Regeneration and propagation of Ariocarpus retusus Scheidw.

(Cactaceae) via somatic embryogenesis

Author(s): Wolfgang Stuppy & Walter Nagl
Source: Bradleya, 10():85-88.
Published By: British Cactus and Succulent Society
https://doi.org/10.25223/brad.n10.1992.a7
URL: http://www.bioone.org/doi/full/10.25223/brad.n10.1992.a7

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Regeneration and propagation of Ariocarpus retusus Scheidw.
(Cactaceae) via somatic embryogenesis
Wolfgang Stuppy & Walter Nagl
Department of Biology & Biotechnology Program,
University of Kaiserslautern, W-6750 Germany
Sum m ary. Seeds of Ariocarpus retusus Scheidw. were
germinated under sterile conditions on solid Murashige and
Skoog (MS) medium containing 20 g/1 sucrose and 20% (v/v)
coconut water. The seedlings produced callus on the same
medium, and after 3-4 m onths the first somatic embryo
appeared. However, massive production of pale yellow somatic
embryos started only after 14—16 months of culture. Calli were
subcultured on to fresh medium every eight weeks. For
subculture, the same medium was used but with only 15% (v/v)
coconut water. For further growth, somatic embryos were
placed on hormone-free MS medium or Schenk & Hildebrandt
(SH) medium. The seedlings could then be grafted on
Pereskiopsis velutina for further development.
Zusammenfassung. Samen von Ariocarpus retusus Scheidw.
wurden unter sterilen Bedingungen auf festem M urashige und
Skoog (MS)-Medium, das 20 g/1 Saccharose und 20% (v/v)
Kokos-Milch enthielt, gekeimt. Die Keimlinge produzierten auf
diesem Medium Kallus, und nach 3-4 Monaten erschienen die
ersten somatischen Embryonen. Die massive Produktion von
hellgelben somatischen Embryonen begann allerdings erst nach
einer K ulturdauer von 14—16 Monaten. Die Kalli wurden alle
acht Wochen auf frisches Medium passagiert. Fur diese
Subkulturen wurde das gleiche Medium verwendet, allerdings
nur m it 15% (v/v) Kokos-Milch. Fur die weitere Entwicklung
wurden die somatischen Embryonen auf hormonfreies MS-
Medium oder Schenk & Hildebrandt (SH)-Medium iiberfuhrt.
Die Keimlinge konnten dann zum weiteren W achstum auf
Pereskiopsis velutina gepfropft werden.
In trod u ction
Cacti are usually propagated by seeds and rooted
offshoots. These conventional methods of propagation
are not always satisfactory, especially for threatened or
endangered species that are very often slow-growing
and produce ju st a few or even no offshoots. Because
these species are mostly very attractive to hobbyists,
there is a great danger of extinction through illegal
collection of wild plants. New methods of propagation
could help to lim it the trade in imported plant
material. Tissue culture techniques offer an oppor­
tunity to propagate rare cacti much faster than
conventional propagation methods can. There are three
basic morphogenetic processes by which in vitro
multiplication of plants can be accomplished:
Accepted for publication: 15 Aug. 1991
Bradleya 10/1992
Bradleya 10/1992
pages 85-88
a) production of m ultiple shoots from existing
meristem s,
b) production of adventitious shoots from callus,
c) somatic (asexual) embryogenesis in callus or
suspension cultures.
Most publications on tissu e culture of cacti describe
the formation of m ultiple shoots from existing apical or
axillary meristem s (Mauseth 1979, Lazarte et al. 1982,
Vyskot & Jara 1984, Starling 1985, Escobar et al. 1986,
Ault & Blackmon 1987). Organogenesis has been
observed from callus cultures of M am m illaria elongata
var. tenuis (Mehra & Cheema 1980), M. woodsii (Kolar
et al. 1976), M. elongata (Johnson & Emino 1979) and
Echinocereus sp. (Hutabarat 1986).
This report describes the formation of somatic
embryos in callus cultures of Ariocarpus retusus
Scheidw., a threatened species from north-eastern
Mexico, which is included in Appendix I of the
Convention on International Trade in Endangered
Species (CITES). According to our knowledge this is the
first report of somatic embryogenesis in the Cactaceae.
M aterials and m ethod s
Seeds of Ariocarpus retusus Scheidw. (obtained from
Mesa Garden, Belen, New Mexico) were surface
sterilized with 70% ethanol, thoroughly washed with
sterile distilled water and germinated aseptically in
baby food jars on solid MS medium (Murashige &
Skoog 1962) containing 20 g/1 sucrose, 10 g/1 agar and
20% (v/v) coconut water. The pH of the medium was
adjusted to 5.9. For callus production, seedlings were
left on the sam e medium for 4 months. After that time
the callus cultures were subcultured every 8 weeks on
to the sam e medium but with only 15% (v/v) coconut
water. For germination, somatic embryos were placed
on both hormone-free MS medium and SH medium
(Schenk & Hildebrandt 1971). The cultures were grown
under constant environmental conditions (27°C, and a
16 hour photoperiod of 1200 lux illum ination by cool-
white fluorescent tubes).
R esu lts
After germination, the seedlings became swollen and
quickly grew into pea-like bodies; finally the epidermis
85
burst and the growth of green callus started. After 3 -4
months of culture the first somatic embryo appeared,
showing the two characteristic cotyledons. These
somatic embryos reached a size of about 5 mm and
then also burst and produced callus. During the first 12
months in culture only a few somatic embryos appeared
on this callus. They all burst and again formed callus
when they reached a size of more than 5 -7 mm.
After about 14-16 months the calli started to
produce many small somatic embryos, often clustered
together (Fig. 1). In contrast to the few early, green
somatic embryos described above, the later ones were
formed in large numbers and were pale yellow in colour
in their early stage. The embryos remained attached to
the calli at a site close to the radicle. Later the
embryos formed green ball-like structures, 3 -4 mm in
diameter, which som etim es already showed areoles
with spines. A very conspicuous feature was the
frequent appearence of more than the regular two
cotyledons (Fig. 2). Four or even five cotyledons could
be observed (seedlings grown from seeds sometimes
exhibit three cotyledons).
If the pale yellow somatic embryos were removed
from the callus and placed on hormone free MS-
medium or SH-medium, they behaved like sexual
embryos that had germinated from a normal seed.
After two days they started to produce a hairy tuft
around the base, as is typical for a young cactus
seedling (Fig. 3). When a cluster of somatic embryos
w as placed on hormone-free medium together with the
adjacent part of the embryogenic callus, the formation
of somatic embryos continued (Fig. 4).
However, in most cases the somatic embryos did
not undergo a normal development on the hormone free
medium. Rather, they became green and burst again,
and then produced callus tissue within 3 -4 weeks. A
few somatic embryos, however, went dark green and
produced a normal root system. Efforts to establish
them in soil are being attempted.
The somatic seedlings can be easily grafted on
Pereskiopsis uelutina. (Fig. 5). This method greatly
speeds up their further development.
D iscu ssion
The present study shows that, in Ariocarpus retusus,
the growth of callus and the formation of somatic
embryos can be initiated by supplementing the culture
medium with coconut water (the liquid endosperm of
the seeds of Cocos nucifera L.). Coconut water contains
a number of (in part little analyzed) components,
among them growth-promoting substances, especially
the cytokinins zeatin and zeatin riboside (van Staden
& Drewes 1975). For basic studies of tissue cultures,
such complex and undefined media should be avoided,
but in many cases the effect of coconut water cannot
yet be replaced by certain concentrations and
combinations of defined phytohormones. Therefore,
coconut water is frequently used in cactus callus
cultures (King 1957, Steinhart 1962, Colomas 1971,
Minocha & Mehra 1974, Mehra & Cheema 1980).
Regeneration via a long period of callus stage
(organogenesis or somatic embryogenesis) may lead to
spontaneous genetic mutations, called somaclonal
variation (Larkin & Scowcroft 1981, Lee & Phillips
86
1988). Therefore, plants which originated from long­
time callus cultures are often not genetically identical.
In contrast, plants regenerated in vitro from apical or
axillary meristems, as well as plants produced by short
term somatic embryogenesis, are considered to be
genetically rather stable (Hu & Wang 1983, Gray &
Purohit 1991). These techniques allow, therefore,
micropropagation of cacti with a high degree of
phenotypic uniformity (Mauseth 1979). Therefore, they
are valuable for commercial aspects, e.g. the trade with
rare species, as they can be used to produce m ultiples
of a single genotype at low costs. The fact that early
maturation of the somatic embryos is often
circumvented as they disorganize to form more
proembryonal callus tissue, and then develop multiple
embryos again, represents an additional advantage for
reducing culture time and costs. The possibility of
grafting somatic seedlings of Ariocarpus retusus on
Pereskiopsis velutina for rapid development, represents
an additional plus in cactus seed technology.
Future experiments will show whether somatic
embryos of Ariocarpus retusus can also be transferred
to soil for further growth and development.
A cknow ledgm ent
We thank Mr Reiner Voss for valuable help with the
macrophotographs.
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Fig. 1. Group of somatic embryos on a piece of callus 14 months old of Ariocarpus retusus.

Fig. 3. Somatic embryos on hormone free medium showing

Fig. 2. Somatic embryo, showing four cotyledons. the characteristic hairy tuft around their base.

Bradleya 10/1992 87
Fig. 4. Embryogenic callus of Ariocarpus retusus on hormone free medium, displaying continuous formation of somatic embryos.

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88 Bradleya 10/1992