appreciatealllife: IN VITRO COLLECTING (IVC). I. THE EFFECT OF COLLECTING METHOD AND ANTIMICROBIAL AGENTS ON CONTAMINATION IN TEMPERATE AND TROPICAL COLLECTIONS

SUMMARY

In vitro collecting is the process of initiating tissue cultures in the field. In order for in vitro collecting to be broadly available as a technique for collecting plant germplasm, the levels of contamination in such cultures must be controlled. Two techniques for in vitro collecting were compared: leaf punch and needle collecting. The effectiveness of these methods for collecting leaf and stem tissues from plants at tropical and temperate sites was compared. Stem tissue collected by the needle collecting method gave cultures with an average contamination percentage of 31% and 16%, from the tropical and temperate sites, respectively, while with the leaf punch method, average contamination percentages were 90% and 69%. The effectiveness of antimicrobial agents in reducing contamination in leaf punch cultures was evaluated. Addition of the fungicide benlate and the antibiotics, cefotaxime and vancomycin, to the leaf punch collections reduced contamination to an average of 30% in the tropical collections and 35% in the temperate collections. Over 90% of both tropical and temperate species collected in multiple samples of 10 or more had at least one clean sample using this medium. The use of either the leaf punch method in combination with a fungicide and antibiotics or the needle collecting technique yielded a high percentage of clean tissues for study and growth.

Key words: antibiotics; benlate; fungicide; leaf culture; tissue culture.

INTRODUCTION

The collecting of seeds and spores has traditionally played an important role in the e% situ preservation of living plant germplasm (Guarino et al., 1995). However, with some species, the viahility or longevity of seeds or spores may be poor. Additionally, seeds or spores may not be available at the time of collecting, particularly from wild species. When collections are done in remote areas, revisiting the site at the time of seed production may not be feasible, and it may be difficult to plan a trip precisely to correspond with seed maturity. Because of these limitations, cuttings or even entire plants are sometimes collected for growth e% situ. However, these often present challenges in the potential volume of plant material, in maintaining the viability of plant material in transport, and in restrictions on their movement across international borders.

In vitro techniques, developed for a variety of species over the past several decades, can be used to further broaden the possibilities for collecting living tissue (Withers, 1995, 2002; Pence, 1996, 1999, 2002). In vitro collecting (IVC), or the initiation of tissue cultures in the field, allows immature seed and non-seed materials to be collected and can avoid a major disturbance to the plant in situ. In general, in vitro materials are also subject to fewer restrictions in crossing international lines than materials that may carry soil or fauna, although they are still governed by any relevant regulations for phytosanitary certificates or import permits.

In vitro collecting has been reported for embryos of the orchid Zygopetallum maxillare (Warren, 1983) and of Cocos nucifera and other palms (Sossou et al., 1987; Assy Bah et al., 1987, 1989; Engelmann, 2002), plantlets of the grasses Digitaria eriantha ssp. pentzii and C.ynodon ddctylon (Ruredzo, 1999), and nodes arid buds of Theobroma cacao (Yidana et al., 1987; Silvana Alvarenga et al., 2002), Gossypium spp. (Altman et al., 1990), species of Prunus and Vitis (Elias, 1988), Ipomoea batatas (Huaman et al., 1995), Manihot e.sculenta (Chavez, in Huaman et al., 1995), Musa, Coffea and Citrus (Withers, 1995; Montoya Henao et al., 2002; Lozoya Saldana et al., 2002; Brenes Hines et al., 2002), Colocasia esculenta (Taylor, 2002), the timber species Shorea leprosula (Krishnapillay et al., 2002), and Persea americana (Sandoval and Villalobos, 2002). In these cases, techniques were tailored to specifie target species or genera, but for broad application, methods are needed which can be used successfully for a high percentage of species.

The first step in working toward this goal is the control of contamination (Pence and Sandoval, 2002). Preliminary studies on in vitro collecting in this laboratory primarily used apical and axillary buds. However, it was often difficult to obtain enough buds of a similar developmental stage from a wide range of wild species for experimental protocols. Thus, a series of studies using the simpler, more abundant leaf and stem tissues was undertaken to address the question of contamination in broadly applied IVC of temperate and tropical species. The specific objectives of the study were to compare the effects of two different collecting methods done at two different collecting sites (temperate and tropical) with and without antimicrobial agents on the subsequent contamination levels in the cultures. Results from the early part of this study have been briefly summarized elsewhere (Pence, 1996, 1999).

MATERIALS AND METHODS

Preliminary experiments. In a series of preliminary experiments, bud and leaf tissues were collected from a variety of species in Florida (February 1988), Tonga (February 1989), Cincinnati, Ohio (March 1989; September 1992), Costa Rica (July 1990), and Trinidad (September 1992). Tissues were surface-sterilized by incubating for 30min with water purification tablets [Halazone, p-N,N-dichlorosulfamy-benzoic acid] dissolved in water (4 mg per 10ml), using the procedure of Yidana et al. (1987), and transferred to vials of medium, as used for later experiments (see below). Some media contained iungicides, either benlate [benomyl; methyl l-(butylcarbomoyl)-2 benzimidazolecarbamate| (Hi Yield Chemical Co., Bonham, TX) or Daconil 2787� [ehlorothalonil(tetrachoroisophthalonitrile)] (Ortho, Chevron Chemical Co., Orlando, FL). Results of these early experiments led to the experiments described below, designed to compare contamination percentages in temperate and tropical IVC.

Plant material and collecting schedule. A series of collections was made to compare contamination from two areas, one tropical and one temperate. Tropical collections were made at the CRFSTT (Centre for Research of Endangered Wildlife of Trinidad and Tobago) Environmental Research Station at Morne Catherine, Chaguaramas, in northwest Trinidad, with the exception of the Tl collections, which were done near that station on Morne Catherine and at a site near Sangre Grande in eastern Trinidad. Temperate collections were made at the Cincinnati Zoo and Botanical Garden and at a nearby site containing a variety of wild species. Eighty-five species from the tropical sites were collected over the course of 3yr during four collections: Tl, April 1994; T2, August 1994; T3, January 1995; and T4, May 1996 (Table 1). Temperate collections were made from 49 species during three seasons: 1994, Cl; 1995, C2; and 1996, C3 (Table 2). Data from three collecting dates during each of the Cl and C3 seasons were used.

Many species were collected on more than one occasion, but because of differences in tissue availability at different times, not all the species were replicated in each collection. Young, green stem tissues were used for needle collecting, while young leaves, approximately one-quarter to one-half expanded were used for the leaf punch collections. In some cases, other young tissues were collected, as indicated.

IVC techniques. Since Halazone had become unavailable since the time of the preliminary studies, tissues were surface-sterilized by swabbing with cheesecloth soaked in 70% ethanol, and all instruments were soaked in 70% cthanol before use. Two techniques for tissue collecting were compared: leaf punch and needle collecting. For leaf punch collecting, a standard one-hole paper punch was used to punch leaf discs, 6 mm in diameter, and these were transferred with forceps to vials of media. For needle collections, a 3- or 5-ml syringe with a no. 21 gauge needle was used to puncture through the stem and collect a tiny cylinder that was a cross-section of the stem tissues. This was expelled into a vial of medium.

Other tissues were collected, including immature flower parts, immature embryos, tendrils, anthers, and young leaves that had not yet uncurled. In these cases, the tissues were isolated, soaked briefly in 70% ethanol (< 1 min), cut with scissors, and transferred to vials of media.

All operations were done in the open air. In most cases the tissues were disinfested and placed on medium out-of-doors at the collecting site, but in some cases materials were collected and held in plastic bags with moist paper towels and processed indoors within a few hours of collecting.

Media and containers. Basal medium consisted of the salts of Murashige and Skoog (1962) with minimal organics (Linsmaier and Skoog, 1965), with 2.2 �M benzylaminopuriric (BA), 2.7 �M ot-naphthaleneacetic acid (NAA), 3% sucrose, and 0.22% Phytagel (Sigma Chemical Co., St. Louis, MO) (LP medium). In leaf punch experiments, some media also contained 1OO mg 1 active benlate added before autoclaving (LP + B medium). When antibiotics were used (LP + A and LP -j- B + A media), they were preweighed and carried into the field in powder form. Just before use, they were dissolved in a measured volume of sterile water and filter-sterilized through a 0.2 �m cellulose acetate membrane filter into a sterile container to give a solution containing Smgml^sup -1^ cefotaxime and 0.25 mg ml^sup -1^ vancomycin (Sigma Chemical Co.). Then, 50 �l of the antibiotic solution were pipeted on top of each tissue piece, using a micropipeter and alcohol-sterilized tips. Antibiotics were prepared and added after all collections were made each day.

For leaf punch experiments, 7-ml borosilicate scintillation vials with polyethylene screw caps were used as containers, with 2.5 ml of medium in each, and were sterilized by autoclaving. The caps were sterilized in 70% ethanol, rinsed in sterile water, and then placed onto the vials. For all needle collections except T1, 1.5-ml polypropylene microcentrifuge tubes with snap caps were used. These were autoelaved before receiving 0.5 ml of sterile medium.

Culture conditions. In the field, culture vials and microcentrifuge tubes were grouped in plastic bags with zip-type closures, which were carried inside opaque cloth bags or trunks. No attempt was made to provide aeration or light to the cultures while in transit. Although cultures were not exposed to direct sunlight, they were subject to ambient temperatures in the field and were checked as luggage during air transport. Upon return to the laboratory, cultures were maintained in the plastic bags on the benchtop until they were observed for contamination.

Evaluation of cultures. Cultures were evaluated 7 -14d after initiation using a dissecting microscope. Any contamination observed was designated as fungal if fungal hyphae were visible and as bacterial when hyphae were not present. Thus, some of the designations made as bacteria may have included non-filamentous fungi, as well. It is likely that many of the cultures contaminated with filamentous fungi were also contaminated with bacteria. However, if fungus was present, no evaluation was made for bacteria, since the fungus often made visual evaluation of bacterial contamination impossible.

The source of contamination was also evaluated. Generally, contamination arose from the explant, but if contamination was observed growing on the medium apart from a clean expiant, this was noted. Air-borne microorganisms were assumed to be the source of this type of contamination. Generally, 10 vials, one expiant per vial, were initiated per treatment per species for leaf punch experiments, while the number of vials initiated for needle collecting treatments ranged from 10 to 100 with different experiments. The results for each species were averaged for each treatment for each collection. Data were analyzed for standard errors and ANOVA (Tukey-Kremer post-hoc test) using StatView 5.0.1. All cultures were autoelaved at 105 kPa and 250�C for 15min before discarding.

RESULTS

Preliminary experiments. Using the Halazone sterilizing procedure on leaf and bud tissues of several species and a medium with no antimicrobial agents, contamination in preliminary experiments ranged from 30 to 100%. Some of these experiments also compared the two fungicides, benlate and Daconil. There appeared to be no difference between them in controlling contamination. Thus, benlate was chosen for future experiments, since it was more easily handled than the very viscous Daconil. In addition, since leaf and stem tissues were often more plentiful than bud tissue, they were subsequently used as experimental models.

Tropical collections. In collection Tl, a comparison of the levels of contamination from the leaf punch and needle collecting techniques was made using control medium (no fungicides or antibiotics) (Table 3). Three of the species were collected by both methods. Contamination with fungus was significantly greater in the leaf punch collections compared with the needle collections. Percentages of bacterial contamination were not significantly different, but because of the differences in fungal contamination, a much higher percentage of clean-appearing expiants was recovered using the needle collecting technique.

Needle collections made in collections T2, T3, and T4 produced similar results, although the percentage of cultures with bacteria and no fungus was slightly lower than in the first needle collection experiment (Table 4). There was no significant effect of the time of year on the contamination in needle collection cultures from this site.

Because of the high fungal contamination with the leaf punch technique in the first collection, benlate was incorporated into some of the media for collections T2 and T3. This significantly decreased the amount of fungal contamination (P < 0.05) (Fig. 1). However, at the same time, the apparent bacterial contamination increased significantly (P < 0.05), keeping the percentage of clean-appearing cultures to less than 30%, with no difference between the two treatments.

For collection T4, antibiotics were tested in order to control bacterial contamination. A solution containing cefotaxime and vancomycin was added on top of the expiant within a few hours of collecting. When antibiotics alone were added to the control medium, fungus was the predominant contamination, as it was in control expiants (Table 5). As before, the addition of benlate alone resulted in a decrease in the fungus but a significant increase in the apparent bacterial contamination. However, when benlate and antibiotics were added together, both the fungal and bacterial contamination levels were suppressed, resulting in 70% of the cultures appearing clean.

In addition to the overall levels of contamination, collections Tl -T4 were also evaluated for the percentage of species for which at least one expiant remained clean for an individual collecting experiment which included replicate collections. When benlate and antibiotics were both used in the T4 collecting medium, over 90% of llir species produced at least one clean expiant from the leaf punch collections. In comparison, only 25-55% of the species collected onto control medium in collections T1-T4 had at least one clean expiant. In contrast, needle collections averaged at least one clean expiant from over 90% of the species on control medium.

Temperate collections. In collection Cl, a comparison of the levels of contamination from the leaf punch and needle collecting techniques was made using control medium (Table 6). Twenty-one species were collected by both methods. Contamination with fungus was significantly greater in the leaf punch compared with needle collections. Percentages of bacterial contamination \vere not significantly different in the paired species, but, as with the tropical collection, the percentage of clean-appearing expiants was much higher using the needle collecting technique.

When benlate was added to the medium, fungal contamination in leaf punch collections decreased (Table 7). At the same time, the apparent bacterial contamination increased somewhat. These changes were not significantly different in the combined Cl and C2 collections, but were in collection C3. As with the tropical collections, the addition of antibiotics to the benlate medium reduced the percentage of bacterial contamination, giving 70% clean-appearing cultures. There was a trend for slightly more contamination later in the season compared with early collections, although the differences were not statistically significant (data not shown).

Collections Cl-C3 were also evaluated for the percentage of species for which at least one expiant remained clean for a given collecting experiment. Despite the lack of fungicide or antibiotics, 77-92% of the species cultured on control media had at least one expiant that appeared clean. When both fungicide and antibiotics were included in the medium in collection C3, 97% of the species had at least one expiant that remained clean.

Collecting other tissues. During the T2 collection, a variety of I issues other than leaf and stem were collected and placed onto control medium lacking benlate or antibiotics. The results were highly variable, ranging from O to 49% of the cultures remaining clean, depending on the tissue. Immature embryos (six collections) had the highest percentage of clean cultures, with almost half the expiants remaining uncontaminated.

Observations of other contamination effects. In the course of these studies, several species were collected more than once, and there was significant variability in contamination from one collection to the next. For example, although henlate controlled fungal eontamination in most cases, in the T3 collection of Sida acuta, half of the pieces were contaminated with a benlate-resistant fungus. Bacterial contamination in the absence of fungus could also he quite variable, ranging from O to 100% in separate Flemingia slrobilifera collections.

Contamination which was separate from the tissue pieces was included in the total eontamination reported, but was also recorded separately. Levels of separate contamination were very low in the case of leaf punch tissues, ranging from 2% of the total fungal contamination to < 1% in different collections. In the ease of needle collecting, contamination apart from the tissue was more common, as high as 16% in one experiment, and was often found near the lip of the microcentrifuge tubes. In all but one case, contamination detected apart from the expiant was fungal.

DISCUSSION

These results indicate that a high percentage of contamination can he controlled in IVC from hoth temperate and tropical locations, and that this technique can he used to successfully collect leaf and stem tissues from a variety of species for culture e.% situ. One-half to three-quarters of the contamination in cultures collected in these studies was controlled by surface sterilization and by the use of a fungicide and antibiotics in the medium. Field-collected materials have often been noted for their high levels of contamination when cultured in vitro, even when disinfested under laboratory conditions (Duhem et al, 1988; Enjalric et al., 1988; Leifert et al., 1994). Enjalric et al. (1988) reported higher contamination in buds of Hevea brasiliensis when they were taken during the rainy season. We have not observed any similar correlation with season in the contamination of leaf or stem cultures from northwest Trinidad.

Observations in these studies indicated that contamination often appeared to arise from internal sources, such as the cut end of a vein, suggesting that internal contamination was also present even after surface sterilization. The presence of endophytic fungi in leaves has been demonstrated in a number of species (White and Cole, 1985; Carroll, 1988), and it is thought that this may be a relatively common occurrence in plants (Petrini, 1986). Bacteria can also have a close association with leaves (Hirano and Upper. 1986), and it may be that some of the fungal and bacterial contamination observed in these experiments resulted from such associations. In addition, these cultures were not indexed but were only evaluated visually, and thus, it is possible that some contaminants remained in the tissues. Subsequent growth was observed, however, in many of the cleanappearing cultures, indicating that generally such microbes were not inhibiting further development.

The fungicide benlate, which has been widely used agriculturally, was effective in lowering the percentage of fungal contamination in IVC leaf cultures. In aqueous solution benlate breaks down into two compounds, methyl-2-benzimidazolecarbamate (MBC) and butyl isocyanate (BIC), which are active against fungi (Hammerschlag and Sisler, 1973). Benlate has been used in plant tissue cultures with little toxicity (Thurston et al., 1979; Haldeman et al., 1987), although some species may show sensitivity (Das Gupta and Hadley, 1977). There have also been several reports of cytokinin-like activity from benlate (Skene, 1972; Thomas, 1974). However, benlate has become less available in recent years and other fungicides and antimicrobial agents, such as Plant Preservative Mixture (PPM(TM), Plant Cell Technology, Inc., Washington, DC), may provide similar results.

The antibiotics cefotaxime and vancomycin were chosen after preliminary experiments testing several antibiotics with leaf discs of Lonicera maackii indicated that this combination appeared to be effective against some bacterial contaminants while at the same time presenting a low toxicity. Cefotaxime is a broad spectrum antibiotic, and has been used to eliminate bacterial contamination from plant tissues in vitro (Savela and Uosukainen, 1994; Chanprame et al., 1996) as well as to eliminate Agrobacterium. tumefaciens in transformation protocols (Shackelford and Chlan, 1996). It is generally of low toxicity (Thurston et al., 1979; Pollock et al., 1983) and has been shown to stimulate or inhibit growth or organogenesis, depending on the tissue and the concentration of cefotaxime (Mathias and Boyd, 1986; d'Utra Vaz et al, 1993; Rugini and Caricato, 1995; Rao et al., 1995; Tosca et al., 1996). Vancomycin, which is effective against Gram-positive bacteria, has also shown growth regulator-type activity in vitro (Chang and Schmidt, 1991). Because of its importance in medicine, however, its use should be limited, and it should be possible to substitute other antibiotics for vancomycin for use in IVC.

Although the tropical collections had more contamination on control media than the temperate collections, the tropical fungi were slightly more responsive overall to the addition of benlate than the fungi in the temperate collections. Cultures that became contaminated in the presence of fungicide and antibiotics may have contained strains resistant to these antimicrobial agents. However, in some cases, fungus grew on the tissue only, spreading only very slowly onto the medium, suggesting that the benlate was not always transported throughout the tissue. With some species, callus growth began in the presence of bacteria. Usually, the callus was overcome by the bacteria after one or more subcultures. However, in one case, Paullinia sp. (Sapindaceae), the callus became established and bacteria could not be visually detected after several subcultures (Pence, 1999).

The needle collecting technique had much lower contamination levels than the leaf punch technique. This may be due to the larger size of the leaf disc, as well as the much higher proportion of surface area in the leaf disc sample. However, the use of a fungicide and antibiotics brought the percentage of contamination in the leaf discs down to a level comparable to that of the needle collecting. Leaf disc tissue is more likely to be available, since stems need to be of a sufficient diameter, but still young, green and relatively soft, to be effectively collected by the needle technique. Stems that are too small for needle collecting can be sliced using scissors. Attempts to culture stem slices suggested that their level of contamination is comparable to that of the leaf discs.

Subsequent growth was obtained in a number of species from both leaf disc and needle collecting, with plants regenerating from both types of tissues (Pence and Plair, 1995; Pence, 1998), and this will be reported in detail elsewhere. The success of leaf and stem IVC is dependent on the ability of the tissue to regenerate shoots and, ultimately, whole plants in vitro, and this is dependent on the species. However, growth from preformed meristems, such as apical or axillary buds, generally produces fewer of the genetic changes which can occur in vitro than adventitious growth (Larkin and Scowcroft, 1981), and methods which minimize genetic change during in vitro manipulation are preferred in any IVC protocol. Results from preliminary experiments suggest that methods that control contamination in leaf tissue should be adaptable to lnul tissue as well. Studies in this laboratory also suggest that the fungi contaminating leaf and bud cultures are similar, and IVC methods are now routinely used by this laboratory to collect buds and leaves from endangered plants (Pence et al., 2002). Other reports of in vitro collecting have been directed at bud collecting, and protocols similar to the ones described here have been successful in recovering clean tissues (Yidana et al., 1987; Altman et al., 1990; Ruredzo, 1991). However, when seeds are not available and suitable bud tissue is limited, the collecting of leaf and stem tissues offers a supplemental method for collecting living plant germplasm.

The approach taken in these studies has been to examine a basic technique of IVC for broad application to many species. Undoubtedly, no one method will be suitable lor every species and every situation. However, this approach should provide a method that can be used with a certain number of species in a variety of circumstances and should provide a basis for modifying the technique for more specific applications.

ACKNOWLEDGEMENTS

The author gratefully acknowledges the generosity of the: late Mr. Marion Mulford for funding the August 1994 eolleeting trip to Trinidad, and of Eliza Biederman for funding the 1990 trip to Costa Rica for preliminary studies; the excellent coordination oi the Tnnidad trips by Bernadette Plair; the excellent technical assistance in the lab and in the field of Bernadette Plair, and for the assistance of Natasha Cavanaugh, Nancy Wickemeyer, Susan Charls, and Virginia Geiger during the Trinidad trips; the Centre for the Rescue of Endangered Species of Trinidad and Tobago (CRESTT), Janice Hernandez, former Director, who provided us access to the Environmental Research Station on Morne Catherine, and who also provided lodging; the National Herbarium of Trinidad and Tobago, Yasmin Comaeu, Director, who, along with her staff, identified plant materials collected during the Trinidad trips and who provided excellent field assistance; those who assisted with food, transportation, and lodging in Trinidad; Allison Flege for assistance in the early development of the technique and for making the Tonga collections; the numerous volunteers and several student assistants who assisted in making media and who assisted with the temperate collections; Betty Myers for assistance in the preparation of Tables 1 and 2; and John L. Caruso, Department of Biological Sciences, University of Cincinnati, in whose laboratory preliminary experiments, prior to June 1991, were done.

[Reference]

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[Author Affiliation]

VALERIE C. PENCE*

Center far Conservation and Research of Endangered Wildlife, Cincinnati Zoo and Botanical Garden, 3400 Vine. Street, Cincinnati, OH 45220

(Received 25 June 2004; accepted 22 November 2004; editor K. Dixon)

[Author Affiliation]

*Author to whom correspondence should be addressed: Email valerie. pence@cincinnatizoo.org

IN VITRO COLLECTING (IVC). I. THE EFFECT OF COLLECTING METHOD AND ANTIMICROBIAL AGENTS ON CONTAMINATION IN TEMPERATE AND TROPICAL COLLECTIONS