PLANT CHIMERAS IN TISSUE CULTURE: A REVIEW
Mary F. Pogany and R. Daniel Lineberger
List of References Cited
IntroductionTissue culture has become an increasingly important propagation tool during the past 15 years. However, observations have been made in research studies and during commercial practice of micropropagated plantlets which differ from the original parent phenotype (see Table 1). Variation in propagules has a major impact on the commercial application of in vitro technologies. It is not clear in some systems whether multiple shoots arise via axillary buds or adventitious buds. When micropropagating chimeral plants, this difference in bud origin can be ascertained by the appearance of adventitiously formed variant shoots. In addition, it is possible to study the number of cells or cell layers involved in the formation of adventitious shoots in vitro based on the resultant plantlet phenotype. The intent of this review is to present a discussion of the literature concerning in vitro culture of plant chimeras and to examine the role that these studies play in advancing the understanding of the ontogeny of shoot meristems in vitro.
Apical OrganizationThe apical organization of most dicots follows the tunica-corpus pattern described by Schmidt (96). According to this conceept , the meristematic region above the youngest leaf primordium is organized into two zones of cells that differ in the plane of cell division occurring within them. The outer zone may have one or several tunica layers in which cell division occurs in an anticlinal orientation. The forming cell plate is oriented perpendicular to the meristem surface and the integrity of each of the tunica layers is maintained. The inner zone, or corpus, is not layered as is the tunica, since the initials divide in both anticlinal and periclinal planes (Figure 1). The tunica may vary from one to several layers depending on species ( ). The initials in the tunica contribute derivative cells to surface growth of the shoot, while the corpus initials contribute derivatives to volume growth of the shoot. The genotypes within the layers, or histogens, are usually stable. However, the genotypic organizational pattern may change due to occassional periclinal divisions within the tunica. The L. I genotype may displace into L. II, L. II may displace into L. I, and so on (Stewart). The location within the meristem at which the displacement event occurs determines the extent of the phenotypic change induced. If displacement occurs near the apical dome, the change may be incorporated into the subsequent flow of cells resulting from division of the apical initials, and the entire phenotype may change. However, if displacement or rearrangement occurs at the flanks ofthe meristem where the rate of cell division has slowed, then the phenotype may change in only one sector of the shoot, or leaf, or area of one leaf. The same secnario could be described for the differentiation of adventitious shoot meristems. The meristem of a shoot may include all, some, or none of the cells of either of the component gentypes of a chimera. Such conditions can produce periclinal, mericlinal, or sectorial chimeras in the adventitious shoots; alternatively shoots which are not chimeral may arise from either of the acomponent genotypes. Herein lies of the power of chimeral analysis; for detectable chimeras, it is a tool to study the site of shoot histogenesis.
Types of Chimeras, Layer TerminologyChimeral plants may originate by grafting, spontaneous mutation, induced mutation, sorting-out from variegated seedlings, mixed callus cultures, or protoplast fusion (112). One of the earliest described cases of a graft chimera was the 'Bizzaria' orange, which arose after a scion of sour orange had been grafted onto a seedling of citron late in the 17th century (112). The vast majority of variegated-leaf chimeras have arisen by spontaneous nuclear or plastid mutation (56). Colchicine has been widely used to induce cytochimeras of fruiting plants (32). Structural classification of chimeras includes involves periclinal, mericlinal and sectorial chimeras. Periclinal describes the stable, "hand-in-glove" arrangement of the tunica-corpus region; mericlinal, describes a type of periclinal where only part of a layer is mutant, and sectorial, describes a form where a solid sector through all apical layers is mutant. The conventional method of describing the genotypes of the tunica and corpus regions is the use of the abbreviations L.I, L.II, and L.III which represent the outermost layer, the next tunica layer in, and the corpus, respectively (95) (Figure 1). A plant chimeral for ploidy level, or a cytochimera, with a diploid L.I, tetraploid L.II, and tetraploid L.III would be 2-4-4. A variegated chimeral plant possessing a mutant chlorophyll deficient (albino) outer tunica layer overlying normal inner tissue would be labeled a WGG chimera (W indicating white, or albino, tissue; G indicating green tissue); while a plant with the outer layer normal, the next layer in mutant, and the inner corpus normal, would be designated GWG, and so on. Such designations are, in the case of chlorophyll chimeras, generally based on the appearance of leaves and other organs produced by derivatives of the apical meristem, and thus may not refer to precise meristem cell layers, since chlorophyll is not synthesized and therefore is not detectable in the tunica and corpus cells of the meristem itself (32).
Development of the Plant"The fact that branch apices on periclinal chimeras maintain the hierarchy of apical layers of the terminal apical meristem means that derivatives of the apical layers have maintained their position down through the region of leaf initiation" (105) (Figure 1). The significance of this phenomenon is that certain visible chimeral traits, such as ploidy changes, mutant plastids, and "thornless" cells, can very effectively act as developmental markers with which to follow the trail of cell derivatives throughout the development of the primary body of the plant. Studies of cytochimeras have been very useful in showing the ontogenitic origin of various tissues and organs of the primary plant body arise. These studies have been reviewed in detail by Dermen (32) and Tilney-Bassett (112), and the present discussion does not attempt to repeat that discusssion. Although some species-dependent variations are observed, several generalities can be drawn from the study of these cytochimeras regarding the ontogeny of leaves, stems, floral organs, and roots. The L.I layer of the dicot tunica typically produces only the epidermis. The L.II derivatives produce the gametes, and to contribute to the formation of floral organs. Leaves may receive variable contri- butions from L.II either alone, or together with L.III. Stem and root tissues arise endogenously from L.III.
Effect of Shoot Origin on Propagation of ChimerasApical buds give rise to axillary buds in such a fashion that all three histogen layers are maintained (Figure 1). Adventitious buds differ in that they originate, by definition, in any tissue other than a previously organized meristem (37). Thus, it is highly important in maintaining a periclinal chimeral plant through vegetative propagation to use techniques involving axillary buds (32, 47). Production of adventitious shoots by conventional propagation methods has long been known to result in separation of the component genotypes of a chimera. Bateson (8) stated: "Whenever therefore plants grown from root-cuttings differ from those grown from stem-cuttings, we may infer that the plant is a periclinal chimera" (8). Among other evidence Bateson cited several cases where plants resulting from forcing shoots from root cuttings of Bouvardia and Regal geraniums were nonchimeral derivatives of the corpus genotype (L.III) of the original chimera with respect to doubleness or singleness of flowers or flower color. After removing all the axillary buds from one-year-old trees of cytochimeral 2-4-4 Malus 'Kimball Giant McIntosh' adventitious bud growth was forced from internodal regions producing some diploid shoots and one tetraploid shoot (31). Based on this evidence, Stewart recharacterized these chimeras as 2-4-2, stating that the trees "could not have been 2-4-4 chimeras since only homogeneously tetraploid shoots can develop endogenously from this type". Thus, it is seen that the forcing of endogenous buds from stem internodes also yields information about the identity of L.III, as does obtaining shoots from roots. Considerable heterogeneity was observed in fruiting trees produced by forcing adventitious shoots from disbudded trees of several Malus cultivars (29). Both 'Richared' and 'Bridgham Red Delicious' appeared to revert to the original 'Delicious', with respect to fruit pigmentation, suggesting that L.I mutations had originally given rise to these cultivars. Most of the 'Redspur' adventitious trees were very similar to the source variety, except for three trees, of which two produced darker red pigmented fruit and one was extremely dwarf. A 'McIntosh' striped sport propagated by adventitious shoots resulted in trees yielding fruits with an entirely blushed color, indicating an L.I mutation had likely given rise to the striped sport. Although the strains of 'Delicious' were easily induced to form adventitious shoots, three years of effort with 'Golden Delicious' produced no buds. The ease with which 'Delicious' produces adventitious buds, particularly in response to heavy pruning, was given as one possible explanation for many of the large number of extant 'Delicious' sports. Removal of all axillary buds of 16 Chrysanthemum 'Indianapolis' cultivars, followed by study of the resulting adventitiously produced shoots revealed that twelve of the cultivars were periclinal chimeras; additionally, a number of these shoots arose from at least two different histogen layers in that they also were periclinal chimeras (106). While adventitious buds often originate from a single cell, or from a single cell layer (explaining their nonchimeral nature) (6, 16, 103), in this work, Stewart and Dermen (106) obtained 27 of 80 adventitious shoots that were still chimeral, stating "the swelling, within which all the adventitious shoots were organized, was formed by divisions of cells derived from all three apical layers". When immature leaves from plastid chimeras or cytochimeras of tobacco were rooted by conventional methods, only nonchimeral plants were produced. Adventitious shoot bud formation occurs in this case from derivatives of either "a single cell or a small focus of cells of the cortical parenchyma (L-II or L-III)" (18). The nonchimeral nature of all the plants produced indicated that derivatives of only a single apical cell layer were involved in the formation of adventitious shoots. The forcing of adventitious buds from eye-excised tubers has been used as a method for characterizing the constitution of L.III in potato chimeras (48). Pinwheel flowering chimeras of Saintpaulia are known to produce almost exclusively nonchimeral off-type progeny from adventitious shoots produced on leaf cuttings (39). Extensive tissue culture studies have been done on pinwheel flowering cultivars of Saintpaulia.
In Vitro Manipulations of Chimeral PlantsTissue culturists have known of the propensity of periclinal chimeras to segregate in vitro for some time( 43). Micropropagators are interested in avoiding chimeral segregation so as to maintain true-to-type progeny. Plant breeders view chimeral segregation as a useful way to obtain novel genetic rearrangements (88). Many chimeras show a marked tend to separate or rearrange in vitro (Table 1). A number of different chimeral rearrangements may be obtained from a single cultivar. These chimeral rearrangements facilitate ontogenetic studies and may themselves be useful as new clones A somewhat smaller body of literature exists on the purposeful attempts to synthesize chimeras using in vitro techniques.
Separation of Chimeras, Variants, Rearrangements: A Case Study Approach
Begonia.Cultured leaf pieces and flower peduncles of two Begonia x hiemalis cultivars produced plantlets via direct shoot formation. 'Aphrodite Pink' expressed little variation but 'Schwabenland Red' expressed nearly 45% phenotypic variants after three cycles of propagation (114). A higher percentage of these variants occurred when smaller shoots were selected for propagation suggesting the possibility that such shoots may have been adventitious segregants. Bigot (11), however, did not obtain variation during in vitro culture of 'Rieger' or 'Schwabenland'.
Chrysanthemum.One of the early works on tissue culture of a periclinal chimera was that of Bush et al. (19), who worked with Chrysanthemum morifolium 'Indianapolis'. After culturing petal segment, petal epidermis, and shoot tip explants, Bush et al. found much more variation in the petal segment and epidermal cultures than in the shoot tip cultures. They suggested that L.I had displaced L.II in approximately two-thirds of the shoot tip-derived plants and in all of the plants obtained from a callus culture, as shown by the presence of carotenoids in petal mesophyll of the regenerated plants (compared to the source variety which has anthocyanins and carotenoids in L.I, but a non-pigmented L.II). Paramutation, true mutation, and environmental effects were cited as additional possible reasons for the observed variation, but Bush et al. also stated that "there is almost certainly a rearrangement of chimeral layers, which may involve differences in genes other than those for color". A further note on the Chrysanthemum study of Bush et al. (19) is that they presented data on a very limited number of original explants; one basal petal segment yielding 102 plants, one petal segment yielding 114 plants, and the shoot tip explant numbers were unspecified. In cell suspension culture of C. morifolium 'Indianapolis Pink', 37% (93/249) of the regenerated plants were variant (91). As an explanation for the 63% pink regenerants, these authors suggested that a possible six genotypes could all result in a phenotypically identical appearance. It was further suggested that adventitious buds in this system could be of multicellular origin. Cassells and Kelleher (22) regenerated plants from C. morifolium flower petals and postulated that adventitious buds were initiated in L.II from a multicellular origin. Nine years after being placed into culture, regenerants from leaf callus of 'Indianapolis White Giant No. 4' were observed to express various abnormalities including aberrant form, apical bud proliferation, variable leaf shape, and stunted growth (109). Phenotypic variation observed in plantlets of C. morifolium differed depending upon explant source, with shoot tips being most stable, capitulum explants next, and stem segments least stable (75).
Dianthus.An early observation of in vitro chimeral separation was made on cultures of Dianthus caryophyllus 'William Sim' from shoot apices (43). No data were presented regarding the frequency of off-types, merely a mention that the cultivar "sometimes reverts" to the inner layer genotype. Further work on both chimeral and nonchimeral cultivars of D. caryophyllus resulted in chimeral separation when meri- stem and macerated shoot tips were cultured (51). In contrast to the observations of Hackett and Anderson (43), adventitious shoots appeared to originate from L.I in this case.
Nicotiana. Variegated chimeras of Nicotiana have separated into green and white segregants in vitro (83). A relatively low percentage (8%) of chimeral regenerants, however, was recovered from leaf disc culture of N. tabacum, N. glauca and interspecific periclinal chimeras of the two species (64). Four different rearrangements were observed in this work indicating that any or all histogenic cell layers could participate in the formation of adventitious buds (95 and 70 explants were cultured on BA and kinetin, respectively; 37 chimeras/266 nonchimeras were formed on BA and 14 chimeras/341 nonchimeras were obtained)on kinetin). After culturing thin cell layers from the apical dome and nine axillary buds of a single sectorially mutated ruffled leaf shoot of Nicotiana tabacum, two of 61 plants regenerated had ruffled leaves. This indicated that adventitious shoots of the smooth phenotype presumably had a single layer origin from L.I (54). The ruffled-leaf mutation appeared to reside in L.II and/or L.III. Thin cell layer explants were used because the plant was infected with tobacco vein mottling virus (TVMV), and healthy plants were desired.
Pelargonium.Pelargonium chimeras have been separated by suspension cultures of leaf protoplasts (53), callus cultures (21, 23, 100) and shoot tip culture (23). Cassells (21) used tissue culture results as evidence that the cultivar under study was actually chimeral. Both of the variegated chimeral Pelargonium cultivars studied by Cassells and Minas (23) underwent chimeral separation upon callus culture, giving rise to entirely albino and entirely green progeny; none were variegated. 'Mme Salleron' could be propagated true-to-type from shoot tips, while 'Mrs Cox' produced chimeral rearrangements. The authors observed that shot tip culture under their conditions resulted in "precocious axillary bud proliferation". Working with callus cultures of scented geranium cultivars, Skirvin and Janick (100) observed high variability among the "calliclones", attributing the variation to one or more factors, including chimeral separation, euploid changes, chromosomal changes, or gene mutations. A new cultivar derived from this work had a doubled chromosome number compared to the original stock material and was released. This may be the first cultivar to have been developed in tissue culture (101).
Rubus.McPheeters and Skirvin (70) proliferated over 900 plants of Rubus laciniatus 'Thornless Evergreen' from shoot tips, obtaining 53% thornless chimeral plants and 47% dwarf, pure thornless plants. The mutant layer in 'Thornless Evergreen' resides in the L.I such that the derivatives are unable to produce prickles as are the derivatives of L.II and L.III. McPheeters and Skirvin were surprised not to have obtained a certain proportion of nonchimeral thorny shoots (from endogenous L.II and/or L.III derivatives) and concluded that the tissue culture conditions must not have been conducive for such bud formation. From the fact that thorny shoots are more vigorous-growing than thornless (in the field), it is surprising that at least some did not arise. Under their conditions, L.I participated in all shoot formation, axillary and adventitious, while L.II and L.III evidently were only involved during axillary bud formation. The possibility exists that epidermal tissue in direct contact with the medium may have responded so rapidly in forming shoots that the endogenous tissues were left behind. Hall et al. (44) induced callus formation on meristem explants of Rubus sp. 'Thornless Loganberry' in a purposeful effort to separate the chimeral tissue layers with the goal of retrieving an entirely thornless plant (44). Only 3 shoots were regenerated from the callus, of which one survived to produce a plant which was entirely thornless. Of 100 offspring produced by seed, 63 were thornless, giving evidence that at least L.II (as well as L.I) of the regenerated plant is genetically thornless.
Saintpaulia.Considerable controversy surronds the ontogeny of adventitious shoots in Saintpaulia. Naylor and Johnson (78) obtained results indicating adventitious shoots derive from one epidermal cell, though they also stated that "adjacent epidermal cells and parenchyma cells within the petiole contribute to its (the shoot's) final formation". These authors stated that in conventional propagation from both petiole and leaf lamina tissue, adventitious shoots originate in epidermal cells. Tissue culture of Saintpaulia has been widely used to test this "single-cell" origin hypothesis. Norris, Smith and Vaughn (81) claimed that "adventitious shoots [of Saintpaulia produced in vitro] are of multicellular origin", and that "all layers of leaf tissue are involved in adventitious bud formation". Large numbers of variegated progeny from putatively chimeral plants of the cultivar Tommie Lou "all ... were identical to the original chimera". The conclusions reached by Norris et al. (81) have been questioned by several authors. On the basis of inheritance of variegation, anatomical sections of leaves and petioles, and the observed pattern of phenotypic regeneration, Marcotrigiano and Stewart (68) refuted these conclusions, arguing that "the cultivars used by Norris et al. were not periclinal chimeras" and that "their results give unequivocal evidence that the same genetic information controlling the pattern of leaf variegation is in all cells in all layers of the leaf", i.e., that 'Tommie Lou'-type variegation is due to genetic expression, and not due to chimerism. Sunblade and Meyer (108) also tissue cultured leaf tissue of 'Tommie Lou' and obtained edge variegated plants, but concluded that these results "may mean that the leaf patterning systems in some of the gesneriads are under genetic control even though the patterns look like a chimera". In their objections to the conclusions reached by Norris et al. (81), Broertjes and van Harten (17) stated that "it appears most improbable that propagation of real periclinal chimeras in vitro, using explants without buds, results in true-to-type vegetative offspring only. One would rather expect a considerable proportion of non-chimeric plants, with the genetic constitution of one of the composing layers of the original chimera". Preil (88) also doubted that the plants described by Norris, Smith and Vaughn (81) were true periclinal chimeras, noting that "it is surprisingly[sic] that from a chimera uniform (chimeral) progenies could be obtained via adventitious buds, all of multicellular origin". Results of Peary et al. (85) showed that the leaf variegation pattern of both 'Tommie Lou' and 'Candy Lou' (a pinwheel flowering cultivar with 'Tommie Lou'-type leaf variegation) was stable through tissue culture of over 1300 plants from leaf, petal and subepidermal explants, but that the pinwheel pattern of 'Candy Lou' was regenerated in a low percentage (3%), indicating that the flower color pattern was chimeral but the leaf pattern was nonchimeral. Interestingly, the flower color patterns of other pinwheel flowering cultivars of Saintpaulia were also unstable from these explants, but whole inflorescences did produce true-to-type plants (60). There apparently are vegetative buds in the axils of Saintpaulia inflorescences, which maintain the chimeral organization.
Other Herbaceous Chimeras.Shoots excised from runner tips of Fragaria vesca 'Albo-Marginata'produced 86.9% or more phenotypically variant plantlets when placed on concentrations of benzyladenine greater than 1.3 uM (67). Histological studies were performed to learn whether shoots arose from axillary or adventitious buds but it was impossible to distinguish a chlorophyll chimera from a nonchimera by examination of the shoot apex (32). The possibility of some variant shoots arising from leaf axils by the outer cell layers periclinally displacing the inner layers was suggested. A striking aspect of the Fragaria system is that this plant so readily forms phenotypic variants on relatively low concentrations of cytokinin (1.3 uM BA). On 4.4 uM BA, 5 original explants proliferated 310 plants in two 5-week subcultures, a remarkably high multiplication rate, theoretically rapid enough to produce one billion shoots in one year from one explant. It appears very likely that a considerable adventitious bud formation must have taken place, to produce an average of an 8 fold proliferation rate every 5 weeks. Few reports on herbaceous chimeras other than Begonia, Chrysanthemum, Dianthus, Nicotiana, Pelargonium, and Saintpaulia chimeras have quantitatively examined the phenomenon of chimeral separation in vitro. Separation into component genotypes has been observed in the variegated bromeliads Ananas comosus 'Variegatus', Cryptanthus 'It', and Aechmea fasciata 'Albo-marginata' (52), in Episcia 'Ember Lace' and 'Cleopatra' (12), Ajuga reptans 'Variegata' and 'Burgundy Glow' (117), Dracaena marginata 'Tricolor' (26), and a blue flowering variety of Freesia (9). Chin (25) divided leaves of Episcia cupreata 'Pink Brocade' into red, white, and green tissues, obtaining green plants and white plants. Plants produced from axillary shoots of Hosta decorata 'Thomas Hogg' which had lost the characteristic white leaf margin during in vitro culture regained it after 5 months storage at 3 to 6oC, but plants of adventitious origin were not mentioned (84). Pierik and Steegmans (87) noted that chimeral separation occurred in shoot cultures of a varie- gated, yellow leaf margined form of Yucca elephantipes when BA levels were too high, resulting in green shoots. In contrast to the observation by Zilis et al. (117) that lowering the cytokinin level resulted in fewer off-types of Ajuga reptans cultivars, Lineberger and Wanstreet (61) found no significant difference in percent phenotypic variants of 'Burgundy Glow' when comparing two growth regulator treatments, obtaining about 30% off-types on either treatment. Most of these off-types were the "pink over green" or "bronze" sport, with some entirely pink plants also observed.
Other Woody Chimeras.A very sparse literature exists on the tissue culture of woody chimeral plants. Rubus, a biennial woody genus, has been discussed previously. Culture of apical fragments of Vitis vinifera 'Meunier', a periclinal chimera possessing a tomentose genotype in L.I, resulted in the development of direct adventitious shoots (98). Of 134 resulting plants, 52.2% were sectorial chimeras with hairless sectors, while one entirely hairless plant arose, presumably from endogenous tissues. The other plantlets phenotypically resembled 'Meunier'. At least in the case of the sectorial chimeras, adventitious shoots in this system must have had a multicellular origin. During adventitious shoot formation on recultured leaf explants of Liquidambar styraciflua 'Variegata', three new variegation rearrangements were observed, of which two have been rooted and outplanted (14). It was further noted that the three new rearrangements, 'W', 'G/W', and 'W/G' all expressed their leaf patterns in vitro, whereas cultures of 'Variegata', normally a mottled yellow and green pattern, appeared green while in culture.
In Vitro Synthesis of ChimerasConsiderable interest in plant chimeras came about in the late 1800's - early 1900's because of unusual "graft hybrid" cases such as the 'Bizzaria' orange and Laburnocytisus adamii (112). In the area of experimental synthesis of graft chimeras, most work has been done on species in the Solanaceae (115, 116). To produce graft chimeras, a scion is grafted onto an understock, the scion is carefully trimmed until only a thin layer remains, callus formation follows, and then shoots form. Some of these adventitious shoots may be chimeral (79). Graft chimeras are chimeral for numerous traits. The potential exists for the exploitation of this method to vegetatively create disease or insect resistant plants, as shown by the synthesis of a whitefly resistant Solanum pennellii-Lycopersicon esculentum graft chimera (27). Research exploring the use of tissue culture to synthesize chimeras has also focused on species in the Solanaceae. A requirement in either the grafting or tissue culture technique is that adventitious buds must arise coordinately from separate genotypes in order to produce a chimeral meristem. Carlson and Chaleff (20) cocultured chimeral callus of Nicotiana tabacum and an amphiploid hybrid between N. glauca and N. langsdorfii, regenerating about 7000 shoots. Most of the regenerated shoots were likely of unicellular or few-celled origin (nonchimeral), but a low percentage (28/7000, 0.4%) of chimeras from multicellular origin were obtained Marcotrigiano and Gouin (65), working with albino and green cell lines from N. tabacum, found that callus from mixed filtered cell suspensions allowed for the most effective mixing of the cell lines. However, few chimeras were regenerated (4 of 1321 total plants). They postulated that either a low number of cells, or perhaps ultimately one cell, was probably involved in the formation of adventitious buds or a chimeral meristem may have formed initially and then one genotype may have been eliminated by diplontic selection. Marcotrigiano and Gouin (66) obtained no chimeral shoots of 871 shoots regenerated from chimeral callus, but recovered 3 interspecific mericlinal chimeras out of 209 adventitious shoots produced at the graft union of grafted plants. They stated that this "absence of chime- ras from tissue culture suggests that shoot organization in vitro may proceed in a different manner than that occurring in vivo". It may be that graft union shoots are more likely to arise from a multicellular origin, but the in vitro environment may allow such rapid cell division rates that rapid formation of homogeneous clusters of cells pre- cludes formation of chimeral meristems.
SummaryTissue culture methodology provides a useful way to separate plant chimeras into their component genotypes. Conditions which favor adventitious shoot formation (leaf or callus culture, suspension culture, extremely rapid shoot proliferation rates) encourage genotypic segregation. Genotypic segregation can confirm the chimeral nature of the cultivar in question, and can allow conclusions to be drawn about the ontogeny of in vitro adventitious shoot formation. Reliable micropropagation of chimeras, though difficult, can be accomplished under the appropriate conditions. Rearrangement of existing chimeras and synthesis of new chimeras are infrequently obtained by in vitro methods, but may provide the opportunity to create novel phenotypes by asexual methods.
List of References Cited