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21
Micropropagation of Cactus Plants (Cactaceae)

Carlos Ramirez Serrano1* • Jaime A. Teixeira da Silva2

1 Departamento de Botánica y Zoología, CUCBA, Universidad de Guadalajara, Km 15.5 Carretera a Nogales, 45101 Las Agujas Nextipac Zapopan Jal. Mexico
2 Kagawa University, Faculty of Agriculture and Graduate School of Agriculture, Department of Horticulture, Miki cho, Ikenobe, 761-0795, Kagawa ken, Japan
Corresponding author: * Carlos.Ramirez.Serrano@cucba.udg.mx

Keywords: apical dominance, areole, cacti, phylloclade

ABSTRACT

The micropropagation of cactus has been carried out over three decades of research, where the application of cytokinins and auxins to media
culture as well as the use of phylloclades as explants are the two main keys to promote areole activation, growth and development. Most
procedures are highly specific and poorly competent. In this chapter we present a brief review on the propagation of cactus plants – mainly of
horticulture interest or needed for conservation – by tissue culture. We also attempt to identify innovation in technology and novelties in
techniques that would allow for the methodologies to be widely applied to all members of the Cactaceae.

1. INTRODUCTION
The cactus family has its origin in America, in which all kind of climates may be found, but are, however, found in arid and semiarid lands (Cornet
1985; Valles 1997). In Mexico, in particularly, 68% of 1,500 species of Cactaceae may be found (Valles 1997; Glass 1998). Cactus plants have a
distinct anatomy and physiology as gross shape; buds that turn into areoles, which are the main character of cacti, and CAM metabolism (Bravo-
Hollis 1997). Besides, cacti have evolved to resist arid weather, and since these plants can preserve and maintain water in their parenchyma,
since they contain fewer respiratory structures, produce serum secretions, and have plunged stomas; they are thus considered xerophytic plants
(Bravo-Hollis 1978). As mentioned above, areoles are cacti-specific structures found on the ribs or tubers as a group of wooly hairs or spines,
which can develop buds, flowers or branches (Glass 1998). Mexico has a rich culture of cacti since those plants are used as medicinal remedies,
food, construction, rituals, and mostly ornamentals (Valles 1997). This use has been increasing since Columbus brought these plants to Europe,
and later Linneus named them as cacti (Bravo-Hollis 1997). However, the ornamental demand has provoked an excessive ransacking and
disappearance of many cacti, by which IUCN (International Union of the Conservation of Nature) since 1997 have been reported more than 200
Mexican cactus species as indeterminate, rare, vulnerable, and endangered; however in 2007 only 157 species (mostly Mexican species)
appear in the red list (IUCN 2007), and CITES 2007 (Convention International of Threatened and Endangered Species) list 6 genera and 29 taxa
in appendix I (Table 1; Sajeva et al. 2007); specifically in Mexico 217 cacti species belonging to 24 genera are protected according to Mexican
Law (Glass 1998), several of which are listed in Table 1.
With this precarious perspective, propagation should be a tool to maintain these valuable genetic resources. However, seed propagation
does is insufficient to cover the demand for cactus due to limited production, availability and viability of this reproductive material. Moreover,
vegetative propagation is insufficient. Consequently, in vitro techniques are fundamental tools to satisfy demand. More importantly, since
numerous cacti species are threatened or endangered, in vitro micropropagation has become, since Mauseth’s work in 1976, the primary tool for
preserving these valuable genetic resources. Cacti can be propagated via rooting of cuttings, sprouts and grafts; however, some wild species
with monopodial development present serious difficulties by vegetative propagation, mainly due for few or a lack of production of sprouts which
grow slowly and, in some cases, the small size of seeds, and low viability (Soltero 1996). The cactus family develops a particular structure, the
areole, which can be the meristem and the organ that becomes leaves, flowers, new stems and spines (Bravo-Hollis 1978). Areoles therefore
have the competence to produce phylloclades which develop into whole plants.
With this review we will demonstrate the methods for mass production of phylloclades developed for cactus species by stimulating dormant
meristems (i.e. cactus micropropagation), whose competence is related to their genetics and physiology. In vitro cactus present monopodial,
cespitous or proliferous, or even branched development (Fig. 1). We also focus on fundamental issues in cactus micropropagation, viz. plant
growth regulators, their combinations and concentrations, as well as various types of explants to achieve successful micropropagation protocols.

Abbreviations: 2,4-D, 2,4-dichlorophenoxiacetic acid; 2iP, N6–(2- isopentyl)adenine; BA, 6-benzyladenine; GA3, gibberellic acid; IAA, indole-3-acetic acid; IBA, indole-
3-butyric acid; KIN, kinetin; NAA, -naphthalene acetic acid; PGR, plant growth regulator; TDZ, thidiazuron; ZEA; zeatin
Ramirez Serrano and Teixeira da Silva 220 Micropropagation of cactus plants

Table 1 List of cacti species that are threatened to extinct in Mexico (IUCN2007, CITES 2007 and Glass 1998).
Species Status Species Status
Acharagma aguirreana (Glass et Foster) Glass Highly endangered Mammillaria hernandezii Glass et Foster Threatened
Ariocarpus agavoides * ** (Castañeda) E F Anderson Highly threatened Mammillaria herrerae Werdermann Highly endangered
Ariocarpus bravoanus** Hernandez et EF Anderson Extremely endangered Mammillaria longiflora (Britton et Rose) Berger Threatened
Ariocarpus fissuratus var hintonii** W Stuppy et NP Taylor Highly endangered Mammillaria louisae Linsay Threatened
Ariocarpus kotschoubeyanus** (Lemare) K Schumann Endangered Mammillaria mathildae Krähenbül et Krainz Endangered
Ariocarpus kotschoubeyanus ssp albiflorus** (Backeberg) Endangered Mammillaria luethyi GS Hinton Highly endangered
Glass
Ariocarpus scaphirostris** Bodeker Highly threatened Mammillaria marcosii Fitz Highly threatened
Ariocarpus trigonus** (Weber) K. Shmann Threatened Mammillaria napina Purpus Endangered
Astrophytum asterias* ** (Zuccarini) Lemaire Rare and endangered Mammillaria oteroi Glass et Foster Threatened
Astrophytum capricorne (Diedrich) Britton et Rose Threatened Mammillaria pectinifera** Weber Threatened
Astrophytum myriostigma Lemaire Threatened Mammillaria plumose Weber et Bois Threatened
Astrophytum ornatum (De Candolle) Weber ex Britton et Threatened Mammillaria saboae Glass Threatened
Rose
Aztekium hintonii Glass et Fitz Endangered Mammillaria saboae ssp goldii (Glass et Foster) D Hunt Threatened
Aztekium ritteri** (Boedeker) Boedeker ex Berger Endangered Mammillaria sanchez-mejoradae R Gonzalez Endangered
Backebergia militaris (Audot) Bravo ex Sanchez- Threatened Mammillaria schenvariana R Ortega-Varela et Glass Probably extinct
Mejorada
Cochemiea maritima Lindsay Threatened Mammillaria schiedeana ssp dumetorum Purpus) D Hunt Threatened
Coryphantha pulleineana (Backeberg) Glass Threatened Mammillaria schwarzii Shurly Threatened
Echinocactus grusonii Hildmann Endangered Mammillaria solisioides** Beckberg Threatened
Echinocactus parryi Engelmann Threatened Mammillaria tayloriorum Glass et Foster Threatened
Echinocereus knippelianus ssp kruegeri (Glass et Foster) Threatened Mammillaria tezontle Fitz et Fitz Extinct
Glass
Echinocereus lindsayi Meyran Threatened Mammillaria theresae Cutak Threatened
Echinocereus nivosus Glass et Foster Threatened Obregonia denegri* Fri Threatened
Echinomastus unguispinus ssp laui (Frank et Zecher) Threatened Pelecyphora aselliformis** Ehrenberg Threatened
Epithelantha micromeris spp polycephala Endangered Pelecyphora strobiliformis** (Werdermann) Fric et Schelle Endangered
Escobaria henricksonii Glass et Foster Endangered Stenocereus eruca (Brandegee) A Gobson et Horak Threatened
Escobaria loredoi (Glass et Foster) NP Taylor Threatened Strombocactus disciformis** ssp esperanzae Glass Grass et Extremely
Arias threatened
Ferocacatus fordii (Orcutt) Britt et Rose Threatened Thelocactus conothele ssp argenteus Glass Threatened
Ferocactus heamatacanthus (Salm-Dick) Borg Threatened Thelocactus conothele ssp aurantiacus Glass Threatened
Ferocactus viridescens (Nuttall) Britton et Rose Threatened Thelocactus garciae Glass et M Mendoza Highly endangered
Ferocactus viridescens var litoralis Lindsay Threatened Thelocactus hastifer (Werdermann et Bödeker) Knuth Threatened
Geohintonia mexicana Glass et Fitz Endangered Thelocactus rinconensis ssp hintonii Lülhy Threatened
Hematocactus uncinatus spp crassihamatus (Weber) Threatened Turbinicarpus alonsoi** Glass et Arias Highly threatened
Glass
Leuchtenbergia principis Hoker Threatened Turbinicarpus dickisoniae** (Glass et Foster) Glass et Threatened
Hofer
Lophophora williamsii (Lemaire ex Salm-Dyck) Coulter Threatened Turbinicarpus gielsdorfianus** (Werdermann) John et Riha Highly endangered
Mammillaria albiflora (Werdermann) Backeberg Threatened Turbinicarpus knuthianus** (Bödeker) John et Riha Threatened
Mammillaria angelensis Craig Threatened Turbinicarpus laui** Glass et Foster Threatened
Mammillaria anniana Glass et Foster Threatened Turbinicarpus lophophoroides** (Werdermann) Buxbaum et Threatened
Backeberg
Mammillaria aureilanata Backeberg Threatened Turbinicarpus mandragora** (Fri ex Berger) A Zimmerman Extremely
threatened
Mammillaria aureilanata ssp. alba (Backeberg) Glass Threatened Turbinicarpus pseudomacrochele**ssp krainzianus Glass Threatened
Mammillaria blossfeldiana Bodeker Threatened Turbinicarpus pseudopectinatus** (Backeber) Glass et Threatened
Foster
Mammillaria boolii Linsay Threatened Turbinicarpus saueri** (Bödeker) John et Riha Threatened
Mammillaria brandegeei spp glareosa (Bödeker) DR Hunt Threatened Turbinicarpus schmiedickeanus** spp flaviflorus (Frank et Highly endangered
Lau) Glass
Mammillaria crinita f zealmanniana (Bödeker) Glass Highly threatened Turbinicarpus schmiedickeanus** spp gracilis (Glass et Threatened
Föster) Glass
Mammillaria deherdtiana Farwig Extremely endangered Turbinicarpus subterraneus**(Bakeberg) A Zimmerman Threatened
Mammillaria deherdtiana ssp. dodsonii (Bravo) D Hunt Threatened Turbinicarpus valdezianus** (Möeler) Glass et Foster Threatened
Mammillaria fittkaui Glass et Foster Threatened Turbinicarpus viereckii** ssp major (Glass et Foster) Glass Threatened
Mammillaria giselae JG Martinez-Avalos et Glass Highly threatened Turbinicarpus ysabelae** (K Schlange) John et Riha Highly endangered
Mammillaria guelzowiana Werdermann Threatened Turbinicarpus zaragozae** (Glass et Foster) Glass et Hofer Highly threatened
*Red list UICN 2007, **Appendice I CITES 2007

2. CACTI PROPROPAGATION AND MICROPROPAGATION DISTINCTIVENESS

Through in vitro techniques the massive and commercial propagation of species of horticultural interest is achievable (Thorpe 1981; Teixeira da
Silva 2006), and plant biotechnology improves as scientific knowledge in genetics and plant physiology increases. Micropropagation is
considered a great tool for mass propagation, which only needs cells or a small portion of plant tissue cultured in vitro under aseptic conditions,
providing nutrients and proper photoperiod and temperature. Through tissue culture it is also possible to acquire a large and quick multiplication,
better clone quality by selection of stocks, maintenance of endangered species, a propagation plan, generation of a germplasm bank,
homogeneous and healthy plants, and only in case, cacti breeding (Boutherin and Bron 1989). Areoles are the intrinsic character of cacti that

Floriculture, Ornamental and Plant Biotechnology Volume V ©2008 Global Science Books, UK
Ramirez Serrano and Teixeira da Silva 221 Micropropagation of cactus plants

Apical Root differentiate into new plants, or plant tissues.

meristem meristem Besides, the physiological stage is under
produces produces genetic control, particularly in species that
auxins that cytokinins, present apical dominance because many
Cactus with Each bud can
maintain which can Few or
single stem areoles in a root and grow cacti species growth one single stem in their
activate single buds separately whole lives, and produce branches or cla-
or dormant state areoles at the develop from the
monopidial dodes eventually when they are damaged in
apical area mother plant
stem the main apex of growth. Farmers have
after damage
taken advantage of this phenomenon, and
have traditionally propagated species with
these characteristics by cutting the edges of
A growth (Fig. 1A). However, this is a slow
process and the amount of new individuals
that can be produced is limited; besides in
species that present discoid stems or sun-
ken apices, this method is practically impos-
Apical sible (Soltero 1996). Amongst cactus plants
dominance Multiple Each bud Multiple
Cactus with has no effects can grow there is cespitous or proliferous develop-
buds buds
proliferous ment as in some species of genus Mam-
on areoles at develop and separately develop and
or cespitous the base that from the millaria, which produce buds at the base
grow as a grow as a
stem are activated mother (Fig. 1B), which means endogenous auxin
mass mass
by cytokinins plant activity does not affect areoles at the bottom
(base) of the plant; moreover, cacti also
present branching development where indi-
B viduals grow like trees, e.g. in Stenocereus
and Opuntia genera (Fig. 1C), where apical
dominance does not exist, endogenous
cytokinin activity can activate areoles at any
part of cladodes or phylloclades. It is remar-
kable that micropropagation of cespitous or
Cactus with There is no Multiple proliferous cactus implies less complexity as
branched stem apical buds Each bud Multiple buds they naturally produce phylloclades, mean-
dominance, develop can root begin to develop ing micropropagation is facilitated and
cytokinins and grow
activate separately where the application of cytokinins syner-
areoles from the gies the endogenous cytokinins resulting in
mother multiple areole activation that will develop
plant into plants (Ramirez-Serrano and Soltero
2007). This is the main tool for cacti micro-
C propagation.
We provide a brief background of cacti
micropropagation in which previous re-
search was conducted. King (1957) reported
Fig. 1 Types of genetic growth and propagation in cactus by apical dominance. (A) Cactus with monopodic
better induction of callus in tissues from
development, apical dominance where auxins have full control of areola dormancy stage. (B) Cactus with
cespitous or proliferous development where auxins have no control on areolas at the base of stems. (C) Cactus several genera such as Nopalea, Willcoxia,
with branched development where apical dominance does no exist, where cytokinins can activate areolas at any Cereus, Pereskia and Echinoceresus in
part of cladodes. With’s medium supplemented with 20%
coconut milk and 5 mg of 2,4-D (2,4-dichlo-
rophenoxiacetic acid). Later, Sachar and
Iyer (1959) studied the effects of plant growth regulators such as the auxin IAA (indole-3-acetic acid) 2 mg /l, the cytokinin KIN (kinetin) 0.5 mg/l
and GA3 (gibberellic acid) 1 mg/l, in Opuntia dillenii that produce embryo-like structures. Later, Steinhart (1962) induced callus in different cacti
by applying 2.5 mg/l 2,4-D and 10% of coconut water. Colomas (1971) established the in vitro cultivation in Pachycereus pringlei with coconut
water. Minocha and Mehra in 1974 produced callus in Neommammillaria prolifera, by applying 10 to 20 mg/l of 2,4-D, plus 1 mg/l KIN and 20%
of coconut water. One year later, Mauseth and Halperin (1975) reported organogenesis in Opuntia polyacantha through the action of 10 mg/l of
BA (6-benzyladenine) produces shoots, 20 to 100 mg/l of GA3 develop spines, and 1 to 50 mg/l of NAA (-naphthalene acetic acid) produces
roots, however the interaction among these three regulators produced only spines. On the other hand, the first regeneration process via
organogenesis for cactus (Mammillaria woodsii) was achieved when MS medium (Murashige and Skoog 1962) was supplemented with 2 mg/l of
IAA and 2 mg/l of KIN, and the rooting occurred through the application of a commercial product and by transferring to a perlite substrate (Kolar
et al. 1976). Instead of regeneration processes, multiples studies have been performed to propagate cactus via activation of areole structures
where the somaclonal variation is likely to not be significant as it is via organogenesis (Larking and Scowcroft 1981; Machado and Prioli 1996).
Micropropagated plants should not display significant genetic variation and be obtained after several steps that define the successful
establishment of a culture, including the selection of plant material, multiplication, rooting and hardiness, plant development, and acclimatization
under greenhouse conditions (defined according to Mauseth (1979) and Johnson and Emino (1979a)), as is shown in Fig. 1 with Mammillaria

Floriculture, Ornamental and Plant Biotechnology Volume V ©2008 Global Science Books, UK
Ramirez Serrano and Teixeira da Silva 222 Micropropagation of cactus plants

geminispina. The first step, i.e. step 0 is crucial, where the plant material must be healthy and competent in order to establish a culture and
demonstrate the desired response, and most of the selected plant material must be juvenile from axillary or apical meristems (Hartmann and
Kester 1987), or from seedlings germinated in vitro (Soltero 1996; Perez-Molphe-Balch and Davila-Figueroa 2002). Step 1 is so named since it
defines the first step in the establishment of culture where the plant material is disinfected and transferred onto sterile medium, normally by
activity of sodium or calcium hypochlorite, alcohol etc., to make the surface of the explant cleaner; however the intrinsic high toxicity restricts the
application of some of these disinfectants (Arias 2002). Regarding multiplication, this step consists of uniform and multiple cloning, where
subculturing is the time needed until the next clone production, that period depending on species and process; the multiplication rate requires
containers that should be as large as the type of clone, and in the case of cacti this requires space for their multiplication (Molphe-Balch and
Davila-Figueroa 2002). Rooting and hardiness of all micropropagated cacti plantlets before adaptation under greenhouse conditions is a
desirable requirement for a high percentage of adaptation (Hurtado 1987); elongated shoots of Schlumbergera form roots in vivo treated with IBA
at 10-3 μM (Kristensen et al. 2005), however hardiness treatment (applying in vitro stress) allows autotrophic activity to develop as
photosynthesis and rooting begin to occur. The last step is adaptation under greenhouse conditions, where the fully developed plantlets or
vigorous buds will become normal autotrophic cacti plants, this step is crucial as it could be the cause of loss of the highest percentage of clones
if the temperature, humidity, sanity and substrates utilized are inadequate (George 1993). For micropropagation explants with multiple areoles
are desirable, although when cacti species have few of these structures, the regeneration process should be performed as somatic
embryogenesis; such as in Aztekium ritteri, which could only form embryo-like structures (Rodríguez-Garay and Rubluo 1992), and in Ariocarpus
retusus were obtained somatic embryos that were grafted in patterns of Pereskiopsis velutina for further development (Stuppy and Nagl 1992).
Infante (1992) described the same phenomenon in Mediocactus coccineus.
2.1. Advances in cactus micropropagation
Before achieving micropropagation, in 1976 it was reported that dormant meristems or areoles in Opuntia polyacantha could be stimulated to
grow and develop (Mauseth 1976). The same author (Mauseth 1977a), observed that cytokinins promote the formation of normal photosynthetic
leaves, whereas the giberelins induce the development of spines. Under this background cytokinins are fundamental for breaking the dormancy
of areoles, which are the physiological structures through which the main apex of growth suppresses the secondary meristem development in
monopodial stems (George 1993). Most of the reports on cacti micropropagation, such as that by Johnson and Emino (1979a) in Opuntia
polyacantha, Mammillaria elongata, Hylocereus calcaratus, Rhipsalis teres, Weberocerus biolleyi, Erythrorhipsalispilocarpa, Epiphyllum
grandiflorum and E. phyllahthus researched the stimulation in areoles by certain cytokinins in combination with other plant growth regulators
(PGRs) such as auxins, where auxin:citokinin balance per specie was a requirement. Later Vizkot and Jara (1984) propagated cacti through
axillary buds such Mammillaria carmenae with 1 mg/l of NAA and 2 mg/l of BA and M. prolifera using same regulators in concentration from 0.5
to 1 g/l, however Astrophytum myriostigma and Tichocereus spachianus were micropropagated with 5 mg IAA and 0.5 mg/l of KIN, the latter
developing monopodially, that means extremely difficult to propagate vegetatively. Starling (1985) worked with Leuchtenbergia principis that was
micropropagated with 10 mg/l of BA and 0.1 mg/l of NAA, but phylloclades did not root; Escobar et al. (1986) worked with Opuntia sp., which
formed branched stems that were well rooting with 0.05 μM of IBA (indole-3-butyric acid); Ault and Blackmon (1987), in Ferocactus acanthodes,
produced with 10 mg/l of KIN and 1 mg of NAA 6.6 shoots per explant that rooted in medium lacking PGRs; Martinez-Vazquez and Rubluo
(1989), in Mammillaria san-angelensis, utilized a combination of longitudinal explants and BA at 1 or 2 mg/l to force the development of areoles
into phylloclades, that rooted simultaneously in presence of NAA; Clayton et al. (1990) developed an applicable method for the subtribe Cactinae
by exposing shoots tips of Escobaria missouriensis, E. robbinsorum, Sclerocactus spinosior and Toumeya papyracantha, and mature tissue from
Mammillaria wrightii, Pediocactus bradyi, P. despainii, P. knowltonii, P. paradinei. P. winkleri, and Sclerocactus mesae-verdae to 22.8 μM of ZEA
(zeatin) with or without of 11.4 μM of IAA, whereby the phylloclades formed roots spontaneously on a medium lacking PGRs, and plants could
adapt to greenhouse conditions; in Mediocactus coccineus the apex of the explants was eliminated and with the simultaneous application of 4.4
μM of BA and 0.27 μM of NAA (Infante 1992) multiple phylloclades were produced, since endogenous auxin activity suppresses the activation of
areoles in Pelecyphora aseliformis (Fig. 2A). In Melocactus bellavistensis, the proliferation of buds with 5 mg/l of BA and 1 mg/l of NAA was
achieved (Hernández et al. 1993). Ortiz-Montiel and Vargas-Figueroa (1995) obtained several phylloclades in Heliocereus elegantissimus var.
elegantissimus with 0.5 to 2 mg/l of KIN and 0.5 mg/l of NAA although they did not mention the acclimatization of phylloclades; Machado and
Prioli (1996) micropropagated Cereus peruvianus by means of apical and lateral explants with 1 mg/l of BA and 1 mg/l of ANA or IAA. Giusti et al.
(2002) tested axillary shoot production and organogenesis protocols to propagate Escobaria minima, Mammillaria pectinifera and Pelecyphora
aselliformis, succesfully micropropagated by 0.54 μM of NAA and 2.27 μM of TDZ (thidiazuron), and regenerated with exception of first one by
2.27 μM of TDZ, where all types of shoots rooted. Kristiansen et al. (2005) tested temperature, photon flux density, auxin treatment (IBA) in
order to improve the in vivo rooting of Schlumbergera truncata as well propagation. It is remarkable that in studies performed in Strombocactus

Active areoles Developing buds

A B C
Fig. 2 Case 1: Activation of endogenous cytokinin by cutting the apical meristem. Areoles are stimulated to form buds in Pelecifora asceliformis which is a cactus
with monopodic development (bar = 5 mm). (A, B) Explant after 2 weeks without apical meristem. (C) Development of buds after 2 months in culture.

Floriculture, Ornamental and Plant Biotechnology Volume V ©2008 Global Science Books, UK
Ramirez Serrano and Teixeira da Silva 223 Micropropagation of cactus plants

Developing buds

A B C D
Fig. 3 Case 2: Effect of exogenous cytokinin. Areoles are stimulated by the activation of endogenous cytokinins in Turbinicarpus pseudomacrochele (monopodial stem),
and Echinocereus viridiflours (branched stem) (bar = 1 cm). (A) and (C) Explants used to start micropropagation of both species in medium supplemented with cytokinins,
where the base and roots are eliminated. (B) and (D) Buds developed after 2 months in in vitro culture.
Developing buds
Explants

A B C

Fig. 4 Case 3: Exogenous cytokinin plus endogenous cytokinin without an endogenous auxin effect. Areoles are stimulated in Turbinicarpus pseudomacrochele
which is a cactus with monopodic development (bar = 5 mm). (A) Explant divided into explants to eliminate auxin action. (B) Explants after 2 months in culture. (C)
Production of shoots on each explant.

disciformis and Turbinicarpus pseudomacrochele (Soltero 1996), Epithelantha micromeris (Velazquez-Enciso and Soltero 2001), and
Pelecyphora strobiliformis (Arias 2002), the cytokinins 2iP (N6–(2-isopentyl)adenine) and KIN were better than BA for stimulating phylloclade
production. Most of these results are summarized in Figs. 3 and 4, where exogenous PGRs neutralize endogenous hormones in order to
produce new buds.
Fay and Gratton (1992) concluded that cytokinins promote a response in cactus tissues, a conclusion that had been previously evaluated by
Nava-Esparza and Yánez (1984) in Cephalocereus senilis, although the effect of KIN and 2,4-D they achieved was not the production of plants,
but rather callus. Havel and Kolar (1983) also achieved the same effect in tissues obtained from an adult plant through a syringe or hypodermic
tool. Johnson and Emino (1979b) proposed that the concentration balance between auxins and cytokinins is specific for the induction of
phylloclades via organogenesis to each species of Cactus, where they achieved an increase of phylloclades in 4 out of 8 species studied
(Mammillaria elongate, Opuntia polyacantha, Hylocereus calcaratus and Rhipsalis teres). In the other hand was reported by Mauseth (1979) in
10 species through the effect of 1 to 10 mg/l of BA, which eliminated the apical dominance to produce axillary buds in Chamaceresus sylvestrii,
Epiphyllum hybrid, Hatiora salicornioides, Lobivia Binghamiana, Mammillaria elongate, Opuntia basilaris, O. polyacantha, Pachyceresus pringlei,
Pereskia aculeate and Selenicereus grandiflorus. However, more factors must be taken into account in order to increase the competence of cacti
micropropagation and to elucidate the main factors that determine the growth of areoles and promote shoot proliferation in the basal part as did
Erschov (1991) who developed an ex vitro method for the cultivation of cacti that consist in dividing the stem to form a top and a base, where the
top section is grafting or rooting and growing shoots from the base; later Infante (1992) performed the same strategy by in vitro culture of
decapitated shoots of Mediocactus coccineus achieving an increasing of number of proliferating shoots. In Pelecyphora aseliformis and P.
strobiliformis, Perez-Molphe-Balch and Davila-Figueroa (2002) evaluated the proliferation of explant or shoots divided transversally and could
thereby produce 128 and 136 phylloclades per explant respectively. Recently in Schlumbergera truncata, the effecto of IBA on rooting, axillary
bud growth and shoot development were evaluated under in vivo contidions, where the main effect is in rooting, however in growth and
development of shoot had negative effects (Kristiansen et al. 2005), and Ramirez-Serrano and Soltero (2007) developed a method applicable to
any cactus by eliminating endogenous auxins effect, causing synergies among endogenous and hexogen cytokinins to force multiple shoot
proliferation. The biotechnology of Schlumbergera and Rhipsalidopsis were recently reviewed by Sriskandarajah et al. (2007).
2.2. Success in acclimatization
In order to acclimatize micropropagated cacti plants, is desirable to induce rooting under in in vitro conditions in media lacking of PGRs or with
low concentration of auxin other than 2,4-D (0.1 to 0.01 mg/l), after that the micropropagated plantets are transferred to containers with peat
moss and ground-sand (1:1), or ground-sand and soil (1:1), covered with plastic bags during 2-3 weeks to avid desiccation and encourage
acclimatization, as were reported the phylloclades’ adaptation to greenhouse conditions for Soltero-Quintana (1996) in Strombocactus
disciformis and Turbinicarpus pseudomacrochele stands out; Epithelanta micromeris by Velázquez-Enciso and Soltero (2001); Turbinicarpus

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Ramirez Serrano and Teixeira da Silva 224 Micropropagation of cactus plants

schmiedickeanus by Arias and collaborators (2001), and Pelecyphora strobiliformis by Arias (2002), also Perez-Molphe-Balch and Davila-
Figueroa (2002) developed a method later for P. aselliformis. Phylloclades during the adaptation period to greenhouse conditions always regain
their characteristic shape depending on the species.

3. ANALYSIS OF PROTOCOL SUCCESS

In this review we focussed on many approaches that define the background of cactus micropropagation. However, under the same experimental
parameters differences arise from genetic differences, and this is of concern where ideally we would want to avoid this genotype effect under the
same culture conditions (Graffiths et al. 1993). Herein lies the main problem that needs to be solved in order to establish a process which is to be
widely competent over a range of genotypes (Ramirez-Serrano 2003). So, most of the processes developed for cactus plants are specific with
low competence among species as was reported by Vyskot and Jara (1984). Having this in mind, the route to improve a cactus’ response has
only been achieved in two ways: by adjusting the explant type, and by assessing the balance of PGRs in the medium. As cactus plants have
monopodial, cespitous or proliferous, or even branched stem development, only intrinsic difficulties show up in the first type (Ramirez-Serrano
and Soltero 2007), whereas proliferous and branched development give an advantage to propagate branched cacti such as Sclumbergera
(Kristiansen et al. 2005). Taking these facts into consideration, the primary hurdle is to break down apical dominance (George 1993). In most
published papers the best explants for cactus micropropagation are the apical shoots (Mauseth 1976; Clayton et al. 1990; Gay and Graton 1992),
or the tangential cutting of buds as performed by Rubluo and Martinez-Vazquez (1989), resulting in the covering generally by multiple areoles
that can develop into phylloclades if cultured under proper conditions (Mauseth and Halperin 1975; Mauseth 1979; Vyskot and Jara 1984; Fay
and Gratton 1992; Soltero 1996; Arias 2002). However, even when cutting the main apex, most of the areoles are still inactive (Infante 1992).
Perez-Molphe-Balch and Davila-Figueroa (2002) also used transversal explants in Pelecyphora asceliformis and P. strobiliformis, and tested
several treatments of cytokinins (BA) and sugar concentrations (30 or 50 g/l), establishing thus a good micropropagation process, however they
could not clarify the interaction between tested variables and the observed results, i.e. the suppression of auxin activity. Each species has its

A A B
A

A
A

D E

Fig. 5 Micropropagation in Mammillaria geminispina with cespitous development as sample of case 3 of exogenous cytokinin plus endogenous cytokinin
minus auxin effect. (bar = 1 cm). (A) Full explant (bar: 5 mm); (B) Divided explant after 4 weeks without apical meristem. (C) Development of buds after 2 months in
culture (bar: 2 cm). (D) Rooted plant before hardiness (bar: 5 mm). (E) Plants after 6 months in greenhouse conditions (bar: 2 cm).

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Ramirez Serrano and Teixeira da Silva 225 Micropropagation of cactus plants

specific PGR requirements as observed in most published papers from Johnson and Emino (1979b) to Kristiansen et al. (2005) in which apical
dominance is partially broken by most of the areoles of the explant which remains dormant. Several cytokinins (BA, KIN, Zea or 2iP) were
probed, these being the main PGRs applied with a very low auxin (IAA, NAA or IBA) concentration in order to balance mitosis and cell elongation,
i.e. implying phylloclade development (Vyskot and Jara 1984; Ault and Blakmon 1987; Rubluo and Matinez-Vazquez 1989; Clayton et al. 1990;
Hernandez et al. 1993; Ortiz-Montiel and Vargas Figueroa 1995; Perez et al. 1995; Ortiz-Montiel and Alcantar 1997; Velazquez and Soltero
2001; Arias 2002). In contrast, species with monopodial development are not completely recalcitrant, but mostly of them show a low response in
vitro such as Strombocactus disciformis (Soltero 1996), Epithelantha micromeris (Velazquez and Soltero 2001), Pelecyfora strobiliformis (Arias
2002) wherein the main restriction is the endogenous auxin that is controlling the main area of each explant inhibiting the activity of endogenous
and hexogen cytokinins (Ramirez-Serrano and Soltero 2007). Actually, all cacti can survive damage independent of the type of development
(monopodic, proliferous or branched), and this characteristic can be enhanced by sizing the explants to allow endogenous cytokinin activity in
the upper part and activity of hexogen cytokinins at or below area that is inserted into the culture medium (Ramirez-Serrano and Soltero 2007)
(Fig. 5) allowing multiple shoot proliferation from 80 to 100% depending on the quantity of areoles, subcultures and species.

4. CONCLUSIONS
Specific methods have been developed in order to propagate highly demanding cactus plants. A widely competent method is applicable to the
Cactaceae family in which clones can remain stable since micropropagation by stimulation of areoles does not promote significant somaclonal
variation. The unique limitation is the quantity of areoles per plant utilized; this in turn has genetics implications. Cloning by seeds can be a tool
for their breeding, eliminating segregation in the progeny. This practice can become available as a result of advances in cacti physiology.
Incorporating mutants can also contribute to developing new cultivars from varieties that are locally adapted, increasing the diversity of cacti
ornamental plants.
ACKNOWLEDGEMENTS
Carlos Ramirez Serrano thanks the Program for Support of Scientific Research of the Universidad de Guadalajara P3E 2005 and 2006, projects 32285 and 49948,
respectively for providing research grants.

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