, fractionation does occur (Figure 7); however, the gradient besides separat- ing given chromosome types also separates the different degrees of condensation within a given type. If chromosome fractionation is to have any practical significance, the influence of differential condensation must be less than the influence due to true chromosomal size dif- ferences. An experiment can test this difference. An ideal marker is the chromosomal region called the nucleolar organ- izer. In daylilies it is a secondary constriction located on only one chromosome type. In this region, the genes for the 40 oom.NH mamdom moEOmoEouso Ham How cowumowMchmE m£H ”ouoz mcowuwmoa uaouommao um oSOmoEouno oEmm onu .m a .m mcowufimoa uGouommap um oEomoEouso mEMm oSD .Q d .0 mcowuflmoa uCoummmap um oEomoBouso oEwm onu .m w .< ”moEOmoEouzu cowco .owuouflz .momaopcoo maamwucmHoMMflm .c ouswwm 42 mcowuowum monnu cw oEoonOHfio memos onu .m a .Q ..o x comm um muouxwa moumcowuomumca .m x com um ououxwe moDNCOfluomumcS .< ”coaumCOfluomHm oEOmoEouso maflaxma .m ohdwfim 43 44 258, 183 and 5.88 rRNA are located. These rRNAs were ex- tracted from ribosomes and radioiodinated to 100,000 dmp/ug. Likewise, DNA from each chromosome fraction was isolated, denatured and bound to nitrocellulose filters. The radio- active RNA was then used as a probe to determine which frac- tion or fractions contained the chromosomes with nucleolar organizer. The data is presented in Figure 8. Several things can be observed. First, the rRNA cistrons appeared in all but two of the seven fractions which contained chro- mosomes. The first and last two fractions did not contain any chromosomes. Second, the rRNA cistrons seemed to be more prevalent on the medium to large chromosomes. This is in agreement with the 13 3133 cytogenetic evidence. This experiment suggests that it is not possible to completely fractionate higher plant chromosomes using buffer #13 and the above chromosome isolation procedure. Chromosomes can efficiently be fractionated in size, but the resulting 13 31333 size does not completely reflect the 13 3133 size because of a lack of uniform condensation during the isolation procedure. Some method needs to be developed which either condenses the chromosomes more uniformly or synchronizes the cells more efficiently. At- tempts to modify the buffers after isolation either have no effect or lead to complete decondensation. Another method, besides a long colchicine pretreatment, is needed to increase the mitotic index and initially condense the chromosomes. A similar problem, although not quite as 45 cowumuucoocoo 0mm .0 mxmums unmouoa .m> 0mm cw cowumosocw mo mafia .m oxmum: ucooHom .m> Gowumuucoocoo oEOmOEouso .< ”mummamououm mp moEOmoEoun0 mo oxmuaz Hmeumo How mcowufimco0 .HH ouswam 3.0 :E\ 0 .U 2 O U AEEV .0200 ON n. mirr mzowczOmIU Ow 0m Om. mm [mN ON 0.. Aw. km: 00. 54 BMVld n % BNVldfl 96 — _L - .v 3xvran % 55 seen that the highest uptake was obtained after a 20 minute incubation in PEG. The uptake was still higher after a longer exposure; however, the morphology of the cells deteriorated considerably. The last variable was the concentration of PEG. The maximum uptake appeared after an exposure to 35 percent PEG (Figure 11 C). The optimal conditions for the uptake of lily chromosomes by tobacco mesophyll protoplasts are an incubation for 20 minutes in 35 percent PEG at a chromosome- to-protoplast ratio of 10:1. A tobacco protoplast which has taken up a large lily chromosome has not been seen to divide; even though PEG- trated control protoplasts which have not incorporated a chromosome divide. This does not seem unreasonable, for the foreign chromosome is almost as large as the entire host nucleus. A more physiological situation would have been to incorporate smaller chromosomes into tobacco protoplasts; however with the DAPI-chlorophyll system it is below the limits of resolution to see small green chromosomes in large red protoplasts. Although the large chromosomes when incor- porated represent an unphysiological situation, this system does give one a rough idea as to the optimal conditions needed for a successful transformation. We now have a system for isolating chromosomes from almost any higher plant species, as well as, a procedure for introducing them into protoplasts. The next step will be to attempt transformation. In mammalian cell culture there are numerous examples of successful chromosomedmediated 56 transformation (Willecke, 1978; Shows and Sakaguchi, 1980). In some instances large foreign chromosome fragments (about 1 percent of the genome) can become stablily integrated into the host genome. The frequency of such an event is about 6 50 percent of all the transformants or between 5 x 10- and 5 x 10-8. If all the transformants, both the stable and unstable, are cultured under selective conditions for a pro- longed period of time, up to 95 percent of the foreign chro- mosome fragments can become stablily integrated into the host genome (Klobutcher and Ruddle, 1978). Whole chromosomes, instead of fragments, can be transferred when liposomes or artificially created lipid vesicles are used (Mukherjee, 33 31., 1978). When the vesicles are made in a chromosome suspension, chromosomes are entrapped as the vesicles form. These vesicles are then fused with recipient cells in a manner similar to somatic hybridization. Besides increasing the size of the introduced genetic material, liposomes also allow a higher frequency of transformation. With cultured mammal- ian cells a frequency of l x 10's, as compared with l x 10-7 for non-liposome mediated gene transfer, can be obtained (Mujkerjee, 33 31., 1978). It is also possible to transfer unselected genes (Wigler, 33 31., 1979). If mammalian cells are exposed to a mixture of two types of DNA, about 95 per- cent of the transformants in which one of the DNAs was sel- ected for will contain the other DNA. A similar system also operates in chromosome-mediated transformation (Klobutcher and Ruddle, 1978). 57 Although chromosome-mediated genetic transformation has not yet been attempted in plant systems, I fully expect the phenomenon to occur. One problem in looking for trans- formation is the lack of suitable genetic markers; however, there might be a marker. In Agrobacterium-transformed strains of tobacco, tissue-cultured cells can be produced which synthesize the unusual amino acid derivatives octopine and nopaline and which do not require an exogenous source of hormones for growth. In such transformed cells part of the bacteria's tumor inducing plasmid is integrated into the tobacco genome (Chilton, 33 31., 1980). This system could be used to study chromosome-mediated transformation. For example, chromosomes isolated from.a Agrobacterium-transformed strain of tobacco could be introduced into non-3grobacterium- transformed tobacco cells. Chromosome-transformed cells would be expected to synthesize n0paline or octopine and be able to grow in the absence of hormones. Chromosome-mediated transformation should alleviate many of the problems plant breeders will face in the future, for it allows the breeder to overcome many of the difficulties of developmental incompatibility and extensive introgression faced when making either somatic or sexual wide hybridiza- tions. Before chromosome-mediated transformation can have an impact upon plant breeding several things are needed. First, one needs to be able to regenerate whole plants from single protoplasts. It is still not possible to produce mature plants from.the protoplasts of many of the most economically important horticultural and agronomic species. Second, there 58 needs to be a more indepth, biochemical analysis of all the horticultural and agronomic traits. The molecular geneti- cist cannot select mutants 13 31333 if the desirable charac- teristics are only defined at a whole plant level. Finally, there needs to be a serious attempt at finding new agronomic and horticultural characteristics which can be expressed 13 31333, for many of the important traits are tissue speci- fic and are not expressed at the tissue culture level. Let us hope that chromosome-mediated genetic transformation will be of more use than the current, molecular genetic technology in future plant breeding. BIBLIOGRAPHY Bak & Zeuthen, 1977. High order structure of metaphase chromosomes. Cold Spring Harbor Symposium 42:367. Barz, Reinholt & Zenk, ed., 1977. Plant Tissue Culture & Its Bio-Technical Application. Springer-verlag, Gérmany. Blumenthal, Dieden, Kapp & Sedat, 1979. Rapid isolation of metaphase chromosomes containing high molecular weight DNA. J.Cell Biology 81:255. Briggs & Knowles, 1967. Introduction to Plant Breeding. Reinholt Press, New York. 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