Journal of Horticultural Science

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In-vitro mass propagation of the near-extinct

Mammillaria san-angelensis Sánchez-Mejorada

O. Martínez-Vázquez & A. Rubluo

To cite this article: O. Martínez-Vázquez & A. Rubluo (1989) In-vitro mass propagation of the
near-extinct Mammillaria san-angelensis Sánchez-Mejorada, Journal of Horticultural Science, 64:1,
99-105, DOI: 10.1080/14620316.1989.11515933

To link to this article: https://doi.org/10.1080/14620316.1989.11515933

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Journal of Horticultural Science (1989) 64 (1) 99-105

In-vitro mass propagation of the near-extinct Mammillaria

san-angelensis Sanchez-Mejorada *
By 0. MARTINEZ-VAZQUEZ and A. RUBLUOt
Plant Tissue Culture Laboratory, The Botanical Garden of the Institute of Biology, U.N.A.M.
Mexico 04510 D.F., Mexico

SUMMARY
The genus Mammillaria includes many valuable species some of which are near to
extinction. The beautiful white-spined M. san-angelensis has been reported to be extinct.
Using the related species M. haageana in preliminary trials, the in-vitro mass propagation
from young seedlings of the near-extinct M. san-angelensis has been achieved. Mor-
phogenetic responses in M. haageana and M. san-angelensis were similar. The results
were influenced mainly by two factors: the presence of the cytokinin 6-benzyladenine
(BA) and the origin of the explant. Callus formation rarely occurred; however, shoots
and multiple shoots were obtained when tip (apical) explants were exposed to different
concentrations of BA and naphthalene acetic acid (NAA). Only multiple shoots were
apparent when lateral explants were exposed to similar experimental conditions. Mass
regeneration was most successful when lateral explants were subjected to three treat-
ments: BA alone at 0.1 mg J- 1 ; BA 0.1 mg J- 1 combined with NAA at 0.01 and BA
1.0 mg J- 1 ; or in combination with NAA 0.01 mg J- 1 •

THE genus Mammillaria contains many species
valuable to amateur and professional cactus
collectors. Because of the great demand for
these attractive plants, many Mexican species
have been over-collected so that many mem-
bers of the genus Mammillaria are in danger of
becoming extinct (Sanchez-Mejorada, 1982;
IUCN, 1985). The beautiful white spined M.
san-angelensis Sanchez-Mejorada has been
described (Sanchez-Mejorada, 1981) as
endemic to the Pedregal de San Angel area in
southern Valley of Mexico. Recently it was
reported almost extinct as a result of over-col-
lection, and to the expansion of Mexico City
during the past 50 years. The author regrets
'The fact that in the National Herbarium of the
University of Mexico there is only one single
specimen of this species that grew so freely in
the campus area'. In the IUCN endangered
cacti list for Mexico (IUCN, 1985), this plant
was reported to be extinct.
A desirable action for safeguarding cacti is to
improve, in any way possible, propagation
techniques. During the last few years, plant
tissue culture has emerged as a powerful tool
for the effective propagation of many species.
Compared with other taxonomic groups, the
Cactaceae have attracted little attention from
those concerned with plant tissue culture
(Narayanaswamy, 1977), although since 1962
several workers have achieved varying degrees
of success (Steinhart, 1962; Kolar et at., 1976;
Johnson and Emino, 1979; and Krulik, 1980),
and recently there have been encouraging
results (Starling and Dodds, 1983; Starling,
1985).
The objective of this investigation was to
study the morphogenetic responses of in-vitro
cultured explants of young seedlings of the
near-extinct M. san-angelensis as influenced by
growth regulators. A major goal was to attempt
mass propagation as a basis for an effective
recovery programme.

• Paper dedicated to Dr Helia Bravo-Hollis on the occasion MATERIALS AND METHODS

of her Honoris Causa doctorate from the Nat10nal Umver-
slly Autonomous of Mexico. An Ecological Reserve within the National
t To whom requests for repnnts should be addressed University of Mexico protects five plants of M.
100 In-vitro propagation of Mammillaria
san-angelensis. Fifteen seeds from these speci-
mens were provided for this study.
It is known that the in-vitro responses of
explants from different plants is in most cases
highly specific, even at the variety level
(Rubluo and Kartha, 1985) particularly in the
genus Mammillaria (Johnson and Emino,
1979). To conserve the scarce supply oJ M. san-
angelensis seeds, preliminary experiments were
carried out with the phylogenetically closely
related species M. haageana (Bravo-Hollis,
pers. comm.) for which adequate supplies of
material were available.
Preliminary trials with Mammillaria haageana
Seeds of M. haageana, kindly donated by the
Cactaceae Laboratory of the Botanical Garden
of the National University of Mexico, were
sterilized in 6% sodium hypochlorite for 20
min, washed at least three times in sterile
double-distilled water and cultivated asep-
tically in hormone-free MS medium
(Murashige and Skoog, 1962) with 3% sucrose.
The pH of the medium was adjusted to 5.8 prior
to adding the agar and the medium autoclaved
for 15 min at l20°C. Seeds were incubated
under three different conditions: (a) continu-
ous light (3.2 wm-2, (b) darkness and (c)
photoperiod of 16 h light (3.2 Wm- 2) and 8 h
darkness. In all the cases the temperature was
27°C ± 1. Although germination occurred in all
these conditions, best results were obtained
with the photoperiod (c). After five months the
M. haageana seeds developed to plantlets
(5-7 mm long) and were ready to be used as a
source of explants, using two methods of pre-
paring the explants for in-vitro culture
(Figure 1).
The M. haageana explants were sown on MS
medium with some added growth regulators:
6-benzylaminopurine (BA); kinetin (kin) and
2,4 dichlorophenoxyacetic acid (2.4-D), and
then incubated (see Table I for details). The
conditions which gave the best plantlet yield
with M. haageana were chosen for the experi-
ments with M. san-angelensis.
Experiments with Mammillaria san-angelensis
Fifteen seeds of M. san-angelensis were care-
fully sterilized in sodium hypochlorite solution
and washed three times in sterile double-dis-
tilled water. They were then grown individually
in test tubes to diminish the risk of loss due to
contamination with hormone-free MS medium,
3% sucrose and incubated at constant 27°C ± 1
and a photoperiod of 16 h light (3.2 Wm- 2).
From the original15 seeds of M. san-angelen-
sis, eight germinated and all grew into seed-
lings. After six months these original seedlings,
5-7 mm tall, were ready to be used as explant
sources and were prepared as in Figure 1, B.
Under aseptic conditions the explants were cut
and separated into tip and lateral segments.
They were inoculated onto MS medium which
included 1.0 mg 1- 1 BA, and finally incubated
under the same temperature and photoperiod.
From these preliminary trials sufficient biologi-
cal material was obtained for experiments on
mass proliferation induction.
TheM. san-angelensis explants were exposed
to various concentrations of BA and NAA (see
Tables II and Ill for details). All results were
recorded after four months in culture.
RESULTS AND DISCUSSION
Experiments with Mammillaria haageana
Table I summarizes the results obtained with
M. haageana. In general the response was poor
and in most cases there was no growth after four
months in culture. However, in some of the
experimental conditions there were mor-
phogenetic responses. The best media for
regeneration contained only BA (1.0 and
2.0 mg 1- 1). The source of the explant was also
important and good results were obtained for
type B especially with the lateral explants.
Apart from these findings, morphogenetic
responses were poor in this experiment
(Table I).
Initiation of callus is generally due to tissue
damaged in in vitro culture, particularly under
the influence of auxins (Steinhart, 1962;
Minocha and Mehra, 1974; and Silverstein et
at., 1983). In our results callus formed only
rarely (Table I). These results are probably due
to the absence of auxins in most cases, although
profuse callusing occurred in the presence of
BA (Table I). Similar results were reported by
Cheema and Mehra (1985) who failed to induce
callus using several auxins and cytokinins on
young cactus seedlings. However, an actively
growing callus was obtained in M. haageana
when 2.4-D was present especially in type B
 0 . MARTfN EZ-VAQU EZ and A. Ruswo 101

..... t,.
'•'

..
1

5
FIG . 1
T he source of expl ants forM. haageana . T = tip (apica l) , L = lateral.

FIGS.2-6
Multiple shoot fo rmatio n from la teral expla nts o f M. san -angelensis (ba r = 10 mm ) , incubated at 27°C 16 h light (3.2 W m- 2)
and 8 h darkness , 4 months in culture . FIG. 2 Me dium cont ainin g: 10 mg 1- 1 BA . Note the compact ca lli . FIG. 3 Me di um
containing: 0.0 1 mg I- 1 BA and 0.0 1 mg I- 1 NAA. Note that calli exist o nly on the base a nd very scarce . FIG. 4 Medium
containing: 0. 10 mg I- 1 BA and 0.10 mg 1- 1 NAA. FIG . 5 Medium containing: 1.00 mg I- 1 BA and 0.0 1 mg I- 1 NAA . No te that
call i is not appare nt but it was present on the base and very scarce. FIG. 6 Medium co ntaining: 1. 00 mg I- 1 BA a nd 1.00 mg I- 1
NAA . No te the presence o f buds (less than 5 mm) .
102 In-vitro propagation of Mammillaria

TABLE I
The effect of growth regulators on the morphogenetic response of several sources of explants of Mammlllana haageana cultured
at 27'C and a 16 h photopenod

Growth Response
Source of regulators Differentiation multiple
explant<•! (mgll) Callus single shoots shoots<•!

A BA
tip 1.0 9/18
a 1.0 no growth, explants turned purple
b 1.0 no growth, explants turned purple
c 1.0 no growth, explants turned purple
tip 2.0 3/18 no growth, explants turned purple
a 20 no growth, explants turned purple
b 2.0 no growth
c 2 0 2,40 +kin no growth
Up 1.0 + 4 0 18/18 yellow1sh, profuse calh
a 1.0 + 4.0 no growth, explants turned purple
b 1.0 + 4.0 no growth, explants turned purple
c 1.0 + 4.0 6/18 greening, friable calli

B BA
tip 1.0 12/18
Ll 1.0 6/18
L2 1.0 9/18
Up 2.0 6/18
Ll 2.0 18/18 3/18 profuse, friable calli
L2 2.0 2,4-D + kin no growth
Up 1.0 + 4.0 12/18 profuse, friable calli
Ll 10 + 4.0 12/18 profuse, friable calli
L2 1.0 + 4.0 15/18 profuse. fnable calli

(a) See Matenals and Methods and F1gure I for source of explants.
(b) Based on six plants per treatment with three replications after four months' culture

explants (Table I). These results are in agree- lations of vigorous callus was influenced by
ment with those of other authors using various 2,4-D as a growth regulator. Minocha and
cacti. Mehra (1974) and Johnson and Emino (1979)
Steinhart (1962) reported that the stimu- found that optimal callus proliferation occurred
TABLE II
Morphogenetic responses of aptcal explants'"' ofMammallana san-angelensis cultured at 27'C photoperwd exposed to different
concemratwns of BA and NAA

Growth regulators (mg/1)

% differentiatiOn responses<•>
BA NAA single shots multiple shoots<'!
0 01 0 00 60 40 (*) malformed buds<dl
010 0.00 20 (*) muluple buds
10 00 0.00 20 very slow growth
0.01 0 01 40 (**) formation of roots
0 10 0.01 100 (**) vigorous shoots
1.00 0.01 100 (****) multiple buds, long roots
10.00 0.01 very slow growth
0.01 0.10 20 some roots
0.10 0.10 40
I 00 0.10 60 20 (**) root formation multiple buds
10.00 0.10 20 20 (*) very slow growth
0.01 1.00 60 40 (*) abundant roots
0.10 1.00 20 40 (***) few roots
1.00 1.00 40 (***) multiple buds
10.00 1.00 20 few roots

(a) Explant type B (see Matenals and Methods)

(b) Based in five plants per treatment With three replications after four months' culture.
(c) Number of shoots per explant with at least 5 mm· 3-7 (*), 8-12 (**); 13-20 ('**); 21-35 (****).
(d) Buds are less than 5 mm.
 0. MARTfNEZ-VAQUEZ and A. RuBLUO 103

TABLE III
Morphogenetic responses of lateral explants!•J ofMammallaria san-angelensis cultured at 27"C with a 16 h photoperiod exposed
to different concentrations of BA and NAA

Growth regulators (mg/1) DifferentiationlbJ

BA NAA Callusi'l Multiple shoots idJ

O.Ql 0.00 100 (*) malformed budsl<l

0.10 0 00 100 (**) 100 (****) multiple buds
10.00 0.00 90 (*) 90 (*) green, compact calli
0.01 0.01 30 (*) 50 (**) formation of roots
0.10 0.01 100 (***) very vigorous shoots
1.00 0.01 100 (*) 70 (****) multiple buds, long, strong roots
10 00 0.01 90 (*) 20 (*) slow growth, green, friable calh
0.01 0.10 30 (*) 80 (**) root formation
0.10 0 10 40 (*) 70 (***) green, fnable calli
1 00 0.10 80 (*) 80 (***) green, friable calh, roots
10.00 0.10 40 (*) 40 (*) slow growth
0.01 1.00 40 (*) 80 (**) slow growth, abundant roots
0.10 1.00 60 (*) 60 (**) few roots
1.00 1.00 80 (*) 80 (***) multiple buds
10.00 1.00 70 (*) 10 (*) few roots
(a) Explant type B (see Matenals and Methods).
(b) Based in five plants per treatment with three rephcauons.
(c) Calh: poor(*); abundant(**); nil(-).
(d) Number of shoots per explant with at least 5 mm: 3-7 (*): 8-12 (**); 13-20 (***); 21-35 (****).
(e) Buds are less than 5 mm.

in response to 2,4-D combined with kinetin.
However, in our results the origin of the explant
also influenced the proliferation of callus
(Table I). Few shoots were produced in M.
haageana (single or multiple) (Table I), but a
satisfactory response was recorded in explant
type B with BA at 1.0 and 2.0 mg 1-I, especially
when lateral explants were exposed to
1.0 mg 1- 1 BA, which produced multiple shoots
and a good yield of shoots per explant.
In view of these results an investigation of
effects of BA at several concentrations, and in
combination with NAA was attempted with the
near-extinct M. san-angelensis. The combina-
tion of BA with NAA is known to give good
morphogenetic responses in a wide range of
families (Evans, 1981). The choice of explants
for M. san-angelensis was also based on results
with the related M. haageana as mentioned
previously.
Experiments with Mammillaria san-angelensis
When explants type 8 of the eight young
seedlings of M. san-angelensis were cultured in
the presence of 1.0 mg 1- 1 BA, under environ-
mental conditions similar to those previously
described, single shoots were obtained in tip
explants (30%) and in laterals, callus produc-
tion with multiple shoots (40%, 21-35 shoots
per explant) was recorded. These tests pro-
vided enough material to conduct mass propa-
gation experiments with M. san-angelensis.
Results in Tables II and III show the mor-
phogenetic responses after four months of
incubation of the selected explants of M. san-
angelensis, and the major differences between
the two types of cultured explants. When tip
(apical) explants were exposed to the culture
media, there was no callus formation and plant
regeneration was recorded as single and mul-
tiple shoots (Table II). However, when lateral
explants were incubated in the same condi-
tions, callus formation occurred in almost all
the tested situations, although it was usually
poor (Table III). The dominant response was
multiple shoot formation with varying degrees
of yield, and in this case there were no single
shoots (Table III).
It has been proposed that, within the Cac-
taceae (Krulik, 1980; Vyskot and Jara, 1984;
Cheema and Mehra, 1985) mechanical severing
of the shoot apex breaks or weakens apical
dominance and axillary buds can be activated to
form new buds. This effect is probably due to a
change in hormone balance within the plant
allowing dormant buds to grow (Krulik, 1980).
This observation explains our results in
Tables II and III. However, in some cases even
in the presence of the shoot apex (tip explant),
there was some multiple shoot formation
104 In-vitro propagation of Mammillaria

(Table II); this can probably be attributed to
the explants being from young seedlings, and
that the axillary buds could proliferate in new
shootlets giving clusters with multiple shoots.
Similar results have been obtained by Starling
and Dodds (1983) in M. glassii and by Starling
(1985) with Leuchtenbergia princips.
Our results were striking when using lateral
explants (Table III); apical dominance disap-
peared completely and consequently all the
explants produced multiple shoots, with vary-
ing degrees of efficiency (Figures 2-5).
In some hormone combinations and in both
types of explants, multiple buds (shorter than
5 mm) were recorded (Figure 6) suggesting a
great potential for improving the final yields of
shoots. As a consequence of the presence of
NAA, root formation appeared in some shoots.
Similar results were reported by Mauseth and
Halperin (1975) on Opuntia, but in our results
there was no clear physiological connection
between the induced root and the shoots.
The effect of BA on the capacity to induce
plant regeneration in members of the Cac-
taceae has been reported previously (Mauseth
and Halperin, 1975; Maushet, 1976; Starling
and Dodds, 1983) and the combined action of
BA and NAA has also proved successful for
plant regeneration (Lazarte et al., 1982) Star-
ling and Dodds, 198;3; Vyskot and Jara, 1984;
Starling, 1985). Our results (Tables II and III)
agree with all these authors.
Mammillaria has been successfully explored
for in vitro plant regeneration: M. wooedssii
(Kolar et al., 1976), M. elongata (Johnson and
Emino, 1979), M. glassii (Starling and Dodds,
1983), and M. carmenae and M. prolifera
(Vyskot and Jara, 1984). Johnson and Emino
(1979) working with several species of
Mammillaria cultured in the same conditions
found that results were very variable: ranging
from totally successful plant regeneration (M.
elongata) to no responses (M. eichlamii). Their
results suggested that each Mammillaria
species might require a specific and critical
auxin-cytokinin ratio for specific responses. In
our case closely similar results were recorded
forM. haageana and M. san-angelensis (Tables
I, II and III) allowing the efficient micro-
propagation of the near-extinct M. san-angelen-
sis. Moreover, the population obtained through
in vitro tissue-culture will allow the reintroduc-
tion of this species into its natural environment.
This study was supported in part by a grant
from Consejo Nacional De Ciencia y Tec-
nologia (CONACyT) PCCBBNA-021701. The
authors wish to thank Dr Graham Pattison and
Dr Robert Bye Jr for revising the paper, and Mr
Vfctor Chavez and Mrs Carmen Loyola for
excellent technical assistance.

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(Accepted 1 July I988)