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MICROPROPAGATION AND COMPARATIVE GROWTH ANALYSIS OF IN VITRO AND E X VITRO CULTURED CACTI by Guadalupe Malda de Suzan
A Dissertation Presented in Partial Fulfillment o f the Requirements for the Degree Doctor o f Philosophy
ARIZONA STATE UNIVERSITY December 1996
UMI Number: 9707395
UMI Microform 9707395 Copyright 1996, by UMI Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code.
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UMI
MICROPROPAGATION AND COMPARATIVE GROWTH ANALYSIS OF IN VITRO AND EX VITRO CULTURED CACTI by Guadalupe Malda de Suzan
has been approved August 1996
APPROVED: .Chairperson
L il
iupervisory Committee
s t'l.'U l, artment Chairpersoj
Dean, Graduate College
jJ L
ABSTRACT Conventional propagation methods are not suitable in most cases for rare cacti because they usually have limited reproductive capacities and show extreme slow growth. Research in this dissertation was devoted to applying in vitro culture techniques to develop a micropropagation and regeneration system for three rare and endangered cacti species. Regenerated and re-established plants were obtained for Coryphantha minima and Obregonia denegrii. Combinations of 0.5 mg I'1 benzyl adenine (BA) with 0.1 mg I'1 naphtalene acetic acid (NAA) or 10 mg I'1 kinetin (KIN) with 1 mg I'1 indole acetic acid (IAA) resulted in superior shoot proliferation, hi addition, massive production of somatic embryos with BA-NAA combinations was observed in Obregonia denegrii which could also be established into foil grown plants. Although spontaneous in vitro rooting occurred with both species, ex vitro root induction offered superior results for subsequent acclimatization of shoots. Unfortunately, micropropagation was unsuccessful for Ariocarpus agavoides due to poor and inconsistent morphogenetic responses during in vitro culture. In developing micropropagation systems for cacti, certain variables were considered so that these techniques could be used in less developed countries where endangered species often occur. This included varying the levels of sugars and nutrients in the culture medium and reducing the relative humidity inside culture vessels. Effects of these variables on growth and photosynthetic activity in vitro were analyzed and compared to cacti cultured ex vitro. Unlike normal, C3 plants, cacti possess a CAM physiology which alters patterns
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o f carbon uptake that can afreet growth responses in tissue culture. In vitro derived plantlets o f Coryphantha minima were seven fold larger than plants cultured ex vitro during the same time period. Gas exchange analysis and daily fluctuations of malic acid revealed that the in vitro environment stimulated photosynthetic activity, with cacti showing a net increase of CO2 uptake in the dark and light. It was also shown that high humidity in the culture vessel affected transpiration rates and, consequently, opening, which resulted in greater CO2 uptake of cacti cultured in vitro. The acclimatization of in vitro derived cacti to ex vitro conditions was facilitated by plant succulence which delayed excessive water loss. Additionally, tissue cultured cacti retained the capacity to generate normal levels of epicuticular wax upon removal from the culture vessel which prevented drastic water loss and aided acclimatization stomatal
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For my parents: Manuel Malda and Guadalupe Barrera. Specially for Humberto, Katia and Gabriela
ACKNOWLEDGMENTS I w ould like to express my profound g ratitude to my com m ittee chair, Dr. Ralph Backhaus, for his patience, advice and friendship. I particularly recognize Dr. Chris M artin who helped me to understand essential aspects o f my experim ents; and my friend, Dr. Gary Nabhan who always m otivate me in the pleasure o f science. I also appreciate the rest o f my com m ittee members, Dr. Jean Stuz and Dr. Donald Pinkava for their valuable guidance every tim e I needed it. I wish to express a special recognition for two persons whom encouraged me to accomplish this work: Hum berto Suzan for his im portant advice in data analysis and constant support and Katia, who helped me to take care o f my plants. Finally, I appreciate the help from Linda Prittchet, Liz E cker and the s ta ff o f the Desert B otanical Garden.
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TABLE OF CONTENTS Page LIST OF TABLES .......................................................................................... LIST OF FIGURES ......................................................................................... INTRODUCTION ............................................................................................ BACKGROUND INFORMATION .............................................................. Cacti tissue culture ............................................................................... T ransplant and survival after tissue culture .................................. E ffects o f the tissue culture environment on plant grow th ...................................................................................................... M ETHODOLOGY ............................................................................................ 1. DEVELOPM ENT OF MICROPROPAGATION AND REGENERATION SYSTEMS ........................................................ Species selected for m icropropagation .................................. Seed germ ination and aseptic initiation o f culture (STAGE I) .................................................................................. Shoot proliferation (STAGE II) ................................................ R ooting o f shoots (STAGE III) ................................................. R eduction o f m icrobial contam ination ..................................... A cclim atization o f ro o ted plants (STAGE IV) ....................... 2. PHYSIOLOGY OF THE ENHANCED GROWTH RESPONSE .......................................................................................... G row th m easurements .................................................................. Photosynthetic activity ................................................................ ix xiv 1 6 7 8
9 13
13 13
16 18 19 20 20
21 23 24
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3. MORPHOLOGICAL AND PHYSIOLOGICAL CHANGES ASSOCIATED WITH TRANSFER OF IN VITRO TO EX VITRO CONDITIONS ..................................................... T ranspiration and w ater loss .................................................... E picuticular wax analysis ......................................................... RESULTS AND DISCUSSION ................................................................... 1. DEVELOPMENT OF M ICROPROPAGATION AND REGENERATION SYSTEM S......................................................... Culture initiation (STAGE I) ..................................................... Shoot proliferation (STAGE II)................................................ Rooting o f new shoots and development o f som atic embryos (STAGE III)............................................... Reduction o f m icrobial contam ination..................................... Acclim atization o f rooted plants............................................... 2. PHYSIOLOGY OF ENHANCED GROW TH.............................. Growth analysis.............................................................................. Photosynthetic activity..................................................................... 3. MORPHOLOGICAL AND PHYSIOLOGICAL CHANGES ASSOCIATED WITH TRANSFER OF IN VITRO TO EX VITRO CON DITIO NS...................................... Changes in acid accum ulation........................................................ Changes in transpiration and w ater lo ss..................................... CONCLUSIONS.................................................................................................. REFERENCES....................................................................................................... viii
29 29 30 32
32 32 35
50 57 62 74 74 90
104 104 105 113 115
LIST OF TABLES Table 1 2 E ffects o f some features on tissue c u ltu r e .................................... O utline o f experim ents fro grow th analysis o f C oryphantha m inim a .................................................................................................. 22 3 Com parison o f germ ination percentages in C oryphantha m inim a, O bregonia denegrii and A riocarpus agavoides cultured in vitro and ex v i tr o ......................................................... 4 Morphogenetic response observed for Ariocarpus agavoides under different IAA - BA combinations supplemented during Stage II of micropropagation.................................................................................... 5 Shoot proliferation response o f Coryphantha m inim a in different concentrations o f BA and NAA..................................... 6 Shoot proliferation response o f C oryphantha m inim a in different concentrations o f IAA and K IN ..................................... 7 R esponse o f type explant in C oryphantha m inim a cultured w ith different cytokinins and a u x in s ......................................... 8 E ffects o f different combinations o f BA and IAA during in vitro shoot proliferation in O bregonia den eg rii , cultured on MS medium supplem ented with 3% sucrose. (D ata from com binations th at produced callus are not show n)................... 45 41 39 38 36 34 Page 11
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Table 9 Shoot proliferation response o f O bregonia den eg rii cultured w ith different concentrations o f BA and NAA........................... 10 R esponse o f Coryphantha minim a shoots subcultured for in v itro ro o t induction in hormone free MS medium and MS
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supplem ented with 1 mg I'1 IB A ....................................................... 52 11 R ooting percentage o f Coryphantha m inim a and O bregonia d en eg rii cultured in MS basal medium with different auxin sources and concentrations............................................................... 12 N um ber o f prim ary roots developed in vitro on Coryphantha m inim a , as a result o f different concentrations o f IBA and su cro se.................................................................................................... 13 Ex vitro rooting o f Coryphantha m inim a and O bregonia den eg rii w ith different auxin sources applied in two su b stra te s............................................................................................... 14 P ercentages o f the incidence o f m icrobial contam ination during experim ents o f in vitro culture w ith C oryphantha m inim a and O bregonia denegrii at different culture stages... 15 E ffects o f sucrose concentration on m icrobial contam ination and plant grow th in culture vessels opened tw ice for 3 m inutes, over a 1 month period....................................................... 61 60 58 54 53
Table 16 E ffects o f an upper layer o f paraffin-lanolin m ixture over the grow th medium in the incidence o f m icrobial contam ination in v itro .................................................................................................... 17 R e-establishm ent percentages o f C oryphantha m inim a after 4 weeks o f acclim atization, in relation to previous rooting treatm en ts.............................................................................................. 18 Re-establishm ent percentages o f O bregonia denegrii after 4 weeks o f acclim atization, in relation to previous rooting treatm en ts.............................................................................................. 19 Survival o f C oryphantha minima 10 m onths after
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acclim atization...................................................................................... 67 20 R e-establishm ent o f Coryphantha m inim a in relation to their in vitro culture origin........................................................................ 21 W ater loss after 20 days at the acclim atization stage o f C oryphantha minima in relation to previous rooting treatm en ts.............................................................................................. 22 Survival o f O bregonia denegrii 4 m onths after acclim atization...................................................................................... 72 71 68
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Table 23 Comparison o f volume and fresh weight increases in a 4 m onths period in C oryphantha minima cultured in v itro in horm one-free M urashige-Skoog media and supplem ented with 10 mg I '1 KIN and 1 mg I '1 IAA............................................. 24 Fractional increase in volum e and fresh weight in a 45 days period in C oryphantha m inim a cultured under different
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levels o f light and ventilation in the culture vessel.................... 81 25 Effects o f M urashige-Skoog nutrients concentration on volume and fresh weight o f Coryphantha m inima cultured during three m onths in v itro .* ........................................................ 26 Effects o f different sucrose concentrations supplem ented to the grow th medium in volum e and fresh weight o f Coryphantha m inim a ........................................................................ 27 Fractional grow th in a th ree m onths period o f O bregonia denegrii p lantlets derived from somatic em bryogenesis and from shoot m ultiplication................................................................ 28 E ffects o f different sucrose concentrations on plant grow th and root developm ent in Coryphantha minima new shoots m aintained at Stage III o f m icropropagation for three m onths.................................................................................................... 88 87 85 83
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Table 29 Percentages o f w ater loss one and 20 days after acclim atization in C oryphantha m inim a derived from different rooting treatm ents. Survival percentages data correspond to day 20........................................................................
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LIST OF FIGURES Figure 1. 2. 3. 4. Selected species for m icropropagation system .............................. M icropropagation system fo r endangered cacti............................ Assim ilation cham ber for gas exchange m easurem ents.............. Shoot pro liferatio n response o f different explant types and sizes o f C oryphantha m inim a subcultured in 10 mg I '1 KIN combined with 1 mg I '1 IAA (P (f) = 0.0011)............................. 5. Shoot proliferation response o f different explants o f O bregonia denegrii cultured in MS supplem ented with 10 mg 1 I '1 KIN and 1 mg 1 F 1 IA A ........................................................ 6. Effects o f different types o f explant on shoot pro liferatio n o f O bregonia d enegrii cultured in MS medium supplem ented with 0.5 mg 1 I '1 BA and 0.1 mg 1 T1NAA.................................. 7. E ffects o f the presence/absence o f IBA (3 mg 1 I*1 ) on in vitro rooting in different sizes o f shoots and som atic embryos o f O bregonia denegrii. Plants were m aintained in MS medium supplem ented with 3.0% sucrose............................. 8. E ffects o f different concentrations o f IBA and sucrose on root developm ent in vitro in Coryphantha m inim a cultured in MS medium...................................................................................... 56 55 49 47 43 Page 14 17 26
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Figure 9. E ffects o f an upper layer o f paraffin-lanolin m ixture over the grow th medium on plant grow th in a four months period. Plant grow th is referred has grow th index (fractional increase volum e).................................................................................. 10. R ates o f w ater loss (based upon fresh weights) after one week in acclim atization stage, from Coryphantha m inim a plants rooted in vitro and plants in an ex vitro rooting treatm ent sim ultaneous to acclim atization.................................. 11. G row th comparison o f a new shoots produced in vitro and seedlings cultured in vitro and ex vitro in C oryphantha m inim a ................................................................................................... 12. D evelopm ent of a new tubercles and increase in volum e during seedling grow th o f O bregonia denegrii cultured in vitro and ex vitro................................................................................. 13. Size com parison betw een different ages and culture conditions in C oryphantha m inim a ................................................ 14. Com parative average sizes in volume o f shoots produced in MS basal medium supplem ented with 0.05 mg I '1 NAA/1 mg I ' 1 BA and 2 mg T 1 IAA/4 mg I ' 1 KIN.........................................
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Figure 15. Effects o f previous rooting treatm ents on fresh weight increase o f Coryphantha m inim a after five months o f acclim atization........................................................................................ 16. Comparison o f malic acid daily fluctuations in Coryphantha m inima plants cultured in vitro , plants acclimatized for 2 months and wild plants m aintained in greenhouse conditions. 17. Diurnal and nocturnal CO 2 exchange in Coryphantha m inim a cultured in vitro com pared to m icropropagated plants maintained ex vitro for six m onths, and to wild plants maintained in greenhouse..................................................................... 18. Malic acid daily fluctuations in Coryphantha minima derived form in vitro culture, 2 and 12 m onths after acclim atization, compared to wild plants m aintained in greenhouse..................... 19. Comparison o f malic acid daily fluctuations in Coryphantha minima plants cultured in v itro at shoot proliferation (Stage II), rooting-elongation (S tage III) and callus derived from Stage II..................................................................................................... 20. Diurnal and nocturnal gas exchange o f Coryphantha m inim a cultured in vitro under different sugar concentrations supplemented to the grow th m edium ...............................................
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Figure 21. Com parison o f transpiration rates o f in vitro cultured plants o f C oryphantha m inim a and wild plants m aintained in greenhouse. Data o f in vitro plants are from Stage II, shoot elongation under low sugar concentration (1.5% ) and one m onth after acclim atization outside the culture vessels 22. Malic acid daily fluctuations in Obregonia denegrii som atic em bryos and new shoots produced in vitro, com pared to one year old seedlings, grown in vivo...................................................... 23. Com parison o f malic acid daily fluctuations o f one year old seedlings grown ex vitro and plants produced in vitro after 3 m onths acclim atization in Obregonia d enegrii ............................. 24. Changes in fresh weight during acclim atization in C oryphantha m inima derived from: In vitro rooting treatm ents using M agenta boxes with norm al lids (St. Ill); in vitro rooting treatm ents using M agenta boxes with vented lids (S t. I ll vented), and plants in trays covered with plastic to keep m oisture for ex vitro rooting............................................ 25. Am ount o f epicuticular waxes in Coryphantha m inima from different culture c o n d itio n s............................................................
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INTRODUCTION The family Cactaceae possesses one o f the highest number o f threatened and endangered species (International Union o f Nature Conservation, 1983; World Wide Fund for Nature (W.W.F.), 1986; Sanchez>Mejorada, 1987). The loss o f many species is due to habitat destruction and to the harvest and illicit trade of these species by collectors (Anderson, 1982). This is particularly true for plants from Mexico. An analysis o f horticultural species compiled in Hortus III (Bailey and Bailey, 1976) reveals that 9% o f all cultivated species o f temperate origin are native to Mexico (Bye, 1993). The most highly represented families of this group are the Cactaceae and Orchidaceae. In a recent analysis (Hernandez and Godinez, 1994) it was shown that Mexico has the highest concentration o f genera and species o f Cactaceae on the American continent. Moreover, about 197 o f these species are endangered, which is the highest number for any country in the Western Hemisphere. The importation and trade o f endangered, wild Mexican cacti began in the middle of the nineteen century and has contributed significantly to their loss. From then until the 1930's, when trading became illegal, the Maison Vandermaden Co. o f Brussels, and the J.A. McDowell & Co. of Mexico and New York exported large numbers of Mexican cacti to Europe, Japan and the United States (Bravo and Sanchez-Mejorada, 1991). Although these companies eventually discontinued operations, illegally obtained rare cacti continue to generate high demand and
exorbitant prices throughout the world (Oldfield, 1985). Unfortunately, many mature specimens are still collected from wild populations (Bye, 1993). An important advance for plant conservation has been Mexico's participation in CITES (Convention o f International Trade in Endangered Species o f Wild Flora and Fauna), which regulates importation and exportation o f wild plants between member nations. Plants listed by CITES (Appendix I) are prohibited from international trade unless artificially propagated. O f the 64 cacti species currently listed, 46 come from Mexico (Hunt, 1992). In Mexico, an industry is currently under development to cultivate these species which should provide additional protection by diverting market pressure away from depleted, wild populations. To date, the rescue o f Mexico's rare populations has largely been unsuccessful because o f habitat destruction and over collection, with losses exceeding the natural rate of regeneration in the wild. It is widely agreed that rare cacti could be cultivated and reintroduced back into the wild under certain situations (Starling and Doods, 1983; Sanchez-Mejorada, 1987; Martinez and Rubluo, 1989). However, conventional propagation methods using seeds or cuttings are much too slow. Rare cacti usually have limited reproductive capacities and often exhibit difficulty in flowering, seed production, germination and offset production. One alternative, is to propagate cacti by tissue culture. This allows for the rapid and continuous production o f plants throughout the year. Large-scale production could also provide commercial incentives for economically depressed areas o f Mexico, where many o f these species occur.
Tissue culture has already proved useful for several cacti species (Johnson and Emino, 1979; Mauseth, 1979; Ault and Bagamon, 1985; Escobar et al., 1986; Clayton et al., 1990; Hubstenberger et al., 1992). These include endangered species from Mexico, such as Mammillaria sanangelensis Sanchez-Mejorada, Leuchtenbergia principis Hooker and Astekium ritteri Boedeker (Starling, 1985; Martinez and Rubluo, 1989; Rodriguez and Rubluo, 1993). However, little information is available concerning the survival of reintroduced species into the wild following their propagation by tissue culture (Rubluo et al., 1993). The major objective o f my thesis was to develop a micropropagation and regeneration system for three rare and endangered species o f cacti, two o f which are endemic to Mexico. Certain aspects, such as the reduction o f microbial contamination, rooting o f new plants under ex vitro conditions and the use of minimal amounts o f growth regulators were also considered, in order to adapt the system for low technology conditions that might be expected in remote, rural areas o f Mexico. As morphological and physiological characteristics associated with in vitro culture could also afreet the quality, quantity and survival o f cacti transferred into the wild, a second objective was to evaluate these features during in vitro culture and establishment. A third objective was to characterize the growth o f cacti in tissue culture. It has been frequently observed that cacti grow much more rapidly in vitro compared to ex vitro (i.e., "normal" greenhouse or outdoor) conditions. This is especially
true for plants undergoing culture in Stage II shoot proliferation media. Also, the difference in growth rates between in vitro and ex vitro cultured cacti are substantially greater than those observed for non-succulent species. Why cacti exhibit such exaggerated growth is unknown. For Aztekium ritteri, a naturally slow-growing cacti, it was shown that tissue cultures produce 5-7 new offshoots after 11 months while ex vitro specimens never produce offshoots (RodriguezGaray and Rubluo, 1993). It has also been observed that Mammillaria woodsii Craig plants grown from seed require at least a year or more to develop to the same size as plants regenerated after a few months in tissue culture (Vystok and Jara, 1984). Similar observations were reported for Mammillaria prolifera Miller (Minocha and Mehra, 1974), Esposotoa huanucoensis Ritter (Angris and Mehra, 1982) and Cephalocereus senilis (Haw.) Pfeiffer (Bonnes et al., 1993). Despite these recurring observations it is unclear why this occurs. It may be that cacti do not experience quiescence during in vitro conditions or that the external carbon sources used in culture media favor continuous rather than episodic growth. Growth regulators may also play a role. Dabekaussen et al. (1991), showed that Sclerorebutia alba Raush produced a 15-fold increase in fresh weight when the concentration o f 6-benzylaminopurine (BA) was raised from 0 to 1 mg I '1. High nitrogen concentration in the medium may also be a factor. Nobel (1983) showed that elevated nitrogen content in hydroponic solutions increased growth rates o f seedlings cacti.
5 In contrast to cacti, non-succulent species rarely exhibit higher growth rates in vitro compared to ex vitro conditions. The difference may be related to some basic physiological phenomenon. The most obvious is carbon acquisition. Succulents, including cacti, possess the crassulacean acid metabolism (CAM) while non-succulents possess C-3 or C-4 photosynthesis. This fundamental difference in carbon metabolism may affect the growth of these plants under in vitro conditions and is one o f the questions explored in this thesis. This was done by comparing the growth rates and photosynthetic activity o f micropropagated versus ex vitro cultured cacti. A better understanding of this enhanced-growth response could be important for culturing other types o f cacti in vitro.
BACKGROUND INFORMATION
The Cactaceae are native to America with three centers o f origin and distribution: Mexico, Peru and Brazil (Gibson and Nobel, 1986). Cacti reach their maximum diversity, abundance and importance in Mexico. Approximately 900 species are restricted to Mega-Mexico 1 which includes Mexico, Arizona, New Mexico and Texas (Rzedowski 1993). Most cacti inhabit arid and semiarid regions where the scarcity o f water constrains plant survival. To cope, cacti have developed a number of special features to conserve water and restrict vegetative growth. These include the development of spherical to cylindrical, photo synthetic stems and the modification o f leaves into non-transpiring spines (Gibson and Nobel, 1986). The stem cortex possesses parenchyma with a leaf-like structure, having a palisade layer and epidermal stomata at densities o f 18.3 to 31.1 per mm2 (Sajeva and Mauseth, 1991). The epidermis also possesses a thick cuticle rich in waxes which limits water loss. Wax contents o f 70 to 250 mg per cm have been reported for Opuntia engelmanii Salm - Dyck (Wilkinson and Mayeux, 1990). Stems can also store water, enabling maintenance o f physiological processes during periods of prolonged drought (Jordan and Nobel, 1981). Typically, when cacti are fully hydrated, water constitutes 90-94% of their weight. This is eventually lost by transpiration under dry conditions (Gibson and Nobel, 1986). The spherical stem also greatly reduces the surface-to-volume ratio and minimizes evaporative
surfaces (Barsikowski and Nobel, 1984). In order to maintain this morphology altered patterns o f stem architecture are necessary. Lateral branches have evolved into unique meristematic structures called areoles, which bear spines, axillary branches and flower buds (Gibson and Nobel, 1986). Areoles can be distributed to form specialized protrusions known as tubercles on globular cacti or ribs on columnar cacti (Bravo and Sanchez-Mejorada, 1991). Photosynthetic carbon acquisition for most Cactaceae is by CAM. Such plants exhibit high water-use efficiency because stomata open only in the dark. This greatly minimizes transpirational water loss due to the reduced water vapor diffusion gradient, which is lowest at night when carbon dioxide uptake occurs (Gibson and Nobel, 1986).
Cacti tissue culture. Cacti have been successfully propagated in vitro using adventitious shoots from callus (Minocha and Mehra, 1974; Johnson and Emino, 1979; Starling and Doods, 1983), axillary shoots from the tips o f young plants or the excised areoles o f mature stems (Mauseth, 1979; Viztok and Jara, 1984; Ault and Bagamon, 1985; Escobar et al., 1986; Martinez and Rubluo, 1989). Clayton et al. (1990) described tissue culture procedures for 35 cacti species from 20 genera and demonstrated that axillary shoots can proliferate at a rate o f 5 to 8 shoots per explant per month. The most common culture media employ MS (Murashige and Skoog, 1962) salts supplemented with sugars, vitamins, hexitols and growth regulators. Low auxin levels include indole-3-acetic acid (IAA), 1-
naphthaleneacetic acid (NAA) or indole-3-butyric acid (EBA) combined with the cytokinins, 6-benzylaminopurine (BA), 6-dimethylallyl-aminopurine (2iP), kinetin (KIN) or zeatin (Starling and Doods, 1983; Ault and Bagamon, 1985; Clayton et al., 1990; Hubstenberger et al., 1992). Culture environments typically range from 24-29 C using a 16 h photoperiod provided by cool white fluorescent lamps with a photon flux density o f 40-80 mmol ^m 'V 1 (Starling and Doods, 1983; Viztok and Jara, 1984; Sanderson et al., 1986; Clayton et al., 1990; Hubstenberger et al., 1992). Rooting in vitro has been satisfactory in the majority o f these studies. However, little is known about the acclimatization and re-establishment of micropropagated cacti following transfer from in vitro conditions (Martinez and Rubluo, 1989; Clayton et al., 1990; Hubstenberger et al., 1992; Rubluo et al., 1993).
Transplant survival after tissue culture. Most studies of cacti tissue culture terminate with the transplantation o f rooted plants into the greenhouse. However, plant re-establishment following greenhouse culture is not guaranteed. Hubstenberger et al. (1992) reported that Sclerocactus mesae-verdae L. Benson (Boissevian and Davidson,) is very difficult to root and does not become re established outside o f tissue culture. In contrast, Pediocactus desnainii Welsh and Goodrich, Toumeya papyracantha (Engel) B&R and two Sclerocactus species show re-establishment rates o f 51%. Effective re-establishment of 80-100% has been reported for more than eight different species of the genera Coryphantha,
Mammillaria, Ferocactus, Opuntia, Echinocereus, Pediocactus or Astrophytum (Ault and Bagamon, 1987; Martinez and Rubluo, 1989; Clayton et al., 1990). However, studies on the survival o f such propagules after transplanting are uncommon. Rubluo et al. (1993) analyzed the performance and survival o f Mammillaria sanangelensis in the wild after its acclimatization in the greenhouse and reported survival rates o f 91% after four years in the field. These high survival rates were due to the reintroduction into the same protected, microhabitats of the original population and because transplanting was performed at the beginning o f the rainy season. The importance o f seasonal factors for transplantation was emphasized by Olwell et al. (1987) in a reintroduction of Pediocactus knowltonii L.Benson propagated by cuttings. Cacti planted in spring did not establish well because o f severe stress the following summer. Returning in vitro derived plants to their original habitats is considered difficult (Fay, 1988). However, with tissue culture, variables can be manipulated to produce large plants that can overcome environmental stresses in the wild. One distinct advantage of tissue-cultured cacti is that slow-growing species develop more rapidly, in quality and size, than conventionally grown seedlings (Hubstenberger et al., 1992).
Effects o f the tissue culture environment on p la n t growth. Most studies on the effects o f tissue culture environments have been conducted on non-succulent plants. Generally, in vitro conditions are detrimental to acclimatization o f plants for ex vitro conditions. Conditions which contribute to this are low air exchange,
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low light intensity, constant day and night temperatures and growth media containing low oxygen and high sucrose (Kozai, 1991a; 1991c). The effects o f some o f these properties are summarized in Table 1. The culture vessel restricts C 0 2 and air exchange and increases the relative humidity and ethylene concentration which can all affect plant growth in vitro. A reported decrease o f C 0 2 from 350 ppm outside to 70- 80 ppm inside the culture vessel (DeYue and Desjardins, 1993) can reduce net photosynthesis. Low air exchange can also reduce epicuticular wax formation which leads to plant desiccation. This and high humidity can lower stomatal density and reduce stomatal functioning (Grout and Ashton, 1978; Sutter and Langhans, 1982; Short et al., 1987; Smith et al., 1990) which can cause difficulty later in rooting and nutrient uptake. Tissue culture plants are also exposed to reduced light and are grown in sucrose-rich media which can affect later acclimatization and survival (Capellades et al., 1990). Low light reduces photosynthesis and suppresses growth, but not completely. This is because sugars in the medium act as the primary carbon and energy source (Capellades et al., 1990; Grout and Ashton, 1978; Kozai, 1991c; Langford and Wainnwrit, 1988). These sugars permit some growth but suppress autotrophic growth and increase the likelihood o f microbial contamination and vitrification. However, in all of these investigations, C-3 rather than CAM plants were studied. In contrast to C-3 plants (Kozai, 1991c), growth rates o f cacti in tissue culture are greatly enhanced over their ex vitro counterparts. Since nearly
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Table 1. Effects of some features on tissue culture.
_____________ CAUSE__________
1. Enclosed culture vessel Low air exchange rate High humidity
EFFECT
Net photosynthesis altered Absence or low epicuticular wax development Lower stomatal density Malfunction of stomata (occasional incomplete closure) Difficulty in nutrient uptake Morphological changes Difficulty in re-establishing autotrophy Difficulty in re-establishing autotrophy Microbial contamination Vitrification Difficulty rooting High dark respiration
High ethylene concentration 2. Low light intensity 3. Sugar supplemented to the medium
4. Low dissolved oxygen in the medium 5. Constant air temperature
12 all members o f Cactaceae utilize the CAM pathway, one suggestion is that this metabolic process affects growth in vitro. Little information is currently available on this. CAM plants, such as Agave deserti Engelman, are normally slow-growing but show enhanced growth when well-watered. This enhanced growth coincides with a metabolic shift from CAM to daytime CO2 uptake (Harstok and Nobel, 1976). This shift may be responsible for the enhanced growth observed in tissue cultured cacti and is investigated in this project. The survival rate o f rare cacti transplanted into the wild is typically low, especially for seedlings (Ecker, 1989). This is surprising because despite having an abundance o f water in their tissues, cacti still undergo transplant shock. Water stress and a delayed resumption o f photosynthesis in shoots with a concomitant absence o f root growth may also contribute to low survival. It is not known if these observations hold for micropropagated cacti as they do with seedling cacti. For tissue cultured cacti, measurements of epicuticular wax formation, photosynthetic activity and water loss as a function o f culture conditions have never been determined. However, these aspects are evaluated in this project. It is also unclear how in vitro conditions might affect growth following transplantation to the outside. These questions are also addressed herein. A better understanding o f these conditions could help to improve the propagation o f other endangered cacti for future recovery efforts.
METHODOLOGY.
1. DEVELOPMENT OF MICROPROPAGATION AND REGENERATION SYSTEMS
Species selected f o r micropropagation. Three species o f cacti were selected for tissue culture studies. All are rare and occur in extremely limited areas o f the Chihuahuan Desert. They have been listed as endangered by CITES (Sajeva and Orlando, 1989; Hunt, 1992) and are classified as "difficult-to-cultivate" (Fig. 1). Obregonia denegrii Fric is a plant 8-20 cm in diameter with a grayish-green stem and triangular tubercles arranged in a rosette. Plants possess apical areoles with 1-3 small spines and have white flowers and pale-brown fruit (Bravo and Sanchez-Mejorada, 1991). This species is endemic to northeastern Mexico and has always been considered rare. It is highly valued in the commercial market (International Union o f Nature Conservancy, 1983) and has been over collected in alarming numbers. No efforts are currently underway to propagate this for the horticultural trade. Reproduction is only by sexual outcrossing and propagation by seed has been limited to amateur enthusiasts in Europe (World Wide Found for Nature, 1986; Sanchez-Mejorada, 1987). Ariocarpus agavoides (Castaneda) E.F. Anderson is a grayish-green plant, 2-6 cm in height, with a strongly tuberculate body and elongated tubercles 5-7 cm
Ariocarpus agavoides
Coryphantha minima
Obregonia denegrii Fig. 1. Selected species for micropropagation system.
15 long. Areoles do not have spines and bear purple flowers (Bravo and SanchezMejorada, 1991). It is endemic to northeastern Mexico and several populations o f this species are known to be extinct. Ariocarpus is highly popular in the horticultural trade because o f its bizarre appearance. It grows very slowly from seed which is scarcely available. For this reason, most plants sold are field collected (International Union for Nature Conservation, 1983; World Wide Fund for Nature, 1986; Sanchez-Mejorada, 1987). In nature, this species reproduces only by seeds and successful germination in situ depends on particularly favorable climatic conditions (World Wide Fund for Nature, 1986). There are no reports of its cultivation. Coryphantha minima Baird is a simple or branching, ovoid plant, 1.2 to 2.5 cm in length and 1.2 cm in diameter. Areoles are somewhat woolly with dense spines, ca. 20 per areole. Spines are ashy gray or pink and arranged in a series of 2 or 3. Flowers are rosette-purple with green filaments and pale orange anthers (Benson, 1982). This species is native to the Chihuahan desert and grows only in two known sites, along novaculite ridges, in Brewster county, Texas (Benson, 1982). Coryphantha minima is relatively easy to cultivate from seed (Sajeva and Orlando, 1989). However, seed germination and seedling survival in natural populations are unknown. In addition to tissue culture studies, Coryphantha minima will be used for physiological studies in this project because o f its enhanced growth characteristics in vitro and because its high proliferation capacity allows destructive sampling for morphological analysis.
Reproduced with permission o f the copyright owner. Further reproduction prohibited without permission.
16 Seed germination and aseptic initiation o f culture (STAG E I). For micropropagation, meristems cultured from areoles excised from shoot apices were harvested from aseptically-grown seedlings. Plants were then regenerated by direct morphogenesis (see Fig. 2). Seeds o f Coryphantha Minima and Obregonia denegrii were obtained from the Desert Botanical Garden, Phoenix, AZ while seeds o f Ariocarpus agavoides were obtained from the Instituto de Ecologia y Alimentos, Tamaulipas, Mexico. For in vitro culture, seeds were disinfected by immersion in a sodium hypochlorite solution (commercial bleach diluted in distilled water, 1:4) for 15 min., followed by three rinses in sterile, distilled water. Seeds were germinated in 0.5X MS salts supplemented with 15 g I'1 sucrose and 8 g I*1 TC agar (Hazelton, Lenexa, KS) dispensed in Magenta GA? vessels at 75 ml per vessel. The media was adjusted to pH 5.7 prior to adding agar and autoclaving at 121 C and 103 kPa for 15 min Seedlings were maintained in growth chambers at a (day/night) temperature range o f 25-27 C. Light was provided by cool white-fluorescent and incandescent lamps ( 90% and 10%, respectively) which provided a total irradiance o f 120-130 mmol m'2 s '1 . For ex vitro culture, seeds o f Coryphantha minima and Obregonia denegrii were sown on coarse sand and irrigated three times a day by mist. Afrer two weeks, seedlings were fertilized biweekly with 0. 5X strength Miracle Grow liquid fertilizer (8-7-6). Two month-old seedlings were transplanted to a soiless potting mixture containing a (1:1:1) mixture o f peat, perlite and coarse
STAGE I
STAGE II
STAGE n i
STAGE IV
Direct Aseptic germination morphogenesis

In vitro
TRANSFER OF ROOTED
V
Apical tips of 2 months old seedlings Shoot proliferation
Rooting of new shoots
SHOOTS TO SOILESS CULTURE
Ex vitro
Fig. 2. Micropropagation system for endangered cacti.
18 sand (Sanderson et al., 1986). Plants were maintained in the same culture room as in vitro plants and watered every five days with tap water and every fifteen days with 15 ml of 0. 5X MS basal inorganic salt solution. Distilled water was used every third irrigation to leach out salts. Germination rates, germination velocity, seedling size, seedling weight and the formation of new spinate areoles were measured for in vitro and ex vitro cultures over time. Growth comparisons were analyzed by a t student test using an Arc- Sine transformation when necessary (Zar, 1984).
Shoot proliferation (STAG E II). Excised tubercles yielded axillary meristems (areoles) that generated large numbers o f new shoot tips. This was particularly useful for endangered species. Young tubercle explants possessing three areoles were best (Dabekaussen et al., 1991) because basipetal areoles (older tubercles) may not regenerate well as a consequence of apical dominance (Hubstenberger et al., 1992). Apical portions of 2-3 month-old seedlings were cultured in basal media containing IX MS basal salts. Treatments included the cytokinins, BA and KIN, at concentrations ofO, 2.5, 5, 7.5 and 10 mg I'1 in combination with the auxins, NAA and IAA at concentrations o f 0, 1 and 3 mg I'1. The experimental design was a full factorial with three factors (explant origin vs. cytokinin vs. auxin) at different levels. The effect of different sucrose levels and increased air exchange in the culture vessel on shoot growth and proliferation were evaluated on Coryphantha
19 minima cultures, using the optimal hormone concentrations from the previous shoot proliferation experiments. Sucrose was evaluated at concentrations o f 0%, 1% 1.5% and 3%. Air exchange in culture vessels were evaluated as a function o f the type o f closure. The common closures for Magenta culture boxes were compared to modified closures having a 6 mm diam. vent covered with a 0.22 pm microporous filter (Sigma # C3430) that permitted greater air exchange. Data o f this experiment was evaluated in a full factorial design. The number o f new shoots produced in all experiments, on different shoot proliferation stages were measured by counting only shoots which were suitable for rooting which are over 0.3 mm in height.
R ooting o f shoots (STAGE III). The evaluation o f in vitro rooting was compared using the auxins IAA, IB A and NAA at concentrations of 0, 1, 3 or 5 mg I'1. The auxin treatment giving the best results was then used in combination with sucrose at 0%, 1.5% and 3% in a full factorial experiment. One disadvantage o f shoots rooted in vitro was that residual agar could interfere with root development (Brochard, 1991) and its removal from shoots was time consuming. Therefore, ex vitro rooting was also evaluated using horticultural grade sand as the rooting medium in combination with commercial rooting powder containing 0.2% NAA and 4% thiram (Rootone). Appearance o f well developed root primordia was used as an indicator o f rooting response.
Reproduced with permission of the copyright owner. Further reproduction prohibited w ithout permission.
20 Reduction o f microbial contamination. Plantlet loss by microbial contamination could be affected by sucrose content in the medium. Microbial contamination was monitored in all experiments using sucrose and in a separate experiment with callus cultures in medium containing 0%, 1% and 2% sucrose in the media. Culture vessels were opened twice in one week, for a period of 3 each, to increase the risk o f contamination. With this in mind, a protective overlayer containing a mixture o f autoclaved paraffin-petroleum jelly (2:1) was tested as a physical barrier for microbial contamination. Under a laminar flow hood, the warm, liquid wax mixture was dispensed onto the culture medium using a 5 ml syringe. The medium surface was covered with a 1-1.5 nun thick layer of this wax mixture. Microbial contamination was then monitored on two treatments 1) those where the paraffin mixture was dispensed on media containing plants and 2) those where plants were transferred into the overlayered medium, working 2a) inside and, 2b) outside the laminar flow unit. The presence or absence of contamination was evaluated in all cases.
Acclimatization o f rooted plants (STAGE IV). Rooted plants had to be protected from water stress when removed from in vitro culture. The in vitro environment also provided sterile conditions so plants could be more susceptible to fungal pathogens. Therefore, it was necessary to gradually reduce relative humidity and, at times, to drench plants with a fungicide when transferring them to pots (Fay and Gratton, 1992).
21 The micropropagation system used for Coryphantha minima and Obregonia denegrii used in vitro rooted plants, ex vitro rooted plants and reestablished new shoots. For plants rooted in vitro, roots were thoroughly washed with tap water to remove any residual agar residue. In all cases, plantlets 1.5-5 cm in height were transplanted into a sterilized potting mixture (same composition used for ex vitro culture) dispensed in 10 cm x 10 cm x 5 cm deep cells in plastic trays. A high relative humidity was maintained for the first three days by covering the trays with a transparent plastic lid. The lids were raised 3 cm over the next three days to gradually reduce humidity. The lids were then removed and plants were maintained the same conditions as the plants initiated by ex vitro culture. Plants at the acclimatization stage were evaluated for survival percentages and growth over a period of time up to six months following transplanting.
2. PHYSIOLOGY OF THE ENHANCED GROWTH RESPONSE Shoot proliferation o f cacti growing under in vitro conditions exhibit enhanced growth rates compared to cacti cultured under ex vitro conditions. Little quantitative data is available and few physiological explanations for this enhanced growth have been reported. To verify that this occurred, growth rates o f Coryphantha minima and Obregonia denegrii were compared between in vitro and ex vitro conditions. Growth comparisons were made on shoots over 1 cm in height. For Coryphantha minima, a series o f experiments (Table 2) was designed
T a ble 2.
Series of experiments in Coryphantha minima for growth analysis.
Experiment (Tissue Variables Experimental culture stage)_______________________________________ design

1) Stage II (Shoot proliferation). -Growth regulators 0.05 NAA/1.0 IBAand 2 IA A / 4 KIN. (mgl1) -Ventilation o f culture vessel -Suspr concentration -Rooting in vitro/ex vitro. -Sugar concentration in vitro (0, 1.5, 3%) Full factorial
2) Stage m (Rooting).
Full factorial
3) In vitro shoot elongation phase.
-Sugar concentration (0, 15,3,4%) -MS basal salts concentration (0.5, 1, 1.5 X) -Ventilation in the culture vessel Ligjit levels (120-130 and 390-400 pmol nr2 s'1 ) -Rooting in vitro - Rooting ex vitro.
Full factorial
4) Stage IV (Acclimatization)
Media comparison
5) Ex vitro culture
-Greenhouse maintenance -Growth chamber maintenance
Media comparison
23 that included Stages II, III and IV of in vitro culture, versus ex vitro culture. Shoot elongation that occurred between Stage II and III was also included to evaluate shoot growth. Shoot proliferation and rooting in Obregonia denegrii produced a high rate o f somatic embryos. Therefore, growth analysis focused on the development and growth o f somatic embryos compared to new shoots produced by in vitro conditions versus those o f seedlings cultured ex vitro. Growth o f Obregonia denegrii was compared, I) during seedling development under in vitro versus ex vitro conditions, 2) during the formation o f somatic embryos and when new shoots were produced in vitro using 0, 1.5 or 3% sucrose, 3) under different nutrient levels containing 0.5X, IX and 1.5X MS salts.
Growth measurements. Nobel (1983) defined growth in cacti as the fractional increase in volume calculated from the product o f the cacti height and diameter squared. For Coryphantha minima, growth rates were determined as shoot volume and fresh weight over time. Volume was calculated from height and diameter measurements taken with a micro - caliper; and the assumption that plants approached a cylindrical shape. Stem surface area was calculated in a similar way, and this information was used to calculate photosynthetic rates. The effects of tubercles on the total volume and surface area was also taking into account to make more accurate estimates. Four cross sections from stems were taken in ten randomly selected plants. Diameter was measured for each section
24 and the circumference was calculated from imprints made from each section using a fine cotton thread surrounding each imprinted contour. The thread length was used to as the sectional circumference. Imprint diameters o f each section were then recalculated by geometric formulae from these circumference values. A regression analysis was performed to adjust the diameter values with the micro caliper-to-imprint diameter values. Because o f the irregular shape of Obregonia denegrii plants, the number of new tubercles was used as the growth parameter instead o f shoot volume. Measurements were taken monthly on a m inim um o f four replicates per treatment. For ex vitro cultured plants, data were recorded one day after watering, to maintain uniformity. Volume and fresh weight data were subjected to analysis o f variance using a SPSS program. Initial volumes and fresh weights were also considered as co-variables to detect any possible effects.
Photosynthetic activity. CAM physiology, as found in the Cactaceae, is a means o f concentrating CO2 to allow plants to utilize both atmospheric and internal C 0 2. Under normal conditions, CAM plants exhibit a very short transition from nocturnal to diurnal C 0 2 assimilation. At the end o f the dark period CAM plants utilize both the C 0 2 taken up from the atmosphere and C 0 2 released by the decarboxylation o f malic acid which is re-fixed via Rubisco (Griffits, 1988). It is possible that in vitro cultured cacti assimilate carbon constantly, resulting in continuous growth. High humidity in the culture vessel may also allow cacti
25 stomata to remain open, permitting continuous CO2 fixation. To address some o f these questions, a comparison of photosynthetic rates was made between in vitro and ex vitro cultured specimens o f Coryphantha minima by measuring the photosynthetic activity both by gas exchange and daily malic acid fluctuations. In situ gas exchange was determined with a portable LiCor Li-6200 photosynthesis system using modified culture vessels as assimilation chambers (see fig. 3). The gas analyzer sensor head was attached to a modified Magenta GA7 culture vessel with a Magenta polypropylene coupler (Sigma # C-0667). The coupling seam was covered with parafilm to seal the system and, by monitoring CO 2 concentration changes, no external air flow was detected inside the chamber. Within each assim ilation chamber a brush-less micro-cooling fan (Radio Shack, #273-244) was installed to circulate air for good gas exchange. The amount of agar medium in each vessel was increased from 75 to 200 ml to reduce the air volume in the assimilation chamber to 558.06 cm3 and to increase air circulation within the chamber. One hour prior to taking measurements, the agar medium was covered with a layer o f the paraffin-petroleum jelly (2:1) mixture to reduce water evaporation from the agar and eliminate CO 2 diffusion due to root respiration. An average o f 3.5 min was required for the adjustment o f the chamber, equilibration and data recording. A 2 ppm change in C 0 2 concentration was the criterion for reading one observation and a time period o f 200 sec was required to reach that change. Three lectures were subsequently recorded every 128 seconds
26
Sensor head
Fan
Infrared gas analyzer
Coupler for Magenta boxes
Paraffin-lanolini layer
Growth medium
Fig. 3. Assimilation chamber for gas exchange measurements.
27 approximately, after this time period. Temperatures were uniform during all measurements (28.6 - 29.5C). The assimilation chamber was always installed in the same position in the culture room, so irradiance levels were similar for all measurements The same system was used for measuring photosynthesis in ex vitro cultured plants. In order to minimize possible effects o f a change in environmental conditions, one week before measurements pots with ex vitro plants were moved from greenhouse to the culture room. One day prior to measurements ex vitro plants were transplanted into horticultural grade sand in the bottom half o f disposable polystyrene Petri dishes (60 x 15 mm).and irrigated with 5 ml distilled water. One hour before being moved into the assimilation chamber for gas exchange lectures, the Petri dish and sand were sealed with the same parafBnpetroleum jelly mixture to prevent water and gas exchange from the roots.. For all, in vitro and ex vitro plants, gas exchange measurements were taken on each o f three replicates. Data were recorded in two times: one during the middle of the dark period and other at the middle of the light period. Photosynthesis was measured on plants in the following treatments: 1) in vitro culture; 2) elongation stage in MS medium supplemented with 10 and 3% sucrose; 3) acclimatized plants which had been grown for 6 months outside the culture vessel and; 4) "wild" plants under cultivation at the Desert Botanical Garden, Phoenix, AZ.
28 Photosynthetic activity was also determined by measuring diurnal fluctuations in tissue acidity. Samples o f shoot tissue weighting 1 g each were obtained at different times during the day from three separate replicates, in the beginning, middle and end o f the dark period and in the middle o f the light period. Each sample was stored in sealed plastic bags and immediately frozen. At the end o f the sampling period, individual tissue samples were thawed and ground with sand in 10 ml distilled water, and then boiled for 15 min. Once samples were cooled to room temperature, 1 ml o f extracted sap was titrated with 0.02 N NaOH to an end point o f pH 7.0. The molar concentration o f malic acid (a diprotic acid) was obtained by dividing by 2 the number of ml o f NaOH used (Pearcy et al. 1989) to determine the acid concentration per unit o f fresh weight in meq/g. For Coryphantha minima, measurements of malic acid fluctuations were made with callus tissues and with new shoots that arose during the shoot proliferation stage. Tissues were harvested from plants grown in the following conditions: 1) in vitro elongation stage in the presence o f 0, 15 and 30 % sucrose, 2) rooted plantlets acclimated for 20 days, 3) tissue cultured propagated plants that had been growing one year in ex vitro conditions, and 4) "wild" plants cultivated at the Desert Botanical Garden, Phoenix, AZ. For Obregonia denegrii, titrations were from 1) plants at the shoot proliferation stage, 2) somatic embryos growing in vitro; 3) plantlets acclimatized for 6 months, and 4) ex vitro cultured plants that were more than 1 year old.
3. MORPHOLOGICAL AND PHYSIOLOGICAL CHANGES ASSOCIATED WITH TRANSFER OF IN VITRO TO E X VITRO CONDITIONS.
In general, most plants cultured in vitro frequently have difficulty adjusting to outside conditions (Jackson et al., 1991; Kozai, 1991b) and successful propagation depends on the ability o f plants to acclimate and survive the transfer to soil. One o f the principal transplant shocks for in vitro cultured plants is due to excessive water loss because o f the partial or complete absence o f epicuticular waxes (Brained and Fuchigami, 1981). Although the succulent nature o f cacti lessens this problem, this is an important consideration when transplanting cacti to the wild. Transpiration rates, water loss and epicuticular wax content were measured during the acclimatization stage in Coryphantha minima and Obregonia denegrii to assess the effects o f in vitro conditions on cacti culture and to evaluate re establishment during acclimatization.
Transpiration and water loss. Transpiration was monitored simultaneously with photosynthesis using the LiCor 6200. In order to accurately measure transpiration, it was necessary to determine air and cacti surface temperatures as well as boundary layer conductance in the assimilation chamber. Temperatures were measured in the culture room with one thermocouple inside the assimilation chamber and other in contact with the stem surface o f the cacti. Data were recorded using a Micrologger 21X (Campbell Scientific Inc.). Boundary layer
30 conductance in the chambers depended on cuvette volume, fan speed and cacti volume and shape. Measurements were made according to Parkinson (1985), using wet filter papers. Boundary layer conductance was based on the energy balance o f the filter paper using differences in relative humidity and temperatures inside the cuvette. Calculations were computed with the LiCor system. Filter papers were prepared in a spherical shape to resemble the shape o f the cacti. Water loss o f the cacti was analyzed by differences in weight over time (Wardle et al., 1983; Short et al., 1987; Smith et al., 1990). Fresh weights were recorded on five replicates per treatment at 1, 5, 10, and 20 days after removing plants out o f the culture vessel. Percent water loss was calculated according to Brained and Fuchigami (1981) using the equation: percent o f water loss = (ft. wt. - wt. after holding) (ft. wt. - oven dry wt.J
Initial fresh weights were used as weight after holding. Dry weight was determined by drying ground residue at 80 C for 24 hr.
Epicuticular wax analysis. Epicuticular wax content was determined by a colorimetric method according to Ebercon et al. (1977). Surface waxes were extracted by dip ping the plants into 15 ml distilled chloroform for 20 sec. Individual samples consisted of one or two shoots with 5 - 10 cm surface area. The extract was evaporated to dryness in a boiling water bath and was followed by adding 5 ml o f acidic Samples were returned to the boiling water bath
31 for 30 min and then 12 ml o f distilled water was added. After cooling and color development by oxidation o f waxes, the optical density o f each sample was read spectrophotometrically at S90 nm. Comparisons were made against standard wax solutions o f known concentrations prepared from pure paraffin. Wax content was expressed as mg cm'2 o f shoot area. Because relative humidity may afreet on epicuticular wax formation, wax content was compared between plants cultured in vitro under normal conditions and on plants grown in the ventilated closure system (Sigma, # C-3430).
RESULTS AND DISCUSSION
1. DEVELOPMENT OF MICROPROPAGATION AND REGENERATION SYSTEMS Culture initiation (STAG E I). Aseptic germination and culture initiation from mature areoles was achieved with no difficulties. Surface sterilization procedures were effective in all cases. Mature areoles o f Coryphantha minima and Obregonia denegrii developed some callus at the base after one month in culture, and in general their size was increased. In contrast, it was not possible to initiate cultures from mature areoles o f Ariocarpus agavoides. After three weeks in culture, explants became oxidized at the base and green tissues turned brown and died. Important differences between species were observed during seed germination. According to Sajeva and Orlando (1989) Coryphantha minima is relatively easy to cultivate from seeds. This was consistent with in vitro (94%) and ex vitro (82%) germination rates. In contrast, Ariocarpus agavoides and Obregonia denegrii reflected the difficulties common for most endangered cacti. In both cases, germination percentages were lower than 45% (Table 3). To increase germination, chemical scarification using a 3 min soak in concentrated hydrochloric acid followed by a 15 min. rinse in 0.5 M potassium nitrate was tested for Obregonia denegrii and Ariocarpus agavoides.
A favorable response was obtained only for Obregonia denegrii, which increased the germination percentage from 20% (no scarification) to 73% with scarification (Table 3). Germination o f Ariocarpus agavoides was not affected by scarification treatments. For all three species, aseptic germination in vitro was slightly higher than non-aseptic, ex vitro germination, regardless o f scarification treatments (Table 3). Following germination, seedlings were maintained for two months in the same culture media. During this time, the epidermis o f Obregonia and Ariocarpus seedlings ruptured, perhaps due to excessive succulence, and seedlings developed large, green callus growths. Callus occurred in approximately 40% o f Obregonia denegrii and 50% of Ariocarpus agavoides seedlings. In Ariocarpus, the callus developed purple areas caused by betalain pigment accumulation, which is common in some cacti (Gibson and Nobel, 1986). A similar phenomena was reported for Ariocarpus retusus Scheid. seedlings cultured on MS medium supplemented with 20 g I '1 and 20% (v/v) coconut w ater (Stuppy and Nagl, 1992). Obregonia and Ariocarpus are genera known as "living rocks" that contain numerous alkaloids and live half buried in soil with only the tips o f their tubercles exposed. They tend to be spineless and are among the slowest growing cacti, with very low vegetative reproductive capacities (Gibson and Nobel, 1986).
34
T able 3. Comparison o f germination percentages* in Coryphantha minima, Obregonia denegrii and Ariocarpus agavoides cultured in vitro and ex vitro.
Coryphantha minima
Obregonia denegrii scarified
Ariocarpus agavoides scarified 42% 45%
In vitro
94%
20%
73%
Ex vitro
82%
17%
27%
41%
40%
35 Shoot proliferation (STAG E II). Shoot proliferation as a consequence o f growth regulator treatment varied considerably for all three cacti. Ariocarpus agavoides presented the greatest difficulty in producing new shoots. Almost all of the combinations tested were unsuccessful (Table 4). The effect o f BA and IAA on axillary meristem production varied, yielding callus, isolated areoles, or roots (Table 4). The most common initial growth response was callus proliferation. Alter two months in culture, formation o f new, isolated areoles was observed in two treatments, one containing 5 mg I*1 BA plus 1 mg I'1 IAA, other containing 7.5 mg I'1 BA plus 1 mg I'1 IAA and 10 mg I '1 BA plus 3 mg f 1 IAA. In another species, Opuntia polycantha Haw, the formation o f isolated areoles occurred only when gibberellic acid was included in the growth medium (Mauseth and Halpering, 1975). Cytokinins or auxins did not induce such a response. The effect of exogenous gibberellic acid treatments on Ariocarpus agavoides was not examined in this study. Some of the new areoles developed into single tubercles, which were separated and subcultured for further shoot proliferation experiments. In only one treatment, that contained 2.5 mg I 1 BA, did new shoots regenerate from a single tubercle and this was observed on only two replicates. Other growth regulator combinations were tested on Ariocarpus agavoides. These included 0.5- 1 0 mg Tl KIN combined with 0.1-5 mg I'1 IAA or NAA. However, in all cases, no initiation o f new shoots was observed. The only responses observed were the formation of callus or an increase in the size o f the explant.
36
T a ble
4. Morphogenetic response* observed for Ariocarpus agavoides under different
IAA - BA combinations supplemented during Stage II of micropropagation.
Growth regulator combination # New shoots* ( mg I '1) 0.33 0.33 1 IAA 1 IAA - 2.5 BA 1 IAA - 5 BA 1 IAA - 7.5 BA 3 IAA - 2.5 BA 3 IAA 2.5 BA 10 BA 10 BA

Proliferation of Isolated areoles* 1 1 3 2 3
Development of rootsb 3

2 1

1.2 0.32
* Callus proliferation, as a morphogenetic response was persistent in all cases, including when other growth regulators combinations were supplemented. Data are not shown in table. 3 Values are mean shoot number standard error b Values are: 1 - few, 2 - regular, 3 - abundant
37 Coryphantha minima exhibited excellent shoot proliferation in treatments containing different concentrations and combinations o f BA plus NAA, or KIN plus IAA (Tables 5 and 6). In general, all BA treatments produced higher numbers of new shoots than KIN treatments. Callus was a byproduct in most of the cultures and the number o f healthy, vigorous shoots varied among treatments. Vitrification was observed, especially in treatments with higher BA and NAA concentrations and occasionally, new shoots were malformed and fasciated. The treatment yielding the highest number of normal-sized shoots with the fewest abnormalities (22.3 shoots per explant) was that containing 10 mg f ' BA plus 0.05 mg I'1 IAA (Table 5). A higher shoot proliferation rate was observed in treatments containing 0.25 mg I'1 BA, which yielded a mean o f 25.3 new shoots per explant; however, 12.3% o f these new shoots were abnormal. Thus, a similar number of normal, healthy shoots (22.19) was observed with this different treatment. In contrast, a decrease in this proliferation rates was observed using a different cytokinin, KIN, combined with another auxin, IAA. The treatment yielding the highest number o f large, vigorous shoots was that containing 10 mg Y 1 KIN plus 1 mg I'1 IAA (Table 6). However, although proliferation rates were lower, new shoots produced with KIN-IAA tended to be larger and have stronger spines than those produced in BA-NAA. Also, the frequency o f abnormal vitreous or malformed shoots was considerably lower in KIN-IAA than BA-NAA treatments.
38
T a b l e S.
Shoot proliferation response of Coryphantha minima in different
concentrations of BA and NAA.
Growth regulators combinations (me I'1 )
# New shoots*
Control (hormone - free) NAA (0) -B A (0.25) -B A (0.5) - BA(0.75) -B A (1.0) NAA (0.05>BA(0.25) -B A (0.5) - BA (0.75) NAA (0.1)-B A (0.25) - BA (0.75) -B A (1.0) NAA (0) - BA (2.5) - BA (5.0) - BA (7.5) -B A (10) NAA (0.05) - BA (2.5) - BA (5) -B A (10) NAA (0.1)- B A (2.5) - BA (5) -B A (10) NAA (1.0)-B A (4) - BA (5) -B A (7.5) NAA (3.0)-B A (4.0) -B A (5.0)
25.3 9.0 18.0 16.0 3.1 2.0 10.0 17.1 14.3 3.0 9.1 13.6 13.2 13.4 7.6 6.9 22.2 9.1 11.5 15.4 1.3 3.0 4.7 1 2
m
0.058
Abnormal shoots * / b
Callus production0
In vitro flowering 1 1
1.78 0.90 1.13 2.03 0.07 0.05 1.10 1.29 3.24 2.01 2.54 1.19 2.09 2.46 0.23 1.65 1.57 1.54 1.25 1.58 0.05 0.54 1.16 0.31 0.54
0 12.3 0 27.6 13.7 0 0 3.8 17.2 5.6 0 66.6 44.8 45.2 7.6 56.3 36.7 0 33.5 41.2 9.5 0 25.4 67.1 0 0
2 1 2 1 2 3 2 1 3 1 1 3 1 1 3 2 3 0 2 2 0 3 2 2 3 3
0 1 0 1 0 0 0 0 0 0 0 1 1 1 1 0 0 1 0 0 0 0 0 1 0 0
0.078
0.35
a - Values are mean shoot number standard error, b - Vitrified, fasciaied and/or malformed new shoots, c - Values are: 0 - none; 1 - few, 2 - regular, 3 - abundant. d - Values are: 0 - no flower development; 1 - flower development in at least two replicates. c- Profuse flower production in all replicates.
39
T able
6.
Shoot proliferation response of Coryphantha minima in different
concentrations of IAA and KIN.
Growth regulator combination (mg I ) IAA (0) - KIN (2.5) u - KIN (5.0)
ii
New shoots means 6.8 0.63 7.2 1.35 7.7 1.06 9.2 1.76 0.8 1.70 1.2 0.90 3.6 0.80 15.9 1.79 1.7 0.40
Abnormal shoots (%) 0 1.5 0 4 0 6.5 2.7 0 9.6
Callus 2 1 2 1 3 2 1 0 2
- KIN (7.5) KIN (10)
ii IAA (l) if if ii IAA (3)
- KIN (2.5) - KIN (5.0) - KIN (7.5) - KIN (10) - KIN (10)
40 In vitro flowering was an event observed for Coryphantha minima in treatments containing BA This response occurred on two different occasions using BA in combination with NAA (Table 5). It is noteworthy that in vitro flowering always occurred at the same time o f the year, in October-November. Deliberate attempts to obtain in vitro flowers during other months were unsuccessful. The cause of this response is not understood. Growth regulators apparently had an influence as flowering only occurred on plants in tissue culture, and not on ex vitro plants growing in chambers. A seasonal effect also could not account exclusively for this effect as natural flowering time for this species is May (US. Fish and Wildlife Service, 1984); and environmental conditions in the culture room were uniform throughout the year. It might be o f particular interest to further explore this response since most Coryphantha minima only bloom only after a minimum o f 3 to 4 years o f age (US. Fish and Wildlife Service, 1984). In these experiments, in vitro flowering occurred on plants less than one year old. Flowering is quite uncommon for plants cultured in vitro because tissues typically remain in a juvenile state, which is contrary to what is required for flower induction to occur in most other plant species. The large number o f shoots afforded by Coryphantha minima cultures permitted the testing o f different explants types for adventitious and axillary proliferation (Table 7). Shoot proliferation as a consequence of different explant sources varied with the growth regulator combinations used. Apical tips were the
41
T able 7. Response of type of explant in Coryphantha minima cultured with different cytokinins and auxinsa.
Growth regulators (mg I'1) BA (1) BA (10) K IN (10) 2iP (10) BA(1) - NAA(0.1) BA (10)-NAA (0.05) BA (10)- IAA (1)
Callus
------
Areole
11 1.85 67%
-------------
1.73 0.40 87.5%

-------------
3.1 0.47 2.7% 5.42 1.43 74.6%
2.7 0.89 0% 3.7 2.8 21.3% 2.4 1.37 10.8% 4.88 0.99 17.8%
Apical tip 16 1.05 1.4% 10.2 2.25 3.8% 9.2 1.76 0% 7.8 1.02 38.7% 3 1.35 0% 24.5 0.5 0%
Vitrified shoot 3 0.85 0% 11.5 2.33 53.8% 4.3 1.08 23.7%

-----
1 1.9 0% 19.6 1.18 0.7%
11.37 1.75 14.75 1.43 0% 61.5% ----6.3 0.98 4.02 1.03 KIN (1 0 )-NAA (1) 17.3% 67.8% 15 1.79 5.75 0.76 KIN ( 1 0 ) -IAA (1) 0% 2.5% --------2.5 1.53 2iP(10)- IA A (l) 18% a Values are mean shoot number standard error and % of abnormal shoots (vitrified and malformed)
42 best explants in all cases, even when vitrified. Vitrified apical shoot tip explants subcultured in 10 mg I'1 BA plus 0.05 mg I'1 IAA produced a mean o f 19.6 new shoots per explant. These explants originating from vitreous shoots produced normal adventitious new shoots which arose from callus that grew at the base of the explants during the first weeks in culture. Although a high number o f adventitious shoots arose from callus cultured in 10 mg I'1BA, a high proportion o f them were abnormal (Table 7). When areoles were used as explants the number of shoots arose in Coryphantha minima (4.88 0.99) was best with 10 mg I'1 KIN plus 1 mg I*1 IAA than with other treatments (Table 7), however, this same number o f shoots produced, was far less than when other explant types were used. This cytokininauxin combination seemed to yield better results on isolated areoles than other growth regulator combinations, which generally resulted in very poor responses. In general, it was observed that smaller explants yielded larger numbers o f new shoots than larger explants (Fig. 4) when the "optimum" combination (containing 10 mg I'1 KIN plus 1 mg I*1 IAA) was used for Coryphantha minima. This may have been due to the higher regenerative capacity o f the smaller shoots. Unlike the other two cacti species used, Obregonia denegrii afforded different morphogenetic responses following culture in Stage II media. The most sticking difference was that Obregonia consistently produced callus that contained a large number of somatic embryos. In addition, Obregonia gave rise to new
43
25
20
15 -
10
rA B C D
TYPE OF EXPLANT
A= B= C= D= E=
Complete, detached new shoots, > 1 cmhgt. Portions 0.7 - 1 cm long from new shoots. Portions 0.3 - 0.5 cm long from new shoots. Apical tips from new shoots. Apical tips from 3 months old seedlings
Fig. 4. Shoot proliferation response of different explant types and sizes of Coryphantha minima subcultured in 10 mg f 1 KIN combined with 1 mg I'1IAA. ( P (f) = 0.0011) New shoots produced in vitro were subcultured in hormone free medium during four weeks before explanting for this experiment.
44 shoots, isolated areoles and, occasionally, produced spines on some areoles (a condition uncommon for this species). The ability to form somatic embryos or shoots varied with BA and IAA levels (Table 8). On occasion, when areoles where produced, they were carefully subcultured to hormone-free medium and grown. In most cases, somatic embryos arose from these areoles after 3 to 4 months in hormone-free culture. When BA was combined with NAAa more uniform response in shoot proliferation and somatic embryogenesis resulted (Table 9). In one experiment, somatic embryogenesis was significantly higher (P (f) = 0.019), producing 5.33 embryos per explant with a single treatment containing 5.0 mg I- I BA alone, than when used in combination with NAA or other high BA concentrations (Table 9). However, in another experiment which employed lower concentrations o f growth regulators (Table 9), somatic embryogenesis yielded better values, specially with concentrations of 0.5 mg I'1 BA alone or combined with 0.1 mg Tl NAA. Thus, somatic embryogenesis can be induced with low growth regulators concentrations. Somatic embryogenesis also varies considerably with the explant source (age and regenerative capacity). O f the other cytokinin-auxin combinations tested, those containing KIN never induced somatic embryogenesis, only callus regeneration and formation of new shoots was observed. The influence o f explant type on Stage II proliferation was determined for a number o f growth regulator combinations. A treatment containing 10 mg I 1 KIN
I Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
45
T a ble
8. Effects of different combinations of BA and IAA during in vitro shoot
proliferation in Obregonia denegrii, cultured on MS medium supplemented with 3% sucrose. (Data from combinations that produced callus are not shown).
GROWTH REGULATOR COMBINATION (mg I 1) 2.5 BA - 1 IAA 7.5 BA - 1 IAA 10.0 BA - 1 IAA
MORPHOGENETIC RESPONSE Development o f isolated areoles Proliferation of new spines in the areoles Development o f isolated areoles Production of 3 somatic embryos and 5.2 new shoots per explant* Production of 2.3 new shoots per explant * Production of 6.3 new shoots per explant *
7.5 BA - 3 IAA 10.0 BA - 3 IAA * Data are mean numbers.
46
T able 9. Shoot proliferation response of Obregonia denegrii cultured with different concentrations o f BA and NAA.
Growth regulator combinations (mg I'1)

Experiment 1 0.25 BA 0.25 B A -0 .1 NAA 0.5 BA 0.5 B A -0 .1 NAA 0.75 BA 0.75 B A -0 .1 NAA 1.0 BA 1.0 B A -0 .1 NAA Experiment 2 2.5 BA 2.5 BA - 1.0 NAA 5.0 BA 5.0 BA - 1.0 NAA 7.5 BA 7.5 BA - 1.0 NAA 10.0 BA 10.0 BA - 1.0 NAA
New Shoots A
1.7 0 .8 7 9.2 8.59 1.0 1.00 11.3 6.23 2.5 2 .1 7 6.2 5.60 12.0 4.32 9.5 4.71 6.6 1.66 6.6 3.18 3.3 0.32 2.6 0.33 5.6 1.85 6.6 0.66 9.0 5.57 7.3 4.84
Somatic embryos A Callus8

1.5 1.5 0 9.0 5.2 9.2 5 .3 5.5 5 .5 5.7 4 .2 1.5 1.5 0.7 0 .7 1.3 0 .8 0.6 0.6 5.3 0 .3 1.6 1.6 0 1 .0 1.1 1.6 0 .8 2.3 0 .3 2 2 2 I 1 2 I 1 2 1 2 3 3 2 2 3
P (0
shoots
0.629
embryos
0.102
P (0
callus
0.816
P (f)
0.395
0.019 .
0.214
A Values are means standard errors. B Values are: 0= none, 1= few, 2= regular, 3= abundant.
47 plus 1 mg I'1 IAA resulted in at least 4 shoots per explant derived from callus (Fig. 5). However, much better proliferation was obtained for Obregonia denegrii using apical tips cultured from 3 months - old seedlings on 0.5 mg I'1 BA plus 0.1 mg I'1 NAA (Fig. 6). With this combination, however, it was possible to obtain shoots from callus. The shoots derived from the apical meristems were axillary while those from callus were adventitious. One advantage o f axillary shoots is that they are easier to root. They also survive better outside the culture vessel than adventitious shoots. Adventitious shoots tend to exhibit abnormal growth, being less symmetrical. Adventitious shoots also produce somatic embryos at the base o f their tubercles in vitro. This suppresses elongation and makes rooting difficult. Callus that afforded somatic embryos subcultured onto fresh, hormone-free medium, continued to produce somatic embryos for several months. An identical response occurred with adventitious shoots. As embryos enlarged, the overgrowth o f somatic embryos formed more callus which appeared to produce pro embryonal tissue. Somatic embryos arose from callus initiated on culture medium containing BA plus NAA that was subcultured onto a hormone-free medium. However, callus initiated and cultured for 15 months on hormone-free never gave rise to somatic embryos. BA and NAA at callus initiation seems to be required for the formation o f somatic embryogenesis in Obregonia denegrii. Similar findings were reported by Stuppy and Nagl (1992) for Ariocarpus retusus Scheidw., another endangered cacti from northeastern Mexico. In this study, callus formed
48
O
2
>
1 0
A B EXPLANT TYPE C
A - Apical tips from 3 m onths old seedlings B - Callus portions ( 1 cm.) C - Apical portions from new shoots produced in vitro.
Fig. 5. Shoot proliferation response of different explants of Obregonia denegrii cultured in MS medium supplemented with 10 KIN and 1 IAA (mg I'1 ).
49
B Type of explant
Explants: A - Apical tips from 3 m onth-old seedlings B - Callus portions C - Apical portions from shoots produced in vitro. D - Complete, detached new shoots produced in vitro. Fig. 6. Effects of different types of explant on shoot proliferation of Obregonia denegrii cultured in MS medium supplemented with 0.5 mg I'1 BA and 0.1 mg I*1 NAA. P(f) for # of new shoots = 0.006. P (f) for # of somatic embryos = 0.08
50 directly from young seedling tissue gave rise to somatic embryos by supplementing the culture medium with coconut water, which is rich in growth regulating substances such as zeatin. Unfortunately, the organic components in the coconut water were not identified (Stuppy and Nagl, 1992). Non-viable embryos were also reported by Rodriguez-Garay and Rubluo (1993) for another endangered Mexican cacti, Aztekium ritteri. In this case 2,4-D was used in combination with KIN to induce embryos. However, excessive growth o f woolly hairs also occurred because o f high auxin concentration. Results obtained for the three species described here show that BA used alone or in combination with NAA favors the morphogenetic expression o f cacti cultured in Stage II. This result is consistent with others (Starling, 1985; Martinez-Vazquez and Rubluo, 1989; Clayton et al., 1990; Rodriguez-Garay and Rubluo, 1994).
Rooting o f new shoots and development o f somatic embryos (STAG E III). Shoots produced in Stage II were used for root induction experiments in Coryphantha minima and Obregonia denegrii. Rooting was evaluated on both axillary and adventitious shoots. These were individually transferred to hormonecontaining or hormone-free MS medium to observe growth and root initiation. Shoot proliferation o f Ariocarpus agavoides shoot was limited and rooting could n o t , be evaluated for this species.
51 Coryphantha minima showed a higher capacity to produce roots in hormone-free medium compared to medium containing 1 mg I*1 EBA (Table 10) in contrast to observations by Clayton et al. (1990) for other cactus species, in which rooting was increased using an auxin source. Shoots transferred to hormone-free media continued to produce shoots (Table 10), suggesting a carry over o f growth regulators from Stage II cultures. This may have influenced shoots transferred to IBA, which continued to produce new shoots and arrested root development. To overcome carry over, shoots were transferred from Stage II to a hormone-free, 0.5X MS medium for one month prior to rooting in Stage III media. This suppressed shoot proliferation and increased rooting percentages. The effect o f IAA, NAA and IBA on rooting percentages o f Coryphantha and Obregonia (Table 11) showed no differences with the type or concentration of auxin used. Rooting quality (root number and length) was also evaluated for Coryphantha and Obregonia cultured in different concentrations o f IBA (Table 12). For both species, IBA concentrations of 3 and 5 mg I'1 resulted in high rooting percentages and good root development. Higher concentrations, up to 10 mg I'1 IBA, did not afford any advantage. Shoot size also influenced rooting of Obregonia, with shoots larger than 1 cm yielding better root induction (Fig. 7). The effect o f sucrose was examined on rooting o f Coryphantha minima in the presence of IBA (Fig. 8). When sucrose was absent or above the usual
52
T able 10.
Response of Coryphantha minima shoots subcultured for in vitro root
induction in hormone free MS medium and MS supplemented with 1 mg I'1IBA.
Treatment Murashige - Skoog, 3.0% sucrose and 1 mg I1IBA Murashige - Skoog 3.0 % sucrose
Rooting %
# new shoots per plant 4.3
Vitrification % in shoots 89
37.5
58.0
2.7
24
53
T a b le 11.
Rooting percentage o f Coryphantha minima and
Obregonia denegrii
cultured in M S basal medium with different auxin sources and concentrations.
Auxin source and-i concentration (mg 1 ) 1 IAA 3 IAA 5 IAA 1 IBA 3 IBA 5 IBA 1 NAA 3 NAA 5 NAA
Rooting Coryphantha 78.0 83.2 87.5 95.3 92.8 89.1 79.6 77.6 83.9
percentages Obregonia 43.7 58.9 57.0 76.6 79.2 64.3 48.7 47.4 57.8
54
T a ble 12.
Number of primary roots developed in vitro on Coryphantha minima, as a
result of different concentrations of IBA and sucrose.
% Sucrose / mg I* 1 IBA
Mean number of primary roots ( a) 1 0.45 0.7 0.36 0.4 0.04 4.7 1.87 2.0 1.03 1.9 0.51 2.5 1.30 1.0 0.74 3.5 1.21 1.4 0.84 0.8 0.58 0.2 0.20 1.3 1.00 1.4 0.92 4.5 1.31 2.8 0.78
0 - 0 IBA 0 - 1 IBA 0 - 3 IBA 0 - 5 EBA 1.5 - 0 IBA 1.5 - 1 IBA 1.5 - 3 IBA 1.5 - 5 IBA 3 - 0 DBA J - 1 IBA 3 - 3 IBA 3 - 5 IBA 4.5 - 0 IBA 4.5 - 1 IBA 4.5 - 3 IBA 4.5 - 5 IBA
55
0 <
U U C ta O 2 P O O C
MS A B C D
O.
MS with IBA
ORION AND SIZE OF PLANT*
* A - Somatic em bryos with no root developed B - Shoots < 0.5 cm C - Shoots 0.7 -1 .0 cm D - Shoots > 1.0 cm
Fig. 7 . Effects of the presence/absence of IBA (3 mg I'1 ) on in vitro rooting in different sizes of shoots and somatic embryos of Obregonia denegrii. Plants were maintamed in MS medium supplemented with 3.0% sucrose.
56
15
30
45
Sucrose (g l'1 )
Fig. 8. Effects of different concentrations of IBA and sucrose on root development in vitro of Coryphantha minima cultured in MS medium
concentrations (30 g I'1), rooting percentages tended to decrease. Rooting was best at sucrose levels o f 13-30 g I'1. Root number was also examined as a function o f sucrose (Table 13). Treatments that gave higher rooting percentages (Fig. 9) yielded lower numbers o f roots. Occasionally, better root systems resulted on treatments having low rooting percentages. The effect o f sugar content on rooting is consistent with others (Wainwright and Scrace, 1989; Maene and Debergh, 1985; Hyndman et al., 1982). Root induction in Coryphantha minima cultured ex vitro was highest with sand compared to the perlite/cacti mix (Table 14). A comparison shows that rooting is favored by in vitro over ex vitro conditions for both species. The high humidity of in vitro conditions may enhance rooting while the incidence of fungal invasion is diminished under ex vitro conditions. However, damping-off was observed when powdered auxin and fungicide (Rootone) were used.
Reduction o f microbial contamination. Microbial contamination was a minor but persistent problem of in vitro culture. A compilation of all in vitro cultures showed that reduced sugar concentrations lowered contamination (Table 15). When cultures were exposed a brief moment (3 min.) to ambient air, microbial contamination was suppressed in sucrose-free medium, however, plant growth was adversely affected showing a net decrease in tissue volume (-0.3 mm3
58
T a b le 13.
Ex vitro rooting o f Coryphantha minima and Obregonia denegrii with
different auxin sources on tw o different substrates.
Substrate and treatm ent Sand + distilled water Sand + 5 mg I '1IBA Sand + Rootone * Perlite/Cactus mix ** + distilled water Perlite/Cactus mix ** + 5 mg 1' IBA Perlite/Cactus mix ** + Rootone *
Rooting percentages Coryphantha Obregonia 0.7 13.6 61.6 77.5 2.4 41.8 51.5 47.3 53.7 0.0 14.6 21.3
* Rootone - Commercial rooting powder with 0.20% NAA and 4% fungicide Thiram. ** Cactus mix - Peat, perlite and coarse sand (1:1:1).
59
450 400 350 H
e X
V
300 250 200 150

100
-
.5
T 3
50-
041 2 3 4 5
Months in culture
- No over-layer Over-layer
Figure 9. Effects of an upper layer of paraffin-lanolin mixture over the growth medium on plant growth in a four-months period. Plant growth is referred to as a growth index.
60
T a ble
14. Percentages of the incidence of microbial contamination during experiments
of in vitro culture with Coryphantha minima and Obregonia denegrii.
Sucrose concentration in the culture medium 0 gf

io g r' is g r 1 20 g r 1 30 g r'
Percentage of contaminated culture vessels 5.8 %

6.6 %
37.6 % * 17.8 %
22.6 %
* In a set of experiments using 15 g I'1 sucrose, the presence o f mites was detected in the culture room, which increased contamination problem.
61
T able
15. E ffects o f sucrose concentration on microbial contamination and plant
growth over a 1-month period, in culture vessels opened tw ice for 3 minutes.
Sucrose concentration in the growth media o g r'

10 g r' 20g r 30 g r
Percentage of contaminated culture vessels

8.2 % 39.6 % 65.0 % 87.8 %
Growth indexa
- 0.3 mm3 0.8 mm3 5.8 mm3 7.1 mm3
a Growth index is the fractional increase in volume of the cactus, in a one moth period. Data are only from non contaminated culture vessels.
62 fractional change in volume in one month) (see Table 16). In contrast, good plant growth was observed when sucrose was present, as expected. When a physical barrier layer o f autoclaved paraffin-petroleum jelly was applied to the growth medium, microbial contamination was notably reduced. Different ways o f embedding plant tissues into the agar through the "wax" layer were evaluated. The easiest procedure was to carefully dispense the warm, liquefied wax onto medium that already contained plants (Table 17). This resulted in the lowest contamination and allowed culture vessels to be opened under non aseptic conditions. The use of such wax layers offers a viable option for in vitro culture under certain circumstances where the risk o f microbial contamination is a problem. The wax barrier also offers a way to reduce the relative humidity inside the culture vessel which can be a benefit to tissue culture under certain circumstances (discussed in the following chapter). Despite these advantages, wax layers appear to reduce plant growth (Fig. 9). Growth o f Obregonia denegrii in wax layers was similar to wax-free treatments during the first two months of culture. However, growth was markedly reduced after that. It may be prudent to subculture every 2 months if wax layers are used.
Acclimatization o f rooted plants. Obregonia denegrii is a species that, under natural conditions, only reproduces by sexual means. In contrast, Coryphantha minima has the ability to produce offshoots which easily give rise to roots. This
63
T a b le
16. Effects o f an upper layer o f paraffin-lanolin mixture over the grow th medium
in the incidence o f microbial contamination
in vitro.
Treatment No paraffin-lanolin layer Paraffin mixture dispensed with plants
Contamination % in non opened vessels 7.9 0
Contamination % in opened vessels. * 87.2 3.2 26.4 5.1
Paraffin mixture dispensed without plants: Sowing outside the laminar flow unit Sowing inside the laminar flow unit
28.6 0
Vessels were opened every three days for 3 hours in the culture room in a 2-months period.
64
T a ble
17. Re-establishment percentages of Coryphantha minima after 4 weeks of
acclimatization, in relation to previous rooting treatments.
Rooting treatment_______________ Re-establishment percentage In vitro treatments Hormone-free rooting. 92.1 Hormone-free rooting with a paraffin98.9 lanolin superficial layer. 85.2 Induced rooting with IBA. Induced rooting with IBA and a paraffinlanolin superficial layer. 93.8 Ex vitro treatments Sand and DBA solutions. Sand and Rootone . Perlite-cactus pot mix and IBA solutions. Perlite -cactus pot mix and Rootone . 18.6 96.0 34.5 81.4
65 vegetative reproductive capacity mimics its ability to become acclimated and establish itself outside, following in vitro culture (compare Tables 18 and 19). Re establishment and survival for Coryphantha minima is almost 6 fold greater than for Obregonia denegrii. Acclimatization o f Coryphantha was high for all plants rooted in vitro (Table 19). However, for Obregonia, plants were better adapted to ex vitro conditions if in vitro rooting occurred in the presence o f IBA cultured in wax layers. Acclimatization o f Obregonia denegrii was far better with ex vitro than with in vitro rooting, especially when plants were rooted in sand or perlite using Rootone as opposed to IBA in solution (Table 18). Spontaneous rooting o f Coryphantha in hormone-free media resulted in plants with the same survival potential as plants rooted with IBA. Thus, rooting in hormone-free media appears to be more prudent. Acclimatization o f Coryphantha was affected by branching habit o f the plantlet (Table 19). Plants with single stems, rooted in vitro in hormone-free media, were less suitable than plants with multiple stems or ramets. However, rooting was no different for single or multi stemmed plants rooted in vitro with IBA or LAA. Acclimatization in relation to sucrose and salt content and relative humidity during in vitro culture was evaluated (Table 20). Coryphantha plants grown in the absence o f sugar did not survive at all. Plants grown in the presence o f 30 g I'1 sucrose were best. The effects of MS salt mixtures and relative humidity, appeared to have little effect on acclimatization.
66
T a ble
18. Re-establishment percentages o f Obregonia denegrii after 4 weeks of
acclimatization, in relation to previous rooting treatments.
_________ Rooting treatment_____________ Re-establishment percentage In vitro treatments Sp ontaneous rooting. 1.7 Spontaneous rooting with a paraffinlanolin superficial layer. Induced rooting with EBA. Induced rooting with DBA and a paraffinlanolin superficial layer. Ex vitro treatments Sand and IBA solutions. Sand and rooting powder. Perlite-cactus pot mix and IBA solutions. 0 2.3 5.5 0 15.8 0
Perlite-cactus pot mix and rooting powder.___________________________________________ 10.2__________
67
T a b le 19. Survival o f Coryphantha minima 10 months after acclimatization.
Plant Category I - Rooted in vitro with IBA and IAA Single, non-branched plants Branched plants (cespitose) Spontaneous in vitro rooting Single, non branched plants Branched plants (cespitose) II - Rooted ex vitro (Single and branched plants) Rooted in perlite/cactus pot mix Rooted in sand
Survival Percentage
89 % 73 %
57 % 92 %
10 % 90 %
68
T a ble
20.
Re-establishment of Coryphantha minima in relation to their in vitro cultural
origin.
In vitro cultural origin Sucrose Concentration (e I'1): 0 15 30 40 Nutrients Streneth: 1/2 X Murashige-Skoog medium IX Murashige-Skoog medium Relative Humiditv: Normal culture vessel Culture vessel with vented closures Growth medium with paraffin over-layer
Re-establishment percentage 0.0 68.9 92.7 41.5 85.3 87.7 77.7 77.9 89.1
69 Water loss during acclimatization was demonstrated for Coryphantha minima (Fig. 10). Between 24-33% water is lost from these succulent species over an 8 day period. In comparison, several non-succulent species experience more than 4S% water loss in a period o f few hours (Brained and Fuchigami, 1981; Short, et al., 1987; Smith, et al., 1990). It is clear that succulence and high mucilage content in cacti is a beneficial feature for acclimatization. The extreme water losses observed in non-succulent plants cultured in vitro occurs in the first few hours after transfer. In cacti, these same water losses require several days. The small difference between in vitro rooted versus non-rooted cacti (Fig. 10) reveals that roots developed in vitro are functional during the first few days in the potting mixture. Rooting treatments have an effect on water loss during acclimatization (Table 21). The temporary passage o f plants into a growth medium covered with a wax layer benefits acclimatization. These plants exhibit reduced water losses when transplanted outside the culture vessel. Plants rooted in ex vitro conditions were exposed to relatively high humidity to promote rooting. Thus, ambient humidity was higher in these plants than for plants grown under wax layers in vitro. This is why plants rooted in ex vitro conditions showed higher water losses. Tissue culture o f Obregonia denegrii resulted in the massive production of somatic embryos and the production of some shoots. Acclimatization of these two propagules were evaluated and no apparent differences in survival were evident
70
35 30
25
20
'Z
b a 5
15
10
0 0
5
8
Days
' Rooted In vitro O Rooted ex vitro/Acdimatiiatioii
Figure 10. Rates of water loss (based upon fresh weights) after one week in acclimatization stage, from Coryphantha minima plants rooted in vitro and plants in an ex vitro rooting treatment simultaneous to acclimatizaton.
* Water loss was indirectly calculated from fresh weights, measured always one day after irrigation. Plants were irrigated every other day
71
T able
21. Water loss after 20 days at the acclimatization stage o f Coryphantha minima
in relation to the previous rooting treatments.
Rooting treatments Spontaneous in vitro rooting Spontaneous in vitro rooting with a paraffin-lanolin superficial layer Induced in vitro rooting Induced in vitro rooting plus a paraffinlanolin superficial layer Ex vitro rooting in sand and rooting powder
W ater loss percent * 42.3 8.4 57.1 8.7 19.7
* Water loss was indirectly calculated in at least 5 replicates per treatment. Initial and final fresh weights were recorded one day after irrigation with 10 ml water.
72
T able 22. Survival of Obregonia denegrii 4 months after acclimatization. Plant Origin Seedlings 6 months old derived from somatic embryogenesis. In vitro spontaneously rooted plants, 8 months old Ex vitro rooted plants, 5 months old Single plants Multi-branched plants Survival Percentage 93 %
98 %
45 % 75 %
73 (Table 22) . Seedlings derived from somatic embryos exhibited the same survival as spontaneously rooted new shoots. However, new shoots rooted in ex vitro conditions showed reduced survival rates, specially for single-stem plants. As in the case o f Coryphantha minima, in vitro- produced plantlets with multiple branches or ramets had better re-establishment and survival rates than did singlestem plants.
2.
PHYSIOLOGY OF ENHANCED GROWTH
Growth analysis. Enhanced growth o f in vitro cultured cacti was only observed during Stage II shoot proliferation. During germination and seedling development o f Stage I, no obvious differences between seedlings grown in vitro and ex vitro were observed in growth measured by volume or by fresh weight increases (Figs. 11 and 12). However, during Stage II in vitro shoot growth was seven-fold greater than ex vitro growth o f Coryphantha minima (Fig. 11). A size comparison between in vitro cultured shoots, seedlings o f different ages and offshoots produced on large mother plant in greenhouse conditions reveals that 3month-old in vitro shoots were larger than one-year-old, ex vitro cultured seedlings and seedlings of the same age. It was noted that 15-month-old offshoots on large mother plants were comparable in size to Stage II, in vitro derived shoots (Fig. 13). Apparently, mother plants furnish hormones or other nutrients that stimulate vegetative growth. This may be similar to the effect of synthetic growth regulators in Stage II culture media. A comparison o f volume and fresh weight increase in shoots cultured in hormone-free media versus media supplemented with a cytokinin and auxin (Table 23) shows that the hormone-supplemented media results in greater growth o f Coryphantha. Enhanced growth could also be related to higher relative humidity in the culture vessel. Reduced relative humidity was achieved by using ventilated caps.
75
1.6
1.4
1.2
i s V
0.6
0.4
0.2
12
15
Months In vitro new shoot o In vitro seedling Ex vitro seedling
Fig. 11. Growth comparison of new shoots produced in vitro and seedlings cultured in vitro and ex vitro in Coryphantha minima. Growth is evaluated as increase in volume, calculated with geometrical formulae.
76
0.7
" o 6 w i5 5
2
<u
c /a
-0.5 - 0.4 0 .3
s 4
3 2

>
0.2
0.1
2 u 0 ) 2 3 O
1 0 1
2
Time (months)
A - - Tubercles in vitro - - X- - Tubercles ex vitro -A Volume in vitro K Volume ex vitro
Fig. 12. Development of new tubercles and increase in volume during seedling growth of Obregonia denegrii cultured in vitro and ex vitro.
C Plant characteristics
A 12-months-old seedling, grown ex vitro. B - 3-months-old new shoot produced in vitro. C 12-months-old new shoot produced and maintained in vitro, subcultured every 3 months on hormone free, MS basal medium supplemented with 15g I'1 sucrose. D 15-months-oId approximately offshoot developed ex vitro, still attached to the mother plant E - 5-yr.-old mature plant, maintained in greenhouse.
Fig. 13. Size comparison between different ages and culture conditions in Coryphantha minima, 5-yr.-old
Note that bars O and E represent reference data of the natural sizes developed in mature plants maintained in greenhouse.
78
Table
23. Comparison of volume and fresh weight increases in a 4 months period in
Coryphantha minima cultured in vitro in Murashige-Skoog media hormone-free and supplemented with 10 mg I'1KIN and 1 mg I'1 IAA. Values are media standard error. No covariant effects of initial volume and fresh weight were detected (P(f = 0.975/1,20) = 0.335).
Volume (cm3) MS supplemented with KIN / IAA 1.560 0.335 0.240 0.106
Fresh Weight (g) 1.63 0.76 0.251 0.098
MS hormone-free
79 This caused a significant reduction in plant volume compared to plants cultured in Magenta vessels capped with normal closures (Fig. 14). Similar observations were reported by Sallanon and Maziere (1992) for rose plants. When relative humidity was reduced, leaf area, shoot length and shoot multiplication rates were decreased, suggesting that water stress was responsible. In contrast, GenoudGourichon (1993) demonstrated that growth o f potato plantlets was greater in ventilated vessels. Nevertheless, this study compared growth in hermetically sealed vessels where air exchange and CO 2 fixation was totally suppressed. Photo synthetic rates were 1.7 times greater in plants in ventilated closures and this may have been the reason for the higher growth rates. However, in Coryphantha higher growth rates could also be attributed to greater water availability. The influence o f light and ventilation also proved to have an effect on in vitro growth o f Coryphantha (Table 24). Increasing light intensity stimulated volume and fresh weight gains, as did non-ventilated closures. Growth enhancement by high light intensity is related to increased photosynthetic activity. Net photosynthetic rates o f Cymbidium orchid plantlets (a CAM plant) increased when irradiation was raised from 35 to 102 and 206 pmol m'2 s"1 (Pospxsilova et al.,1992) Similarly, high light intensity in combination with high CO 2 levels, enhanced in vitro shoot elongation and leaf development of cacao shoots (Figueira et al., 1991). Thus, high light intensities appears to influence in vitro growth
80
2.5 -
s Q
Low relative humidity (vented vessels)
High relative humidity (common vessels)
Figure 14. Comparative average sizes in volume of shoots produced in MS basal medium supplemented with 0.05 mg I'1NAA/1 mg I'1BA and 2 mg I'1 IAA/4 mg F1KIN. P ( f = 4.98/1,21) = 0.037
81
T able 24. Fractional increase in volume and fresh weight in a 45 days period in Coryphantha minima cultured under different levels of light and ventilation in the culture vessel.
Environmental variable Light 120 - 300 pmol m'2 s'1 390 - 400 pmol m'2 s'1 Ventilation Culture vessels with normal lids Culture vessels with vented lids Source of variation Light Ventilation
Increase in volume (cm3) Increase in fresh weight (g) 0.192 0.394 0.22 0.45
0.401 0.182 Fresh weight P(f=6.58/l,80)=0.012* P(f=l 1.94/1,80)=0.001*
0.49 0.20 Volume P(f=2.17/l,80)=0.144 P(f=3.78/1,80)=0.055
P(f = 0.69/1,80)=0.408 Sugar in the medium P(f=1.03/1,80)=0.312 2 ways interactions: P(f=3.28/1,80)=0.074 P(f=0.029/1,80)=0.866 Sucrose-Ventilation Light-Ventilation P(fr=2.12/1,80)=0.144 0 P(f=5.52/l,80)=0.021* Covariates (Initial fresh weight): P(f=22.67/1,80)=0.000
*
82 despite the commonly held view that photosynthesis is not supposed to affect the growth o f so called "heterotrophic" plants. Enhanced growth of Coryphantha in vitro also appears to be influenced by photosynthetic activity, as it will be discussed in next chapter. The effect o f nutrient concentration on growth was examined (Table 25). Raising or lowering the MS salt concentration by 50% generally caused a reduction in growth. Growth reduction was more severe with 1.5X MS that with 0.5X MS. Little is known concerning the effect o f inorganic salts on cacti. Nobel (1988) suggested that cacti are tolerant to elevated levels o f certain heavy metals. Raising Cu and Zn levels 1000 times in hydroponic Hoagland's solution inhibited root and shoot growth by only 17% for two cacti species. Increasing boron 150 times in hydroponic Hoagland's solution, inhibited cactus growth by 30-50%. Growth inhibition by other elements has not been reported. Nutrient salt levels have important consequences for carbon assimilation by cacti. Nocturnal acid accumulation was greater in Carnegiea gigantea, Ferocactus acanthodes (Lemaire) B.& R. and Trichocereus chilensis when tissue nitrogen levels were higher (Nobel, 1983). For these species growth was greatest in soils containing higher levels of N, Mg and Fe. Maximum growth o f two hydroponically grown cacti, Ferocactus acanthodes and Opuntia ficus-indica (Linn.)Miller occurred at or near full-strength nitrate levels in Hoagland's solution (Nobel, 1988). Increasing the P, K and N levels from 0.01 to 1 times resulted in
83
T a ble 25.
Effects of Murashige-Skoog nutrients concentration on volume and fresh
weight of Coryphantha minima cultured during three months in vitro*
Nutrient strength 0.5 X MS solution 1.0 X MS solution 1.5 X MS solution
Increase in volume (cm3) 0.190 0.076 0.219 0.057 0.137 0.045
Increase in fresh weight (g) 0.09 0.008 0.15 0.04 0.07 0.02
*P (f = 3.29/2,79) = 0.042
84 20%, 61% and 100% gains in shoot dry weight, respectively. Likewise, seedling growth o f another CAM plant, Agave deserti, was enhanced when grown in the elevated N levels o f hydroponics (Nobel and Hartsock, 1986). Thus, growth of most CAM plants is increased by nitrogen fertilization (Nobel, 1988). The MS salts used in micropropagation media contain high levels o f nutrients and should promote the high growth rates observed for cacti cultured in vitro. Altering the sucrose concentration also affects growth in vitro (Table 26). Growth was greatest at 30 g I*1 (3.0%) sucrose, the standard concentration. Raising or lowering the sucrose concentration generally caused a reduction in growth. Plants did not survive in the absence o f sucrose. Growth was severely reduced at 0.5% sucrose, and slightly reduced at 1.5% and 4.5% sucrose. The absence o f sucrose from the medium also killed rose shoots cultured in vitro (Langford and Wainwright, 1988). In tobacco, reductions o f sucrose in the media cause a pronounced reduction o f root, stem and leaf dry mass (Pospisilova et al., 1992). In contrast, elevated sugar concentrations o f 4.5% probably reduce growth by increasing osmotic pressure in the agar medium (Pospisilova et al., 1992). For Coryphantha, portions o f explants under high sucrose concentrations turned brown and stopped growing. Pospisilova et al. (1992) indicated that elevated sucrose reduced net photosynthetic rate in Rose and Digitalis grown in vitro. Obregonia denegrii did not exhibit the enhanced growth, in terms of biomass that Coryphantha did. This was because Obregonia produced somatic embryos
85
T able 26. Effects of different sucrose concentrations supplemented to the growth medium in volume and fresh weight of Coryphantha minima, after three months in culture.
Sucrose concentration % 0 0.5 1.5 3.0 4.5
Increase in volume (cm3) + + 0.043 0.248 0.262 0.208
Increase in fresh weight (g) + + 0.002 0.098 0.187 0.06
**Plants did not survived under this treatment
86 rather than shoots, and plantlets derived from embryos that tend to be much smaller than even ex v/7ro-cultured seedlings. If growth is measured in terms o f new propagules, then somatic embryos are produced at about the same number o f new shoots cultured in vitro (Table 27). However, biomass increases were much greater for Coryphantha. With Obregonia somatic embryos, no significant differences were detected between sucrose or MS concentration treatments (Table 28). The initial number o f tubercles produced by both explants was detected as a covariant (P(f=4.71/1,45)=0.032) for subsequent production of new tubercles. The enhanced growth response disappeared after rooting occurred. Decreases in fresh weight and volume in Coryphantha and Obregonia coincided with root development after plants were transferred to Stage III media. Root development percentage and fresh weight was highest at sucrose concentrations of 3.0%, however, increase in plant volume was less (Table 28). Plants rooted in vitro versus ex vitro showed no statistical significant differences in rooting percentage, however, subsequent growth after 5 months o f acclimatization was lower for Coryphantha minima plants derived from in vitro rooting treatments (Fig. 15). Plants destined to ex vitro rooting were transferred out o f their culture vessels approximately one month before plants rooted in vitro were transferred for their acclimatization treatments. Thus, data in Fig. 15 suggests that stress may affect plants after they are removed from culture vessels.
87
T able 27. Fractional growth in a three months period of Obregonia denegrii plantlets derived from somatic embiyogenesis and from shoot multiplication
Plantlet origin Somatic embryos Shoot proliferation
Increase in number of tubercles* 0.88 0.039 0.05 0.007
Increase in fresh weight (g) 0.849 0.056 - 0.062 0.034
* Initial number of tubercles was detected as a covariant: P(f = 4.71/1,45) = 0.032
88
T able 28. Effects of different sucrose concentrations on plant growth and root development in Coryphantha minima new shoots maintained at Stage EH of micropropagation for three months.
Sucrose concentration 1.5 g l'1 3.0 gT1

4 .5
Increase in volume (cm3) 0.248 0.163 0.111 0.146 0.253 0.166
Increase in fresh weight (g) 0.09 0.006 0.17 0.110 0.05 0.012
Root development percentage 50.00% 72.16% 61.11%
g r1
89
1.2 -|
1-
3 08
1
|
^5
'
0.6 -
V I V 0.4 -
In vitro Previous rooting procedure
Ex vitro
Fig. 15. Effects of previous rooting treatments on fresh weight increase of Coryphantha minima after five months of acclimatization P ( f = 8.75/1,12)= 0.012
90 In sum, growth analysis indicate that certain conditions, such as relative humidity in the culture vessel, sucrose and nitrogen levels have a more profound effect on in vitro growth than other factors.
Photosynthetic activity. Observations o f Coryphantha minima in tissue culture suggested that photosynthetic activity might play a role on that enhanced growth response. To examine this, photo synthetic rates were compared in Stage II cultures, in acclimatized plants and in wild collected plants maintained in the Desert Botanical Garden, Phoenix, Arizona. Photosynthesis was measured by CO2 gas exchange or malic acid fluctuations. Daily malic acid fluctuation reflects photo synthetic activity in CAlM plants. Titratable acidity o f Coryphantha (Fig. 16) shows that the night-time peak is greatest for in vitro versus ex vitro plants. This indicates that nocturnal carbon fixation was actually higher during in vitro conditions. This was an unexpected result. However, the same result was obtained when photosynthesis was measured in terms o f gas exchange (Fig. 17). Here in vitro cultured plants also demonstrated greater CO2 uptake rates (1.20 pmol m '2 sl ) than did ex vitro plants (0.79 pmol m*2 s*1) in the dark. However, an even more surprising result was that plants cultured in vitro showed a net C 0 2 uptake during the light period compared to ex vitro plants which showed a respiratory loss o f C 0 2. This was unexpected, because it showed that this species o f cacti acted as an autotroph in both the light and dark period during in vitro
91
100 i
9S.6 90 80 70 * 60 50 403020-
if
i' '3
n a
10-
Light
Dark 1
Dark 2
Dark 3
Light
- -O - - in vitro 0 wild - - x- - - acclimatized
Figure 16. Comparison of malic acid daily fluctuations in Coryphantha minima plants cultured in vitro, plants acclimatized for 2 months and wild plants maintained under greenhouse conditions.
92
1.4 1.2 I
m vttro
o U
0.8 0.6
I
E
Light period
I wild
wild
v_ 0.4
5.
0.2
0
-
Dark period
0.2
-0.4
-
ex vitro
0.6
Figure 17. Diurnal and nocturnal CO2 exchange in Coryphantha minima cultured in vitro compared to micropropagated plants maintained ex vitro for six months, and to wild collected plants maintained in greenhouse.
93 culture. In contrast, ex vitro cultured cacti exhibited the normal CAM pattern during the light and dark periods. It is possible that the increased photosynthetic uptake in vitro could account for the enhance growth response. Upon transfer to ex vitro conditions, plants revert to normal CAM metabolism as shown by acid fluctuations in plants after 2 and 12 months of acclimatization compared to mature plants under greenhouse conditions (Fig. 18). The nocturnal acid peak drops from 95.6 peq g '1 FW for in vitro conditions, to 46.3 peq g '1 FW in 2-month acclimatized plants. Nocturnal acid is 71.2 peq g 1 FW in 12-month acclimatized plants and 57.7 peq g 1 FW in wild plants maintained in greenhouse. The influence o f culture type (i.e., Stage II vs. Stage III vs. callus culture) on acid fluctuation shows that Coryphantha callus cultures have altered CAM physiology and no nocturnal peaks are observed (Fig. 19). This is similar to previous work reporting that diurnal fluctuations o f titratable acidity were absent in Chamaecereus sylvestrii cell suspension cultures (Seeni and Gnanam, 1980). It was proposed that the alteration in cultured cells was due to the formation of small vacuoles that did not store as much organic acid at night. The absence of nocturnal acid accumulation in callus suggests that callus is truly heterotrophic and does not possess any CAM physiology. It also suggest that the absence of well developed stomata on callus may be related to such a lack o f CO 2 uptake cycle. After shoots form, peaks in nocturnal acidity become evident, indicating
94
so
70 60 71.02
u cs ti 40 z OS cs u 30 H
20
10
50 -
0
Light Dark 1 Dark 2 Dark 3 Light - - wild plants
- 2 months acclimatization
12 months acclimatization - -
Figure 18. Malic acid daily fluctuations in Coryphantha minima plants derived from in vitro culture, 2 and 12 months after acclimatization, compared to wild plants maintained in greenhouse.
95
100
90 80 70 60 50 40 30
20
10
95.6
0
Light Dark 1 o Stage II Dark 2 Stage III Dark 3 Callus Light
Figure 19. Comparison of malic acid daily fluctuations in Coryphantha minima plants cultured in vitro at shoot proliferation (Stage II), rooting-elongation (Stage m ) and callus derived from Stage II.
96 the establishment o f CAM physiology. In other cacti species it has been shown that seedlings undergo a conversion from C-3 to CAM as seedlings germinate and shoots develop (Altesor et al., 1992). A similar process may occur in tissue culture as shoots arise in vitro. The most surprising finding is that photo synthetic C 0 2 uptake of Coryphantha shoots is greater as sucrose concentrations increase from 1.5 to 3. 0% (Fig. 20). Although the nocturnal peak is still observed in 3 % sucrose, it is noteworthy that C 0 2 uptake is greater in both light and dark periods in 3% versus 1.5% sucrose. Thus, sucrose appears to stimulate autotrophic growth in vitro. It is generally assumed that plants cultured in vitro cannot achieve full photosynthesis because of sub-optimal light and C 0 2 concentrations in the culture vessel (Capellades et al, 1990, Kozai, 1991 a, 1991 c). This is why sucrose is furnished, as a supplemental carbon source, in the growth medium. The addition of sucrose is also assumed to favor heterotrophic over autotrophic growth. Observations of two C-3 plants, Rosa and Pieris, support this, where photo synthetic rates are decreased as sugar concentration increased above 1% (Capellades et al, 1990). This is direct contrast to the observations made here with Coryphantha shoots. With CAM species, such as Coryphantha, C 0 2 fixation appears to be enhanced in vitro and autotrophic abilities appear to be stimulated with elevated sucrose concentrations. One possible explanation is that C 0 2 uptake in Coryphantha increases because stomatal opening is greater in 3 % than 1.5%
97
o
^
'in rt c
0.8 -
> 3%
0.4 0.2
1.SOH
-
Light 3% 1.50% 3%
Dark 1.50%
Sucrose concentration in the growth medium
Figure 20. Diurnal and nocturnal gas exchange of Coryphantha minima cultured in vitro under different sugar concentrations supplemented to hormone - free growth medium.
98 sucrose treatments. Because o f stomatal opening, plants absorb more CO 2 . This is supported by transpiration measurements showing that transpiration is higher in 3% than 1.5% sucrose (Fig. 21). CAM plants appear to offer some plasticity with changes in water availability (Griffiths. 1988). Although the enhanced growth response was not as pronounced in Obregonia denegrii, photosynthetic activity was also evaluated in this species in vitro. Daily malic acid fluctuations clearly show a nocturnal acid accumulation in somatic embryos and new shoots, and nocturnal peaks being greater for in vitro tissues than ex vitro seedlings (Fig. 22). During the light period, there is a notable increase in titratable acidity in somatic embryos compared to shoots or seedlings. During the acclimatization and conversion o f Obregonia somatic embryos to plantlets, acid levels remain elevated compared to ex vitro seedlings (Fig. 23). These in vitro derived plants were also much larger, after three months, than were 1-year old ex vitro seedlings. Together these data suggest that in vitro conditions, especially during Stage II culture, enhance the ability of Obregonia somatic embryos to fix CO 2 in comparison to plants grown in ex vitro conditions. This is analogous to that observed for Coryphantha Stage II cultures and suggests that the enhance growth response of CAM species may be linked to the improved photosynthesis that is exhibited by these species when they are cultured in vitro. What is particularly surprising, is that elevated CO 2 fixation is observed in both dark and light periods
99
12
-i
10
O
X
f/i
c e
In vitro/ Stagell
In vitro/Ioiv sugar
Acclimatized plants
Figure 21. Comparison of transpiration rates of in produced vitro cultured plants of Coryphantha minima and wild plants maintained in greenhouse. Data of in vitro plants are from Stage II, shoot elongation under low sugar concentration (1.5%) and one month after acclimatization outside the culture vessels.
100
160 140 E L 120 so
154.57
108.5
S T ioo
a.
2* 80 ( O 60
1 3 40
20
A* -
_A
0
Light Dark 1 Dark 2 shoots Dark 3 -Light
embryos
-A - -seedlings
Figure 22. Malic acid daily fluctuations in Obregonia denegrii somatic embryos and new shoots produced in vitro, compared to one-year-old seedlings, grown in vivo.
101
90 -,
_ 80 X 81.94
5 - 70 'sO
91
c 60
. ./9.9<J
t 50
1
CB
40-
O
|
e B
20
-
X*
10-
Light
Dark 1
Dark 2
Dark 3
Light
-X-- -Acclimatizednew shoots
Ex vitro seedlings
Figure 23. Comparison of malic acid daily fluctuations of one-year-old seedlings grown ex vitro and plants in vitro after 3 months acclimatization in Obregonia denegrii.
102 o f these CAM species when they are cultured in vitro. Under normal circumstances, one would expect that CAM species show a net uptake o f C 0 2 in the dark and a net release o f C 02, due to respiration, in the light. However, when cultured in vitro, CAM plants show a net uptake o f C 02 in the dark and light. This is unique and may explain the phenomenon o f enhanced growth. It may also explain why enhanced growth is not observed in C-3 species. These plants appear to maintain their normal diurnal cycle in vitro, where the net uptake o f C 02 occurs in the light and the net release o f C 02, due to respiration, occurs in the dark. Thus, in vitro conditions do not alter the ability of C-3 plants to have a net uptake o f C 02 in both the light and dark conditions as CAM species now appear to do. In hindsight, it is now apparent that, photoperiod might have a profound effect on the enhanced growth phenomenon. If, as we now suspect, enhanced growth occurs because of increases in C 02 fixation, then growth might be further enhanced by placing these plants in an extended dark period. At first glance, this seems preposterous, to put plants in the dark to make them grow more. However this data suggest that as long as sucrose, mineral salts and hormones are provided, and by extending the dark period, it may be possible to obtain even greater amounts of growth o f CAM plants in vitro. It is not known what the optimum day/night cycle would be. However, photoperiod is known to be crucial for
103 growth o f cacti. Nobel (1989) showed that, under greenhouse conditions, a photoperiod increase from 6 to 8 hours, increased growth o f Agave deserti by 33 %, o f Ferocactus acanthodes by 81 % and o f Opuntia ficus-indica by 51%. Similar observations were made for Opuntia which produced more dry weight under long photoperiods (Sanderson et al., 1986). The length o f the light/dark cycle used in the current project were 16/8 h. It is not known how an adjustment in this cycle would affect in vitro growth o f cacti, but it may have a pronounced affect. The response might be the opposite that would be expected for ex vitro growth. For endangered CAM plants, in vitro culture appears to offer an optional and advantageous means o f preserving or rescuing certain species. Propagation of threatened CAM species by conventional procedures is not easy and growth is, naturally, very slow. However, when CAM plants are cultured in vitro, they exhibit the unique ability to grow and photo synthesize at extremely accelerated rates. This enhanced growth ability was not widely recognized, and the improved photosynthesis was not discovered until now. Because o f these features, micropropagation o f endangered CAM plants is rapid and results in increased biomass production o f these species. In situations were these species are imperiled, micropropagation offers a viable alternative despite the elevated costs or the genetic limitations afforded by cloned plants.
3. MORPHOLOGICAL AND PHYSIOLOGICAL CHANGES ASSOCIATED WITH TRANSFER OF IN VITRO TO E X VITRO CONDITIONS
Changes in acid accumulation. Diurnal acid fluctuations indicate that plants transferred from culture vessels to the acclimatization stage experience a decrease in daily CO 2 fixation, especially during the dark period. In Coryphantha, acid levels drop from 95.6 peq H* g '1 FW for in vitro plants to 46.3 peq H* g 1 fresh weight after 2 months of acclimatization (Fig. 16). After six months of acclimatization Coryphantha plants still have lower rates than those observed in vitro. Obregonia exhibits a similar fate. Titratable acidity o f plantlets transferred to acclimatization conditions show a decrease in nocturnal acid levels, from 108.5 peq H* g '1 fresh weight in new shoots, to 81. peq H* g '1 FW in plantlets after 3 months o f acclimatization (Figs. 22 and 23). Unlike C-3 species, photosynthesis in Coryphantha and Obregonia actually decreases during acclimatization. Most non-CAM species show an increase in photosynthesis as they adjust to ex vitro conditions, when plants switch from heterotrophic to autotrophic growth (Donelly and Vidaver, 1984; Capellades et al., 1990; Kozai, 1991b; DeYue et al., 1992). For example, Pospisilova et al., (1992) reported that net photosynthesis increased from 5.34 mmol CO2 m * 2s 6.48 mmol CO 2 m 2 s *' on in vitro tobacco plants acclimated after three weeks. CAM plants do not appear to make the switch very quickly. It is possible that the resumption o f photosynthesis is due to the withdrawal of sucrose, which appears to
105 to stimulate photosynthesis or to the removal o f N. It has been shown that N levels affect C 0 2 fixation in agaves and cacti, measured as a decrease in nocturnal acid accumulation (Nobel, 1988). Nocturnal acid levels are low in Agave utahensis which contains 0.9% N by DW and are maximal in tissues containing 1.5% N (Nobel, 1988). Plants transferred out o f tissue culture experience a sudden decrease in nutrients, which may reduce photo synthetic activity. This has been observed with several C-3 species (Bhojwani and Dbawan, 1988). Despite these problems CAM plants eventually improve and reach acid accumulation values that approach mature, wild plants, however, recovery is slow. Coryphantha requires up to a year to reach acid accumulation levels comparable to wild plants (Fig. 18). Similar delays are observed with Obregonia (Fig. 23). Differences in C 0 2 fixation, measured as accumulated acids, may also be associated with plant size. Larger plants may accumulate more acid than small plants. Altesor et al. (1992) found that the surface-to-volume ratio of Opuntia pilifera, Neobuxbamia tetetzo (Coulter)Backeberg and Ferocactus recurvus seedlings, affected malate accumulation. Because in vitro derived plants have higher surface-to-volume ratios than ex vitro plants, acid accumulation levels should be higher in vitro.
Changes in transpiration and water loss. Maximum water loss was observed for Coryphantha plants cultured in Stage II conditions (Fig. 21).
106 However, one month after plants were acclimated, water loss dropped (Fig. 21). It is believed that in vitro cultured plants possess abnormally functioning stomata that constantly stay open, in contrast to greenhouse grown plants (Sallanon and Maziere, 1992; Diettrich et al., 1992). Such plants cannot regulate water loss unless they are adapted to lower relative humidity. This has been achieved in Dianthus and geranium tissue cultures where humidity was decreased using lids with higher moisture vapor transmission rates. These reduced stomatal apertures and transpiration rates (Cassels and Walsh, 1994; De Yue et al., 1993). Observations with Coryphantha showed that day and night time CO 2 uptake was higher under in vitro conditions, indicating greater and continuous stomatal opening than under ex vitro conditions. Water loss monitored during acclimatization was most rapid during the first 5 days after transfer (Fig. 24). Plants rooted under ex vitro conditions showed less water loss than plants rooted in vitro with vented or normal lids (Fig. 24). However, the amount o f water lost after 20 days did not affect plant survival (Table 29). Plants cultured in vented closures appeared to adapt better to ex vitro conditions, having slightly better stomatal control. However, this did not offer any distinct survival advantage in the long run. The decrease in carbon fixation activity observed during acclimatization in Coryphantha minima plants (see Fig. 16) might suggest certain stomatal closure as an immediate response to the variation associated with the transference to a
107
0.95 0.9 0.85

!S> 0.8
JZ 0.75 V I
ep 0.7 5
<
g 0.65
0.6
0.55 0.5 day 1 day 5 day 10 day 20
Days after acclimatization Ex vitro - - * - - Stage HI o Stage HI vented
Fig. 24. Changes in fresh weight during acclimatization in Coryphantha minima derived from: In vitro rooting treatments using Magenta boxes with normal lids (Stage m ); in vitro rooting treatments using Magenta boxes vented lids (Stage m , vented), and plants in trays covered with plastic to keep moisture for ex vitro rooting.
108
T able
29. Percentages of water loss 1 and 20 days after * acclimatization in
Coryphantha minima grown under different rooting treatments. Survival percentages data correspond to day 20.
WATER LOSS Plant origin Stage HI (typical)a Day 1 25.6 87.42% Stage HI (vented)b 18.06 90.06% Ex vitro rootingc 11.70 89.23% a - In vitro rooting treatments using Magenta boxes with normal lids. b - In vitro rooting treatments using Magenta boxes with vented lids. c - Plants in trays covered with plastic to retain moisture. * Calculations were on the basis of initial fresh weight plants had when they were transplanted to acclimatization treatments. Data are from at least 10 replicates randomly selected, on each plant origin. 26.54 29.17 Day 20 34.79 Survival
109 less humid environment. This is indirectly validated by the low transpiration rates detected during the first month o f acclimatization (see Fig. 21). Although stomatal control may be important, water losses in Coryphantha during the first days o f acclimatization still were considerable, suggesting that tissue cultured plants had poor epicuticular wax development. Wax analysis o f plants in each stage o f tissue culture confirmed this (Fig. 25). Moreover, epicuticular wax content in plants transferred to ambient conditions improved 5-fold after 3 months achieving wax levels very close to those o f mature plants in greenhouse (Fig. 25). The effect o f in vitro culture on epicuticular wax formation is well documented. Low light intensity and low photosynthetic rates are thought to impair wax synthesis (Kerstiens, 1994). However, in vitro cultured cacti presented low wax content despite their improved photosynthesis. Therefore, other factors than reduced photosynthetic activity in vitro might be related to this decrease in wax synthesis. In addition, no correlation has been found between survival rates and wax content o f in vitro derived plants (Sutter, 1988; Kerstiens, 1994; Dietrich et al., 1992). Wax synthesis in cacti is also related to plant age. In Opuntia engelmanii wax content varies from 70 pg cm'2 in buds to 136 pg cm'2 in secondary cladodes and 250 pg cm'2 in older cladodes in plants under natural conditions (Wilkinson and Mayeux, 1990). As it was explained above, the grater water loss occurred during the first days o f acclimatization and meanwhile, it tended to stabilize (see Fig. 24).
110
0.07 -i
0.06 -
0.05 -
! 0.04-
0.03 -
0.02
0.01
AC 1M
AC3M
AC6M
EX 1YR
WILD
Fig. 25. Amount of epicuticular waxes in Coryphantha minima from different culture conditions: Stage II - in vitro shoot proliferation; Stage with IB.-in vitro rooting; Stage m V - in vitro rooting with vented closures; Ac lm - one month after acclimatization; Ac 3m - three months after acclimatization; Ac 6 M - six months after acclimatization; Ex 1 yr. - one year after acclimatization; Wild - wild specimens maintained in greenhouse.
Ill However, the recuperation o f normal wax levels was apparent until the 3rd. month o f acclimatization. Therefore, in spite o f the effects o f in vitro culture on wax synthesis, no direct relation between low wax and ex vitro desiccation has been found. This could be in accordance to Santamaria and Kerstiens (1994), whom indicated that the main factor affecting water loss during acclimatization appears to be abnormal closure o f stomata. In contrast, carbon dioxide fixation and transpiration rates show a possible stomatal closure as it was also explained above. Regardless these contradictions, it is evident that abnormal stomatal behavior is associated to in vitro derived plants during their acclimatization stage. The survival of acclimatized Coryphantha plantlets was not affected by excessive water loss, demonstrating that body succulence allowed cacti to survive and recover after a considerable degree o f desiccation. The morphological and physiological effects o f acclimatization on non-succulent plants has been documented. The photosynthetic contribution o f in v/Yro-formed leaves on raspberry plants was small or negative at acclimatization, while the first new leaves formed ex vitro had intermediate photosynthetic capability (Donnelly and Vidaver, 1984). It was concluded that acclimatization required the production of new leaves formed in the new environment. It has also been suggested that leaves formed in vitro are unable to develop further in ex vitro conditions and after a few weeks are replaced by newly formed, normal leaves possessing stomata that function correctly with normal amounts of epicuticular wax (Diettrich et al.,
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112
1992). However, leaves formed in vitro are still important for acclimatization. Leaves formed in vitro support the expansion o f new leaves and are necessary for the normal growth o f plantlets (DeYue et al., 1993). Thus, success during acclimatization depends on new growth initiated after transplantation. Acclimatization of cacti may depend on other attributes because cacti have photosynthetic, succulent stems instead o f leaves (Gibson and Nobel, 1986). Thus, acclimatization does not depend on the formation o f new leaf primordia when adapting to a different, ex vitro environment. Rather, the whole cacti body must acclimatize to these changes, in steps, until complete acclimatization is reached. The high plasticity of CAM physiology allows cacti to tolerate extremely dry conditions, closing stomata and recycling endogenous CO 2 (Griffits, 1988). It is possible that transplant stress during acclimatization resemble this situation. When not under water stress plants show a net CO2 uptake during the day (Harstock and Nobel, 1976) or to shift to a C-3 pathway in early ontogenetic stages (Altesor et al., 1992) or in callus cultures (Seeni and Gnaman, 1980). Thus, CAM plants possess unique features that can adapt them for in vitro culture conditions and further acclimatize them out the culture vessels.
CONCLUSIONS Micropropagation system. A diverse morphogenetic response was obtained for each cacti species: Shoot proliferation and flowering induction in Coryphantha minima; somatic embryogenesis and shoot proliferation in Obregonia denegrii or callus and isolate areoles development in Ariocarpus agavoides. These responses were observed in all treatments using a BA-NAA combination during Stage II of micropropagation. Recalcitrant species like Ariocarpus agavoides still needs further study to define a reliable method for in vitro propagation. The selected cacti species showed high capacity to spontaneously develop roots in vitro without the addition of any auxin to the growth media; however in vitro root development was increased in the presence of 3 to 5 mg I'1IBA. Root induction ex vitro was not as effective, but performance of these plantlets during acclimatization was superior. An upper layer o f a mixture of paraffin - lanolin over the culture media reduced considerably microbial contamination and played some beneficial role for the further acclimatization of the plantlets. However, the use o f this over-layer affected plant growth. Growth analysis. There is an effective growth enhancement in cacti cultured in vitro compared to ex vitro culture. Growth regulators added to the growth media during Stage II of micropropagation play a definitive role on such growth increase. However, typical conditions of the in vitro environment like high relative humidity in the culture vessels and the supplementary carbon source in the culture medium were found to be important factors related to the growth enhancement observed in vitro.
114 Analysis of the C 02uptake and daily fluctuation o f acids revealed that cacti cultured in vitro maintain higher photosynthetic rates than rates observed for same plants under natural conditions. The continuous CO2 fixation during fight and night periods observed for.in vitro cultured cactus demonstrate a total autothrophic growth. This helps to understand why such an improved growth occurs. Once the in vitro derived plants are transferred out the culture vessels, their metabolism returns to its typical rates. Sucrose supplemented to the growth medium stimulates autotrophic growth of Coryphantha minima, in contrast to other observations for C-3 plants cultured in vitro, where sucrose favors hererotrophic growth. The high humidity in culture vessels limits the normal development of epicuticular waxes; however, during acclimatization cacti regenerated epicuticular waxes to regular levels. Specific attributes of cacti, like succulence and CAM physiology induce a specific growth response in cacti cultured in vitro, contrasting to growth observed for other C-3 or C-4 species under same circumstances. Results obtained in this dissertation allude to a high plasticity of CAM physiology that leads to favorable growth responses during in vitro culture in plants possessing such photosynthetic pathway. Therefore, in vitro mass propagation can be a reliable alternative to produce vigorous, healthy and big plantlets of a considerable number of endangered species which possess CAM physiology.
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INFORMATION TO USERS

ARIZONA STATE UNIVERSITY December 1996

UMI Number: 9707395

300 North Zeeb Road Ann Arbor, MI 48103

has been approved August 1996

s t'l.'U l, artment Chairpersoj

Dean, Graduate College

104 104 105 113 115

Table 1. Effects of some features on tissue culture.

4. Low dissolved oxygen in the medium 5. Constant air temperature

1. DEVELOPMENT OF MICROPROPAGATION AND REGENERATION SYSTEMS

Obregonia denegrii Fig. 1. Selected species for micropropagation system.

Direct Aseptic germination morphogenesis

Rooting of new shoots

SHOOTS TO SOILESS CULTURE

Fig. 2. Micropropagation system for endangered cacti.

Series of experiments in Coryphantha minima for growth analysis.

Experiment (Tissue Variables Experimental culture stage)_______________________________________ design

3) In vitro shoot elongation phase.

-Greenhouse maintenance -Growth chamber maintenance

Infrared gas analyzer

Coupler for Magenta boxes

Fig. 3. Assimilation chamber for gas exchange measurements.

RESULTS AND DISCUSSION

Obregonia denegrii scarified

Ariocarpus agavoides scarified 42% 45%

4. Morphogenetic response* observed for Ariocarpus agavoides under different

IAA - BA combinations supplemented during Stage II of micropropagation.

Proliferation of Isolated areoles* 1 1 3 2 3

Shoot proliferation response of Coryphantha minima in different

concentrations of BA and NAA.

Growth regulators combinations (me I'1 )

Shoot proliferation response of Coryphantha minima in different

concentrations of IAA and KIN.

Abnormal shoots (%) 0 1.5 0 4 0 6.5 2.7 0 9.6

- KIN (7.5) KIN (10)

ii IAA (l) if if ii IAA (3)

1.73 0.40 87.5%

3.1 0.47 2.7% 5.42 1.43 74.6%

Vitrified shoot 3 0.85 0% 11.5 2.33 53.8% 4.3 1.08 23.7%

1 1.9 0% 19.6 1.18 0.7%

8. Effects of different combinations of BA and IAA during in vitro shoot

7.5 BA - 3 IAA 10.0 BA - 3 IAA * Data are mean numbers.

Growth regulator combinations (mg I'1)

Somatic embryos A Callus8

Response of Coryphantha minima shoots subcultured for in vitro root

induction in hormone free MS medium and MS supplemented with 1 mg I'1IBA.

# new shoots per plant 4.3

Rooting percentage o f Coryphantha minima and

cultured in M S basal medium with different auxin sources and concentrations.

Number of primary roots developed in vitro on Coryphantha minima, as a

result of different concentrations of IBA and sucrose.

ORION AND SIZE OF PLANT*

Ex vitro rooting o f Coryphantha minima and Obregonia denegrii with

different auxin sources on tw o different substrates.

450 400 350 H

300 250 200 150

14. Percentages of the incidence of microbial contamination during experiments

of in vitro culture with Coryphantha minima and Obregonia denegrii.

Sucrose concentration in the culture medium 0 gf

Percentage of contaminated culture vessels 5.8 %

15. E ffects o f sucrose concentration on microbial contamination and plant

Sucrose concentration in the growth media o g r'

Perlite-cactus pot mix and rooting powder._________________________________ 10.2