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Micropropagation of Turbinicarpus laui Glass et Foster, an endemic and

endangered species

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Micropropagation of Turbinicarpus laui Glass Et Foster, an Endemic and Endangered Species
Author(s): Martín Mata Rosas , Mario Alberto Monroy De La Rosa, Katja Moebius Goldammer,
Víctor M. Chávez Avila
Source: In Vitro Cellular & Developmental Biology. Plant, Vol. 37, No. 3 (May - Jun., 2001), pp.
400-404
Published by: Society for In Vitro Biology
Stable URL: http://www.jstor.org/stable/4293481
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In Vitro Cell. Dev. Biol.-Plant 37:400-404, May-June 2001 DOI:10.1079/IVP2000156
? 2001 Society for In Vitro Biology
1054-5476/01 $10.00+0.00

MICROPROPAGATION OF TURBINICARPUS LAUI GLASS ET FOSTER, AN ENDEMIC AND ENDANGERED

SPECIES

MARTINMATAROSAS1"2,MARIOALBERTOMONROYDE LA ROSA', KATJAMOEBIUSGOLDAMMER',

AND VICTORM. CHAVEZAVILA'*

'Laboratorio de Cultivo de Tejidos Vegetales, Jardin Botdnico del Instituto de Biologia, Universidad Nacional de MWxico,Mixico,
Autdnoma
D.F. 04510
2Jardin Botdnico Clavijero, Instituto de Ecologia, A.C., Mixico

(Received 22 April 1999; accepted 16 November2000; editor R. H. Smith)

SUMMARY

In a short period of time, a large number of adventitious shoots were regenerated from longitudinal sections of in vitro-
germinated seedlings of the endangered Mexican cactus, Turbinicarpus laui. The induction medium consisted of
Murashige and Skoog salts, supplemented with 6-benzylaminopurine (BA) and oL-naphthaleneacetic acid (NAA) in a wide
range of combinations. The most effective concentrations of growth regulators for shoots initiation were 8.8-13.32 VpMBA
with 0-2.68 pM NAA. After 3-4 mo., individualized shoots were rooted on half-strength Murashige and Skoog medium
and then transferred to soil to acclimatize under greenhouse conditions. In vitro strategies play a key role in the
conservation and propagation of this commercially important endangered species of cactus.

Key words: cacti; tissue culture; organogenesis; conservation.

INTRODUCTION
Turbinicarpus laui Glass et Foster (Cactaceae) is an endemic
species restricted to a small area of central Mexico in scattered
populations (Fig. la). Due to its high ornamental value, individuals
and seeds are illegally collected from the wild populations. Its
critical situation has been recognized by the Mexican government,
and it is classified as a threatened species (N.O.M., 1994; Vovides
et al., 1997), and also listed in Appendix I of CITES (Franco
Martinez, 1997), the most restrictive listing status. Fragmentation of
habitat, reduced population size and isolation could lead to a fast
loss of genetic variability (Wright, 1969, cited by Ledig et al., 1997)
and eventually the extinction of T. laui.
Micropropagation is widely recommended as a biotechnological
tool to study and conserve endangered species (Bonness et al.,
1993; Carneiro et al., 1999). Two comprehensive reviews about in
vitro propagation of cacti were published by Hubstenberger et al.
(1992) and Fay and Gratton (1992). There are a number of reports
on in vitro regeneration initiated from aseptically germinated
seedlings that include: Ariocarpus trigonus (Web.) K. Schum. and
Leuchtenbergia principis (Hook.) (Starling and Hutson, 1984),
Cephalocereus senilis (Haw.) Pfeiff. (Corona and Yafiez, 1984),
Astrophytum asterias (Zucc.) Lem. (Starling, 1985), Mammillaria
san-angelensis Sinchez Mejorada (Martinez-Vazquez and Rubluo,
1989), Aztekium ritteri (Boed.) Boed. (Rodriguez-Garay and Rubluo,
1992), Ariocarpus retusus Scheidw. (Stuppy and Nagl, 1992; Olguin,
1994), and 18 species of several other genera (Coryphantha,
Echinocactus, Echinocereus, Ferocactus, Mammillaria, Stenocactus)
*Authorto whomcorrespondenceshould be addressed:Email victorm@
ibiologia.unam.mx
(P6rez et al., 1998). As far as we know, there are only two studies,
cited by Fay and Gratton (1992) for the genus Turbinicarpus: a
personal communication (with Ronse) referring to T. lophophoroides
(Werd.) Buxbaum et Backbg. and in vitro propagation of T.
pseudomacrochele (Backeberg) Buxbaum et Backbg. at the Royal
Botanic Gardens, Kew.
In the present study, we describe the micropropagation of T. laui
from sections of in vitro-germinated seedlings and their establish-
ment in soil.
ANDMETHODS
MATERIALS
Adult individualsof Turbinicarpus laui confiscatedfromillegal collectors
by the Mexicangovernmentwere incorporatedinto the Botanical Garden's
collection at the National University of Mexico. The seeds that were
producedunder greenhouseconditionsfor some individualswere surface-
sterilizedin a 30% (v/v) commercialbleach solution (sodiumhypochlorite),
containing two drops of Tween 80 100 ml for 30 min. Seeds were rinsed
twice with sterile distilled waterand sown in 125 ml wide-mouthjars (three
seeds per jar; 10 jars per treatment)containing25 ml of culture medium.
Three different media were employed for the germinationof seeds: (1)
inorganicsalts and vitamins of MS (Murashigeand Skoog, 1962), glycine
2 mg 1-1, myo-inositol100 mg 1-1, sucrose 30 g 1-1 (basal MS medium);
(2) modifiedMS (MMS)consistedonly of the inorganicsalts of MS, without
organic compoundsand only 10 g 1-1 sucrose; (3) half-strengthbasal MS
medium(MS 50%). Results were recordedat weekly intervalsfor 5 wk.
Five-week-old in vitro-germinatedseedlings were dissected into two
longitudinal sections (explants) under sterile conditions. Explants were
cultured on basal MS medium with all possible combinations of 6-
benzylaminopurine(BA; 0, 2.2, 4.4, 8.8, 13.32 1PM)and ot-naphthalene-
acetic acid (NAA;0, 0.54, 2.68 jLM)(inductionmedia).
Each treatment consisted of two explants (coming from different
individuals)per jar, with three repetitions. Cultureswere maintainedfor
1 mo. on the inductionmedia, and then transferredto basal MS medium.
In all experiments,nutrientmedia were adjusted to pH 5.7 with 0.1 N

400
 MICROPROPAGATION OF TURBINICARPUS LAUI 401

FIG. 1. (a) Flowering mature plant of T. laui. (b) Organogenic callus from sections of in vitro-germinated seedlings. (c) Multiple shoot
formation from callus, 8 wk after induction. (d) In vitro rooting of individualized shoots. (e) Shoots rooted in vitro in half-strength MS
medium. (f) Plants of T. laui generated in vitro and growing under greenhouse conditions. Bar = 1 cm.

NaOH and 0.1 N HC1, and 8.5 g 1-F bacto-agar was added before
autoclaving at 105 kPa for 15 min at 121'C. Cultures were incubated at
27 ? 20C under a light regime of 16 h photoperiod provided by cool-white
fluorescent lamps at 50 [pmol m-2 s-1
The formation or absence of callus and the number of shoots from each
explant were recorded, 4 and 13 wk after the induction period. Shoots (0.5-
1.0 cm) were rooted individually in half-strength basal MS medium (MS
50%). These plantlets were transferred to a mixture of equal volumes of fine
gravel, volcanic sand and leaf mould for acclimatization under greenhouse
conditions. For acclimatization, pots covered with polypropylene bags were
used, in order to maintain a high relative humidity (80-90%), at 350C under
low light condition. The bags were removed after 2 wk.
Germinationrate, callus, and shoot production were calculated and
analyzed using a one-way analysis of variance (ANOVA),followed by a
multiple comparisonof means using Tukey'sHSD criterion(P < 0.05).
RESULTS
Germination. Germination frequencies were 41.7% on basal
MS medium, 40.0% on MMS and 28.1% on MS 50% (Table 1).
Although no significant differences were found among treatments,
MS 50% medium appeared to be less effective for germination.
402 ROSASET AL.

TABLE1 Organogenesis. Shoots developed from callus were obtained

from all explants in the different treatments,except in NAA at
TURBINICARPUS
LAUIIN VITROSEED* GERMINATION
0.54 VIMin the absence of cytokinin,in which shootformationwas
PERCENTAGE,RESULTSAFTER5 WK IN CULTURE
not achieved, and on the treatment without any plant growth
Germination(%) regulator,where only 33% of the explants formed callus and only
Time (wk) Basal MS medium MMS MS 50% 1.3 shootsper explantwere recovered(Table2). For the majorityof
1 11.67 ? 16.31 11.67 ? ? 15.75 explants, callus formation was the first response and the
19.57 13.75
2 21.67 ? 24.48 25.00 ? 28.36 20.00 ? 19.19 development of shoots or eventually isolated roots occurred
3 38.33 ? 31.11 35.00 ? 35.00 25.63 thereafter(datanot shown).Abundanceof callus was not correlated
+ 21.12
4 40.00 ? 31.72 38.33 ? 34.67 28.13 ? 26.31 with shoot development(Fig. Ib, c).
5 41.67 ? 33.98 40.00 ? 33.51 28.13 ? 26.31 Although statistically significant differences could not be
* Three seeds per jar, 10 replicates established, the greatestshoot proliferationpotentialwas observed
per treatment. in the cultures with 8.8-13.32 RMof cytokinin with or without
Basal MS medium:inorganicsalts and vitaminsof MS, glycine 2 mg 1-1,
auxin (Table 2). The smallest numberof shoots per treatmentwas
myo-inositol100 mg 1-1, sucrose 30 g 1-1.
ModifiedMS (MMS):inorganicsalts of MS withoutorganiccompounds, obtained on the treatment without plant growth regulators and
10 g 1-isucrose. 2.68 pM NAA in the absence of BA. The highest averagesof shoots
MS 50%: half-strengthbasal MS medium.
per explant registered 13 wk after the induction period were:
269.78 (8.8 pM BA), 146.22 (13.32 pM BA+2.68 p M NAA) and
Germinationoccurredgradually,althoughduringthe first and third 125.78 (13.32 pM BA+0.54 pM NAA).
weeks, higher percentages were registered. By the fifth week, Thirteenweeks after the inductionperiod, when the majorityof
plantlets reached 5-10 mm length and had developed 10-20 shoots had reached 0.5-1 cm in height, they were separatedfor
tubercles. rooting.The rootsappearedafter2 wk in half-strengthMS medium.
Callus development. Callus growthor absence on the explants The establishmentin soil had 94-100% survival(Fig. Id-f).
was determined.Significantdifferenceswere observedbetweenthe
development of callus on the medium without plant growth DIscusSION
regulators,where the callus developmentwas limited comparedto
most cytokinin? auxintreatments(Table2). This seems to indicate Seeds germinatedequally well on all three media examined.
that callus formationis favored in the presence of exogenous However,results seemed more satisfactoryin the media with the
cytokinin/auxin. By the end of the fourth week on induction highest concentration of mineral salts (basal MS medium and
medium, some callus development had occurred in almost all modified MS), as pointed out by Hubstenbergeret al. (1992). For
treatments(Table2). Callus developmentoccurredin 100% of the germinationit seems more importantto maintainthe high mineral
explants in all combinationsand explants when 4.4 pM BA was salt content of the MS medium than to change the organic
included in the medium.Meanwhile,not all combinationswith 8.8 compounds.Olguin (1994) found higher germinationpercentages
and 13.32 pRMBA promoted100% callus growth.In general, in for Ariocarpusretususwith 100% MS mineral salts than in half-
the complete experiment,callus was friable and green to hyaline strength MS with 30 g 1-1 sucrose added, and also described
in color. Callusfromwhich shoots emergedwas not much different asynchronous germination. These results are similar to our
in appearance from the non-shooting callus. Small buds with observationswith T. laui. Higher rates of in vitro germinationof
incipienttuberclesemergedfromslightly darkerand denserregions Mediocactuscoccineusseeds occurredon MS between the second
present in callus. Several weeks passed until the areoles grew and thirdweek of culture (Infante,1992). For T. laui they occurred
thornsand the shoots acquiredtheir darkgreen color. This process between the first and third week.
was continuousin the in vitro subcultures. Our observations throughout germination support Rabenda's

TABLE2

LAUIPERCENTAGEOF EXPLANTS*WITHCALLUSGROWTH,AFTER4 WK IN CULTUREON MS MEDIUMWITHBA AND NAA,

TURBINICARPUS
AND AVERAGESHOOTNUMBERPER EXPLANT(?SD) OF PLANTLETSECTIONSTREATED1 MO.IN INDUCTIONMEDIUM,RESULTSAFTER
13 WK IN BASALMS MEDIUM

NAA (p2M)
0 0.54 2.68
BA (p.M) Callus (%) Shootsper explant Callus (%) Shoots per explant Callus (%) Shoots per explant
0.0 33.3 b 1.3 ? 1.3 50 b 0.0 ? 0.0 50 b 16.4 ? 26.2
2.22 50.0 b 88.4 + 77.4 100 a 45.3 ? 78.5 100 a 95.6 ? 69.4
4.44 100.0 a 99.1 ? 61.6 100 a 95.6 ? 81.6 100 a 50.7 ? 87.7
8.80 83.3 b 269.8 + 213.6 100 a 13.8 ? 12.4 100 a 47.6 ? 82.4
13.32 100.0 a 50.2 ? 64.3 100 a 125.8 ? 126.7 83 b 146.2 + 138.7

* Six explantsper treatment.

Differentletters within columnsindicate significantdifference,P 0.05.
-
 MICROPROPAGATION
OF TURBINICARPUS
LAUI 403

(1990) reportregardingthe naturallatent germinationmechanism 1992). If this study is to encourage an effective means of
exhibited by cactus seeds, showinga discontinuousdistributionof conservation of the species, the next logical step is to try
the germinationtime. When interpretedas an adaptativequality proliferationof new individual shoots without callus formation,
of the species, it would enable cacti to maintaina numberof seeds avoiding potential somaclonal variation induced by the in vitro
on the ground that would not perish, should growth conditions techniques but maintaining genetic integrity to ensure species
change. vigor.
The fact that shoots developed from callus, even in the control Plant tissue culture has been recognizedas a useful tool in the
treatment,indicated(1) thatthe immaturetissue of the plantletshas propagationof cactus (Johnsonand Emino, 1979; Mauseth,1979;
a high numberof cells that expressmorphogeneticplasticityand (2) Starling,1985). Micropropagation representsan alternativefor the
organogenic competent cells were induced and developed in study and maintenanceof valuablegermplasm(Smithet al., 1991),
response to the new plant hormonebalance of the explants. Shoot as well as an aid in solving the problemsof furnishingthe market
regenerationof Mammillariasan-angelensiswas observed on MS with rarespecies of commercialinterest,thus reducingpressureson
basal medium(Martinez-Vazquez and Rubluo, 1989). Minochaand wild populations(Oldfield,1985; Fay, 1994).
Mehra (1974) also found that callus formed in the absence of
cytokininon NeomammillariaproliferaMiller. ACKNOWLEDGMENTS
The first shoots of T. laui appeared2 wk after initiationon the
inductionmedium.A. retususproducedshootswithinthe first month WethankbiologistJer6nimo
Reyesforproviding
theseedsof T.lauiand
of culture(Olguin,1994); Pdrezet al. (1998) recordedshoot growth Dr Andr6sVovides,Institutode Ecologia,A.C. for revisionof the
fromareoles after 6-12 wk. manuscript.
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