GROWTH AND GDH AND AAT ISOENZYME PATTERNS IN TERRESTRIAL AND EPIPHYTIC BROMELIADS AS INFLUENCED BY NITROGEN SOURCE

6 Pages·1997·1 MB·

by  Mercier, Helenice

Selbyana 18(1): 89-94
GROWTH AND GDH AND AAT ISOENZYME PATTERNS IN
TERRESTRIAL AND EPIPHYTIC BROMELIADS AS
INFLUENCED BY NITROGEN SOURCE
HELENICE MERCIER*I, GILBERTO B. KERBAUy1,
Departamento de Botaruca, Universidade de Sao Paulo, c.P. 11461, CEP 05422-970, Sao Paulo,
Sp, Brasil,
MARIA TEREZA V. CARVALH02 AND ERIC DERBYSHIRE2
Centro de Energia Nuclear na Agricultura, Universidade de Sao Paulo, C.P.96, CEP 13400,
Piracicaba, Sp, Brasil
ABSTRACT. Seedlings of three bromeliad species, Pitcairnia jlammea, Vriesea philippo-coburgii and
Tillandsia pohliana, with different growth habits were in vitro cultured for 6 months in a modified knudson
medium containing 8mM nitrogen, such as N03 -, N~ +, NH4N03, glutamine or urea. Growth analysis
showed that all species presented a very efficient uptake and utilization of glutamine and NH4N03• The
presence of N03 - substantially stimulated root development. To examine some possible effects of different
nitrogen sources on nitrogen assimilation, protein was extracted from leaf shoots of three species and
glutamate dehydrogenase (GDH) and aspartate aminotransferase (AAT) activities were determined by gel
electrophoresis. Effects of nitrogen source were shown most clearly by the different mobility of GDH
observed in samples which had been supplied with Ca(N03)2 compared to those found in samples supplied
with (NH.)2S0• . Urea as a nitrogen source depressed AAT activity in the terrestrial species Pitcairnia
jlammea but enhanced it in the epiphyte Tillandsia pohliana. The latter species and the other epiphyte
examined, Vriesea philippo-coburgii, were distinguishable from P. jlammea through their AAT mobility.
while the two epiphytes differed between themselves principally in the level of their AAT activity.
INTRODUCTION teins, nitrate reductase and phosphoenolpyruvate
carboxylase (Sugiharto and Sugiyama 1992). The
Plants depend upon the availability of light,
nitrogen assimilatory pathway involves various
CO2, water, nutrients and favorable temperatures enzymes, including glutamate dehydrogenase
for growth, maintenance and reproduction. Physi
(GDH) which catalyzes the arnination of alpha
ological adaptations to alterations in nutrient avail
ketoglutarate as well as the deamination of glu
ability in the environment may include improve
tamate (Oaks and Yamaya 1990), and aspartate
ments in root nutrient acquisition and assimilation
aminotransferase (AAT) which plays an impor
systems (Redinbaugh and Campbell 1991). Re
tant role in the assimilation of reduced nitrogen
garding N availability much research has focused
into aspartic acid and asparagine from glutamate
on crops, but there are few studies on non-agro
(Gantt et at. 1992). The existence of several iso
nomic species. Nitrogen is taken up by most high
enzymes of GDH has been demonstrated in a
er plants in the form of nitrate or ammonium; the
number of plants; different isoenzymes have been
N form may be important in the habitat preference
associated with physiological function, cellular
of a species (Falkengren-Grerup 1995). Organic
differentiation or intracellular compartmentation
forms of N, such as amino acids, are also utilized
by some species (Chapin et at. 1993). (Gonzalez-Bravo and Maeso 1987). ATT has
Very little is known about physiological and been studied in various plants, and different iso
biochemical responses of bromeliads to nitrogen enzymes have been documented for this enzyme
availability (Mercier 1993). Terrestrial bromeli (1\rrano et at. 1990).
ads use nitrogen taken up from the soil through In the present paper, the effect of five nitrogen
their roots. In contrast, epiphytic bromeliads ab sources on in vitro growth of seedlings of three
sorb nutrients from airborne inputs and canopy bromeliad species with different growth habits
throughfall, through absorbing foliar trichomes. was examined. The alterations in N metabolism
The tank-forming epiphytic species can accu were followed by changes in total-N, arnmo
mulate significant amounts of essential mineral nium-N and GDH and AAT isoenzyme patterns.
elements, including organic nitrogen, in their
leaf bases (Benzing 1980, 1990).
Nitrogen is an important regulator of gene ex Abbreviations: AAT, aspartate arninotransferase;.GDH,
pression of some proteins in higher plants, in glutamate dehidrogenase; NaEDTA, sodium ethylene-di
cluding ChI alb light-harvesting complex apopro- arnine-tetraacetic acid; NEM, N-ethyhnaleirnide; PAGE,
polyacrylamide gel electrophoresis; PMSF, phenyl
methylsuphonyl fluoride; SDS, sodium dodecyl sulfate;
* Corresponding author; E-mail: [email protected] TRIS, trishydroxymethylaminomethane
89
90 SELBYANA [Volume 18(1)
MATERIALS AND METHODS Electrophoresis of GDH and AAT
Plant species and culture conditions Leafy shoot samples (200mg fresh weight!
200f,LI buffer) were homogenized in 0.05M
Seedlings of Pitcaimia fiammea Lindley, a TRIS-base buffer (pH 8.2) containing 6mM
terrestrial species, Vriesea philippo-coburgii NaEDTA, 250mM sucrose, 50mM NaCl, 2mM
Wawra, a tank epiphyte bromeliad and Tilland PMSF and 2mM NEM. The homogenate was
sia pohliana Mez, an atmospheric tank-less ep centrifuged at 15,000 xg for 10 min, and the
iphyte were grown in aseptic Knudson (Knudson supernatant was used for GDH and AAT elec
1946) basal medium modified by substituting the trophoresis assays.
original nitrogen salts by equivalent concentra Non-denaturing PAGE in 7% acrylamine was
tions (8 mM) of Ca(N03)2, (NH4)2S04' NH4N03, performed using TRIS-glycine buffer pH 8.3,
urea or glutamine. Concentrations of all other according to the method used by Davies (1964).
nutrients were unchanged. The micronutrients of Volumes of SOfLl, corresponding to approxi
the Murashige and Skoog (1962) formulation mately l50fLg of protein, were introduced into
were added to these media. CaSOiO.64g1-1) was the slots of each gel; gels were run at a constant
used to replace S04 -2 ions, except for the NH4 + current of 30mA for 5h with constant cooling.
treatment. The culture media were buffered with Proteins with GDH or AAT activity were de
Igl-l CaC03 to maintain the pH near 5.0 (Sal tected by the staining procedures described by
ama and Wareing 1979). The seedlings were ob Shaw and Prasad (1970).
tained from seeds inoculated into 125ml Erlen
meyer flasks containing 50ml each of gel me RESULTS AND DISCUSSION
dium (0.7% agar) and kept under a 16h photo
In vitro growth
period with fluorescent light at lOWm-2 and
temperature of 26 ± 2°C. In vitro germination and aseptic seedling cul
After 6 months of incubation, the seedlings ture were necessary in this nitrogen nutrition
were harvested and evaluated for shoot length study, because contamination by nitrifying or
and number of roots and leaves per plant. The ganisms and the conversion of ammonium to ni
treatments were repeated 3 times with 60 seed trate is a significant problem in ammonium
lings per replication. based solutions used for aeroponic, hydroponic,
and sand culture (Padgett and Leonard 1993).
Total-nitrogen Furthermore, for some tank bromeliads the phy
totelmata is the site of N -fixation by cyanobac
2
The amount of total nitrogen was assayed by teria (Bermudes and Benzing 1991). Another ad
the Kjeldahl method as described in Loomis and vantage of using a sterile gellified medium is
Shull (1937). Samples (500 mg dry weight) of that it allows for both root nutrition, as is the
tissue were digested with concentrated H2S04; case of P. fiammea, as well as the foliar nutrition
aliquots of the digested samples were alkaline of the epiphytic species. The latter possibility
steam distilled with 6N NaOH; NH3 was trapped occurs in the form of a surface film of nutritious
in O.02N Hel. From the HCl volume used and solution which covers the leaves, making it pos
the dry weight of tissue digested, the total re sible for the nutrients to reach the leaf cells
duced N content was calculated. Two indepen through diffusion.
dent samplings were made for each data point The Knudson medium has been successfully
and their values averaged. used in this laboratory for the in vitro germi
nation and culture of bromeliads (Mercier and
Free-NH4+ Kerbauy 1992). Based on these results, a pilot
project was begun in our laboratory about in
Samples (200mg fresh weight) of tissue were vitro nitrogen nutrition in the Bromeliaceae, us
ground in a mortar with Sml distilled water at ing the same concentration of total nitrogen
60°C; 5ml chloroform was added, and the re present in basal Knudson medium (8 mM). Very
sulting extract was then filtered with Whatman little is known on nitrogen availability in native
#1 filter paper. The free ammonium was deter Bromeliaceae habitats. Benzing (1980) showed
mined in aliquots of the upper aqueous phase by some results for Guzmania monostachia, a litter
the phenol-hypochlorite reaction (Weatherburn impounding bromeliad found in southern Flori
1967). Two independent samplings were made da, which contained 197.8 mgl-1 (or 14 mM) of
for each data point and their values averaged. total nitrogen in the tank.
The results were expressed on a dry weight basis In the three studied species, N03 - as the sole
for comparison with total-N determinations as nitrogen source had significantly less effect on the
described above. shoot length, while in contrast, N03 - stimulated
1997] MERCIER ET AL.: GROWTH AND ISOENZYME PATTERNS IN BROMELIADS 91
Pitcairnia Vriesea Tillandsia
5-
I 1
jj ••••
N03 NH4 NH4N03 UREA GLU N03 NH4 NH4N03 UREA GLU
\11111
]1,00
j
~1111.,1
N03 NH4 NH4N03 UREA GLU N03 NH4 NH4N03 UREA GlU N03 NH4 NH4N03 UREA GLU
.•• 4-
3tI
~hl ~Li. :---j~ ---
N03 NH4 NH4N03 UREA GLU N03 NH4 NH4N03 UREA GLU N03 NH4 NH4N03 UREA GLU
Treatment
FIGURE I. In vitro growth expressed as shoot length and leaf and root numbers per seedling of P. jlammea,
v. philippo-coburgii and T. pohliana cultivated for 6 months with five different nitrogen sources. Mean:!: S.E.M.
root fonnation (FIGURE 1). In all three bromeliads, that nitrate has perhaps inhibited the NH4 + up
a decrease of shoot growth accompanied the ~ + take. Similar results were also obtained in Picea
treatment while root development was inhibited in abies seedlings (Aarnes et at. 1995). In relation
the two epiphytic species (FIGURE 1). Many seed to the effect of glutamine as the sole nitrogen
lings died in this treatment. In the case of P. jlam source, it was noted that this amide stimulated
mea, the leaves of surviving plants showed a dark the shoot growth of all three bromeliad species
green color parallel with curled laminae, suggest (FIGURE 1). For T. pohliana no roots were de
ing an apparent ~ + toxicity. High total-N con tected. Organic nitrogen was preferentially ac
centrations were detected in the shoots of all three cumulated in the epiphytes and free-NH4+ con
bromeliads, and, at least for P. flammea and V. tent was very high in the atmospheric bromeliad
philippo-coburgii, the accumulation of free ~ + (FIGURE 2). In nature, organic nitrogen in the
reached very high levels (FIGURE 2). ~ +- toxicity form of amino acids is present in the precipita
might be related to detoxification stress, resulting tion that has previously contacted host tissues
from the requirement for carbohydrates needed to (Thckey 1970). These organic nutrients, dis
assimilate a supra-optimum amount of ~ + (Gi solved in the canopy fluids, can be absorbed
van 1979). The increased demand on the carbon through foliar trichomes. Nyman et aZ. (1987)
supply could become limiting for bromeliad showed net uptake of dissolved free amino acids
growth. In this sense, GDH enzyme could fulfill on an intact epidermal surface of Tillandsia pau
an important function linking carbon and nitrogen cijolia, indicating their active transport and ac
metabolism (Robinson et aZ. 1992). For P. jlam cumulation by specialized trichomes present on
mea and T. pohliana seedlings, the effect of ~ + the leaves. Benzing (1970) showed that gluta
nutrition had a positive response in GDH activity mine induced the best growth result in five spe
(FIGURE 3). cies of bromeliads, including terrestrial and epi
All three bromeliads studied were character phytic species. They were in vitro cultured in a
ized by an efficient utilization of nitrogen as media containing 14 amino acids (2mM) added
NH4N03, evident from the results of leaf and one by one. It was also reported that alanine and
root growth (FIGURE 1). Especially low concen glutamic acid presented opposite effects, inhib
trations of free-NH4 + were detected, indicating iting the growth of them all.
92 SELBYANA [Volwne 18(1)
Pitcairnia Vriesea Tillandsia
120 T
+
~l.L 100 t
:1 ilil 80 t
60 ,
.;0 30
20
20 10
o
N03 NH4 NH4N03 UREA GLU o N03 NH4 NH4NOs UREA GLU . N03 NH4 NH4N03 UREA GLU
::L):_
12 -_J
10
i
I20 1+ :ta_
IS
N03 NH4 NH4N03 UREA GLU N03 NH4 NH4N03 UREA GLU NH4 NH4N03 UREA GLU
Treatment
FIGURE 2. Total nitrogen contents and free-NH. + levels found in shoots of 6 months seedlings of P. flammea,
V. philippo-coburgii and T. pohliana grown with five different nitrogen sources. Mean ± S.E.M.
When urea was used, it was observed that for since it is common to find amphibian organisms
P. jlammea the majority of the seedlings died living among the overlapping leaf bases.
after 1 month of incubation. So we decided to
decrease the urea concentration to a half (4mM) GDH and AAT enzymes
for this species. Even with this reduction, the
GDH activity was detected in all the samples
organic nitrogen source did not produce good
regardless of the nitrogen source used, and in
development of the surviving seedlings, as
each sample it was restricted to a single zone in
shown in FIGURE 1. Apparently the high level of
the gel (FIGURE 3). The mobility of the enzyme
free-NH4+ detected in P. jlammea shoot tissues was not identical in all the samples and varied
(FIGURE 2) caused ammonium toxicity. On the
from Rm 0.17 to Rm 0.19. At least in P. jlam
contrary, the tank epiphyte V. philippo-coburgii
mea and T. pohliana the enzyme was less mo
showed good shoot growth results, even though
bile (Rm 0.17) in treatments with (NH4)2S04
root development was inhibited (FIGURE 1). No
than in, for example, the treatments with
roots were detected also in T. pohliana seedlings
grown in urea medium and shoot appearance Ca(N03)2' T. pohliana supplied with urea gave
a result similar to that obtained from the
was pale green in color. High levels of organic
nitrogen were accwnulated in the shoots of epi (NH.)2S04 treatment. Quantitatively, the slow
phytic bromeliads, especially in V. phiZippo-cob form of the enzyme was more active than the
urgii, while free-NH4+ was present to a lesser fast form, showing NH4 + as a positive effector
extent (FIGURE 2), suggesting different pathways of GDH gene. Many authors have already de
of urea metabolism between terrestrial and epi tected a dependence of the banding pattern of
phytic bromeliads. Urea can be hydrolyzed by GDH activity on nitrogen nutrition. In rice plant
urease, which is widespread in the plant king roots, the addition of NH4Cl to a basal nitrogen
dom, producing two moles of ammonia, or urea deficient mediwn caused the development of a
may be utilized as an intact molecule by con new GDH isoform (Kanamori et al. 1972). In
densation with ornithine to form arginine (Rein maize seedlings, the GDH enzyme could be re
bothe and Mothes 1962). On the other hand, ei solved into only one band. In the seedling root
ther the uptake of urea or the formation of am extract, the effect of N03 - or NH4 + nutrition
moniwn from urea was perhaps slower relative showed that NH4 + had a positive response in
to growth demands of V. philippo-coburgii and/or GDH activity. In contrast, for the seedling leaf
T. pohliana, without the accwnulation of toxic extract, the GDH activity was not altered in re
free-NH. + in shoot tissues. For V. phiZippo-cob sponse to nitrogen sources (Oaks 1994).
urgii the natural occurrence of urea as a form of In relation to AAT, all the samples of P. jlam
tank-dissolved nitrogen may be considered, mea and V. philippo-coburgii showed AAT ac-
1997] MERCIER ET AL.: GROWTH AND ISOENZYME PATTERNS IN BROMELIADS 93
Pitc:airnia -flam- m-ea V.i.na philippo-caburllii Tillandsia pahliona
A
0.18
I
Uu
U
U
1
nn
B HOi NH~'" NH.NOl UREA GLU NOi NH,- NH~3 UREA GLU HO3- NH.+ NH~ UREA GLU
~n l
I '
-1 0.S3
I
0.48
1
FIGURE 3. Effect of nitrogen sources on the GDH and AAT isoenzyme patterns in P. flammea (terrestrial),
v. philippo-coburgii (tank epiphyte) and T. pohliana (atmospheric epiphyte). The isoenzymes were resolved in
7% polyacrylamide gel and all lanes were loaded with 150fLg of protein. Staining intensity of the bands is in
accordance to the relative abundance of the isoenzyme forms. GLU = glutamine.
tivity and in both species it was confined to a from T. pohliana may reflect a role of nitrogen
single band (FIGURE 3). However the mobility of nutrition on the mechanism that regulates the ex
the enzyme differed between the species with pression of AAT isoenzymes, which might be
the terrestrial P. fiammea presenting Rm 0.33 specific for each genotype. Although AAT can
and V. philippo-coburgii Rm 0.48. Qualitatively be considered one of the most important ami
no effect of nitrogen supply was observed. In notransferase, the physiological role of each
contrast to these two species, T. pohliana AAT gene/isoenzyme in aspartate metabolism
showed activity only in the sample grown in the remains elusive (Lam et at. 1995).
presence of urea. The mobility of the enzyme in
this sample (Rm 0.48) was the same as that ob LITERATURE CITED
served in the other epiphyte V. philippo-cobur
AARNES H., A. B. ERIKSEN AND T. E. SOUTHON. 1995.
gii. With the addition of N03 - or urea to P. fiam Metabolism of nitrate and ammonium in seedlings
mea, AAT decreased in activity (FIGURE 3). Both of Norway spruce (Picea abies) measured by in
treatments reduced seedling development, sug vivo 14N and 15N NMR spectroscopy. Physiol.
gesting that aspartate was not sufficiently avail Plant. 94: 384-390.
able to support growth. However our results ATKINS C. AND L. BEEVERS. 1990. Synthesis, transport
94 SELBYANA [Volume 18(1)
and utilization of translocated solutes of nitrogen. MURASHIGE T. AND F. SKOOG. 1962. A revised medium
in Nitrogen in higher plants. Abrol Y.P., ed., Re for rapid growth and bioassays with tobacco tissue
search Studies Press, Somerset. Pp.223-277. cultures. Physiol. Plant. 15: 473-497.
BENZING D. H. 1970. Availability of exogenously sup NYMAN L. P., J. P. DAVIS, S. J. O'DELL, J. ARDITTl, G.
plied nitrogen to seedlings of the Bromeliaceae. C. STEPHENS AND D. H. BENZING. 1987. Active
Bull. Torrey Bot. Club 97: 154-159. uptake of amino acids by leaves of an epiphytic
---. 1980. The biology of the bromeliads. Eureka vascular plant, Tillandsia paucifolia (Bromeli
Printing Company. Eureka, California. aceae). PI. Physiol. 83: 681-684.
---. 1990. Vascular epiphytes. Cambridge Uni OAKS A. 1994. Primary nitrogen assimilation in high
versity Press, Cambridge. er plants and its regulation. Canad. J. Bot. 72:
BERMUDES D. AND D. H. BENZING. 1991. Nitrogen fix 739-750.
ation in association with Ecuadorean bromeliads. ---AND T. Y AMAYA. 1990. Nitrogen assimilation
J. Trop. Eco!. 7: 531-536. in leaves and roots-a role for glutamate dehydro
CHAPIN III F. S., L. MOILANUN AND K. KIELLAND. 1993. genase. in Nitrogen in higher plants, Abrol Y.P. ed.
Preferential use of organic nitrogen for growth by Research Studies Press, Somerset, pp.181-191.
a non-mycorrhizal arctic sedge. Nature 361: 150- PADGETT P. E. AND R. T. LEONARD. 1993. Contami
153. nation of ammonium-based nutrient solutions by
DAVIES B. J. 1964. Disc electrophoresis II. Method and nitrifying organisms and the conversion of am
application to human serum proteins. Ann. New monium to nitrate. PI. Physiol. 101: 141-146.
York Acad. Sci. 121: 404-427. REDDY K. S. AND R. C. MENARY. 1990. Nitrate re
F ALKENGREN-GRERUP V. 1995. Interspecies differences ductase and nitrate accumulation in relation to ni
in the preference of ammonium and nitrate in vas trate toxicity in Boronia megastigma. Physiol. PI.
cular plants. Oecologia 102: 305-31l. 78: 430-434.
GANTT J. S., R. J. LARSON, W. F. MARK, M. W. FARN REDINBAUGH M. G. AND W. H. CAMPBELL. 1991. High
HAM, S. M. PATHIRANA, S. S. MILLER AND C. P. er plant responses to environmental nitrate. Phys
VANCE. 1987. Aspartate aminotransferase in ef iol. PI. 82: 640-650.
fective and ineffective alfalfa nodules: cloning of REIN BOTHE H. AND K. MOTHES. 1962. Urea, ureides
a cDNA and determination of enzyme activity, and guanidines in plants. Annual Rev. PI. Physiol.
protein and mRNA levels. PI. Physiol. 98: 868- 13: 129-150.
878. ROBINSON S. A., G. R. STEWARD AND R. PHILLIPS.
GIVAN C. V. 1979. Metabolic detoxification of am 1992. Regulation of glutamate dehydrogenase ac
monium in higher plants. in Biochemistry of high tivity in relation to carbon limitation and protein
er plants. Stumpf P. R. and E. E. Conn, ed. Aca catabolism in carrot cell suspension cultures. PI.
demic Press, New York 5: 329-357. Physioi. 98: 1190-1195.
GONZALEZ-BRAVO M. P. AND M. MAESO. 1987. Effect SALAMA A. M. S., D. A. EL AND P. F WAREING. 1979.
of nitrate and ammonium on NADH-dependent Effects of mineral nutrition on endogenous cyto
kinins in plants of sunflower (Helianthus annuus
glutamate dehydrogenase activity of detached
roots and cotyledons of Citrullus vulgaris L. J. L.). J. Exp. Bot. 30: 971-981.
SARAVITZ C. H., S. CHAILLOU, J. MUSSET, D. C. RAPER
Plant Physiol. 127: 331-337.
AND J. F. MOROT-GAUDRY. 1994. Influence of ni
KANAMORI T., S. KONISHI AND E. TAKAHASHI. 1972.
trate on uptake of ammonium by nitrogen-deplet
Inducible formation of glutamate dehydrogenase
ed soybean: is the effect located in roots or
in rice plant roots by the addition of ammonia to
shoots? J. Exp. Bot. 45: 1575-1584.
the media. Physiol. PI. 26: 1-6.
SHAW C. R. AND R. PRASAD. 1970. Starch gel electro
KNUDsoN L. 1946. A new nutrient solution for ger
phoresis of enzymes. A compilation of recipes.
mination of orchid seed. Amer. Orchid Soc. Bul.
Biochem. Genet. 4: 297-320.
15: 214-217.
SUGIHARTO B. AND T. SUGIYAMA. 1992. Effects of ni
LAM H. M., K. COSCHIGANO, C. SCHULTZ, R. MELLO
trate and ammonium on gene expression of phos
OLIVEIRA, G. TJADEN, 1. OLIVEIRA, N. NGAI, M. H.
phoenolpyruvate carboxylase and nitrogen metab
HSIEH AND G. CORUZZI. 1995. Use of Arabidopsis
olism in maize leaf tissue during recovery from
mutants and genes to study amide amino acid bio
nitrogen stress. P!. Physiol. 98: 1403-1408.
synthesis. PI. Cell 7: 887-898.
TUCKEY JR. H. B. 1970. The leaching of substrates
LOOMIS W. E. AND C. A. SCHULL. 1937. Methods in from plants. Annual Rev. PI. Physiol. 21: 305-
plant physiology-a laboratory manual and re 324.
search handbook. Sinnott, E. W. ed., New York TURANO F J., B. 1. WILSON AND B. F. MATTHEWS. 1990.
and London, McGraw-Hill Book Company. Purification and characterization of aspartate ami
MERCIER H. 1993. Efeitos de fontes nitrogenadas so notransferase isoenzymes from carrot suspension
bre 0 desenvolvimento, teores hormonais endo cultures. PI. Physiol. 92: 587-594.
genos, perfis polipeptfdicos e isoenzimaticos em ---, --- AND ---. 1991. Rapid purifica
tres especies de bromelias cultivadas in vitro. PhD tion and thermostability of the cytoplasma aspar
thesis. University of Sao Paulo, Sao Paulo, SP, tate aminotransferase from carrot suspension cul
Brazil. tures. PI. Physiol. 97: 606-612.
---AND G. B. KERBAUY. 1992. In vitro multipli WEATHERBURN M. W. 1967. Phenol-hypochlorite re
cation of Vrieseafosteriana. PI. Cell Tissue Organ action for determination of ammonia. Anal. Chem.
Cult., 30: 247-249. 39: 971-974.