Tissue 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 Organization
The 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 Terminology
Chimeral 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).
Photo 2. Citrus fasciata
Photo 3. Citrus fasciata
Photo 6. Citrus Consolei
Photo 7. Citrus medica
Photo 8. Citrus medica
Photo 9. Citrus medica
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 Chimeras
Apical 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 Plants
Tissue 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 Chimeras
Considerable 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.
Summary
Tissue 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.
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