In vitro micrografting and the histology of graft

union formation of selected species of prickly

pear cactus (Opuntia spp.)
A.A. Estrada-Luna
a,*
, C. Lopez-Peralta
b
,
E. Cardenas-Soriano
c
a
Campus San Luis Potos , Colegio de Postgraduados, Iturbide 73, Salinas de Hidalgo SLP 78600, Mexico
b
Laboratorio de Biotecnolog a, Centro de Genetica, Colegio de Postgraduados,
Montecillo, Edo. de Mexico CP 56230, Mexico
c
Centro de Fitopatolog a, Colegio de Postgraduados, Montecillo, Edo. de Mexico CP 56230, Mexico
Accepted 31 May 2001
Abstract
Horizontal and wedge grafts were utilized with micropropagated prickly pear cactus (Opuntia
spp.) to determine the best method for in vitro micrografts. By frequent sampling of the develop-
mental stages of the micrografts we characterized the histological events during graft union
formation. Five Opuntia species (O. streptacantha Lemaire, O. robusta Wendland, O. cochinera
Grifths, O. leucotricha De Candolle, and O. cus-indica Linne (Miller)) were used as rootstocks
and combined with O. cus-indica to produce homo- and heterografts. Total growth was recorded
after 90 days of ex vitro culture to determine the effect of rootstock genotype on scion development.
Our results indicate that the easiest and most successful method for micrografting prickly pear
cactus was the horizontal graft. Within 28 days vascular connections occurred within the callus
bridge between rootstocks and scions in all genetic combinations. Hence, the Opuntia species
stockscion combinations were compatible. Homografts (grafts between same species) grew
signicantly faster than the heterografts (grafts between different species) after 90 days of ex vitro
transfer. Histological observations revealed ve developmental stages of graft union formation: (1)
development of a necrotic layer, (2) proliferation of callus bridge at the graft interface, (3) dif-
ferentiation of new vascular cambium, (4) restoration of the new vascular tissue, and (5) restoration
of the continuity of external epidermal tissue at the union zone. We conclude that the in vitro
horizontal graft is a successful, easy and reliable method to graft micropropagated prickly pear
Scientia Horticulturae 92 (2002) 317327
*
Corresponding author. Present address: Centro de Investigacion y Estudios Avanzados del IPN. Unidad
Irapuato, Km. 9.6 Libramiento Norte Carretera Irapuato-Leon. Apartado Postal 629, Irapuato, Gto., Mexico.
Tel.: 52-462-39634; fax: 52-462-45849.
E-mail addresses: aestradaluna@yahoo.com, aestrada@ira.cinvestav.mx (A.A. Estrada-Luna).
0304-4238/02/$ see front matter # 2002 Elsevier Science B.V. All rights reserved.
PII: S0 3 0 4 - 4 2 3 8 ( 0 1 ) 0 0 2 9 6 - 5
cactus particularly since no contamination or tissue dehydration occurred and no special structures
or adhesives were needed for successful graft union formation. This grafting technique has poten-
tial use in the commercial production of other cactus species. #2002 Elsevier Science B.V. All rights
reserved.
Keywords: In vitro grafting; Graft union formation; Heterograft; Homograft; Micropropagation; Cactus-pear
1. Introduction
Prickly pear cactus (PPC) (Opuntia spp.) is adapted to grow and produce under low
water regimes and poor soils (Nobel, 1994). Because of its high adaptability and multiple
uses, it has been dispersed from its native habitats in North America to the other world
regions (Barbera, 1995). In many countries PPC is considered an important re-vegetation
crop to control wind and water erosion in disturbed areas (Pimienta, 1994). Internationally,
about 100,000 ha are devoted to Opuntia fruit and cladode production in commercial
orchards; however, more than three million hectares of PPC are grown in native habitats
(Barbera, 1995; Pimienta, 1994). Fruits and cladodes are valued because of their high
nutrient, vitamin and medicinal components (Hegwood, 1990). They can be eaten fresh or
processed to produce candies, wine, vinegar, juice, and avorings (Saenz-Hernandez,
1995). Production of the red dye acetocarmine may also be possible through extraction of
the parasitic insect cochineal (Dactylopius coccus Costa), which relies on PPC as the host
plant (Flores-Flores and Tekelenburg, 1995).
PPC is mainly propagated through the rooting of cladodes (Lazcano et al., 1999);
however, new individuals could also be obtained from seeds, fruits, tissue culture and
grafting (Estrada-Luna, 1988). Grafting is an ancient technique (Hartmann et al., 1997),
which is commonly used to propagate cactus species (Cullmann et al., 1986). However,
Opuntia is generally grafted only during studies on virus or mycoplasm transmission or as
ornamental curiosity (Pimienta, 1974). Grafting in commercial propagation has been
restricted because of poor success, which is attributed to difculties in the use of unreliable
techniques, problems with fungi or bacteria contamination and dehydration stress of tissues
in the graft union area (Maldonado and Zapien, 1977). A potential alternative to avoid
some of these problems is micrografting of plantlets, which is performed under aseptic and
high relative humidity conditions (Burger, 1985; Hartmann et al., 1997). Additionally, in
vitro micrografting may provide several advantages such as elimination of viruses,
rejuvenation of mature tissues, year round plant production, enhance compatibility studies
and correlative relations between rootstocks and scions, make specic genotypic combi-
nations to increase plant productivity, and extend ecological limits of a particular plant
species or cultivar to tolerate edaphic conditions (Richardson et al., 1996; Hartmann et al.,
1997). This last aspect is particularly important because the ecological distribution and
growth of PPC is strongly determined by its calcifuge and calcicole nature (Del Castillo,
1996). The objectives of this research were: (1) to determine the appropriate and reliable
micrografting techniques for cactus-pear plantlets; (2) to determine the optimum combi-
nations among ve Opuntia species and (3) to characterize the developmental stages of
graft union formation of PPC.
318 A.A. Estrada-Luna et al. / Scientia Horticulturae 92 (2002) 317327
2. Materials and methods
2.1. Plant material and micropropagation
PPC (Opuntia spp.) plantlets from ve species (O. streptacantha Lemaire, O. robusta
Wendland; O. cochinera Grifths; O. leucotricha De Candolle, and O. cus-indica Linne
(Miller)) were micropropagated following techniques established by Estrada-Luna (1988).
Axillary buds excised from young cladodes (57 cm long) were initially used as explants
and induced to develop shoots using Murashige and Skoog (1962) basal salt formulation
adjusted to pH 5.7 and supplemented with 5% sucrose (Sigma Chemical) and 0.6% bacto-
agar (Difco Laboratories, Detroit, MI). BA [6-benzyl aminopurine] (Sigma Chemical) and
2ip (N
6
-[D
2
-isopentenyl] adenine) (Sigma Chemical) were added at 2.5 and 5 mg l
1
,
respectively, for shoot induction and proliferation cultures. Adventitious roots developed in
a hormone-free medium. All cultures were grown in a culture room with light at
100 mmol m
2
s
1
photosynthetic photon ux density (PPFD) (GTE, Sylvania, USA;
``cool white'' uorescent lamps, 75 W) at plant level and a photoperiod of 16 h.
Temperature was maintained at 27 2

C.
2.2. Micrografting, variables measured and data analysis
Under aseptic conditions, shoots of uniform length and diameter were selected from in
vitro cultures and used as rootstocks and scions in micrografting. In the rst experiment,
wedge and horizontal grafts were tested to determine the optimum union for micrografting
(Fig. 1). The percentage of successful and unsuccessful grafts was determined 30 days after
grafting and in vitro culture. In a second trial, an experiment organized as a completely
randomized design was run to determine the effects of rootstock on scion development.
O. cus-indica was used as the scion and micrografted with the horizontal graft to the ve
different Opuntia species included in this study. The combinations resulted in the
formation of homografts (graft including the same plant species in both rootstock and
Fig. 1. In vitro micrografting of PPC (Opuntia spp.) plantlets with wedge and horizontal grafts.
A.A. Estrada-Luna et al. / Scientia Horticulturae 92 (2002) 317327 319
scion) and heterografts (graft including different plant species for the rootstock and scion).
In all cases, the rootstocks and scions were 1.0 and 0.5 cm in length, respectively, and their
diameters were kept as uniform as possible. Graft survival rate and total shoot length (cm)
were recorded on the micrografts 90 days after ex vitro transfer. Data were statistically
analyzed by analysis of variance (ANOVA), w
2
(chi-square test), and Tukey (a = 0:05%)
for the means separation (SAS, 1996).
2.3. Plant transfer, acclimatization and culture conditions
The successful micrografts were removed from the culture containers (baby food jars),
and rinsed with tap water to remove remaining agar from the root system. Subsequently,
they were transplanted into individual commercial plastic pots (12:5 7:5 cm) lled with
a pasteurized mix of sand, peat moss and vermiculite (1:2:1 (v/v)), covered with plastic
bags and transferred to glasshouse conditions. During the rst 2 weeks, the bags were
gradually punctured to allow air exchange for plant acclimatization. Maximum PPFD at
plant level was 1000 mmol m
2
s
1
, and the average day/night temperature was 32/18 8C,
respectively. Plantlets were irrigated as needed and fertilized once a week with 50 ml of the
Murashige and Skoog (1962) salt formulation with pH adjusted to 5.6.
2.4. Histology of graft development
Three samples of two homo- and heterograft combinations were harvested at 6 and 12 h,
and 1, 2, 4, 8, 12, 16 20, 24, and 28 days after grafting to study the sequence of anatomical
events during the graft union formation. The specimens were trimmed to 5 mm above and
below the graft union (1.0 cm long), xed for 24 h with Craff III (Berlyn and Miskche,
1976), dehydrated through a series of ethyl alcohol dilutions (30, 50, 70, 90, 96 and 100)
and three times in absolute ethanol for 6 h per stage. Tissue was then embedded in parafn
(TissuePrep, Fisher Scientic, Fair Lawn, NJ) in an oven at 55 2

C. Sections of 10 mm
thick from longitudinal and transverse sections were cut using a rotary microtome
(American Optical model 820).
In order to mount the tissue specimens, a drop of Haupts adhesive (Carolina Biological
Supply, Burlington, NC) was applied on a slide and spread evenly across it. Ribbons
containing the tissue sections were cut into suitable length and placed on the slide. Two
drops of oating solution (3% (v/v) formaldehyde) were added before it was placed on a
hot plate maintained at 39 8C. Plant samples were stained with aqueous safranine-fast
green (Sigma Chemical, St. Louis, MO) and mounted with Permount (Fisher Scientic,
Fair Lawn, NJ). Finally, the slides were examined with an Axiophot photomicroscope
(Ziess, West Germany) and the desired sections were photographed.
3. Results
3.1. In vitro micrografting and ex vitro growth
The success of in vitro grafting micropropagated shoots of PPC varied according to the
type of graft tested. In general, the horizontal graft method was signicantly more
320 A.A. Estrada-Luna et al. / Scientia Horticulturae 92 (2002) 317327
successful (90% viability) than the wedge grafting method (30% viability) according the
chi-square test (P _ 0:001). In horizontal micrografts, no problems were observed during
the union formation as long as the contact surfaces were perfectly smooth (Fig. 2a). Only
10% graft failure occurred due to scion displacement. The wedge micrografts were more
difcult to perform and had lower success. Wedge micrografts failed because of problems
with mismatched tissues between the scion and rootstock and tissue oxidation at the union
site (Fig. 2b). Additionally, the development of new shoots in rootstocks close to the union
area caused the scion to be displaced, which resulted in mismatched union between root-
stocks and scions (Fig. 2c). In successful grafts, the re-establishment of growth by scions
was evident 24 days after grafting (Fig. 2d).
After ex vitro transplantation, there was 100% survival rate on the micrografts for all
combinations tested. However, rootstocks had signicant effects on the scion development.
In general, homografts had signicantly greater height than heterografts 90 days after plant
transfer (Table 1). Combinations including O. cochinera and O. leucotricha as rootstocks
signicantly reduced the size of the scion (Table 1).
3.2. Anatomy of the graft union formation
The histology of the graft union formation was similar in all graft combinations.
Our observations revealed ve developmental stages during graft union formation:
Fig. 2. PPC (Opuntia spp.) micrografts: (a) Close-up of the perfect graft union (arrow) of a horizontal homograft
between O. ficus-indica Linne (Miller) (rootstock) and O. ficus-indica Linne (Miller) (scion). (b) Wedge
micrograft showing mismatched union and tissue swelling and oxidation in rootstock. (c) Wedge micrograft
showing the development of a new shoot (arrow) growing from the rootstock and partial detachment of scion.
(d) A 28-day-old horizontal micrograft between O. robusta Wendland (rootstock) and O. ficus-indica Linne
(Miller) (scion) growing under tissue culture conditions. See the graft union formation (arrow).
A.A. Estrada-Luna et al. / Scientia Horticulturae 92 (2002) 317327 321
(1) development of a necrotic layer, (2) proliferation of callus bridge at the graft interface,
(3) differentiation of new vascular cambium, (4) restoration of new vascular tissue, and (5)
restoration of the continuity of the epidermis at the graft union.
During the initial hours after lining-up the rootstock and scion, there was no evidence in
cambial activity. However, the formation of a distinct layer resulting fromthe shrunken and
heavily stained cellsnecrotic layerwas visible 24 h after grafting in both partners
(Fig. 3a). The rst evidence of cell division was the development of the callus bridge at
the interface between scion and rootstock, which corresponded to the initiation of stage two
during graft union formation. This was observed in all combinations between days 1 and 4
after grafting. Areas of parenchyma cells adjacent to the necrotic layer were rst initiated to
divide at different points alongthe inner surface of thewoundedtissues onbothgraft partners
(Fig. 3b). In some regions, the proliferating calli followed a clear pattern of anticlinal or
periclinal cell division; however, a disorganized mass of cells was also observed. After
several days, cortical and pithy parenchyma cells had divided to produce a multi-layer callus
above andbelowof the necrotic zonewas formed tophysicallyjointhe sciontothe rootstock,
which caused a slight swelling of the tissues close to the interface (visual observation).
During the early stages of callus bridge formation, the dividing cells were long and
relatively small as compared to other typical parenchyma cells belonging to the scion and
rootstock tissues (Fig. 3b); however, by day 28 when the callus lled all spaces on the
interface, they had grown and became similar (Fig. 3c). By day 12, there was intense
cellular activity on both the rootstock and scion at the graft interface as indicated not only
by callus proliferation across the graft union but also by the re-absorption of the necrotic
zone that then started to disappear. The wound-repair process of vascular cambium tissues
across the callus bridge (stage three), also started after day 12. Cell differentiation initiated
on parenchyma cells in both stock and scion tissues, which were located close to the
disrupted vascular tissues. The originated cells were elongated and oriented towards the
callus bridge (Fig. 3c and d). Some cells with secondary cell wall thickening were observed
later on showing the same longitudinal orientation in both graft partners (Fig. 3c and d).
Evidence of a functional and compatible graft union was observed 28 days after grafting
and in vitro culture (Fig. 2d), when the scion began a vigorous growth. New vascular
elements including vessels and tracheids were seen to unite across the interface zone. The
newly formed xylem and phloem were longitudinally re-oriented in some cases; however,
this fact did not interfere with the union itself, since the continuity of the vascular system
was attained (Fig. 3e). At this step, the necrotic layer was slightly visible along the
Table 1
Effects of rootstock on scion O. ficus-indica Linne (Miller) development 90 days after ex vitro transplanting
Rootstock Scion length
a
(cm)
O. ficus-indica Linne (Miller) 28.8 a
*
O. robusta Wendland 25.6 b
O. streptacantha Lemaire 24.9 b
O. cochinera Griffiths 18.4 c
O. leucotricha De Candolle 16.3 c
a
n = 20.
*
Means followed by different letters are significantly different (P _ 0:05) as indicated by Tukey test.
322 A.A. Estrada-Luna et al. / Scientia Horticulturae 92 (2002) 317327
wounded zone (Fig. 3e). The epidermis of the stock and scion at the union zone was
completely continuous 40 days after grafting (Fig. 3f ).
4. Discussion
Grafting is a common practice to propagate cacti species. It is used to counteract the
slow development showed by these plants (Cullmann et al., 1986) or to propagate rare
Fig. 3. Longitudinal sections of PPC (Opuntia spp.) of a heterograft between O. streptacantha Lemaire
(rootstock) and O. ficus-indica Linne (Miller) (scion). (a) A 3-day-old micrograft showing necrotic layer (nl) and
interface zone (iz) [100]. (b) A 3-day-old micrograft showing the proliferation of callus bridge cells (cbc), nl,
and iz [400]. (c) A 20-day-old micrograft showing original vascular tissues (ovt), the iz filled-up by a multi-
layer of proliferating callus bridge cells (cbc), and cells in process of differentiation to regenerate new vascular
tissues (nvt) [100]. (d) Close-up of a 16-day-old micrograft showing elongated cells (arrow) in process of
differentiation to regenerate vascular tissues [1000]. (e) A 28-day-old micrograft showing the nl and the
continuity of the regenerated new vascular cells (nvc) across the graft union [400]. (f ) Close-up of a 28-day-
old micrograft showing the scare developed on a successful graft union (arrow) [400].
A.A. Estrada-Luna et al. / Scientia Horticulturae 92 (2002) 317327 323
ornamental species or desirable mutations (Shimomura and Fujihara, 1978; Hartmann
et al., 1997). Establishing an in vitro grafting method for PPC may combine advantages
from grafting with those of micropropagation (Burger, 1985). Performing a horizontal
graft was the most successful and easy method for in vitro micrografting PPC. In
general, the typical problems of fungi and bacteria contamination or tissue dehydra-
tion observed during conventional grafting of Opuntia were minimized. However,
clean and accurate cuts during dissection were critical to have good contact between
scion and rootstock (Fig. 2a and d). Seventy percent of the wedge micrografts failed
to form graft unions. This poor success was because of the problems with partial
or total detachment of the scion, which were produced by tissue oxidation, cell
proliferation and swelling of the tissues injured during grafting in both scion and
rootstock (Fig. 2b).
Development of new shoots from axillary buds in the rootstock was observed in all
wedge micrografts. In 35% of the cases, the growth of several shoots caused the scion to be
detached from the rootstock (Fig. 2c) or its development was drastically limited probably
by deviation of the nutrients from the rootstock to the new shoots.
The Opuntia species included in this study did not have problems with compatibility
during micrografting. Evidence of this is the high percentage of success during grafting, the
establishment of the vascular tissue continuity observed in the histological study, and the
excellent growth observed after 90 days of ex vitro culture. This agrees with the results
obtained from several authors who concluded that incompatibility problems among cacti
genus and species are not common (Shimomura and Fujihara, 1976; Maldonado and
Zapien, 1977; Cullmann et al., 1986). However, combinations including different genera
may show incompatibility that results in stunted growth of the scion and changes in the
morphology (Das and Mukhopadhyay, 1976). The genetics of the rootstock affected the
growth rate of the scion. When O. cus-indica was combined with O. cus-indica during
grafting (homograft), signicantly higher scion development was observed. In contrast,
when O. cochinera and O. leucotricha were used as rootstocks, the scion only grew 57 and
64% as compared to the homograft. This may have happened because the plants more
closely related, are more physiologically compatible and have an increased rate of
regeneration of the micrograft (Moore, 1984).
The graft union formation in micropropagated PPC shoots showed a similar sequence of
events to those observed in graft formations with other herbaceous or woody plant species.
Moore (1984) and Hartmann et al. (1997) have described four general steps during a
compatible graft union formation: formation of the union, development of a necrotic layer
and proliferation of callus bridge at the graft interface, differentiation of new vascular
cambium and restoration of the continuity of new vascular tissue.
In the rst stage after grafting, we observed that the ruptured cells resulting from the
dissection of the scion and rootstock formed a necrotic layer at the graft interface, which
compartmentalize the rest of the plant as a defensive mechanism to eliminate invasion of
pathogens (Hartmann et al., 1997). After the necrotic layer appeared (Fig. 3a), we observed
the initiation of cell division from parenchyma tissue adjacent to the necrotic layer to
develop the callus bridge at the interface zone, between days 1 and 4 after grafting (Fig. 3b),
which was comparable with the results obtained by Shimomura and Fujihara (1976)
grafting different cacti species.
324 A.A. Estrada-Luna et al. / Scientia Horticulturae 92 (2002) 317327
The growth of callus is a key process in the development of the graft union because it
physically joins the scion to the rootstock (Jeffree and Yeoman, 1983; Moore, 1984).
According to our observations, cellular division in surfaces of both rootstock and scion was
initiated in several points by the cells at the most central part of the union, which was
extended through day 28. Similar observations were obtained by Shimomura and Fujihara
(1976) in their study with Notocactus and Hylocereus.
The re-establishment of vascular continuity through the interface zone is the critical
event that determines the compatibility between the stock and the scion on the development
of graft union formation (Esau, 1965; Shimomura and Fujihara, 1978; Yeoman and Brown,
1976; Moore, 1984). This is because the restoration of the vascular bundles ensures the ow
of substances between the stock and the scion (Moore, 1984). For the micropropagated
PPC shoots, the differentiation process started 12 days after grafting when new elongated
cells were observed dividing near the vascular cambium (Fig. 3c and d), which later
differentiated into new vessels and tracheids. This process continued for several days in
prickly pear grafts; however, it may uctuate between 9 and 21 days after grafting
depending to the species examined and type of combination process (Moore and Walker,
1981; Moore, 1984).
The true union is achieved only after the xylem and phloem have made perfect contact
(Moore and Walker, 1981). In our case, the realignment and restoration of the vascular
bundles between scion and rootstock was completed 28 days after grafting and in vitro
culture in all graft combinations tested (Fig. 3e). This was the result of the eventual
differentiation of the elongated cells originated in the cambial area into the new vessels,
tracheary elements and phloemsieves through the callus (Moore and Walker, 1981; Moore,
1984). In some grafts, we observed a slight re-orientation of the new vascular connections,
which is the result of the uneven match at the union of the vascular tissues of stock and
scion (Esau, 1965). However, no delays or unsuccessful grafts were observed.
5. Conclusions
The in vitro horizontal graft is a successful and easy method to micrograft PPC. Growing
the grafts under in vitro conditions avoided problems with fungi or bacteria tissue
contamination or scion stress by excess of dehydration. No special tools to perform
the grafting or structures to hold both partners during union formation were needed. The
histology of the union formation in in vitro grafted PPC is comparable to that observed in
graft formations with other plant species, including ve general stages: formation of the
union, development of a necrotic layer and proliferation of callus bridge at the graft
interface, differentiation of new vascular cambium, restoration of the continuity of new
vascular tissue, and restoration of the continuity of the epidermis at the union zone.
Restoration of the continuity of new vascular tissue was observed 28 days after grafting.
After ex vitro transplanting, the development of scion was affected by the type of
combination during grafting, which was favored when homografts combinations were
formed. Finally, this micrografting technology has the potential for large scale production
of PPC plants and might be extended to the propagation of other micropropagated cacti
species.
A.A. Estrada-Luna et al. / Scientia Horticulturae 92 (2002) 317327 325
Acknowledgements
The authors would like to thank Fred T. Davies Jr., Benton Storey, and R. Daniel
Lineberger from the Horticultural Sciences Department of Texas A&M University for their
critical review and valuable comments and corrections to this paper. A.A. Estrada-Luna is
grateful to the National Council of Science and Technology (CONACYT) of Mexico for
the economical support provided during his graduate studies.
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