Micropropagation of Carnivorous Plants

A simple, efficient system for seed surface disinfestation and in vitro germination was developed for the carnivorous pitcher plants Darlingtonia californica and Sarracenia leucophylla. Of the disinfectants tested, hydrogen peroxide or 10% Clorox® were most effective for disinfesting seeds of D. californica, while concentrated sulfuric acid worked best for S. leucophylla. Differences in the effectiveness of sterilants were associated by differences in seed coat morphology. Seeds of D. californica imbibed at 4-7°C in sterile deionized water with surfactant and gibberellin germinated earlier than seeds without exposure to gibberellin. Unimbibed seeds of S. leucophylla germinated rapidly in sterile water after treatment in concentrated sulfuric acid. Scanning electron microscopy of D. californica seed coats revealed waxy trichomes covering the seed surface. In contrast, the seed coats of S. leucophylla were pitted with surface and sub-surface cells possessing heavily thickened cell walls. These cells were devoid of contents. Fungal hyphae were observed on the seed surface and within empty cells of the integuments. Scanning electron microscopy observations and comparison of seed coat morphology of the carnivorous plant genera Drosera, Dionaea, Sarracenia and Darlingtonia revealed a wide range of differences in structure and ornamentation, which may suggest a species specific approach to surface disinfestation. A simple effective system for the in vitro growth, multiplication and rooting of axenically germinated seedlings of D. californica was developed. Seedlings grown on solid Yi strength Murashige and Skoog medium produced more biomass and more and longer pitcher leaves than seedlings grown on other solid media assayed. Root development on solid media was minimal and usually limited to the seminal root regardless of the medium. Seeds stimulated by gibberellic acid prior to germination and exposed to auxin and cytokinin during early seedling development produced multiple offshoots as well as fibrous root systems when transferred to Y2 strength liquid medium containing charcoal. Similarly treated seedlings transferred to Yi strength liquid media without charcoal produced multiple offshoots but fewer root systems. Seedlings cultured in medium without charcoal produced more but smaller pitchers than seedlings cultured in medium containing charcoal. Multiplication did not occur on solid media, and seedling growth was stunted. Seedling multiplication through offshoots occurred in all liquid media and was both prolific and rapid. Darlingtonia californica was regenerated from whole, in vitro germinated seedlings and excised segments from in vitro generated juvenile pitchers. When incubated on solid Phytomax Orchid Multiplication Medium, seedlings produced protocorm-like bodies and green leafy callus When divided and subcultured in liquid Phytomax Orchid Multiplication Medium, explants of both protocorm-like bodies and green, leafy callus gave rise to multiple shoots as well as more protocorm-like bodies and green, leafy callus. These could be further divided and subcultured. Transverse segments of excised pitcher leaves from axenically-grown seedlings produced shoots and protocorm-like bodies when subcultured in liquid Phytomax Orchid Multiplication Medium. Unlike D. californica, seedlings of S. leucophylla did not readily produce offshoots when incubated on solid media. A protocol for extraction of embryos from selected Sarracenia species was developed. ACKNOWLEDGEMENTS I especially thank my major professor Dr. Alison Roberts for her guidance, patience and vision in helping me bring this project to fruition. I thank her for believing in me, and for keeping me focused during difficult times (of which there were many). This must not have been easy for her. I thank the members of my committee, Dr. Richard Koske for his guidance and advice throughout the years, Dr. Joanna Norris for her exceptional patience, and Dr. Carl Beckman whose spirituality has left its mark on me. I also thank Dr. Larry Englander for acting as chair of my defense. I thank Chris Nerone, Veronica Masson, Marybeth Able, Stacie Paquette, Jane Leary, my sons Kenneth and icklaus Uhnak, John Phillip Jr. , and Judy Gould. I thank Ranger Hall graduate students Ryan Tainsh and Mike Budziszek for helping with statistics and computer problems, Alissa Neill for support, and Mary Beth Hanley for kindly leaving food in the refrigerator on nights I worked until dawn. I thank Dr. Eric Roberts for generously saving me from certain destruction during the final hours. I thank the Alley family for believing in me enough to offer their prayers of support. I thank William Alley, Janice Alley, and William Alley Jr. I also thank Claudia, Becky, Ron and Nana Franke for their much appreciated support and prayers. I thank my parents Dominic and Bernadette for their love and support. A special thank-you is due to my wife, Leslie Anne Uhnak, who not only typed much of this dissertation (more than once), but also endured a somewhat chaotic life during its completion. I also thank her and my stepdaughter, Kelsey Desrosiers, for their support and belief in me during this remarkable ordeal.

longer pitcher leaves than seedlings grown on other solid media assayed. Root development on solid media was minimal and usually limited to the seminal root regardless of the medium . Seeds stimulated by gibberellic acid prior to germination and exposed to auxin and cytokinin during early seedling development produced multiple offshoots as well as fibrous root systems when transferred to Y 2 strength liquid medium containing charcoal. Similarly treated seedlings transferred to Yi strength liquid media without charcoal produced multiple offshoots but fewer root systems. Seedlings cultured in medium without charcoal produced more but smaller pitchers than seedlings cultured in medium containing charcoal. Multiplication did not occur on solid media, and seedling growth was stunted. Seedling multiplication through offshoots occurred in all liquid media and was both prolific and rapid.
Darlingtonia californica was regenerated from whole, in vitro germinated seedlings and excised segments from in vitro generated juvenile pitchers. When incubated on solid Phytomax Orchid Multiplication Medium, seedlings produced protocorm-like bodies and green leafy callus When divided and subcultured in liquid Phytomax Orchid Multiplication Medium, explants of both protocorm-like bodies and green, leafy callus gave rise to multiple shoots as well as more protocorm-like bodies and green, leafy callus. These could be further divided and subcultured. Transverse segments of excised pitcher leaves from axenically-grown seedlings produced shoots  , for early references) .
Their many unique anatomical and physiological adaptations peculiar to the carnivorous habit continue to attract attention from workers in such diverse areas as developmental biology, ecology, cell biology, micropropagation and evolutionary biology.
This artificially grouped assemblage of plants comprises more than 600 species  and has grown considerably since 1989 when Givnish reported 538 .  Budzianowski, , 1995aBudzianowski, , 1995bHook, 2001 ;and Zenk and Steglish, 1969). Additionally, extracts of the roots of Sarracenia jl.ava possess anti tumor activity against certain forms of human cancer cells . Collection pressure and/or habitat destruction has reduced or depleted many natural populations of carnivorous plants in the wild  containing levels of nickel , zinc, chromium and other metals that are generally phytotoxic makes it extremely interesting and appropriate for the study of what may be unique developmental and physiological processes. S. leucophy lla is an American pitcher plant found on the lower Gulf Coast of the United States in Georgia, Florida, Alabama and Mississippi.
Although several species of carnivorous plants have been micropropagated (see appendix C this dissertation), work on members of the Sarraceniaceae has been limited. A search of the literature revealed only two papers on their in vitro culture.  reported qualitative observations on applying the techniques of orchid culture to D. californica and S. purpurea. D. californica also was included in a broad survey of the feasibility of micropropagation of carnivorous plants .
Until recently few reports have addressed the germination of seeds of carnivorous plants, particularly members of the Sarraceniaceae. Prior to the publication of Ellison ' s (2001) extensive study on the germination of 8 species of Sarracenia, the literature contained only two papers (not including Withner's note) on the germination of seeds of only one specie, S. purpurea (Mandossian, 1966;Gotsch and Ellison, 1998). No micropropagation systems for the Sarraceniaceae have been described in the literature. This is surprising since many of the species that are offered commercially have been propagated by tissue culture. The lack of published information on propagation of these species likely reflects the concerns of commercial producers who consider such techniques proprietary (a brief search of the web on 4/ 12/03 found four commercial suppliers offering carnivorous plant species from tissue culture including members of the genera Sarracenia, Heliamphora , and Nepenthes). I feel that development of such systems and publication of the protocols will help alleviate collection pressure in the wild and offer other workers in the field methods that may facilitate the study of developmental , physiological, and ecological questions whose answers may be found within the unique adaptations and habit of this marvelou s group of plants.
This manuscript reports on the initial steps in the establishment of a micropropagation system for members of the carnivorous plant family Sarraceniaceae.
wash, seeds were resuspended in 2-3 ml of sterile deionized water and transferred to sterile glass petri dishes. Fine forceps or a sterile bacterial loop was used to transfer the seeds (one per well) to individual wells of 12 or 24-well sterile plastic culture plates (Sigma, St. Louis, Missouri) with each well containing 2 ml of sterile MYP (malt extract, 7.0 g/l; yeast extract, 0.5 g/I; bacto-peptone, 1.0 g/l; Koske, 1977) and l 0 g/l sucrose. Medium pH was adjusted to 6.5 with KOH before the addition of 9.5 g/l Bacto-agar (Difeo, Detroit, Michigan) and autoclaved at 121°C for 15 min. Fortyeight seeds were used in each treatment. Experiments were repeated twice using two different seed lots for each species. Seeds were purchased in 2001 and 2002 respectively. All seeds were collected by the supplier in the fall of each year and stored at 4-7°C prior to shipment (personal communication, Peter Paul ' s Nurseries, Canadaigua, NY). Culture plates were wrapped in Saran Wrap® brand plastic wrap (Dow Brands, Indianapolis, Indiana), sealed in a Rubbermaid® container (Newell Rubbermaid Inc. , Freeport, Illinois) and placed in a dark growth chamber at a constant temperature of 27°C. Cultures were examined for signs of contamination with the aid of a dissecting microscope daily or every other day for a period of 15 days .
Additionally, 48 seeds of S. purpurea and 24 seeds each of S. flava , S. alata and S. rubra were subjected to the sterile wash series in treatment #5 as described above.

Scanning Electron Microscopy
For surface studies, seeds of Darlingtonia, Sarracenia, Drosera and Dionaea spp. were air-dried for 24 h then sputter-coated with gold during rotation for 90-160 s.
For internal studies, seeds were cut in half or into sections using a razor blade or fine pointed surgical scalpel and a dissecting microscope. Any residual endosperm or embryonic tissue was removed using a fine pointed dental pick. Cut seeds were airdried for 24 h, fixed to double sided tape (Scotch) on microscope coverslips, sputter coated with gold and attached to the stage mount with double sided tape. Seeds of D. californica and S. leucophylla also were treated for 4,8,12, and 16 min in concentrated H 2 S0 4 , rinsed 3 times in sterile deionized water and air-dried for 24 h before being prepared for SEM as described above. Observations were made with an Hitachi 4100 Scanning Electron Microscope (Hitachi, Ltd ., Toyo, Japan). Images were captured using a Sunspark Image Capture System (Sun Microsystems Inc., Santa Clara, California).

Seed Germination Studies
Six methods used to germinate seeds are summarized in table #1. Germination was scored by emergence of the radicle.

Germination of Unimbibed Seeds in Liquid Culture
Seeds of D. californica and S. leucophylla were surface-dis infested for I 0 and 20 min, respectively, in 10% Clorox® solution (Method # 1, Table 1) and rinsed.
Following the last wash, seeds were poured (along with 1-2 ml of water) onto sterile filter paper in a sterile petri dish and transferred, with fine forceps or a bacterial loop, to 50 ml Erlenmeyer flasks (six seeds per flask, six flasks per treatment) containing 1) 5 ml of sterile H 2 0 (pH 5.0) or 2) 5 ml of liquid Y2 strength Murashige and Skoog (MS) salts + MS vitamins + 20 g/ l sucrose . The flasks were placed on a gyrotary shaker at 120 rpm under room temperature and lighting conditions (diffuse light, 23°C ± 3°). All transfers were performed in a laminar flow hood. With the exception of the acid treatment for D. californica, experiments were repeated twice.
Surface-disinfestation time was 10 min in H 2 0 2 . After disinfestation seeds were transferred (25 seeds each) to nine, 15 mm x I 00 mm plastic petri dishes (3 per treatment) containing 25 ml of water agar. Incubation was at room temperature and room lighting conditions as described above. Observations were made daily until first germination was observed, then every other day for a period of 21 days. The experiment was repeated twice.
Germination of Seeds of S. leucophylla, S. alata, and S. purpurea Following Cold-Imbibition and Treatment In Concentrated H 2 S0 4 Seeds of S. leucophylla, S. alata, and S. purpurea were prepared by method #6 (Table 1)

Effect of Scarification on Germination of Seeds of D. californica
The micropylar regions of 30 seeds of D. californica were removed with the aid of a dissecting microscope and fine pointed scalpel, and seeds were then subjected to Method # 3 ( Table 1) and incubated at room temperature and light. The experiment was repeated twice.

Statistical Analysis
Results of sterile H 2 0 wash and surface sterilant treatments are reported as percent contaminated seeds after 15 days incubation on MYP. Treatments were compared using chi-square analysis with Yates correction (Schefler, 1979) of number of seeds contaminated after 15 days incubation and the number of seeds not contaminated. Data for germination in liquid medium were analyzed by converting number of seeds germinated after 21 days to percent. Percentages were transformed using arc sin transformation. T-tests were performed on the transformed data.
Analysis of germination data from solid media experiments was performed with ANOV A followed by Fisher Post-Hoc tests. Comparisons of early and late germination between treatments were made with chi-square analysis of number of seeds germinated on day 15 vs. total number germinated after 21 days. ANOV A, t-test and transformations were performed with Statview 5.0 (Statview: Using Statview SAS Third Edition, 1999). Chi Square analysis was performed with a program provided by Dr. R. Koske (Dept. of Biological Sciences, URI) .

RESULTS: Surface Disinfestation -Sterile Wash
In all species washing with sterile water generally was ineffective for obtaining sterile seed material (Table #2). However, in some trials, surface disinfestation of seeds of D. californica was achieved with a sterile H 2 0 wash ( Table   2, Trials 2,and 3).

Treatment in Surface Sterilants
Seeds of D. californica could be surface disinfested with all sterilants tested using exposure times as low as 4 min (Table 3) . In contrast, seeds of S. leucophylla remained contaminated regardless of the treatment or exposure time ( Table 3 ).
Treatment of S. leucophy lla in concentrated H 2 S0 4 resulted in a consistent reduction in percent of contaminated seeds as exposure time in sterilant increased ( Figure I). A similar linear decrease in contamination was observed in response to treatment with Physan 20, but in much smaller increments, and the contamination rate remained high.
Contamination sometimes spiked as time in sterilant increased (Fig. I). Chi -square analysis of treatment times for S. leucophylla seeds with concentrated H 2 S0 4 suggested that optimum treatment time was between 8 and 12 min (Table 4).

Scanning Electron Microscopy
Dry seeds of D. californica are "tear-drop" shaped with finger-like trichomes on the seed surface ( Fig. 2A) . Erect trichomes (those with their long axis perpendicular to the long axis of the seed) were most numerous at the chalazal end of the seed (Fig. 2B) and diminished in density toward the tapering micropylar region Seed coats of S. purpurea, S. jlava, and S. rubra were similar to those of S. leucophylla in surface topography . In these species fungal hyphae, sporangia and spores were frequently observed on the surfaces of many of the seeds (Figs. 6i and j) or appeared to be growing out of damaged surface cells (Fig. 6J). In contrast, seeds of Drosera species showed considerable interspecific variation in surface topography (Figs. 7 A-J). The surface of Dionaea muscipula seeds (Figs. 8A -H) was relatively smooth (Fig. 8A) with slight bulges in the outer periclinal wall of surface cells giving the seed coat surface a "cobblestone" appearance ( Fig. 8B). The outermost layer of the seed coat was characterized by hollow, heavily sclerified cells ( Fig. 8C) with thick anticlinal walls (Fig. 80). The periclinal surface was covered by a thick, homogeneous, extracellular matrix (Fig. 8E). Although the seed coat layers appeared more impenetrable than in any of the other genera examined, evidence of fungal infection was observed (Figs. 8F and G). When viewed in paradermal section the interior of the seed coat had a honeycombed appearance (Fig. 8H).

Germination Studies in Liquid Medium
Germination of seeds of D. cal(fornica in water (Method # I, Table 1) was significantly greater than in Yi strength MS (P=0.006 and P=0.004respectively for two trials) at 21 days of incubation (Fig. 9). The number of seeds germinated on day 15 vs. the total number germinated by day 21 was significant at P=0.01 (chi square= 8.517) and P=0.05 (chi-square = 6.23) respectively for trials I and 2. Seeds of D. californica treated in H 2 S0 4 (Method #2, Table I) failed to germinate after 21 days of incubation.
Seeds of S. leucophy lla could not be surface-sterilized in quantities required for further experiments. For instance, in one trial , contamination appeared early in 5 of 6 flasks containing 6 seeds each. Appearance of contamination sometimes followed germination and seeds germinated even in contaminated flasks (see Appendix A, Ancillary Results to Manuscript 1 for data on S. leucophylla). S. leucophy lla showed 11.5% germination in H 2 0 after 21 days compared to no germination in liquid Yi strength MS in the only trial that produced usable data. Germination on solid Yi strength MS was not determined due to contamination in all flasks. Further work with S. leucophy lla was limited to work with 3 seedlings that had germinated in one uncontaminated flask of 6 seeds in H 2 0.

Germination on Semi-Solid H 2 0 Agar Medium
The response of seeds of D. californica varied widely between germination Methods #3, #4 and #5. The highest percent germination (~80%) was observed in seeds receiving cold-imbibition and GA 3 stimulation before germination (Fig. 10). All treatments differed significantly from each other (P=0.0005).

Imbibition and Treatment in Concentrated H2S04
Seeds of S. leucophylla, S. alata, and S. purpurea failed to germinate after 60 days of incubation in a growth chamber with temperature and light conditions as previously described.

Effect of Scarification on Germination of D. californica
Germination of scarified seeds was 70% and 77% respectively after 21 days in two trials (data not shown-see Appendix A).

Surface Disinfestation
The very high effectiveness of the variety of sterilants tested for surface disinfestation of the seeds of D. californica was unexpected. SEM observations of the seed coat revealed a surface morphology that presented opportunity for the collection and adherence of spores of microorganisms. Also, seeds of D. californica have been reported as difficult to disinfest . A lack of contamination during incubation of D. californica seeds was observed in preliminary experiments (data not shown). In addition, when data from the sterile wash series were collected, it became apparent that seed surfaces were either remarkably free of microorganisms or could be made free of them with a simple wash. The use of 3 different seed lots in these experiments suggests that this phenomenon is not seed-lot specific. Although all sterilants tested were effective, 10% Clorox® and 3% H20 2 were selected for use in subsequent experiments with D. californica because of their consistent effectiveness.
Physan 20 was rejected after seeds di sinfested in a 10% solution failed to germinate.
Similarly, concentrated H 2 S0 4 was rejected after seeds treated for 4 min failed to germinate (see Appendix A). SEM observations showed that seed coat integrity had been compromised by the acid.
In contrast, seeds of S. leucophylla were difficult to disinfest in quantity using the 4 sterilants tested. Although difficulty in disinfestation of explanted organ tissues from several species of carnivorous plants has been reported , repo11s of diffic ulty in disinfestation of seeds used as initial explant material for culture of carnivorous plants has been limited (see Table 3, Appendix A) . The inability to effectively disinfest seeds of S. leucophy lla may result from endophytes present within the seed coat or pathogens that gained entry during or after its maturation. SEM observations revealed fungal hyphae traversing the seed coat surface of S. leucophylla (Figs. 3F and I). Fungal hyphae and sporangia were also observed emerging from damaged cells of the seed surface of S. purpurea (Fig.   61) and the micropylar region of the seed coat of Dionaea muscipula (Fig. 8E). This implies that they are not in the category of surface organisms, thus rendering surface sterilization insufficient as a disinfestation treatment for these seeds.
The effectiveness of H2S0 4 as a disinfectant for seeds of S. leucophylla may have resulted from its ability to successively dissolve the seed coat layers  and reach previously sheltered endophytes or their spores. In preliminary experiments (data not shown) several species of Drosera and Dionaea muscipula were easily disinfested with 3% H 2 0 2 . Ease of disinfestation was most likely due to the unornamented outer layers of their seed coats as observed in SEM (Figs. 7F and I; Contamination of ex planted tissue of carnivorous plants has sometimes been attributed to the presence of endophytic organisms . Although the literature contains many reports of endophytes in noncarnivorous plants (e.g. White et al. , 1993Carroll, 1988;Hinton and Bacon, 1984), to my knowledge no investigations of endophytes in carnivorous plants have been reported.

Germination Studies
The rapid germination of unimbibed seeds of D. californica in H 2 0 and coldimbibed seeds in H20 agar is both surprising and fortuitous. Horticultural literature suggests that germination of seeds of D. californica requires a cold treatment of up to 2 months (Lecoufle, 1990;Pietropaolo and Pietropaolo, 1986). Earlier observations by  reported the failure of mature seeds of D. californica to germinate under laboratory conditions when sown immediately after collection; successful germination was only achieved through the use of immature seeds from green seed capsules. The research reported in this manuscript shows that cold imbibition of mature seeds of D. californica for 24 h in H 2 0 results in high germination percentages within 21 days. This rapid germination may be the result of the role of cold imbibition in breaking seed dormancy (Vidaver,I 977).
Seeds of S. leucophylla, S. alata and S. purpurea failed to germinate if cold imbibed prior to disinfestation in concentrated H 2 S0 4 . These results confirm observations by Ellison (2001 ). However, unimbibed seeds of S. leucophy lla germinated when treated for I 0 min in concentrated H 2 S0 4 (see Appendix B) and incubated in H 2 0 .
Because sufficient numbers of seedlings can be produced without GA 3 stimulation, use of the hormone is not warranted for the production of seed I ings for research because the effect of GA 3 on seedling morphology and its long-term effect on growth still require investigation. Similarly, because of the labor required, surgical removal of the micropylar pole of seeds was not considered to be worthwhile in es ta bl ishrnent of this micropropagation system.

Summary
Based on the results of this investigation, protocols for the disinfestation and axenic germination of seeds of D. californica have been established. Additionally, this study indicates a possible relationship between seed coat morphology and ease of seed disinfestation. l..O        Root development on all solid media was minimal and usually limited to the seminal root. Seeds stimulated by gibberellic acid prior to germination and exposed to auxin and cytokinin during early seedling development produced multiple offshoots as well as fibrous root systems when transferred to Yz strength liquid medium containing charcoal. Similarly treated seedlings transferred to Y2 strength liquid media without charcoal produced multiple offshoots but fewer roots. Seedlings cultured in medium without charcoal produced more but smaller pitchers than seedlings cultured in medium containing charcoal. Multiplication did not occur on solid media, and seedling growth was stunted. Seedling multiplication through offshoots occurred in all liquid media and was both prolific and rapid .

INTRODUCTION
Manuscript 1 (this dissertation) reported techniques for the effective surface sterilization and subsequent axenic germination of seeds of Darlingtonia californica.
This manuscript reports on the selection of a suitable growth and rooting medium for in vitro germinated seedlings that also allows the induction of clonal multiplication.
Growth and rooting were investigated using both solid and liquid media.
Darlingtonia cal(fornica is a North American pitcher plant naturally occurring in Oregon and California . It is interesting as a study organism not only because of its carnivorous habit but also because it offers the opportunity to study a combination of unique developmental and physiological events. For instance, D. californica usually grows beside cold (below 20°C) running streams in serpentine soils containing levels of nickel , zinc, chromium and other metals that are phytotoxic to many plant species . However, it accumulates only low levels of these metals (Reeves et al. ,I 983). Additionally, it forms two morphologically and anatomically different pitcher types from the same meristem during the course of its development (Frank, I 975). Such a developmental switch offers the opportunity for investigations at both the cytological and genetic levels . Future research utilizing D.
californica as a study organism will require the availability of sufficient amounts of plant material. This may be difficult because commercially produced adult or juvenile plants are seldom easy to obtain. Successful in vivo greenhouse cultivation has been limited to a few private collections .
Although D. californica has been the subject of several histological and developmental investigations for over a century (see  for early references) little work has been done on its culture in vitro.  reported a short series of qualitative observations on the application of orchid tissue culture techniques to carnivorous plants. However, he reported the failure of D. californica to form roots in vitro after two trials. Also, D. californica has been included as part of a broad feasibility survey on the micropropagation of several carnivorous plant genera . Although  reported that few roots were formed in vitro , results were not reported quantitatively.
This manuscript reports on the establishment of a system for in vitro culture of D. californica that provides a continuing source of explant material for subculture and future experimentation.

Plant Material
For growth studies on solid media, seeds of D. californica were surface  Table 1 for medium components;pH 5.0).

Growth Studies on Solid Media
Three basal media were used : MS  at Yi strength salts, Burgeff's N 3 f , and a modified, sphagnum-based medium . Media components are listed in

Growth and rooting in liquid media
Triplicate 250  seedlings with offshoots (9-12 pitcher leaves) that had been grown on solid POMM for 2 months. All media were adjusted to pH 5.0 prior to autoclaving .. Concentrations of sucrose and vitamins were as described for solid media. Flasks were transferred to a growth chamber with light and temp conditions as described above. After 6 12 weeks of culture, plants were harvested and data collected.

Growth on Solid Media
Comparisons of numbers of pitchers produced per seedling between different media formulations were made using ANOVA followed by Fishers Post Hoc test. For comparison of pitcher length, average pitcher lengths per replicate were log transformed then analyzed as described above. Dry masses are reported as percent difference between treatments.

Rooting and Growth in Liquid Media
To determine average pitcher lengths, the longest 25 pitchers in each flask were measured to the nearest mm, and average pitcher length per flask was calculated.
In assessing the total number of pitchers per flask, only pitchers longer than 2 cm were measured. Vitrified or etiolated pitchers were not scored. Total number of roots per flask was determined by scoring the number of roots observed on 17 separate clusters of pitchers (each cluster arising from a single rhizome). Dry weight of total plant material from each flask was obtained as described previously. Differences among treatments were analyzed with ANOVA and Fisher's Post Hoc test.

Growth on Solid Media
Seedlings grown on solid Y 2 strength MS medium produced more pitchers than those grown on either Burgeff s N 3 f medium (P = 0.002), or on sphagnum based medium (P= 0.002, Fig. 1). Although growth of seedlings on Burgeffs medium resulted in the least number of pitchers produced, this difference was not significant compared with those grown on sphagnum medium. The average length of pitchers was significantly greater for seedlings grown on MS medium than those grown in sphagnum medium (P= 0.03) but not significantly different from those grown in Burgeffs medium (Fig. 2). Qualitatively, seedlings grown in 1i MS medium appeared greener and healthier than those grown in the other media formulations (Fig. 3A).
Seedlings grown in Burgeff s medium (Fig. 3B) began to exhibit a yellow appearance soon after transfer from H 2 0 agar. Several began to brown by the end of the experiment, and growth appeared to be arrested. In contrast to seedling growth on both Y 2 MS and Burgeffs, seedlings grown in sphagnum-based medium ( Fig. 3C) occupied an intermediate position and seedling color ranged from green to yellow green. Except for the seminal root, roots were not observed in any of the media tested.
In general, growth was slow on all three media. Averages of two trials for the collective dry weights of all seedlings in a treatment were 0.057 g (Y2 MS), 0.020 g (Burgeffs) and 0.019 g (sphagnum).

Rooting and Growth in Liquid Media
Production of new pitchers was both rapid and prolific in all liquid formulations tested (Fig. 4 ). Clusters of pitchers could be separated (Fig. SA) and were observed to originate from a single central rhizome. The large number of pitchers produced in liquid culture was in stark contrast to that produced from the single seedlings grown on solid Yz strength MS medium for six months (Fig. SB). The number of pitchers was significantly greater without charcoal in either Yi strength or l;4 strength liquid MS medium (P<0.02), but was not affected by medium strength in the presence or absence of charcoal (P>0.93 , Fig. 6). In contrast, pitcher length was significantly greater with charcoal in either Yz and l;4 strength MS (P <0.0001, Fig. 7).
Pitchers produced in medium containing charcoal were brighter green and appeared to be more robust due to an observed (but not quantified) difference in diameter. Root production was greater in Yi strength medium with charcoal than in any other treatment (P=0.0001 , Fig. 8). However, significantly more roots were produced in Yi

DISCUSSION
The object of this study was to select a suitable medium for in vitro growth and rooting of D. californica. The three media assayed were chosen from the literature based on results reported from prior use in related studies. MS medium at Y2 strength salts was selected because reduced-salt MS medium is commonly employed for in vitro culture of many carnivorous plant species (see Appendix B).  used Burgeff s N 3 f medium in his studies on D. californica. However, the formulation is not reported and was obtained from . The components for sphagnumbased medium were based on . Potassium nitrate and myo-inositol concentrations for sphagnum-based medium were based on full strength MS medium .
Media were chosen based on preliminary studies (sphagnum-based medium) or results of previous studies (Burgeffs N 3 fmedium, . However, the growth of D. californica was poor on all solid media tested. Of the media tested, sphagnum-based medium was the least defined. The condition of the sphagnum used in preparation of the medium and the conditions present in the habitat where it was collected, may have affected levels of macro and micronutrients available in the final medium formulation. This may have affected the growth promoting and growth sustaining ability of the medium because certain plants have requirements for specific concentrations of micronutrients (Dodds and Roberts, 1995). Vitamin and sucrose levels were the same in all media. However, nitrogen was supplied as potassium nitrate and casein hydrolysate in sphagnum-based medium, rather than ammonium ion, as in the other media. Growth of plants or plant tissue in culture has been shown to be most rapid when both nitrate and ammonium are available (George and Sherrington, 1984). Similarly, the poor growth on solid Burgeffs medium may have resulted from a deficiency in micronutrients, not present in published formulations   Growth on solid medium, regardless of the formulation, may have been poor because D. californica did not form roots and was therefore unable to absorb enough water or nutrients to allow more vigorous growth. Also , the seminal root may have depleted nutrients in the media surrounding it due to the relatively small volume of media in the tube. In contrast, growth on Y2 strength MS may have been greater than on the other formulations because of its completeness in terms of macro and micronutrients.
One possibility for the prolific growth of D. californica in liquid medium at both Y2 and ';4 strength salts may have been the availability of nutrients to the pitcher leaves. Because of its carnivorous habit, internal zones of the pitcher are specialized for absorption of nutrients from digested prey . In a sense, the liquid medium may have served, literally, as a nutrient soup for the pitcher leaves.
Additionally, incubating plant material in liquid-shaken cultures has been shown to increase the rate of shoot proliferation in some species of non-carnivorous plants .
Increased pitcher production without charcoal in Yz and ~ strength MS media may be related to the endogenous production of hormones which stimulate proliferation of D. californica in its natural habitat. In media containing charcoal phytohormones may have been adsorbed before they could reach levels that stimulate production of new pitchers. Activated charcoal added to media has been shown to adsorb cytokinin (Takayama and Misawa, 1980). Because the seedlings used to initiate this experiment had been exposed to GA 3 and grown on medium containing phytohormones, carry over could have had the ongoing effect of enhancement of pitcher production in charcoal free medium. However, the effect of any carry over of phytohormones to media with charcoal may have been negated due to adsorption of the hormones by the charcoal. In media containing charcoal, pitcher length may have been greater due to adsorption of phytohormones allowing resources to be allocated to the growth of individual pitchers instead of the production of new pitchers.
Increased root production in Yz MS with charcoal in comparison to all other treatments, could have been due to greater nutrient availability in the Y2 MS .
Absorption of nutrients by pitcher leaves of carnivorous plants has been shown to stimulate uptake of nutrients by roots and contribute to greater root length (Adamec, 2002). Additionally, activated charcoal may adsorb toxic substances in the medium resulting in increased root production (Ziv, 1979;Takayama and Misawa, 1980). In the present study, the effect of charcoal on root induction appears to be synergistic with the strength of the medium. Charcoal as an additive for root induction has been used for in vitro rooting of difficult-to-root carnivorous plant species such as Nepenthes ). More work is required in order to clarify the role of charcoal in root induction. .r= (,,) ....

D. californica is a North American pitcher plant endemic to the states of
Oregon and California . S. leucophylla is a southern trumpet pitcher plant distributed along the southeast coast of the U.S. . As with other members of the Sarraceniaceae, these plants have long been objects of interest and study for botanists (see , for early references) . Both species possess several attributes that make them suitable for studies in morphogenesis. The shoot systems of D. californica and S. leucophylla are heteroblastic. In D. californica both juvenile and adult pitcher leaves are tubular (epiasidate). However, juvenile leaves lack the hood, keel and fishtail appendage characteristic of the adult leaves (Frank, 1976). Juvenile pitcher leaves of S. leucophylla differ from the adult form in that they are narrow, and only gradually widen toward the mouth of the pitcher .
They also lack the abaxial rolled margin present on the mouth of the adult form.
Additionally S. leucophylla is the only member of the genus Sarracenia to form a complete new set of pitcher leaves at the end of the growing season .
Members of the Sarraceniaceae have similar pitcher leaf anatomy. The internal surface of pitcher leaves is divided into several morphologically distinct zones that can be distinguished by epidermal structures. While species and genera vary in the number and size of these zones, the zones function similarly in the attraction, capture and retention of prey and also in the digestion and absorption of nutrients . MacDougal (1903) observed that when Sarracenia pitchers were placed in total darkness they doubled in length. He reported that this etiolation was caused by differential changes in zone lengths, with cell numbers increasing in some zones. These early observations indicate that renewed meristematic activity had occurred within some zones. MacDougal ' s observations are the basis of the pitcher segmentation experiments reported in this manuscript. Additionally, development of epiasidate leaves of the Darlingtonia type may be similar to carpel development (Frank, 1975). Carpel margins are formed by the fusion of two epidermal layers that retain considerable meristematic activity following fusion (Walker, 1975). The margins of D. californica pitcher leaves undergo a similar fusion (Frank, 1975), which may be associated with retention of meristematic activity.
In contrast to the above morphological similarities, D. californica and S.
leucophylla differ considerably in their growth habit in the natural environment.
Seedlings of D. californica establish a fibrous root system following germination and during early development of the seedling. Following the production of mature pitchers, the stem becomes plagiotropic, and roots are formed at the nodes (Frank, 1976). It also readily forms offshoots in its natural habitats. On the other hand, S.
leucophy lla reproduces vegetativel y much more slowly in its natural habitats (Schnell, morphologies offer an opportunity to better understand differences and similarities in morphogenic potential across the two genera. Such an understanding may facilitate micropropagation of members of this and other pitcher plant families such as Nepentheaceae and Cephalotaceae in which epiasidate leaves are produced. Additionally, such studies are particularly timely and important since some members of the Sarraceniaceae have been placed on the endangered species list, e.g. , S. oreophila and S. jonesii (Godt and Hamrick, 1996). To my knowledge, the literature does not contain any reports on in vitro morphogenic responses of D. californica or S. leucophylla.

Seed Germination
Seeds of D. californica were obtained from a commercial supplier and germinated using Methods# 2, # 3, #4 and# 5 as described in manuscript 1 (Table 1) this dissertation. Culture of whole seedlings derived from GA 3 stimulated and unstimulated seeds in liquid medium Seeds of D. californica were germinated with and without GA 3 (Methods # 4 and# 5, Table 1, manuscript 1 ). Thirty-two, 18-day-old whole seedlings from each treatment were transferred, 4 each, to eight, 25 ml plastic culture flasks each containing 9 ml of liquid POMM and incubated in a growth chamber as described above.

Culture of whole seedlings in full strength liquid MS medium
To assess the effect of full strength MS medium (Murishige and Skoog, 1962) on the growth and development of D. californica, 12, 25-day-old seedlings (germinated using Method #4, Table 1, Manuscript 1) were transferred to two, 250 ml Erlenmeyer flasks (6 seedlings per flask) containing 75 ml of liquid MS medium without hormones. Flasks were incubated in a growth chamber (described above) and examined after 28 days of culture.
Pitchers from GA 3 -stimulated seeds germinated on solid medium Seeds of D. californica were germinated as described for Method # 5 (Table 1, Manuscript 1 ), except that seeds were exposed to GA3 for 12 days instead of 24 h and forty seeds were transferred to each of two , 250 ml Erlenmeyer flasks containing 75 ml of solid POMM (8 .5 g/l agar) . Following germination, seedlings were allowed to grow for 2 months.

Pitchers from seeds germinated in liquid medium without GA 3
Seeds of D. californica were surface disinfested and germinated using Method # 1 (Table 1, Manuscript 1) in 50 ml Erlenmeyer flasks and placed on a gyrotary shaker (120 rpm) at room temperature and light conditions. Following 3 months of culture, dense clusters of pitchers were removed and divided. Two clusters (each cluster containing approx. 9-12 pitchers) were transferred to each of three, 500 ml Erlenmeyer flasks containing 50 ml of liquid POMM and placed in the growth chamber.

Organogenesis from whole or fragmented pitchers
Pitchers harvested from seedlings derived from GA 3 stimulated seeds cultured on solid medium were cut into fragments approximately 3-5 mm in length. Six to seven fragments (representing a single pitcher) were transferred to each of three, 50 ml Erlenmeyer flasks containing 10 ml of liquid POMM and placed in the growth chamber. Observations were made over a 45 day period.
Whole pitchers ( 1-2 cm long) derived from seedlings not exposed to GA 3 and cultured for 45 days in liquid medium were carefully excised using a fine pointed scalpel, fine and medium pointed forceps and a pair of fine pointed iridectomy scissors. Care was taken to insure that rhizomateous tissue was not included at the base of the excised pitchers. In order to ascertain regeneration potential within anatomical pitcher zones, excised pitchers were subjected to the following treatments: 1) whole pitchers were placed (one each) in each of six, 125 mm X 25 mm Pyrex test tubes containing 8 ml of liquid POMM; 2) whole pitchers were cut into 2-3 mm sections and the fragments of 2 pitchers were placed in each of six tubes; 3) whole pitchers were cut in half and the distal portions placed in one set of six tubes and the basal portions in another set. Additionally, two whole seedlings, 5-months-old and maintained on Y:z strength MS medium, were transferred, one each, to each of 2 tubes.
This experiment was repeated one month later with modifications. Pitchers were excised as described above, but pitcher length was 1.0 -1.8 cm.

Multiplication of S. leucophylla in vitro
Two, 2-week-old seedlings of S. leucophylla that had germinated on MYP (from surface disinfestations experiments Manuscript 1, this dissertation) were transferred to 10 ml of Y2 strength MS medium + vitamins in 125 mm x 25 mm test tubes and placed in a growth chamber. Temperature and light conditions were as described in Manuscript 1, this dissertation. After 30 days they were transferred to solid POMM. After two months of growth they were subcultured to a 500 ml Erlenmeyer flask containing 50 ml of liquid POMM. After 105 more days the remaining tissue was divided into several pieces, each containing a portion of the rhizome and several pitchers, and distributed among three, 250 ml Erlenmeyer flasks , each containing 75 ml of liquid POMM. Four pitchers were excised from rhizomes and cut into seven fragments each and placed randomly in 4, 25 ml plastic culture vessels each containing 9 ml of liquid POMM. Following another 45 days of culture the growth in the flasks was documented photographically.

Extraction of intact endosperm and embryos
Seeds of S. leucophylla and S. purpurea were treated in concentrated H2S04 for 10 min followed by three, 5 min rinses in sterile distilled H 20 and then dissected and the embryos removed. Dissections and extractions were performed with the aid of a dissecting microscope, fine pointed forceps, and micro-dissection needles. A fine pointed hypodermic needle attached to the blunt end of a dental pick served as a micro-scalpel. Seed coats were split, by making a single, incision along the long axis of the seed. Portions of the micropylar and chalazal end of the seed were removed with the micro-scalpel and the coat gently teased away from the endosperm using forceps and micro-dissection needles. After removal of the seed coat, an oblique cut was made across the micropylar region of the endosperm. Embryos were teased out by manipulation and gentle pressure on the chalazal end and/or by carefully removing layers of endosperm tissue using the edge of a micro-scalpel as a micro-scraper.
Alternatively, following treatment in acid, seeds were placed in 3 ml of sterile H 2 0 in 1.5 ml plastic centrifuge tubes and spun on a vortex mixer for 3 min. followed by a change of H 2 0 and gentle agitation with the tip of a sterile Pasteur pi pet. This process was repeated several times until most or all of the seed coats were removed. Residual coat material was removed under the dissecting microscope by gentle teasing using micro-dissection instruments. Embryos were extracted as described above. These procedures were performed on the lab bench or, in order to obtain sterile material for experiment, in a laminar flow hood.

Production of protocorms and callus from tissue derived from whole seedlings by sequential culture on solid and liquid medium
When 4-week-old seedlings were placed with their longitudinal axis flush against the surface of POMM (Fig.IA), seven of 12 began to brown during the first week of culture. These seedlings were completely brown by the end of the second week and produced no new growth for the duration of the experiment. Five seedlings remained green with some yellowing of the hypocotyls and cotyledons (3 in flask 1, 2 in flask 2). Their cotyledonary nodes began to green and swell during the second week, forming hard green callus and/or green leafy callus (GLC) by the end of week 3.
These growths eventually obscured the cotyledonary nodes and also appeared along the hypocotyls proximal to the node. GLC also arose basipetally toward the root tip.
No increase in length of the cotyledons, hypocotyls, or primary root was observed.
After 70 days of culture two hard callus masses resembling orchid protocorms (protocorm-like bodies, PLB) had formed ( Fig.1 B). The GLC remained leafy (Fig. lC) and many small, spherical, bud-like structures appeared on the surface of the tissue. These structures formed on both types of callus and were morphologically similar to developing orchid protocorms ( Fig.1 D). Similar callus forms in leaf cultures of Cattleya orchids (Fig.1 E). When removed for subculture (first subculture) the GLC was more friable than the PLB. After approximately 15 days of subculture, both PLB and GLC began to form new shoots, GLC and spherical bud-like structures, which later formed PLB . PLB could sometimes be separated from parent tissue by gently swirling the medium in the flask, and larger PLB sometimes separated spontaneously in culture. These large PLB resembled mature orchid protocorms (compare Fig.2Aand 2B). After 44 days of subculture, dense clumps of pitchers and callus were produced (Figs. 2C and 2D). The cultures appeared healthy and new growth initiated from the circumference of the collective tissue mass.
When the masses were divided for subculture (second subculture) numerous plantlets lacking roots, large and small PLB, and various amounts of GLC in several stages of development were observed. Tissue masses were divided into several large clusters, but the original explant tissue could not be identified. Yellowing and browning of some tissue during the first week of the second subculture was followed by a 1 to 1 Y2 -week quiescent period before new growth was observed. A color change from yellow-green to green was the first indication that growth had resumed.
After 45 days of growth the flasks were photographed (Fig.3And B). Shoots generated from subcultures of PLB and GLC were morphologically similar to shoots produced from whole seedlings (Fig. 3C). As of this writing the flasks are in a growth chamber and have produced a large amount of tissue for further subculture and experimental use.
Production of callus from whole seedlings derived from GA 3 stimulated and unstimulated seeds and cultured in liquid medium After 36 days of subculture five seedlings in the treatment without GA 3 had swollen nodes and leaf bases and had produced new pitchers (Fig.4A, compare with Fig. 1 A). PLB and GLC were present on only 1 of these seedlings. The others had completely browned by the end of the experiment. In contrast, eight of 16 seedlings from seeds stimulated by GA3 showed new green growth and on 3 of these PLB and GLC could be observed (Fig. 4B). The remaining 8 seedlings either browned or yellowed by the time the experiment was terminated and produced neither PLB nor GLC.
Culture of whole seedlings on full strength liquid MS medium -When 12, 25-day-old seedlings were cultured in full strength MS medium, no evidence of multiplication or morphogenesis was observed after 30 days (Fig. 4C).
One seedling browned, and 11 remained green with some yellowing. Some browning of pitchers was observed.

Organogenesis from whole and fragmented pitchers
When fragments of pitchers harvested from plants grown from GA 3 stimulated seeds on solid medium were cultured in liquid POMM, new growth was observed in 2 of 3 flasks after 45 days (Fig. 5A). One fragment of 7 in flask #2 has formed 5 welldeveloped pitcher leaves (Fig. 5B) and several small PLB were clustered around the center of leaf origin (Fig. 5B). These small PLB could be separated from the parent tissue (Fig. 5C). In flask# 3, two of seven fragments had produced two PLB ( Fig. 4D and E). One PLB had given rise to a cluster of small pitchers (Fig. 4D). In this experiment three of 20 pitcher fragments produced new growth. All six pitcher fragments in flask #1 gradually browned and died.
The pitchers used as source material for subsequent experiments were plantlets derived from non-GA 3 treated seeds and subcultured in liquid POMM for 45 days.
During this time the plantlets multiplied and completely covered the bottom of the flask (Fig. 6A). A few small roots originating from central cluster rhizomes were visible with the aid of a hand-lens. The plantlets appeared chlorotic at the time pitchers were excised and fragmented.
In four of the six tubes in which pitcher fragments were cultured, new growth occurred. (Fig. 68). Eleven of a total of 50 fragments produced new growth, and the rest slowly browned. In tubes one and two, 4 fragments produced new growth, tube three contained 10 fragments with two producing new growth, all fragments browned in tubes four and five, and one fragment in tube six produced new growth. The fragments varied from small spherical bodies (Fig. 68, inset), small PLB, to large hard, dark green callus (Fig. 68). PLB closely resembled orchid protocorms ( show new growth at the end of the experiment. This growth appeared to be from pitcher elongation and was accompanied by dark green coloration. Adventive structures were not observed. The variable results noted above indicate that morphogenic potential of pitcher fragments may depend on the zone of the pitcher from which fragments were derived, so fragments were cultured individually and their position within the pitcher was noted when the experiment was repeated. However, very little regeneration was obtained in this experiment. One of 15 whole pitchers showed new growth from the tip (Fig. 7 A) after 30 days of subculture. One of 15 basal halves of bisected pitchers formed a PLB and leaf-like structures at the distal end. This growth lost its coloration in the last 10 days of the experiment (Fig. 7B). No growth was observed in any other explants.
Whole pitchers usually browned first from their basal ends and after one week of culture, the distal ends appeared larger and more translucent and were the last area to brown. Fragmented pitcher halves were paired so that fragments of the same pitcher were in adjacent tubes. Although all ultimately turned brown, the process of browning commenced at different times during the experiment and appeared synchronized between complementary tubes containing fragments of the same pitcher. The same was true for serially sequenced fragments . All fragments from the same pitcher began to brown at approximately the same time. Browning first appeared in the basal fragments in all treatments.

Multiplication of Sarracenia leucophylla in vitro
Seedlings of S. leucophylla grew slowly on solid Y2 strength MS medium.
After two months of growth they had produced few pitchers and showed little tendency toward adventive multiplication. Transfer to solid POMM did not appear to have much effect. Although they did produce more pitchers and rhizome length appeared to increase slightly. Following transfer to liquid POMM, clusters of pitchers were formed. Pitcher clusters could be divided by rhizome cutting and subculture of clusters resulted in rapid multiplication, continued increase in pitcher height, and developmental movement toward adult pitcher morphology (Fig. 8A, B, and C).
Excised pitcher fragments gradually browned and died. No new growth was observed on any of the subcultured pitcher fragments after 45 days of subculture.

Conditions for the production of protocorm-like bodies and callus
Because whole, axenically germinated seedlings have been used to initiate clonal multiplication through direct differentiation of shoots in several micropropagation systems (Malik and Saxena, 1992;Teo et al., 2001; l 982a), the formation of callus and PLB from whole, in vitro germinated seedlings of D. californica was unexpected. Additionally,  reported the recalcitrance of whole, in vitro generated seedlings of the rare pitcher plant Nepenthes khasiana to produce callus when exposed to phytohormones in culture. In contrast, during this study, D. californica readily produced several types of callus and regenerative tissue distinguishable by differing gross morphology. During the last three decades of tissue culture, hypocotyls and cotyledons have been excised from axenically germinated seedlings of a variety of plant species and subcultured to initiate callus and somatic embryogenesis (see Kohlenbeck, 1978, for early review of somatic embryo genesis).
Similar developmental phenomena also were observed by Truscott (1966) in his studies of morphogenesis in Cuscuta gronovii. He observed that original explants (extracted, intact embryos) showed little development but gave rise to many adventive buds. Callus-like outgrowths formed at the bases of adventive buds and eventually formed many smaller buds. These phenomena are similar to that observed in whole cultured seedlings of D. californica. Additionally, Parliman et al., (1982b) described similar structures arising from cultured leaf tissue of D. muscipula. These structures were considered to be adventitious. Also, they report that in mature cultures, only the first bud appears to be adventitious and the subsequent buds appear to be lateral buds derived in sequence from it. This could also be the case in D. californica since preformed buds most likely do not occur on the hypocotyls and cotyledons.
Although histological studies were not performed during this study, the nature of PLB and GLC can be interpreted based on the anatomy and growth habit of the shoot system of D. californica seedlings. During leaf development in D. californica the newest emerging leaf forms a clasping base that nearly encircles the leaf primordium of the next developing pitcher leaf (Frank, 1976). This imbricate developmental pattern places axillary buds in close proximity prior to internode elongation. If these buds are activated by the presence of phytohormones they may give rise to the structures described in this manuscript as PLB. These structures would appear clustered at shoot tips. Alternatively, PLBs produced along the hypocotyls and cotyledons may be adventitious buds (Goebel ' s "Anlagen" -see Appendix C).
The ability of PLB and GLC to proliferate when divided and subcultured was not surprising since, at the time of division, considerable cytodifferentiation and morphogenesis had occurred. When transferred to fresh medium, the rapid formation of many shoots in dense cluster~ may have been the result of simultaneous activation of the PLB produced during the initial culture period. This rapid formation of shoots in dense clusters supplies evidence that PLB are bud-like in behavior.
The initial yellowing and die back of some pitchers after subculture may have been associated with medium pH. Although pH was not monitored during culture, a change from the original pH of 5.0 may have occurred. Liquid cultures of the Australian pitcher plant Cephalotus follicularis were observed to die back when transferred to fresh medium (Adams et al., 1979). It was suggested that this phenomenon was pH dependent because it occurred when medium at pH 5.7 was used for subculture.
In this study lack of rooting may have been due to high auxin levels. The auxin in the medium coupled with the natural production of auxin by proliferating shoots may have led to levels inhibitory to root development. High auxin levels have been shown to cause callus formation at the shoot base of in vitro cultured plants and inhibit root formation (Lane, 1979).

Effect of GA 3 stimulation on whole seedlings cultured in liquid medium
The lack of response of seedlings, without prior GA 3 stimulation, to incubation in liquid medium with phytohormones is puzzling. Protocorm-like bodies and green, leafy callus were produced by non-stimulated seedlings cultured on the same medium in solid form. Seedlings transferred to solid medium were 21-28 days old vs. 15-18 days old for the seedlings transferred to liquid medium. It has been established that response in culture varies considerably in some species depending on physiological age, endogenous hormone concentrations as well as the history of the explant material (Hu and Wang, 1982).
The death of seedlings incubated in full strength MS medium was not unexpected. In nature, carnivorous plants generally inhabit nutrient poor habitats  and media containing reduced salt concentrations have been successfully employed for in vitro culture of these plants.

Regenerative potential of fragmented pitchers
This series of experiments tested the hypothesis that regenerative potential varies throughout specific zones of pitcher leaves of the Sarraceniaceae. This hypothesis was based on earlier observations on elongation potential of etiolated pitcher leaves of Sarracenia (MacDougal, 1903 ), the mode of leaf histogenesis (Frank, 1975(Frank, ,1976 and carpel development (Walker,1975 1942, pp.19-36). Because pitcher development was continuous in vitro , it was hypothesized that pitchers fragmented to approximate the natural zonation might retain regenerative or developmental potential either at zone boundaries or along the suture line.
The first experiment was performed to quickly assay the ability of randomly fragmented pitchers to produce new growth from the excised fragments. The results of this experiment supported the hypothesis with new growth in the form of PLB, pitcher leaves and one whole plantlet being produced from several of the fragments.
In a second experiment, fragmented whole pitchers produced PLB, small green spherical bodies, and large, dark green callus. Why the cultured whole, distal, and basal halves of pitchers ultimately browned and died is not understood. One possibility is, the fragmented pitchers may be stimulated by a collective wound response to produce callus because of the number of wounded fragments placed together in the same test tube. This response may not have occurred in the tubes containing whole or half pitchers because of a low level of signal molecules since less wound surface area was exposed to the medium. Another possibility is that the tissue zone or zones capable of the response were located interior to the cut and may require being cut in order to initiate the response. These hypotheses could not be investigated because all the fragments from one pitcher were placed collectively in individual test tubes.
Although the results of the last experiment in this series were inconclusive some useful information was gathered. For instance, whole pitchers usually browned from the basal end first with the browning proceeding acropetally. The distal tips were also observed to become enlarged and translucent before ultimately browning.
The only growth to appear from whole pitchers was from the distal end. These three observations, though based on a small sample size, suggest that a closer look at the distal zone may be the first step in planning future investigations. Additionally, because regenerative structures were produced by tissue with prior exposure to GA 3 and tissue without GA 3 exposure, the role of this hormone in the induction of renewed meristematic activity, as observed in these experiments, still remains unclear.

Multiplication of Sarracenia leucophylla in vitro
The slow growth of S. leucophylla on solid medium without hormones may be related to its natural habit. In nature this species does not proliferate by extensive secondary rhizome formation as does D. californica . Instead, it produces new shoots from the primary rhizome. Commercial propagation is accomplished through cuttings taken from the primary rhizome that include at least one root (Pietropaolo andPietropaolo, 1986, D' Amato, 1998). This form of propagation is very slow and faster rates may be obtainable through in vitro culture.
Additionally, cuttings must be taken them just before the new growing season. This restriction does not apply to in vitro culture.
The lack of response of S. leucophylla when transferred to solid POMM containing auxin and cytokinin may have been caused by its previous long period of culture on solid medium. Prolonged culture with depletion of nutrients and leaching of toxic metabolites from the plants into the medium may have caused arrested growth. This hypothesis is supported by its rapid proliferation and growth when transferred to liquid POMM. Serial subculturing can break the recalcitrant state of many species as well as transfer to a liquid shaking system (Hu and Wang, l 984).
Renewed initiation of growth and multiplication of S. leucophylla occurred when transferred to liquid medium without shaking.
Failure of pitcher fragments of S. leucophylla to form new growth in liquid culture could be due to one of several factors including developmental age, an inherent inability to form new centers of meristematic activity, or the sample size may have been too small.

Extraction of intact endosperm and embryos
The removal of seed coats using the vortex mixer method was preferable over the first method outlined. It allowed more seeds to be processed in a shorter time and extensive manipulation of seeds to completely remove the coat was usually not

Sterile Wash
Contamination 24 of 24 seeds contaminated by day 15 after 15 days (fungal mycelia)

Results of Surface Sterilization Experiments
Darlingtonia californica 3% Hydrogen Peroxide DCHPl 48 seeds -one seed per well in plastic well plates (MYP + 10 g/l sucrose (pH 6.5 Total germinated= 3of24 = 12.5% Total Germinated = 1 of 3 6 = 2. 7% Results of Experiment # SLlRl Sarracenia leucophylla -Type ofExperiment-Germination -seeds not stratified -20 minutes in 10% Clorox -6 seeds/flask -6 flasks/treatment 50 ml Erlenmeyer on gyrotary shaker -room light and temperature conditions -Start date 2/28/02. Sarracenia leucophylla -Seed Selection Experiments -When seeds obtained from a commercial source were inspected with the aid of a dissecting microscope they could be divided into four categories; 1) Normal or non-suspect seeds -these seeds exhibit normal morphology, light brown color, outer coat heavily waxed, and no surface detectable imperfections; 2) Infected seeds -these seeds exhibit large areas of seed coat covered by a colored mass, usually gray, green, yellow or orange red, that appears to be tightly adhering fungal hyphae or sporulating structures. They may also have small holes in the seed coat; 3) Suspect seeds -these seeds appear to be normal as far as their general appearance, however on closer inspection, some cells of the outer seed coat may appear black or discolored. These areas may comprise several cells in diameter and can go undetected if the seed is not carefully examined; 4) Abnormal seeds -these seeds present abnormal developmental morphology and they may be very small, round, cup shaped or they can be elongate and narrow. Seeds were divided into the four categories and 20 seeds from each category were weighed.   Results of Experiment DCGEM2002 -Seeds used in this experiment were given three different treatments before plating on three different substrates. One hundred and twenty seeds of D. californica were removed from storage at 4-7°C and surface sterilized. Seeds were soaked overnight in 10 ml of sterile distilled H 2 0 (pH.5.0) + 1 drop of Tween 20 at 4-7°C.

Start Date 7130102
The callus and protocorm-like structures formed in experiment IA were each cut into 3 smaller segments for a total of I 5 segments. Five segments were transferred to each of 3, 50 ml Erlenmeyer flasks containing I 0 ml ofliquid POMM. Flasks were placed in the growth chamber. As of 8115/02 (I 5 days of subculture) the segments in all 3 flasks show growth of shoots from both the segments of protocorm-like bodies and the green, leafy callus. On 9114/02 the flasks were photographed ( 44 days of subculture).

Experiment# Morph lC (continuation of Morph IA and lB)
Start Date 9/15/02 Following 45 days of subculture the clumps of shoots produced in two of the flasks were divided and subcultured again. The remaining flask was sacrificed for drawings and observations. Clumps of plants were evenly distributed among 3, 250 ml Erlenmeyer flasks each containing 75 ml of liquid POMM. The flasks were placed in the growth chamber. During the next 7 days there was an initial die back of pitcher leaves. This was followed by a spurt of new growth. As of 10115/ the plants in the flasks appeared healthy and were continuing to multiply.

Experiment # Morph 2 Start Date 7130102
Three seedlings, 3 months old, from experiment DC5 l that had remained in 50 ml Erlenmeyer flasks containing I Oml of Yi strength liquid MS medium were divided into 2 pieces each (by this time the seedlings had formed dense clusters of pitchers). Two clusters were placed in each of 3, 500 ml Erlenmeyer flasks containing 50 ml of liquid POMM. The flasks were placed in the growth chamber. On 8115/02 one flask was used for experiment Morph 3B.By 9/ 17/02 the plants in the remaining flasks had multiplied. One of the remaining 2 flasks was sacrificed to experiment Morph 3C.

Experiment #Morph 3A Start date -7130102
This experiment was performored to test the regenerative ability of in vitro grown pitchers. Plantlets from experiment DC GA 1 were used. Pitchers were removed under sterile conditions and cut into 2-3 pieces. Pieces were placed in each of 3, 50 ml Erlenmeyer flasks containing 10 ml of MM medium. The flasks were placed in a growth chamber at 27°C with a 16 hr photoperiod.   2-3 week old seedlings from experiment DCGEM2002C, treatment #2 (24 hr soak in H20) and treatment #3 (24h hr in GA3) were transferred from water agar to coming 25 ml polystyrene tissue culture flasks containing 9 ml of liquid POMM. Four seedlings per flask, four replicates from each treatment. This experiment was conducted to gather preliminary observations on growth of D. californica in full strength liquid MS. 3-4 week old seedlings from exp. DCGEM2002C, treatment #2 (24 soak in H20) were placed, 6 per flask, in 75 ml of liquid full strength MS medium in 250 ml erlenmyer flasks and placed in the growth chamber. As of 10/ 15/02 1 has browned and the others remain green but growth is very slow. Photographs were taken on 10116/02.

Experiment Morph #6A Start Date -7130102
Two seedlings of S. leucophylla that had germinated on 3/2/02 on MYP (as part of surface sterilization experiments) were transferred on 3/15/02 to solid Yi strength MS medium in 125 mm X25 mm test tubes. On 4/ 15/02 the two seedlings showed little growth and no multiplication, They were transferred to solid POMM. On 7/30/02 (after 3 112 months of growth), during which they produced few new shoots and increased rhizome length only slightly, they were transferred to a 500 ml Erlenmeyer flask containg 50 ml of liquid POMM. BY 9/ 15/02 they had formed clusters of pitchers with large rhizomes that could be divided, these were divided into several pieces and placed in three, 25 Oml erlenmyer flasks containing 75 ml of liquid POMM and returned to the growth chamber. On 10/20/02 they were photographed, divided and placed in three, 500 ml Erlenmeyer flasks containing 100 ml of POMM.
Experiment Morph # 6B Start Date -9/ 15/02 When 6 month old pitchers from Morph #6A were transferred to new medium, four pitchers were fragmented and seven fragments were placed in each of four plastic tissue culture flasks containing 9 ml of liquid POMM. All were placed in the growth chamber under the same conditions that had allowed regeneration in D. californica fragments. As of 10/15/02, all had turned brown. Two turned slightly red after the first week and appeared to swell but after 30 days they had turned brown and apparently died.

Experiment Morph # 7 Start Date -4129102
Two young pitchers were harvested from mature greenhouse grown plants ( 1 from each plant). One pitcher was approx . 6-7 weeks old and the other approx. 2-3 weeks old The pitchers were cut from the plants at the base of zone 5 using a sterile dissecting scissors. Care was taken not to include any portion of the rhizome. Following surface sterilization (20% Clorox, 10 min, 3, 3 min rinses) sections were cut from each zone with a sterile scalpel. The sections were placed in 250 ml Erlenmeyer flasks containing 75 ml of POMM. Three flasks were used for each pitcher with the sections being placed in the following order: One, unopened pitcher (1.8 cm in height) was harvested from a greenhouse grown plant, cleaned in a mild detergent for several minutes under running tapwater and surface sterilized in 3% H202 for 10 minutes. The pitcher was cut into nine fragments, 2 mm in length, in sequence from zone 5 (#1 fragment) to zone 1(#9 fragment ) and one fragment per tube was placed in each of 9, 125 mm X 25 mm test tubes containing 10 ml of liquid POMM. The tubes were placed in the growth chamber. After, 24 hr. contamination was observed in tube #1. After 48 hrs contamination was observed in tube 2. Contamination was observed in tubes 3 and 4 after 72 hrs . At the end of seven days all had contaminated.

INTRODUCTION
Carnivorous plant species have long been objects of study because they possess unique physiological and anatomical adaptations peculiar to the carnivorous habit (see Lloyd, 1942, for early references). This collection of artificially grouped plants comprises over 600 species  and has grown considerably since 1989 when Givnish reported 538 species. The taxonomic occurrence of these plants is spread over 10 families and 19 genera (tablel). Givnish reported eight families and 18 genera in 1989. This group is important for several reasons. Several members of the genus Drosera and the related single species genus Dionaea are used in the preparation of medicinal compounds and for the extraction of other secondary metabolites Budzianowski, 2001 ;. Collection pressure and/or habitat destruction has reduced or depleted many natural populations of carnivorous plants in the wild . Many species of carnivorous plants are considered threatened or endangered with many currently listed in the list of protected species at either the national or international level by the Convention on International Trade in Endangered Species .
Carnivorous plants are increasingly being used as model systems for the study of fundamental problems in plant development Leichtscheidle, et al., 1989;Samaj, et al. , 1995). germination. While it is not within the scope of these papers to address problems regarding types of dormancy, clarification of the problem will facilitate future research in developing micropropagation protocols for additional species. One problem that has to be addressed is that the use of disinfectants in the laboratory generally negates data from application to the ecology and timing of germination in nature since seeds in nature are exposed to many types of soil microorganisms and environmental factors . However, knowledge of the phenology of the seed phase of the plant life cycle can contribute significantly to successful germination in the laboratory.  suggest that most species of carnivorous plants have dormant seeds with some being non-dormant. According to these authors, physiological dormancy is the most common type of dormancy found within the carnivorous plant group with a few species having morphological or morphophysiological dormancy.
The following selective, chronology details some of the history of laboratory work with carnivorous plants. Data and observations pertaining to seeds and/or surface sterilization of seeds and other explant tissues has been extracted and presented in a series of tables. In summary, any attempt to establish a micropropagation system or micropropagation protocols for selected carnivorous plant species must begin with a survey of the literature.

Burgher (1961) -Burgher reported a method for the sterilization of seeds of
Drosera intermedia, a germination protocol, and a medium that allowed only limited growth of plantlets. Most important were his observations that the highest germination occurred when the seeds were exposed to a cycle of 12 h of light at 3 8°C followed by 12 h of light at l 5°C. No dark period was necessary. However, when seeds were exposed to 12 h of darkness at l 5°C, germination was approximately 13% less than that obtained in constant light. Seeds were illuminated with two, 40 watt, cool-white fluorescent bulbs. Germination occurred after five weeks of treatment.
Burgher reported that the medium used for his experiments was not satisfactory for continued growth and maintenance of the seedlings. He also reported that exposure of the seeds to IAA (indole-3-acetic acid), gibberellic acid, dilute sulfuric acid, high temperature (3 7°C) or freezing followed by either high or room temperature did not enhance germination. He did not report concentrations or exposure times.

Pringsheim and Pringsheim (1962) -These researchers obtained axenic
cultures of Uticularia exoleta in liquid culture using a modification of a medium developed for the growth of the green alga Micrasterias. Paramount among their experimental results are the observations that peptone and meat extract added to the basal medium not only promoted superior growth of the plants but also caused the plants to flower. They reported the concentrations employed as 0.05% each of Difcotryptone and Difeo-beef extract. They also reported that peptone, used alone, improved growth slightly but beef extract was necessary for flower production.
Additionally, they reported that growth of cultured plants was much improved when the medium was prepared by dissolving iron sulfate crystals after the chelating agent (EDT A) was added to the medium and before addition of other trace elements.
Withner (1964) -Withner was the first to report a protocol for the culture of Sterilization time was 15 min. The seeds were next washed with sterile distilled water and transferred to 500 ml flasks containing 180 ml of culture medium as described by Lidell (1953). However, the agar percentage was reduced to 0.65% (w/v). Neither contamination rates nor germination percentages are given. Germinated seedlings were explanted (one per flask) and raised under a bank of fluorescent and grow-lux Lindsmaier-Skoog medium was reported. The medium also contained 30g/l sucrose and the pH was adjusted to 6.5 before adding 6 g/l agar. Leaves placed on full strength medium died. Leaves cultured on this medium without hormones produced an average of three leaves per leaf. Leaves from these aseptic seedlings grown on the above medium were transferred to the same medium containing hormones (0.02 mg/l 6-benzylamino purine (BA) and 0.01 mg/I napthaleneacetic acid (NAA)). This hormonal ratio produced a combined maximum number of plantlets and growth rate.
Plantlet yield was increased by subculturing to higher hormonal levels (BA 2.0 mg/I: NAA 1.0 mg/I) for three to four weeks then plantlets were subcultured back to the lower hormonal concentrations to maximize growth. These workers estimated that a single leaf producing seven to eight, three leaved plantlets could be expected to produce 500 plantlets in six months of culture.
Beebe (1980) -Beebe described aseptic germination, callus formation, and adventitious bud development in Dionaea muscipula. He germinated seeds on full strength MS medium with the following supplements: thiamine, O. lmg/l; nicotinic acid, 0.5 mg/I; pyridoxine, 0.5 mg/l; glycine, 2 mg/l; myo-inositol, 100 mg/I; casein hydrolysate, 1.0 g/l; sucrose, 20 g/l; coconut milk, 1.5% (v/v); and 1.0% agar. The pH of the medium was adjusted to 5.6 before autoclaving. Beebe reported germination and early seedling development to be as described by  for seeds germinated in soil under greenhouse conditions. He stated that over a four -year period, several different seed lots were used with germination ranging from 78% in a sample of 95 seeds to 40% in a sample of 150 seeds. Seeds germinated throughout a 10-55 day period with the majority (over 70%) germinating between 10 and 35 days.
Various concentrations of growth substances were applied to this medium for developmental studies. The growth substances used were 1-napthaleneacetic acid (NAA) and N6-benzyladenine (BA). High ratios ofNAA to BA promoted the production of roots from both aseptically germinated seedlings and from callus tissue.
Equal ratios ofNAA to BA promoted the proliferation of callus and callus buds from both cultured seedlings and callus other callus buds. He also observed that seedlings on mediwn containing I 0-7 M ofNAA and I 0-5 MBA formed large, fleshy adventitious buds at the tips of broad petioles. The petiole margins on these plantlets were ruffled. These buds, which he termed "callus-buds", formed in place of traps at the leaf tips. Beebe also reported that rapid callus formation and bud growth occurred on media containing 10·  mg/l casein hydrolysate, and 30 g/l sucrose were also added to the germination medium. The pH was adjusted to 5.9 before adding agar (6 .7 g/l). Following germination, plantlets were subcultured to Yi strength basal MS medium (including the additional components listed above) plus 1.9 mg/l napthaleneacetic acid (NAA) and 0.2 mg/I 6-benzlamino purine (BA). The pH was adjusted to 4.9 prior to the addition of 6. 7 g/l agar. Ex plants grown on this medium showed a 5-14 fold increase in the number of healthy, vigorous, rooted plantlets in 60 days . Plantlets were next transferred to an acclimatization or pre-transplant medium. The authors state that this medium allowed the rapid increase in size of the subcultured plantlets but did not allow an increase in the number of plantlets. The composition of the medium was Y 2 strength MS medium with organic components as above except the NAA and BA were removed and GA 3 at 0.3 mg/I or 1.0 mg/I was added. The authors reported that after 40 days growth on this medium plantlets were ready to be transferred ex vitro .

Parliman et al., (1982b) -Parliman and co-workers reported adventitious bud
differentiation and development from tissue cultured leaf cuttings of Dionaea muscipula. The source of the leaf cuttings for these experiments was aseptically germinated seedlings as described in their previous work (Parliman et al. , (1982a).
They expected adventitious buds to arise independently from the leaf tissue.
However, secondary buds developed from the rhizomes of primary adventitious buds with each secondary bud initiating another secondary bud in a chain-like fashion.
They termed these secondary buds lateral buds (LB) as opposed to the primary adventitious bud (AB). They presumed that these buds developed from meristematic tissue within the AB rhizomes. They also stated that the origin of the AB buds is unknown (whether from single or multiple cell origin). The medium employed for these experiments was Yi strength MS with organic supplements as described earlier (Parliman et al., 1982a). Hormonal concentrations that produced the greatest number of AB and LB buds were NAA at 1.9 mg/I +6(-y-y-dimethylallylamino)-purine (2iP) at 0.2 mg/I. Additionally, they reported that leaves dipped for 24 h in 2iP at 2.1 mg/I produced the greatest number of adventitious and lateral bud derived plantlets when subsequently cultured on the above medium. with added grapevine exudates (5 %). The authors do not give any references for the preparation of the exudate. Leaves were sampled periodically at 6,9,15 and 21 days for developing organoids (this term is taken to mean new shoot meristematic regions).
Tissue was prepared for both TEM and light microscopy. They reported that visual observations of the tissue on day six revealed no apparent changes. However, serial sections of the tissue revealed that cell division had already begun to take place in specific regions at an earlier time.
They reported the formation of large clusters of dividing cells located in the inner portions of the explant tissue that may be associated with, or lying close to, the vascular system. These cells are described as being highly vacuolate with a welldefined nucleus. They also describe cells entering into the division process that are highly reminiscent of cells found in shoot apical meristems. That is, they apparently have a high affinity for cytological stains and show an increased accumulation of starch. These centers of division are reported to arise as a proliferation of parenchymatous tissue and may include the cells of the subepidermal layer. The authors state that by day 21 these regions have given rise to protuberances that upon histological examination possess distinctly differentiated shoot meristematic apices and leaf primordial. With continued culture, complete plants were regenerated that were able to live under normal conditions (normal conditions are not defined).
10) Adjust pH of medium to 5.8 before adding agar. 11) 8.0 g/l agar 12) Keep cultures in the dark for 3-4 days. 13) Transfer to light conditions of 2500 lux light intensity for 12 hours photoperiod at 26 ± 2°C. 14) After 4 weeks growth, differentiated shoots may be subcultured for further multiplication or transplanted to rooting medium.
Rooting Medium: 1) Yi strength MS medium 2) 2.0 mg/I NAA 3) 0. 1 mg/I kinetin 4) 20 g/l sucrose 5) Adjust pH to 5.8 before adding agar. 6) 8.0 g/l agar 7) Store cultures in the dark for l week. 8) Transfer to light conditions of 2500 lux light intensity at 26 +-2 degrees Celsius. 9) Transfer to strengthening medium for faster growth and development of pitchers. 10) Approximately 80% of the plantlets should develop roots.
Strengthening and pitcher development medium: 1) 1/,i strength MS medium (hormone-free) 2) 20 g/l sucrose 3) Adjust pH of medium to 5.8 prior to addition of agar. 4) 6 g/l agar 5) Light and temperature conditions as above. 6) When plantlets are approximately 5-7 inches in length with well developed roots, remove from flasks , wash thoroughly with tap water followed by sterile distilled water, and then transfer to pots containing vermiculite and drained soil (3: 1) 7) Harden in a growth chamber. 8) Within 4-5 weeks, potted plants should develop pitchers with normal lids.

Additional Notes
Nodal shoot segments were found to produce the most multiple shoots (10-12). Apical shoot explants produced only 6-8 new shoots. Ascorbic acid, citric acid, arginine and adenine sulfate enhanced shoot production ( 12-15 per explant) when added to the multiplication medium. If they were not added the cultures declined in growth and gradually deteriorated . Leaf and root explants did not produce shoots or callus. Hardening before transplantation to a growing medium was essential.
Explant source: Leaves from aseptically grown plantlets of D. rotundifolia. The authors neither state nor reference a protocol for the production of the aseptic plantlets. However, see  and . The authors report that the source plants were grown on MS medium  with a 16-hour photoperiod (PPF of 32uM/m2/s) under cool white fluorescent bulbs at a temperature of24 ± 2°C.
Medium for the induction of adventitious buds: The authors reported testing 49 different media formulations and report best production occurred on liquid MS medium with 10-8M of NAA ( 18.4 plantlets per explant). Solidified MS medium without growth regulators produced approximately 7 adventitious buds per leaf explant. 1) Full strength MS medium (basal salts) 2) 30 g/l sucrose 3) 100 mg/I myo-inositol 4) Adjust the pH to 5.8 before adding agar or use liquid medium (for liquid induction medium add 10-8M ofNAA). 5) 7 g/l agar Medium for the induction of callus: 1) Full strength MS medium and supplements as above for solid medium plus 1 o-6 or 5x10-6 M of BA 2) Place in light and temperature conditions as outlined under Protocol.
Protocol for the generation of adventitious buds: 1) Transfer leaves excised from aseptically cultured plants to either solidified MS medium with no growth regulators (abaxial side in contact with the medium) or to liquid MS medium supplemented with 1 o-8 M of NAA.
2) Place liquid medium cultures on a gyratory shaker at 120 rpm.
3) Maintain both liquid and solid media with a 16-hour photoperiod under cool white fluorescent lights with a PPF of 32 umol/m 2 /s (400-700 nm) . 4) Maintain solid and/or liquid cultures at 24 ± 2°C.

Additional Notes
A change in the leaf pigmentation from green to dark red or red to dark red occurred two days after culture initiation. Media supplemented with 0 to 2 x 10· 5 M NAA induced intensive red pigment formation with some necrosis after 28 days in culture. Optimal callus formation and proliferation occurred in the light only on medium containing 1 o-6 or Sx 1 o-6 MBA. The callus formed was light green and very compact. Full strength, solidified MS medium without hormones produced the most direct shoot formation of all solidified formulations tested. Buds were observed to form on the whole leaf surface, but in particular near the tentacles.

Induction of flowering:
Plants can be induced to flower by subculturing to Y4 strength MS medium supplemented with 0.12uM IBA plus 0.44 µMBA. This medium reportedly induced 100% flowering . Plants subcultured from '!4 strength MS medium at pH 5.7 to Y4 strength MS medium at pH 4.0 obtained the largest diameter (in 8 weeks subsequent growth) in comparison to plants subcultured to Y4 strength media at higher pH values.
Source of explant material: whole leaves excised from greenhouse-cultivated plants.
Surface sterilization of leaves: Whole leaves were excised and surface disinfested in 10% (v/v) Clorox (0.5% sodium hypochlorite) with 1 drop of Tween 20 surfactant added to each 50 ml of 10% Clorox solution. Excised leaves were disinfested for 5 minutes followed by three 5minute rinses in sterile distilled water. Longer time spans ( 10-20 minutes) in the disinfestations solution caused extensive tissue damage and lack of regeneration of adventitious plantlets. Anthony states that approximately 75% of the original cultures contaminated with 95% of the D. rotundifolia cultures were lost to contamination. She suggests that the contamination was due to the presence of symbiotic bacteria and fungi that may play a role in the digestive process (see Chandler and Anderson, 1977).

Medium :
1) Yi strength MS medium  prepared by diluting full strength MS medium containing vitamins. 2) 30 g/l sucrose 3) 0,02 mg/I BA 4) 0.01 mg/I NAA Note: Addition of phytohormones may not be necessary for the induction of adventitious plantlets since Anthony states that on both Yi strength MS medium without hormones and Yi strength MS medium supplemented with phytohormones, the multiple adventitious plantlets entirely covered the leaf surface. However, hormones were required for the induction of flowering in D. binata. 5) Adjust pH to 5.7 before adding agar. 6) 8 g/l agar Protocol: 1) Excise whole leaf from plants. Surface steri lization of seeds: 1) Disinfest seeds (seeds four weeks old were used in these experiments) for 15 minutes in a solution of benzalconium chloride (0 .1 % w/v) and calcium propionate ( 18 mM) . 2) Rinse in sterile distilled water.
3) Transfer to sterile 500 ml flasks containing 180 ml of nutrient medium (Chandler and Anderson used Liddell ' s orchid germination medium and reported lowering the agar concentration to 0.65% w/v). 4) Light and temperature conditions were not stated. 5) Following germination (time required not reported), transfer to fresh medium.
For purposes of their experiments Chandler and Anderson transferred 1 seedling per 500 ml flask. 6) For growth, maintain cultures at 20 °C under fluorescent and Gro-lux tubes (8.5 Wm-2 at the agar surface). Photoperiod not reported .
Note: This is a very early paper in the history of carnivorous plant tissue culture. However, it contains information regarding techniques, media formulations and experimental results that may be useful in designing future tissue culture experiments.
Outline of experiments: 1) Seeds were surface sterilized with chlorine water (specifics not reported), embryos were excised under aseptic conditions and planted on various media. 2) The media employed were: Basal medium = modified White ' s medium + 2% sucrose+ 0.8% agar.
Leaf bases were fleshy and adhered to the stem. No roots developed. 5) Embryos cultured on Medium #4 did not germinate, but formed proliferating dark brown, friable callus after 10 weeks of culture. Patches of pearly white tissue appeared on these brown calli. 6) The white tissue was excised from these calli and subcultured on Medium #4 and basal medium + casein hydrol ysate + kinetin (1 ppm) + IAA (I ppm). Active cell division occurred in both media. 7) Approximately 12% of the subcultures on Medium #4 differentiated into shoots and roots after two weeks of subculture.

Micropropagation Protocol
Surface sterilization of explants: 1) Excise whole leaves from stock plants.
2) Shake for five minutes in 0.1 % Tween 20 surfactant with 3 changes of the solution during this time span (vacuum aspirate and replace with fresh solution). 3) Replace solution with 1% Physan 20 (10% n-alkyl dimethyl benzyl ammonium chloride+ 10% n-alkyl dimethyl ethylbenzyl ammonium chloride) and shake for 5 minutes with 3 changes of solution. 4) Replace solution with 10% Clorox (0.525% sodium hypochlorite) with 0.1% Tween 20 surfactant. Shake for five minutes with changes of the sterilant solution. 5) Rinse three times with sterile distilled water.
Note: Succulent winter leaves survive disinfestations much better than summer leaves. Both leaf forms give rise to adventitious plantlets equally well.
Aseptic culture: 1) Transfer surface disinfested leaves to culture tubes or flasks containing  medium with all salts, thiamine and inositol at 115 strength, 0.02 mg/I BA, 0.01 mg/l NAA, 30 g/l sucrose. 2) Adjust pH of medium to 6.5 before adding agar. 3) 6 g/l agar 4) Leaves cultured on this medium should produce 7-8 plantlets with roots on each leaf exp I ant in approximately 8 weeks. Subculture: 1) For a higher yield of plantlets, subculture the leaves to 115 strength Linsmaier-Skoog medium containing 2.0 mg/I BA and 1.0 mg/I NAA for 3-4 weeks. This results in additional adventitious bud formation . 2) To maximize the growth of each plantlet, subculture again on 1/5 strength Linsmaier-Skoog medium with a reduced hormone level (0 .02 mg/I BA and 0.01 mg/I NAA). 3) Acclimatize the plantlets by planting in limed, sterilized sphagnum moss.
Gradually reduce the relative humidity from 100% to 50% over a period of 4 weeks.
Note: Linsmaier-Skoog medium contains the same macronutrient and micronutrient salts as  medium and differs only in its vitamin composition. Linsmaier and Skoog increased the level of thiamine four-fold from the original MS formulation. Nicotinic acid and pyridoxine were found unnecessary and eliminated from the composition of vitamin mixture.  .

Adventitious or Not?
This appendix contains some thoughts on the formation of adventitious structures in plants.
Traditionally, and for convenience, plant anatomists and morphologists have considered the primary plant body to be composed of three organs: roots, stems and leaves. The stem, leaves and attendant structures are generally referred to as the shoot system. These organs are seen to arise with a predictable, positional regularity and modularity along the root/shoot axis. In other words, the essential entity in development is the dynamic event. The expression of such an event is the pattern .
Any organ that arises outside of this basic developmental model or event sequence/pattern in an abnormal position is termed "adventitious". Gray ( 1887) termed aerial roots of orchids to be "anomalous" structures. Other early literature makes reference to "morphological roots" to describe roots developing from tissues other than the pericycle of the main root (Ames and McDaniels, 1947).  describes adventitious structures in plants as "an organ that developed in an unusual position" . Raven, Evert and Eichorn (1993) describe the term adventitious as "referring to a structure arising from an unusual place, such as buds at other places than leaf axis, or roots growing from stems and leaves". Whether or not any structure is truly adventitious in nature (that is, arising from an unusual place, and not part of the dynamics of a deeper developmental level of pattern expression) is a fundamental question in plant biology.
The phenomenon of adventitious development in vascular plants also belongs to that class of morphogenic events usually referred to as regeneration in the classical botanical literature. These phenomena include, but are not limited to, the formation of adventitious roots, shoots, plantlets and somatic embryos.
Regeneration in plants has long been known and studied. It has been broadly defined as the growth of new plant parts after the removal of corresponding parts elsewhere in the plant , includes references to early studies and reviews). In view of current biochemical and molecular techniques, and physiological and anatomical interpretations, this definition needs amendment to include organogenesis on the plant body as well as organogenesis, histogenesis and somatic embryogenesis in tissue surgically excised from the plant and grown aseptically in vitro.
Therefore, it is suggested that the term "regeneration" be taken to include any cell division and subsequent differentiation sequence that results in the formation of a complete tissue, organ, plantlet, or somatic embryo or embryo-like structure within either the plant body or tissues excised from the plant and maintained in culture. Such excised tissues are generally termed "explants" (Arditti, 1993 ).
A confusing number of descriptive terms have accumulated in the literature regarding all aspects of adventitious development. For instance , in an early review, considered the phenomena of regeneration to imply a development of dormant or latent rudiments, which he termed ' Anlagen' . These Anlagen were already present in the plant, either as dormant, differentiated embryonic tissue -and thus distinguishable either histologically or cytologically from surrounding tissue -or as a tendency or disposition in the tissue toward the formation of new structures. In this latter case, Goebel describes this tissue disposition as being outwardly invisible. Ames and McDaniels (1947), in their text on plant anatomy, state that adventitious roots may arise from meristematic root germs or 'cushions' that are cytologically or histologically distinguishable from the surrounding cells; or, in contrast, from groups of cells that are not distinguishable from the surrounding tissue, though they are still capable of forming new structures. These are additional terms for Goebel ' s ' Anlagen' and ' tissue disposition' .  states that adventitious roots may arise from dormant primordia that had been laid in place earlier in development until stimulated to grow, or from primordia that arise anew in apparently undifferentiated tissue.
Sinnott (1960)  control of differentiation at the biochemical, molecular and physical levels of organization have led to the synthesis of developmental ideas that, when applied to regeneration, necessitate a regrouping of these phenomena under the broader heading of developmental theory. Steeves and Sussex (1989, pg. 336) remark that it is important to recognize that, in de novo regeneration it is not an organized root, shoot or flower that is being initiated but rather a meristem. This meristem may not be determined until sometime after its initiation and prior to its gaining autonomous control over its developmental fate. Thus, the entire realm of adventitious development can be seen as the study of pre-formed (and in some cases, still indeterminate) meristematic centers or primordia, or the study of, what appears to be, the de novo formation of meristematic zones (Anlagen?). In each case, the cells or tissues involved may be seen as being at the center of a morphogenic field, with the meristem being a morphogenic unit and its activities establishing the boundaries of a larger field of developmental influence.
The ideas discussed above were originally written to serve as an introduction to a study of de novo meristem formation with particular emphasis on the role of the epidermis as a potent generative and formative tissue system. Interest in this area is founded on a desire to understand plants in terms of organismal theory. A return to this work is anticipated in the future.