|
Electronic Journal of Biotechnology
Universidad Católica de Valparaíso
ISSN: 0717-3458
Vol. 9, Num. 3, 2006, pp. 210-214
|
Electronic Journal of Biotechnology, Vol. 9, No. 3, Special
Issue, 2006, pg. 210-214
RESEARCH ARTICLE
Bioactivity
of Scytonema hofmanni (Cyanobacteria) in Lilium alexandrae
in vitro propagation
María Cristina Zaccaro*1,
Adriana Kato2, Gloria Zulpa3, Mónica Magdalena Storni4, Noemí Steyerthal5, Karina
Lobasso6, Ana María Stella7
1, 3, 4, 5, 6 Laboratorio
de Fisiología Vegetal y Biología de Cyanobacteria,
Departamento de Biodiversidad y Biología Experimental,
Facultad de Ciencias Exactas y Naturales FCEN,
Universidad de Buenos Aires,
Intendente Güiraldes 2620 Pab. II 4º P Lab 2,
Ciudad Universitaria C1428EHA,
Buenos Aires, Argentina,
Tel: 0054 1145763300/09 ext. 201,
Fax: 0054 1145763384,
E-mail: cyanob@bg.fcen.uba.ar
2 Instituto
de Floricultura,
INTA-Castelar,
Las Cabañas y De los Reseros s/n (1712),
Buenos Aires, Argentina,
Tel/Fax: 5411 4481/3736,
E-mail: akato@castelar.inta.gov.ar
3 Laboratorio
de Fisiología Vegetal y Biología de Cyanobacteria,
Departamento de Biodiversidad y Biología Experimental,
Facultad de Ciencias Exactas y Naturales FCEN,
Universidad de Buenos Aires,
Intendente Güiraldes 2620 Pab. II 4º P Lab 2,
Ciudad Universitaria C1428EHABuenos Aires, Argentina,
Tel: 0054 1145763300/09 ext. 201,
Fax: 0054 1145763384,
E-mail: cyanob@bg.fcen.uba.ar
4 Laboratorio
de Fisiología Vegetal y Biología de Cyanobacteria,
Departamento de Biodiversidad y Biología Experimental,
Facultad de Ciencias Exactas y Naturales FCEN,
Universidad de Buenos Aires,
Intendente Güiraldes 2620 Pab. II 4º P Lab 2,
Ciudad Universitaria C1428EHA,
Buenos Aires, Argentina,
Tel: 0054 1145763300/09 ext. 201,
Fax: 0054 1145763384,
E-mail: cyanob@bg.fcen.uba.ar
5 Laboratorio
deAnálisis Biológicos,
Departamento de Química Biológica,
Facultad de Ciencias Exactas y Naturales,
Universidad de Buenos Aires,
Intendente Güiraldes 2620 Pab. II 4º P Lab 2,
Ciudad Universitaria C1428EHA,
Buenos Aires, Argentina,
Tel: 0054 1145763300/09 ext. 201,
Fax: 0054 1145763384
6Laboratorio
de Fisiología Vegetal y Biología de Cyanobacteria,
Departamento de Biodiversidad y Biología Experimental,
Facultad de Ciencias Exactas y Naturales,
Universidad de Buenos Aires,
Intendente Güiraldes 2620 Pab. II 4º P Lab 2, Ciudad Universitaria C1428EHA,
Buenos Aires, Argentina,
Tel: 0054 1145763300/09 ext. 201,
Fax: 0054 1145763384,
E-mail: cyanob@bg.fcen.uba.ar
7 Laboratorio
de Ecoporfirinas,
Departamento de Química Biológica,
Facultad de Ciencias Exactas y Naturales,
Universidad de Buenos Aires - CONICET ,
Intendente Güiraldes 2620 Pab. II 4º P Lab 2,
Ciudad Universitaria C1428EHA,
Buenos Aires, Argentina,
Tel: 0054 1145763300/09 ext. 201,
Fax: 0054 1145763384,
E-mail: stella@qb.fcen.uba.ar
*Corresponding author
Financial support: UBACyT
X64, Universidad de Buenos Aires, Argentina.
Code Number: ej06027
Cyanobacteria produces bioactive
compounds including plant growth regulators. Naphthalene acetic acid (NAA),
a toxic substance, is a synthetic plant regulator used in micropropagation.
The aim of this work was to evaluate morphogenetic and antioxidant effects
produced by intra and extracellular substances from Scytonema hofmanni (Cyanobacteria)
during the multiplication in vitro of Lilium alexandrae and
to compare them to those produced by NAA. Intra and extracellular cyanobacterial
products increased a) bulblets production reaching 83% and 78% of NAA effect,
respectively; b) the bulblet diameter compared to NAA; and c) the bulblet
survival due to the promotion of antioxidant activity measured as catalase,
ascorbate peroxidase, and glutathione reductase activity. The cyanobacterial
substances stimulated regeneration and delayed bulblet senescence. They
could replace NAA, dangerous for the operator, not only during the regeneration
phase but also during the storage of the viable bulblets cultivated in
vitro.
Keywords: ascorbate
peroxidase, bioactive substances, bulb regeneration, catalase, Cyanobacteria,
glutathione reductase, Lilium alexandrae, Scytonema hofmanni.
Abbreviations: |
BWE: biomass water
extract.
EP: extracellular products.
NAA: naphthalene acetic acid.
MS: Murashige-Skoog. |
Growth
regulators which accelerate the production of a number of agronomical interesting
plants are used in in vitro culture of plants. Growth regulators are
mainly obtained by chemical synthesis. Cyanobacteria produce a variety of
bioactive compounds including growth phyto-regulators (Metting
and Pyne, 1986), that could be used in the in vitro production
of vegetables, fruits, fungi and ornamental flowers. Already in 1979, Zulpa
de Caire et al. (1979) established that Nostoc muscorum Ag. liberated
into the culture medium auxin-like substances. It has recently demonstrated
that a number of Cyanobacteria produce, accumulate, and liberate 3-indol
acetic acid (Sergeeva et al. 2002). Arthronema africanum produces
the cytokinin isopentenyl adenine (Stirk et al. 1999). Stirk
et al. (2002) found auxin and cytokinin activity by three cyanobacterial
strains. It is important to mention that some cyanobacterial products promote
regeneration in Daucus carota (Wake et al. 1992), Santalum
album (Bapat et al. 1996), Oryza sativa (Zaccaro
et al. 2002; Storni de Cano et al. 2003), Lilium
alexandrae hort. Wallace, from Japan, is an ornamental plant, with white
flowers bending down to form an angle of 90º, with respect to the vertical
axis of the plant making this species a very interesting one for the improvement
of other Lilium species (Nakayama, 1989). Naphthalene
acetic acid (NAA) is a synthetic phyto-regulator very much used in micropropagation.
Toxicological information indicates that NAA causes contact irritation of
skin, eyes, mucous membranes, as well as the upper respiratory tract. It
also attacks the central nervous system and can be absorbed by the operators
skin (Sigma Aldrich-Argentina S.A.). The aim of this work was to evaluate
the morphogenetic and antioxidant effects produced by intra and extracellular
substances from Scytonema hofmanni (Cyanobacteria), on the in
vitro propagation of L. alexandrae and to compare them with
those produced by synthetic phyto-regulators.
Materials
and Methods
Explant
obtention
Microscales
(6-8 x 6-10 mm) from bulblets of L. alexandrae (1.8-2.5
g fresh weight and 5-6.2 cm
diameter), were obtained in vitro after 120 days growing on MS medium
(Murashige and Skoog, 1962), with a photoperiod of 12 hrs light
and a light intensity of 45 µmol photon x m-2 seg-1 and
25 ± 1ºC. The
abaxial side of the explant was placed on the culture medium.
Obtaining
of cyanobacterial products
Biomass
water extracts (BWE) and extracellular products (EP). S.
hofmanni Ag. ex Born. et Flahault axenic strain Nº 58, from
the culture collection belonging to Laboratorio de Cyanobacteria, Facultad
de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Argentina,
was cultivated in modified Watanabe medium (Storni de Cano
et al. 2003), photoperiod 12 hrs light, 45 µmol photon x m-2 seg-1,
27 ± 1ºC.
After 30 days growing in that medium, the biomass was separated from the
culture medium by centrifugation at 10.000 xg at 5ºC.
The fresh biomass was homogenized with alumina (1 g biomass / 3 g alumina), and extracted with sterile
distilled water (1 g fresh
biomass / 5 ml water). After centrifugation the supernatant BWE was obtained.
The culture medium contained the extracellular products (EP). BWE and EP
were sterilised by ultrafiltration (0.22 µm).
Obtaining
of bulblets
Treatments: I,
8 ml MS + 2 ml distilled water. II, 8 ml MS + 2 ml EP. III,
8 ml MS + 2 ml distilled water + 0.1 mg/L NAA. IV, 8 ml MS + 2 ml
BWE. The media contained 3% sucrose, 0.7% agar and pH was adjusted to 6.8,
before being sterilized in tubes (14 x
2.5 cm
). The explants were placed in
a dark culture chamber, for three weeks in order to obtain differentiation
and bulblets growth. After that period they were transferred to 16:8 photoperiod,
45 µmol photon x m-2 seg-1 and 25 ±
1ºC
.
Each 5 weeks, they were transferred to fresh medium. At days 20, 30, 40,
50 and 70 the number of bulblets per explant, bulblet longest diameter, bulblet
roots and leaves length and number per explant were measured. At day
260 in
this case without transference
to fresh medium, the survival capacity of the bulblets in each treatment
was established.
Biochemical
parameters
Enzymatic
activity of catalase, ascorbate peroxidase and glutathione reductase were
determined according to Beers and Sizer (1951), Nakano
and Asada (1981) and Schaedler and Bassham (1977),
respectively, in order to establish the bulblets oxidative stress level produced
by the regeneration treatments.
Statistic
analysis
Analysis
of variance was performed for all data, using a completely randomized experimental
design. A one-way ANOVA was performed (PC program GraphPad Prism). A Tukey
HDS test (p < 0.05) was used to compare different treatments for each
day, n = 14.
Results
and Discussion
At
day 50 (Figure 1) S. hofmanni BWE increased
the number of bulblets per explant by 27% comparing with MS, representing
this increment 78% of the bulblets produced by NAA. At day 70 the increment
was 21 y 32% for EP and BWE, respectively, compared to MS. With respect to
NAA, EP produced 78% and BWE 83% of the number obtained with the synthetic
auxin (Figure 2a). No matter higher the number of bulblets
produced by NAA, cyanobacterial intracellular products increased the number
of bulblets thicker than 5 mm as
at day 70 BWE surpassed NAA, MS and EP effect by nearly
30% (Figure 2b). This effect on thickness is important
to survival of the bulblets. The number of roots per explant (Figure 2c) in MS and EP was lesser than NAA by 70% and in BWE by 50%, at day
70. This could mean that BWE favoured the higher thickness of the bulblets,
compared to NAA, at the expense of the number of roots, probably due to a
differential distribution of organic nutrients. The number of leaves per
explant (Figure 2d) was the same in EP and NAA at day
70, showing at the beginning an acceleration of differentiation with EP.
Extracellular products from other Cyanobacteria were shown to replace synthetic
phyto-regulators in the organogenesis of rice calli (Storni
de Cano et al. 2003), to increase the phyto-regulators content of Lupinus
termis (Haroun and Hussein, 2003). Cyanobacterial crude
extracts showed auxinic activity in potato tissue culture (Shanab
et al. 2003). At day 260 the number of bulblets decreased in all the
treatments. In MS 50% bulblets were necrotic and in NAA 100%, but with EP
and BWE 90% were viable. Table 1 shows that EP and
BWE produced an increment of 3.85 and 1.60 fold in catalase activity with
respect to NAA which in turn doubled this activity with respect to MS. Bulblets
ascorbate peroxidase activity increased 4 times with NAA, 3 times with BWE
and 1.67 times with EP. Glutathione reductase activity increased 6.34 times
with EP, 2.64 times with BWE and 1.52 times with NAA comparing with the control
with MS. The activity of enzymes related with reactive oxygen species was
remarkably incremented by cyanobacterial products, surpassing NAA for catalase
and glutathione reductase activity which could explain the higher survival
of bulblet that were not transferred to fresh medium for 260 days remaining
viable by 90%. It is known that the activity of antioxidant enzymes increases
in tissues under different stress conditions, such as sugar beet calli (Hagége,
1996).
Table
1. Activity
of antioxidant enzymes in bulblets at day 260. Different letters
indicate significant differences (p < 0.05). |
Treatment |
Catalase
(umol/min g fresh weight) |
Ascorbate
peroxidase
(umol/min g fresh weight) |
Glutathione
reductase
(umol/min g fresh weight) |
MS |
7.212 ± 0.003 d |
10.604 ± 0.010d |
2.212 ± 0.700C |
MS
+ NAA |
15.511 ± 0.003c |
44.051 ± 0.005a |
3.384 ± 0.050C |
MS
+ EP |
59.852 ± 0.005ª |
16.784 ± 0.005c |
14.034 ± 0.060A |
MS
+ BWE |
24.893 ± 0.004b |
32.623 ± 0.005b |
5.854 ± 0.050B |
Concluding
Remarks
Intra
and extracellular products from S. hofmanni increment L. alexandrae bulblets
production from microscales, comparing with control without synthetic phyto-regulators
as well as bulblets survival in culture because they promote a higher antioxidant
activity. This effect could be due to the production and liberation of plant
growth regulators such as auxin-like and cytokinin-like substances by the
cyanobacterium, which produced similar results than those obtained with the
synthetic auxin NAA and also because the bulblets senescence was delayed,
an effect well-known for cytokinin. Besides, the cyanobacterial phyto-regulators
could replace substances that result dangerous to the operator not only during
the regeneration phase but also during the storage of the viable bulblets
cultivated in vitro.
References
-
BAPAT,
V.A.; IYER, R.K. and RAO, P.S. Effect of cyanobacterial extract on somatic
embryogenesis in tissue culture of sandalwood ( Santalum album). Journal
of Medicinal and Aromatic Plant Sciences, 1996, vol. 18, no. 1, p.
10-14.
-
BEERS,
R.F. and SIZER, I.W. A spectrophotometric method for measuring the breakdown
of hydrogen peroxide by catalase. Journal of Biological Chemistry,
1951, vol. 195, no. 1, p. 133-140.
-
HAGÉGE,
D. Habituation in sugar beet plant cells: permanent stress or antioxidant
adaptive strategy? In vitro Cellular and Developmental Biology - Plant, 1996,
vol. 32, no. 1, p.1-5.
-
HAROUN,
S.A. and HUSSEIN, M.H. The promotive effect of algal biofertilizers on
growth, protein pattern and some activities of Lupinustermis plants
grown in siliceous soil. Asian Journal of Plant Sciences, 2003,
vol. 2, no. 13, p. 944-951.
-
METTING,
B. and PYNE, J.W. Biologically active compounds from microalgae. Enzyme
and Microbial Technology, 1986, vol. 8, p. 386-394. [CrossRef]
-
MURASHIGE,
T. and SKOOG, F. A revised medium for rapid growth and bioassays with tobacco
tissue cultures. Physiologia Plantarum, 1962, vol. 15, p.
473-497.
-
NAKANO,
Y. and ASADA, T. Hydrogen peroxide is sacavenged by ascorbate specific peroxidase
in spinach chloroplast. Plant Cell Physiology, 1981, vol. 22, no.
5, p. 867-880.
-
NAKAYAMA,
S. Genus Lilium L. Lilium alexandrae. In: TSUKAMOTO, Y. ed. The
grand dictionary of horticulture. Shogakukan, Tokyo, Japan, 1989, vol.
5, p. 198-209. ISBN 4-09-305105-4.
-
SERGEEVA,
E.; LIAIMER, A. and BERGMAN, B. Evidence for production of phytohormone
indol-3-acetic acid by cyanobacteria. Planta, 2002, vol. 215, no.
2, p. 229-238. [CrossRef]
-
SHAEDLER,
M. and BASSHAM, J. Chloroplast gluthationereductase. Plant Physiology, 1977,
vol. 59, p. 1011-1012.
-
SHANAB,
S.; SAKER, M.M. and ABDEL-RAHMAN, M. Crude extract of some fresh water
cyanobacteria have auxin like activity on potato tissue culture. Arabian
Journal of Biotechnology, 2003, vol. 6, no. 2, p. 297-312.
-
STIRK,
W.A.; ÖRDÖG, V. and VAN STADEN, J. Identification of the cytokinin isopentenyladenine
in a strain of Arthronemaafricanum (Cyanobacteria) . Journal
of Phycology, 1999, vol. 35, no. 1, p. 89-92. [CrossRef]
-
STIRK,
W.A.; ÖRDÖG, Vince; VAN STADEN, J. and JÄGER, K. Cytokinin- and auxin-like
activity in Cyanophyta and microalgae. Journal of Applied Phycology,
2002, vol. 14, no. 3, p. 215-221. [CrossRef]
-
STORNI
DE CANO, Mónica; ZACCARO, María Cristina; GARCÍA, Ileana; STELLA, Ana María
and ZULPA DE CAIRE, Gloria. Enhancing rice callus regeneration by extracellular
products of Tolypothrixtenuis (Cyanobacteria). World Journal
of Microbiology and Biotechnology, 2003, vol. 19, no. 1, p. 29-34. [CrossRef]
-
WAKE,
H.; AKASAKA, A.; UMETSU, H.; OZEKI, Y.; SHIMOMURA, K. and MATSUNAGA, T.
Promotion of plantlet formation from somatic embryos of carrot treated
with a high molecular weight extract from a marine cyanobacterium. Plant
Cell Reports, 1992, vol. 11, no. 2, p. 62-65. [CrossRef]
-
ZACCARO,
María Cristina; STELLA, Ana María; GARCÍA, Ileana; EBERLE, Ariel; DÍAZ,
Marina; STORNI DE CANO, Mónica and ZULPA DE CAIRE, Gloria. Organogenesis
induction in rice callus by cyanobacterial extracellular products. Phytomorphology, 2002,
vol. 52, no. 4, p. 263-271.
-
ZULPA
DE CAIRE, Gloria; ZACCARO DE MULÉ, María Cristina and STORNI DE CANO, Mónica.
Productos extracelulares de Nostoc muscorum Ag. (cepa 79a) obtenidos
en medios con y sin nitrógenocombinado. I: Sus efectos sobre plántulas
de arroz. Phyton, 1979, vol. 37, no. 1, p. 1-13.
Note: Electronic Journal of Biotechnology is not responsible
if on-line references cited on manuscripts are not available any more after
the date of publication.
©
2006 by Pontificia Universidad Católica de Valparaíso --
Chile
The following images related to this document are available:
Photo images
[ej06027f2.jpg]
[ej06027f1.jpg]
[ej06027t1.jpg]
|