In vitro culture as a potential method for the conservation of endangered plants possessing crassulacean acid metabolism

Scientia Horticulturae 81 (1999) 71±87

In vitro culture as a potential method for the conservation of endangered plants possessing crassulacean acid metabolism Guadalupe Malda1,*, Humberto SuzaÂn1, Ralph Backhaus Department of Botany, Arizona State University, P.O. Box 871601, Tempe, AZ 85287-1601, USA Accepted 22 October 1998

Abstract Rare and endangered plants possessing crassulacean acid metabolism (CAM), such as cacti, usually present limited reproductive capacities and very slow growth rates. The use of in vitro culture can overcome these difficulties. The massive in vitro production of new propagules which result in totally regenerated plants is described for two endangered cacti, Obregonia denegrii Fric. and Coryphantha minima Baird. A comparison of in vitro and ex vitro growth rates demonstrated that the in vitro environment notably accelerates cacti growth. Malic acid titratable acidity indicated that increase of the net carbon dioxide uptake is associated with active growth. This might be related to particular factors of the in vitro environment such as the high relative humidity inside the culture vessels, or growth regulators supplemented to the growth media. In vitro-derived cacti showed a proficient re-establishment capability which could be related to their succulence since water loss during transplantation did not represent a crucial hydric stress. Succulence and plasticity of the CAM metabolic pathway in plants like cactus, represent some possible advantageous for the application of in vitro propagation techniques in a number of endangered, succulent plants like members of the Cactaceae, Agavaceae, Orchidaceae, or Bromeliaceae families. # 1999 Elsevier Science B.V. All rights reserved. Keywords: In vitro growth; Endangered species; CAM plants; Cacti propagation

* Corresponding author. Fax: +52-42-154-7777; e-mail: [email protected] 1 Present address: Escuela BiologõÂa, Facultad de Ciencias Naturales, Universidad AutoÂnoma de QuereÂtaro, Centro Universitario, Cerro de las Campanas s/n, QuereÂtaro, Qro. C.P. 76017, MeÂxico. 0304-4238/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 2 3 8 ( 9 8 ) 0 0 2 5 0 - 7

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1. Introduction Habitat destruction and the collection of wild plants for illicit trade are the main factors affecting nearly all succulent plant species. Of a total of 915 species of endangered flora subject to special protection by the Mexican government (SEMARNAP, 1995), 500 species are succulents likely to be possessing CAM physiology (Crassulacean Acid Metabolism). The majority of these plants are members of the Cactaceae family which are almost all CAM species. This family represents 28% of the total number of threatened and endangered species of Mexican vascular plants. CAM plants are slow growing plants that sometimes have limited reproductive capacities and often have very specific and limited conditions for flowering, seed production, germination and offset production; therefore, conventional propagation methods are much too slow. One alternative is to propagate them by tissue culture, which allows for a rapid and continuous production of plants. Tissue culture has already proved successful for several members of the Cactaceae family (Johnson and Emino, 1979; Mauseth, 1979; Ault and Bagamon, 1985; Escobar et al., 1986; Clayton et al., 1990; Hubstenberger et al., 1992); members of Aizoaceae, Aloaceae, Euphorbiaceae families (Jackobeck et al., 1986; Gratton and Fay, 1990); and certain Agave species (Robert et al., 1987; Powers and Backhaus, 1988; Binh et al., 1990; Castro-Concha et al., 1990). In addition, the high regenerative ability in several species of the Crassulaceae family has been widely proved (Bigot, 1976; Uhring, 1983; Dickens and van Staden, 1988; Gratton and Fay, 1990). It has been observed, particularly for cacti, that in vitro-derived plantlets grow much more rapidly and produce a considerable number of new growths. RodrõÂguez and Rubluo (1993) indicated that Aztekium ritterii Boedeker, a very slow growing cactus, produced 5±7 new offshoots after 11 months in culture, while ex vitro specimens never produce offshoots. Another cactus, Mammillaria woodsii grown from seeds, required at least a year or more to reach the same size as plants regenerated after a few months in tissue culture (Vizkot and Jara, 1984). Comparable observations are published for cactus species such as Mammillaria prolifera (Minocha and Mehra, 1974), Esposotoa huanucoensis (Angris and Mehra, 1982) and Cephalocereus senilis (Bonnes et al., 1993). A quantitative study (Malda, 1996) confirmed such improved growth in vitro in two endangered Mexican cacti. In contrast to cacti, the non-succulent species rarely exhibit such high in vitro growth rates. Several studies have revealed that in vitro culture of non-succulents results in various environmental limitations for optimal plant development, which substantially reduce photosynthetic activity and growth (Capellades et al., 1990; Kozai, 1991; DeYue and Desjardins, 1993). In addition, the in vitro environment generally keeps relative humidity values close to saturation, and as a

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consequence, the plants produced have uncontrolled water loss. Therefore, acclimatization of in vitro-derived plants is critical to prevent this unnatural water loss. Some authors state that this elevated water loss is mainly due to an abnormal functioning of stomata (SantamarõÂa and Kerstiens, 1994); whereas others state that the low epicuticular wax content of in vitro-derived plants is the principal cause for desiccation (Grout and Ashton, 1978; Sutter, 1988). However, most of these considerations refer to plants possessing C-3 or C-4 photosynthesis, whereas cacti utilize the crassulacean acid metabolism (CAM). This is a nocturnal CO2 concentrating process which permits plants to close their stomata during the day (Winter, 1985). Considering that majority of plants cultured in vitro are generally unable to completely close their stomata (Diettrich et al., 1992; Shallanon and Maziere, 1992), it is possible that typical CAM cycle of CO2 uptake may be altered (Malda, 1996). The application of in vitro culture to propagate endangered species has been studied with some species from Mexico, such as Mammillaria sanangelensis (MartõÂnez-VaÂzquez and Rubluo, 1989), Leuchtenbergia principis (Starling, 1985) and Aztekium ritteri (RodrõÂguez and Rubluo, 1993). High survival percentages are reported in all cases however, little information is available concerning the survival of reintroduced species into the wild following their propagation by tissue culture (Rubluo et al., 1993). In addition, requirements for shoot proliferation and rooting vary among species and morphogenetic responses to culture variables are specific. In this contribution, we examine different morphogenetic responses and evaluate plant development during in vitro culture for two endangered cacti: Coryphantha minima Baird and Obregonia denegrii Fric. In addition, we assess the significance of certain attributes such as succulence and CAM on the feasibility of propagating via tissue culture some endangered species of the CAM photosynthetic group. 2. Methodology Seeds of Obregonia denegrii and Coryphantha minima obtained from the Desert Botanical Garden, Phoenix, AZ, USA, were aseptically germinated in 0.5X Murashighe±Skoog (MS) medium (Murashige and Skoog, 1962) supplemented with 15 g lÿ1 sucrose and 8 g lÿ1 agar. The medium was adjusted to pH 5.7 and autoclaved for 15 min. (at 1218C/103 kPa). Seedlings were maintained at 25±278C, under a total photosynthetic photon flux (PPF) of 120± 130 mmol mÿ2 sÿ1, provided by cool fluorescent lamps, in a 16 h period. For a comparative ex vitro culture, seeds were sown in containers with coarse sand, placed in the same culture room as in vitro conditions and irrigated three times a day by mist. After 2 weeks, seedlings were fertilized biweekly with 0.5X

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strength Murashige±Skoog (MS) salt solution throughout their culture. Twomonth old seedlings were transplanted to potting media containing a 1 : 1 : 1 mixture of peat, perlite and coarse sand; and irrigated every 5 days with tap water. Ex vitro plants were maintained under the same light and temperature conditions as in vitro cultures. In vitro proliferation was tested from apical portions (epicotyls) of 2-month old seedlings transferred to MS basal salts containing 0.5 mg lÿ1 6-benzylaminopurine (BA) combined with 0.1 mg lÿ1 1-naphtalenacetic acid (NAA). Root development of the new shoots was tested in the presence of 5 mg lÿ1 indole3-butyric acid (IBA) and NAA. A treatment combining acclimatization and rooting was tested, transferring non-rooted shoots to sand (horticultural grade). Commercial rooting powder containing 0.2% NAA and 4% Thiram (Rootone) was employed to induce ex vitro root formation. Totally regenerated plants were transplanted out of the in vitro environment to the same pot mixture used for ex vitro seedlings and acclimatized by gradually reducing the relative humidity, covering plants with plastic bags (MartõÂnez-VaÂzquez and Rubluo, 1989). The effects of in vitro conditions on cactus re-establishment were analyzed by measuring water loss in plants derived from different culture conditions. Percent water loss was calculated by differences in weight in a 1 h period, as: PCL ˆ

FW ÿ WaH  100 FW ÿ DW

where PCL is the percent water loss; FW represents fresh weight; WaH is weight after holding and DW is oven-dry weight. PCL estimations were then divided by drying time to obtain the rate of water loss, according to Brainerd and Fuchigami (1982). Data were recorded on five replicates per treatment at 1, 5, 10 and 20 days after removing plants from the culture vessel. To examine epicuticular wax formation related to water loss, wax concentration was measured at different phases of in vitro culture and compared to ex vitro cultured seedlings and wild specimens. Waxes were extracted and determined by a colorimetric method (Ebercon et al., 1977). Comparisons were made against standard wax solutions of known concentrations, prepared from pure paraffin. Wax content was expressed as g mÿ2 of shoot area. For plant growth evaluations, the number of new tubercles and increases in plant volume and fresh weight were considered as growth parameters. Measurements were taken monthly on a minimum of four replicates per treatment; and recorded always one day after watering in ex vitro cultured plants. Data were subjected to analysis of variance, considering initial volume and fresh weight values as possible co-variables. Photosynthetic activity was determined by daily fluctuations in tissue acidity. Samples of shoot tissue, weighting 1 g each, were obtained at different times of the day from three separate replicates: at the beginning, the middle and the end of

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the dark period, as well as at the middle of the light period. Each sample was stored in sealed plastic bags and immediately frozen. Tissue acidity was determined from individual tissue samples, thawed and then ground with sand in 10 ml distilled water, and boiled for 15 min. Once samples were cooled to room temperature, 1 ml of extracted sap was titrated with 0.02 N NaOH to an arbitrary pH 7.0 end point. Acid concentration units are expressed as meq gÿ1 per fresh weight, according to Pearcy et al. (1989). Titrations were from plants at different stages of in vitro culture and 1-year old, ex vitro cultured plants. 3. Results 3.1. In vitro propagation O. denegrii had distinct morphogenetic responses in the presence of growth regulators in the culture medium, including: (a) production of callus containing a large number of somatic embryos; (b) development of new axillary and adventitious shoots; and (c) formation of isolated areoles and spines in some cases. The ability to form somatic embryos, as well as new axillary shoots, was consistent with the BA/NAA combination, showing proliferation rates of 9.2 somatic embryos and 11.3 new shoots per explant in a 4-month period (Table 1). When the O. denegrii callus that produced somatic embryos was subcultured on a fresh, hormone-free medium, it continued to produce somatic embryos for several months. As embryos enlarged, the overgrowth of the original callus still presented more pro-embryonic tissue as a continuous process that remained for at least 4 months. Table 1 Effects of 6-benzyladenine (BA) combined with 1-naphtalenacetic acid (NAA) (0.5 and 0.1 mg lÿ1 , respectively) on different morphogenetic responses in Coryphantha minima and Obregonia denegrii cultured in vitro on Murashige±Skoog medium Species and culture treatments

Number of new shoots/explant

Number of somatic embryos/explant

Callus In vitro developmenta flowering

Obregonia denegrii Hormone-free medium BA/NAA

0 11.4  6.2

0 9.2  5.3

3 1

No No

Coryphantha minima Hormone-free medium BA/NAA

1.0  0.1 21.7  1.4

0 0

2 1

No Yes

Values are means  standard errors. Values are: 0 ± none, 1 ± few, 2 ± regular, 3 ± abundant.

a

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In contrast, no somatic embryogenesis, but suitable shoot proliferation rates (21.6 new shoots/explant) and two flowering events were observed for C. minima (Table 1). Based on experiments (Malda, unpublished data), BA applied in the growth medium at different concentrations consistently induced flowering in the young, new shoots. Although the size of in vitro flowers was almost one-half of normal, their morphology was typical for the species. However, in vitro crosspollination was unsuccessful. For both species, in vitro rooting of the new shoots was achieved in treatments containing auxins, even though spontaneous rooting in hormone-free media repeatedly occurred, particularly for C. minima. Although ex vitro root induction presented lower rooting percentages, the values can be considered satisfactory in terms of obtaining totally rooted and acclimatized plants in both species (Table 2). Rate of water loss monitored during acclimatization was high (up to 0.93) during the first 5 days after transfer, but it tended to stabilize within the following 15 days (Fig. 1). The total amount of water loss after 20 days of acclimatization did not affect plant survival, and following this period of time the plants begun to recuperate. The low amounts of epicuticular waxes registered for the in vitro-derived cacti (Fig. 2) did not affect survival, and furthermore, total waxes increased 5-fold after 3 months, reaching wax levels very close to those of mature plants in the greenhouse throughout acclimatization. During acclimatization, the survival percentage was high for plantlets derived from spontaneous in vitro rooting and ex vitro rooting treatments (Table 3). However, total acclimatization of the different kinds of propagules obtained from in vitro and ex vitro cultures in both species presented adequate re-establishment capacities. 3.2. Growth analysis At the time of germination and young seedling growth, C. minima and O. denegrii presented similar numbers of new tubercles and gain in fresh weight, Table 2 In vitro and ex vitro rooting percentages of Coryphantha minima and Obregonia denegrii new shoots produced in vitro Rooting treatments

C. minima

O. denegrii

In vitro IBA, 5 mg lÿ1 IAA, 5 mg lÿ1 Ex vitro rooting In vitro self-rooting

89 87 77 59

64 57 54 28

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Fig. 1. Rate of water loss in a 20-day acclimatization period in C. minima plants derived from in vitro and ex vitro rooting treatments.

Fig. 2. Amount of epicuticular waxes (average, minimal and maximal values) in C. minima plants from different culture stages and conditions: A ± in vitro shoot proliferation; B ± in vitro rooting; C ± ex vitro rooting; D ± 1 month after acclimatization; E ± 3 months after acclimatization; F ± 6 months after acclimatization, and G ± wild specimens maintained in greenhouse conditions.

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Table 3 Survival percentage of C. minima and O. denegrii, 4 months after acclimatization Survival percentage Seedlings 6 months old derived from somatic embryogenesis Rooted in vitro with IBA and IAA In vitro spontaneously rooted plants Ex vitro rooted plants

Coryphantha minima

Obregonia denegrii

±

93

89 92 90

73 98 75

with no apparent differences between in vitro and ex vitro development. However, enhanced growth was observed during the in vitro shoot proliferation stage, when plant regulators (BA/NAA) were supplemented to the growth media (Table 4). To a lesser extent, growth of new plantlets still continued with increased rates during the later phases of the in vitro culture without the influence of an external source of growth regulators. Daily fluctuations of malic acid concentration in the tissues indirectly indicated that photosynthetic activity was improved by the in vitro conditions. Fig. 3 shows that CO2 uptake is higher for in vitro than ex vitro individuals. Upon transfer to the ex vitro environment, in vitro plants reverted to normal CO2 fixation rates, as shown by malic acid fluctuations in plantlets after 2 months of acclimatization. O. denegrii somatic embryos had high acid levels in the tissues, which persisted even after acclimatization, when embryos transformed into complete seedlings. Growth of somatic embryo-derived seedlings was still higher after 3 months of acclimatization compared to growth rates of 1-year old, ex vitro cultured seedlings. Table 4 Increases in fresh weight of cactus cultured in vitro at different stages of micropropagation, during a 4-month period Culture conditions In vitro culture initiation Shoot proliferation stage (growth regulators added) In vitro elongation phase, without growth regulators In vitro rooting stage Acclimatization stage In vitro maintenance of seedlings Ex vitro maintenance of seedlings Values are mean  standard error.

Fresh weight gain (g (4 months)ÿ1) Coryphantha minina

Obregonia denegrii

0.09  0.008 1.90  0.980

0.76  0.10 1.36  0.82

0.25  0.098

0.98  0.56

1.32  0.850 0.49  0.243 0.17  0.131 0.05  0.083

0.03  0.02 0.02  0.03 0.38  0.01 0.33  0.07

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Fig. 3. Comparison of daily fluctuations in malic acid concentrations of in vitro-derived and ex vitro-cultured plants. (a) Somatic embryos, in vitro shoots and 1-year old ex vitro seedlings of O. denegrii. (b) In vitro shoots and wild plants of C. minima.

4. Discussion 4.1. In vitro propagation In vitro proliferation of C. minima and O. denegrii is relatively easy and different kinds of propagules (new shoots or somatic embryos) can be induced

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using BA combined with NAA at low concentrations. Although particular requirements for optimal growth regulator concentrations varies with species, similar observations have reported the effectiveness of BA±NAA combinations to proliferate several cactus species (Starling, 1985; MartiÂnez-VaÂzquez and Rubluo, 1989; Clayton et al., 1990; Hubstenberger et al., 1992; RodriÂguez and Rubluo, 1993) and other succulents like members of genus Euphorbia (Jackobeck et al., 1986), or some members of the Azoaceae, Agavaceae and Crassulaceae families (Gratton and Fay, 1990). It is of special interest that in vitro flowering was observed in C. minima, as most C. minima plants bloom only after 3±4 years of age. In our experiments, in vitro flowering occurred on plants less than 1-year old; and always occurred at the same time of the year (October±November). Deliberated attempts to obtain flowers in different months and using other growth regulators were unsuccessful. Cytokinins like BA apparently had an influence on flowering because it only occurred on plants in tissue culture, and not on ex vitro plants growing in the same culture chambers. A seasonal effect then, could not account for this, as the natural flowering time for this species is May (US Fish and Wildlife Service, 1984); and environmental conditions in the culture room were uniform throughout the year. Flowering is quite uncommon for plants cultured in vitro because tissues typically remain in a juvenile stage, which is contrary to the requirements for flower induction in most plant species. However, if in vitro flowering is achieved in some endangered species, studies of in vitro cross-pollination could be a feasible advance. The two cacti species showed adequate capacities to form new shoots with well-developed roots, even without the action of an external source of auxins as induction agents. The same ability can be effectively used for ex vitro rooting which, in addition, simplifies the in vitro culture procedure because it eliminates an acclimatization phase. The apparent natural ability to develop roots, together with the efficiency of an ex vitro rooting treatment, denote potentialities that considerably reduce time and costs during the cactus propagation system and might guarantee high survival percentages after transplanting. Growth regulators added to the growth media during the shoot proliferation stage played a definitive role in growth enhancement of cacti cultured in vitro. This is consistent with results of Dabekaussen et al. (1991) who found that the cactus Sclerorebutia alba produced a 15-fold increase in fresh weight when the concentration of BA was raised from 0 to 1 mg lÿ1. However, high growth rates were also observed in subsequent phases of in vitro culture, in the absence of growth regulators. The high relative humidity in the culture vessels, the supplementary carbon source and the nutrient culture media (specially nitrogenrich), appear to be important factors related to the observed improvement of growth and photosynthetic activity (Malda, 1996). These factors could be easily

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adapted to conventional, ex vitro propagation methods; so it may be possible to enhance the growth rates of slow growing species, typical of as many CAM plants. Related studies indicate that the application of some cytokinins via superficial sprays enhance growth in three cacti species cultured under typical nursery conditions (Sanderson et al., 1986), whereas the culture of some agaves and cacti under high nitrogen conditions like hydroponics, positively influence their growth (Nobel, 1983; Nobel and Hartsock, 1986). In the present study, malic acid titrations detected a notable increase of CO2 uptake in the plants maintained in vitro. Similar results were observed in a different study, using gas exchange determinations with an infrared gas analyzer (Malda, 1996). It was found that the typical CAM metabolism pattern was actually modified by the in vitro conditions. Therefore, despite possible metabolic alterations, the increased growth observed frequently in cacti cultured in vitro can be explained on the basis of photosynthetic activity improvement. The relevance of photosynthetic activity improvement in C. minima and O. denegrii relies on their CAM physiology. It is probable that, besides cacti, other CAM plants could respond in a similar manner; and that in vitro culture represents an important potential to effectively propagate slow growing and endangered plants with CAM physiology. 4.2. Re-establishment ability of in vitro-derived cacti A crucial aspect of in vitro propagation is to procure totally regenerated plants capable of surviving outside the culture vessels. One of the major problems is that most of the in vitro-derived plants experience a desiccation shock just after transplant (Kozai, 1991), and sometimes this is fatal during the first day of acclimatization. As indicated herein, the greater water loss in the two cactus species occurred during the first days of acclimatization, after which it tended to stabilize. However, water loss was not as rapid and no desiccation shock was observed in the cactus species, contrary to most plant species grown in vitro (Brainerd and Fuchigami, 1982; Bhojwani and Dhawan, 1988; Smith et al., 1990; Diettrich et al., 1992). Also, although the in vitro-derived cacti presented reduced wax levels, no correlation was found between survival percentages and epicuticular wax content, which is in accordance with previous studies for other plant species (Sutter, 1988; Diettrich et al., 1992; Kerstiens, 1994). The water loss recuperation time period does not correspond to the time required for a recuperation of normal wax levels, apparently until the third month, in C. minima. Although water loss was relatively significant during acclimatization, survival of cacti was not affected, suggesting that body succulence allowed plants to survive and recover after a certain degree of desiccation. In relation to the morphological and physiological effects of acclimatization, several authors have suggested that new leaves are required for the optimal re-

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establishment of in vitro-derived plants. Donnelly and Vidvaver (1984) demonstrated that the photosynthetic contribution of in vitro-formed leaves on raspberry plants was small or negative at acclimatization, while the first new leaves formed ex vitro had intermediate photosynthetic capability. In addition, Diettrich et al. (1992) suggested that in Digitalis lanata, leaves formed in vitro are unable to develop further under ex vitro conditions, and after a few weeks they are replaced by newly formed, normal leaves possessing stomata that function correctly and contain normal amounts of epicuticular waxes. Nevertheless, acclimatization of cacti depends on different attributes than those observed for other plant species, because cactus species have photosynthetic, succulent stems instead of leaves. In vitro-derived cacti do not develop new leaf primordia with different characteristics while adapting to a changed, ex vitro environment. Rather, the whole cactus body must gradually adapt, until complete acclimatization is reached. Leaf and stem succulence of other endangered CAM species could also signify positive attributes that could minimize plant stress during one of the most crucial phases of in vitro culture: acclimatization. 4.3. Feasibility of in vitro propagation of endangered cam plant species CAM physiology in several species allows a high plasticity to tolerate different, extreme conditions. For example, Hastock and Nobel (1976) demonstrated that Agave desertii shifted to C-3 metabolism, showing a net CO2 uptake during the day after excessive watering under greenhouse culture. Such a response could be related to the constant day/night CO2 uptake observed in the cactus Coryphantha minima cultured in vitro (Malda, 1996). Furthermore, cell suspension and callus cultures of the cactus Chamaecereus sylvestrii, definitely shifted to a C-3 type pathway under the in vitro environment (Seeni and Gnaman, 1980). The same plasticity also takes place under natural conditions, since it was found that several cacti species exhibit a photosynthetic C-3 pathway during their early ontogenetic stages, even though they are obligate CAM plants (Altesor et al., 1992). The physiological flexibility of plants with CAM physiology makes it possible for these plants to undergo a rapid and productive in vitro culture mass propagation which can be a reliable technique to produce vigorous, healthy and big plantlets. Besides the high number of endangered species of the Cactaceae family, there is a considerable number of other endangered species possessing CAM physiology in both, obligate or facultative mode. Estimations of the vascular plants distributed in Mexico indicate that there are approximately 22 800 species (Rzedowski, 1993); from which a rough 10% probably present CAM metabolism. Such approximations are based on the principle that most members of Agave and Manfreda genera as well as Bromeliaceae, Cactaceae, Orchidaceae, and Crassulaceae families present CAM metabolism (Szarek, 1979; Winter, 1985).

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Based on this same principle, the possible abundance of endangered CAM plants distributed in Mexico, roughly estimated from the most recent list of endangered species (SEMARNAP, 1995), includes 258 species of the Cactaceae family; 180 Orchidaceae; 21 Euphorbiaceae; 18 Crassulaceae, as well as 18 species of genus Agave and 5 species of genus Manfreda (Gentry, 1982; GarciÂa Franco, 1987; Arias, 1993). Like most endangered cacti, many of these endangered, CAM plants have strong pressures on their populations as a result of the frequent depredation for their ornamental attributes. Large-scale production using in vitro culture techniques could provide commercial incentives for economically depressed areas where many of these species are continuously removed. Application of in vitro culture on endangered species, however, is a technique that can be adopted under special circumstances. Plant regeneration from existing meristems is a genetically conservative propagation method that has been used extensively with rare and endangered plants (Fillipini et al., 1994). This is acceptable for plant species with severely damaged populations in which no other propagation method is successful. Considering that artificial propagation is a desirable resolution to reduce external pressures to wild populations on rare and endangered species (i.e. illegal commercial trade), in vitro culture is a promising method. However, it must be applied in accordance with a conservation strategy that maximizes genetic diversity. Axillary shoot proliferation from a single explant (meristem) produces genetically identical plants; therefore, it is not desirable to propagate plants from such a small and genetically restricted sample. However, an appropriated sampling of seeds, seedlings or vegetative material obtained in a non-destructive manner from natural populations must be considered to obtain a set of clones representing a wide gene pool. Adventitious shoot proliferation (e.g. from callus) or somatic embryogenesis frequently cause some degree of somaclonal variation in vitro. This is one aspect that should be carefully pondered for a conservation strategy applying in vitro propagation techniques. Some authors have suggested the possibility of widening the genetic base of a species by inducing somaclonal variation in vitro as a method of generating new vigor into natural populations of endangered species (Bramwell, 1990; Fillipini et al., 1994). Nevertheless this is discussible as in general, somaclonal variants are not genetically stable and the genetic (or epigenetic) changes may not respond positively as the natural genetic variability does in the species. In spite of such controversy, application of in vitro culture using seeds as the source of propagation greatly increases germination percentages, particularly in succulent plants (Hill, 1977; Fay, 1992) and promotes seedling growth, as it was demonstrated for the two succulent species addressed in this paper. Furthermore, horticultural management in some of these endangered CAM plants could be improved by simulating some features of the in vitro culture on

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conventional propagation techniques. Factors like a very humid environment, a nitrogen-rich substrate and an external source of cytokinins could be easily adopted under greenhouse conditions. This kind of practice may help to obtain healthy and vigorous plants for recovery or reintroduction programs. Conversely, mass propagation of certain endangered CAM plants using in vitro culture, may satisfy their normal commercial demand and diminish the exploitation pressure from their natural populations. Acknowledgements We thank Linda Pritchet and the staff from the Desert Botanical Garden, Phoenix, AZ, USA, who kindly provided plant material (seeds and mature specimens). This work was supported by CONACYT (Mexico) with a scholarship to G. Malda. References Altesor, A., Ezcurra, E., Silva, C., 1992. Changes in the photosynthetic metabolism during the early ontogeny of four cactus species. Acta Oecologica. 13(6), 777±785. Angris, R., Mehra, P.N., 1982. An exceptionally fast growing callus tissue of Esposotoa huanucoensis. Biol. Bulletin India 4, 51±54. Arias, M.S., 1993. CactaÂceas: ConservacioÂn y Diversidad en MeÂxico. Revista de la Sociedad Mexicana de Historia Natural, vol. Esp. XLIV. pp. 109±115. Ault, J.R., Bagamon, W., 1985. in vitro propagation of Ferocactus acanthodes (Cactaceae). HortScience 22, 126±127. Bhojwani, S., Dhawan, V., 1988. Acclimatization of tissue culture-raised plants for transplantation to the field. In: Dhawan, V. (Ed.), Applications of Biotechnology in Forestry and Horticulture. Plenium Press, New York, pp. 249±256. Bigot, C., 1976. Bourgeonnement in vitro aÁ partir d'eÂpiderme seÂpare de feuille de Bryophyllum daigremontianum (CrassulaceÂes). Can. J. Bot. 54, 852±867. Binh, L.T., Muoi, L.T., Oanh, H.T.K., Thang, T.D., Phong, D.T., 1990. Rapid propagation of agave by in vitro tissue culture. Plant Cell, Tissue and Organ Culture 23, 67±70. Bonnes, M.S., PareÂ, P.W., Mabry, T.J., 1993. Novel callus and suspension cultures of the `old man' cactus (Cephalocereus senilis). Cactus and Succulent J. (Los Angeles) 65, 144±147. Brainerd, K.E., Fuchigami, L.H., 1982. Stomatal functioning of in vitro and greenhouse apple leaves in darkness, mannitol, ABA and CO2. J. Exp. Bot. 134, 388±392. Bramwell, D., 1990. The role of in vitro cultivation in the conservation of endangered species. In: HernaÂndez Bermejo, J.E., Clemente, M., Heywood, V. (Eds.), Proc. Int. Congress of Conserv. Techniques in Botanic Gardens. Koeltz Scientific Books, pp. 3±15. Capellades, M., Vandershaeghe, P., Lemeur, S., Debergh, P.C., 1990. How important is photosynthesis in micropropagation?. In: Sangwan, R.S., Sangwan-Noreel (Eds.), The impact of Biotechnology in Agriculture. Kluwer Academic Publishers, The Netherlands, pp. 29±37. Castro-Concha, L., Loyola, V.M., Chang, J.L., Robert, M.L., 1990. Glutamate dehydrogenase activity in normal and vitrified plants of Agave tequilana Weber propagated in vitro. Plant Cell, Tissue and Organ Culture 22, 147±151.

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