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42
In Vitro Flowering Studies in
Psygmorchis pusilla
Ana Paula Artimonte Vaz1* • Gilberto Barbante Kerbauy2
1 EMBRAPA Transferência de Tecnologia, Escritório de Negócios de Campinas, Av. Dr. André Tosello 209, Caixa Postal 6062, Campinas, SP, CEP: 13083-970, Brasil
2 Laboratório de Fisiologia Vegetal, Instituto de Biociências, Universidade de São Paulo, Caixa Postal 11461, São Paulo, SP, CEP: 05422-970, Brasil
Corresponding author: * ana@campinas.snt.embrapa.br
Keywords: carbohydrates, hormones, nutrients, Orchidaceae, photoperiodism, temperature
ABSTRACT
In the present study, the effects of medium composition (nutrients, carbohydrates and hormones), photoperiodism and temperature on in vitro
flower formation of Psygmorchis pusilla were investigated. Low nitrogen concentration and high concentrations of potassium and calcium in the
culture medium favoured floral development, with a preferential requirement for ammonium. A positive correlation between flowering and sucrose,
glucose or fructose at different concentrations was also detected. The presence of BA in the culture medium stimulated flowering, but floral buds
developed abnormally. Endogenous levels of Z, ZR and iP increased during the initial development of the floral spike. Long photoperiods did not
affect plant growth and leaf number, but enhanced floral spike development. However, flower anthesis did not occur under photoperiods longer
than 20 hours. The most favourable temperature for Psygmorchis pusilla growth and flower formation in vitro was 27°C. The events of
resumption of floral buds, growth of floral spikes and floral bud anthesis were associated with different optimal media composition, temperatures
and photoperiods.
1. INTRODUCTION
The establishing of a reliable in vitro protocol to promote precocious orchid flowering is an important step for the studies related to the
biochemical, physiological, molecular and genetic mechanisms of flower induction and also for assisting orchid breeding programs (Kostenyuk et
al. 1999; Hee et al. 2007).
Psygmorchis pusilla Dodson and Dressler, a tropical epiphytic species, is especially noteworthy for orchid flowering studies because of the
rapid growth and of the relative short time required to reach maturity. This species is naturally precocious and flowering in flask has been first
obtained by Livingston (1962), but in a sporadically way.
Through a genetic selection and improvement of at least 5 years, abundant and uniform flowered plants have been obtained in flasks,
becoming an useful and favourable material for in vitro studies concerning the biochemical and physiological aspects related to tropical orchid
flower formation.
Various treatments generally related to flowering process were applied to young plants obtained asymbiotically by in vitro germination, such
as temperature, photoperiod, sugars, nutrients and hormones.
2. MATERIAL AND METHODS
2.1. Plant cultures
Seedlings of Psygmorchis pusilla were obtained through asymbiotically germination of immature seeds and grown for 120 days on Vacin and
Went medium (Vacin and Went 1949), modified by substitution of ferric tartarate by 27.8 mg l-1 Fe-EDTA and manganese sulfate by Murashige
and Skoog micronutrients solution (Murashige and Skoog 1962). The medium was added of 6% (w/v) ripe banana pulp, 0.1% (w/v) activated
charcoal and 2% (w/v) sucrose, and solidified with 0.6 % (w/v) agar “Oxoid”.
After 3 months, plants were shifted to fresh medium as described previously but without adding ripe banana pulp and activated charcoal and
submitted to different treatments. The pH of all media was adjusted to 5.8 before autoclaving at 120°C and 1 atm for 15 min. Cultures were
maintained in 250-mL Erlenmeyer flasks containing 80 mL culture medium and sealed with perforated rubber stoppers. The perforations were
filled with cotton on which two drops of a saturated potassium permanganate solution were placed.
®
Abbreviations: ABA, abscisic acid; BAP, benzyladenine purine; GA3, gibberellic acid; IAA, indolyl-acetic acid; iP, isopentenyladenine; [9R]iP, isopentenyladenosine;
NAA, naphthalene acetic acid; TDZ, thidiazuron; Z, zeatin; ZR, zeatin riboside
Vaz and Kerbauy In vitro flowering of Psygomorchis pusilla
2.2. Photoperiod and temperature treatments
To determine the influence of photoperiod on growth and flowering of P. pusilla plants, daylengths of 6, 8, 12, 16, 20, 22 and 24 h (30 μmol m-2 s-1,
Sylvania cool-white fluorescent tubes) and darkness effects were evaluated as described in Vaz et al. (2004).
2.3. Nutritional evaluation
The effects of nutrients on floral spike development were investigated by changing the total mineral salt concentration of standard Vacin and
Went medium. Inorganic N (NH4+ and NO3-), P, K and Ca concentrations were analysed separately as described in Vaz and Kerbauy (2000).
2.4. Hormonal treatments and endogenous quantification
Hormonal solutions of benzyladenine purine (BAP), naphthalene acetic acid (NAA), gibberellic acid (GA3), abscisic acid (ABA) and thidiazuron
(TDZ) were filter-sterilised with 0.45 µm membranes and separately added at 1, 2 and 4 µM to Vacin and Went medium, modified by Vaz (2002)
and supplied with 2% (w/v) sucrose.
Plants were incubated during 48 h in 150-ml Erlenmeyer flasks containing 20 ml liquid culture medium and capped with aluminum foil. After
that, cultures were shifted to 250-ml Erlenmeyer flasks with equal hormone-containing and hormone-free media, solidified with 6% (w/v) agar
“Oxoid” and closed with perforated rubber stoppers. Each treatment involved 3 flasks, each inoculated with 5 plants. The pH of all media was
adjusted to 5.8 before autoclaving at 120°C and 1 atm for 15 min. Tissue culture vials were maintained at a photoperiod of 16 h (Sylvania cool-
white fluorescent tubes, 30 μmol m-2 s-1) and 25 ± 2°C.
After 120 days of culture, floral spike number, leaf number and leaf growth (cm) were scored. Values were compared through one-way
variance analysis and the Bonferroni’s test was used to detect differences (P ≤ 0.05) among treatments.
Endogenous hormonal levels were measured in these plants. Samples of 1g fresh leaves were taken after 10, 20, 30, 40, 50 and 60 days of
culture. Indolyl-acetic acid (IAA), ABA, zeatin (Z), zeatin riboside (ZR), isopentenyladenine (iP) and isopentenyladenosine ([9R]iP) quantification
were based on an indirect ELISA, being the extracts previously purified in reverse-phase HPLC, as described by Peres et al. (1997) and Zaffari
et al. (1998). Hormonal analyses were carried out in triplicate and contents were expressed as the mean ± standard errors. Results were
subjected to one-way variance analysis and Tukey’s test were used to detect differences (P ≤ 0.05) among treatments.
2.5. Extraction and analysis of carbohydrates
Fresh samples of shoot axis and leaves were washed thoroughly, weighted, homogenised and extracted 3 × with 80% (v/v) ethanol at 80°C for 5
min. After centrifugation for 10 min at 3,000 × g, the ethanolic supernatants, consisting of the soluble carbohydrate fraction, were combined and
concentrated under vacuum at 35°C. Total carbohydrate was determined colorimetrically in triplicate using the phenol-sulphuric method (Dubois
et al. 1956). Carbohydrate content was expressed as glucose equivalents. Enzymatic starch extraction and quantification were performed from
the residue by amyloglucosidase-peroxidase method, according to Arêas and Lajolo (1980). Carbohydrate analyses were carried out in triplicate
and contents were expressed as the mean ± standard errors. Results were subjected to one-way variance analysis and Tukey’s test was used to
detect differences (P ≤ 0.05) among treatments.
2.6. Anatomical analyses
Tissues from shoot axis of plants of 7-months-old cultivated on hormone-free Vacin and Went medium were taken after incubation for 60 days, at
10 days intervals. Samples were fixed in FAA50 (formalin-acetic acid, 50% ethanol), dehydrated and embedded in paraffin using standard
methods. Longitudinal sections (12 μm) from lateral meristems were stained with hematoxilin and a solution of safranin 1% and astra blue 1%,
9:1 (v/v) as modified by Kraus and Arduin (1997).
3. RESULTS AND DISCUSSION
Manual cross-pollination was done under aseptic conditions,
allowing the development of normal fruits (Fig. 1) bearing many
fertilised seeds (Fig. 2). The protocorm grows horizontally, for-
ming creeping structures and giving a dorsiventral form to the
embryo (Figs. 3A, 5B). These crests may be modified leaves as
suggested for Phalaenopsis by Nishimura (1981). Many absor-
bent hairs develop on the basal side of the protocorm (Fig. 3C),
showing ramifications at a later stage (Fig. 3D).
This species behave more conspicuously as a quantitative
long-day (short-night) plant in terms of flowering, reflected by
significant increases in floral spike number and endogenous
carbohydrate contents with lengthening light period. Floral spike
formation was also observed under darkness, being this un-
usual response ascribed to sugar absorption from the medium
(Vaz et al. 2004). In this case, a very precocious flower induc-
tion of lateral buds should not be completely discarded, remain-
ing the question involving in vitro flowering induction of P. pusilla.
A negative correlation between mineral salt concentration
and the number of floral spikes was observed. Low concentration of N stimulated flower formation, being this effect more pronounced than that
Fig. 1 In vitro Psygmorchis pusilla flowering plant, bearing a green fruit.
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Vaz and Kerbauy In vitro flowering of Psygomorchis pusilla
observed for the lowest total salt level used. Pre-
sence of NH4+ as the sole nitrogen source in the
medium showed to be essential for plant growth and
development, while NO3- absence depressed flower-
ing. Flower formation was also enhanced by K and
Ca medium enrichment (Vaz and Kerbauy 2000).
Flowering plants showed prominent increases in
endogenous levels of Ca and K when compared to
non-flowering ones, whereas higher contents of Mg
and P were found in these latter. Sulfur level was
similar in both plants (Fig. 4).
In spite of various hormonal approaches repor-
ted to induce in vitro and ex vitro flowering in different
orchid species (Hew and Clifford 1993; Chen et al.
1997; Chia et al. 1999; Kostenyuk et al. 1999; Su et
al. 2001; Ferreira et al. 2006), treatments of NAA,
GA3, ABA and TDZ were inhibitory to reproductive
development of P. pusilla. However, they did not
totally block flower spike formation, suggesting an
interaction with other signals. Again, a very preco-
cious reproductive induction in these plants may be
considered.
A positive correlation between floral spike forma-
tion and BAP was observed, but a pulse of this cyto-
kinin, by means of plant immersion in hormone-con-
taining liquid media for 48 hours, was not
sufficient to enhance floral spike formation
after plants were transferred to hormone-
free media. Continuous presence of BAP in
the medium showed to be important for
flowering process, being 1 µM the most
efficient concentration. Higher concentra-
tions did not lead to a further increase in
floral spike number (Fig. 5).
The requirement of a continuous pre-
sence of BAP in the media to promote floral
spike formation in P. pusilla suggests an
important role of cytokinin in this process.
However, the fact that flower buds were
initiated on media containing no cytokinin
indicates that enhanced levels of these hor-
mones may not be required for floral transi-
tion or development, or alternatively, the
plants could be able to supply enough endo-
genous cytokinin levels for these processes.
ABC
Fig. 2 Psygmorchis pusilla mature and dry seeds. (A) Electronic micrograph of seed (x140). (B) Detail of seed central region (x500). (C) Detail of seed apical region
(x500).
AB
CD
AB
CD
Fig. 3 Electronic micrograph of a 3-months-old protocorm. (A) in frontal (x70), (B) and lateral view
(x50). (C) Development of epidermic hairs (x400), (D) showing ramifications (x500).
0 1020304050
K
S
P
endogenous nutrients
g kg-1(DW)
control flow ering non-f low ering
0,0 0,5 1,0 1,5 2,0
Ca
Mg
Fig. 4 Endogenous nutrient levels (g kg-1) of K, S, P, Ca and Mg extracted from 4-month-old plants of
Psygmorchis pusilla. (A) control, (B) flowering and (C) non-flowering medium.
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Vaz and Kerbauy In vitro flowering of Psygomorchis pusilla
No doubt, the presence of BAP in
the media induced a number of devia-
tions in flower development in P. pusilla
plants, including delay in anthesis, forma-
tion of unusual secondary floral spike and
alterations in floral organs, mainly incom-
plete sepal and labellum formation. Cyto-
kinins could be involved in controlling the
expression of floral meristem genes,
thereby affecting later stages of flower
development and floral organ differentia-
tion, as suggested by Venglat and Sawh-
ney (1996) for Arabidopsis. According to
various authors, higher cytokinin levels
can negatively affect flower development
in orchid plants cultivated in vitro (Hew
and Clifford 1993; Duan and Yazawa
1995a, 1995b; Hew and Yong 1997; Hee
et al. 2007). The failure of most P. pusilla
flowers to reach anthesis at BAP treatments
could result from different hormonal require-
ments at various stages of floral development.
The precise role of hormones in long-lasting
developmental processes is far from being eluci-
dated.
Higher levels of endogenous Z, ZR and iP
were observed during the resumption of floral
meristem development and floral organ differen-
tiation (Fig. 6A). The [9R]iP content was rela-
tively low as compared to the others quantified
cytokinins (data not shown).
In P. pusilla plants, cytokinin is apparently
necessary to resume and to maintain cell divi-
sions at floral meristem and floral organ develop-
ment, as indicated by endogenous levels of Z
and ZR. The relatively slow development of floral
spikes on media without BAP is likely to reflect
the limited ability of plants to produce its own
cytokinin under these conditions. In apical shoot
meristem of tobacco, Dewitte et al. (1999) ob-
served increased cytokinins concentrations
during initiation of petals and sepals and further
development of flowers, while relatively low
levels were detected during the re-organization
of the meristem prior to flowering. The authors
suggested a sink effect of areas with high divi-
sion activity, both mitotic and meiotic.
Our observations support the idea that en-
hanced cytokinin levels in P. pusilla is essential
for cell differentiation during reproductive or-
ganogenesis, but may not be sufficient for floral
evocation in the strictest sense.
Endogenous ABA contents showed constant increases during floral spike development (Fig. 6B). Some authors have related ABA levels to
an improvement of carbohydrate metabolism during plant flowering (Tanimoto et al. 1985; Ofosu-Anim et al. 1996; Ito et al. 1999). A slight
increase in IAA content was also observed during the final floral bud development, but this result is of difficult interpretation (Fig. 6B).
Floral bud development was accompanied by glucose and fructose consumption (Fig. 7). The monosaccharides were obtained by sucrose
hydrolysis and starch mobilization (data not shown), providing energy and structural components to floral organs development. Carbohydrate
metabolism has long been associated with the flowering process, acting both as an energy source and/or as gene regulator molecules.
Flower anthesis was just observed under a distinct culture condition (photoperiod inferior to 20 hours and 27°C temperature), different from
other favourable conditions for previous stages of floral spike development. An adequate balance between sugar, nitrogen and hormones was
essential to reach normal flowers in vitro, as it reduces stress responses, sink competition, increases in water potential and loss of
photosynthetic pigments.
0
2
4
6
8
10
12
14
16
0 102030405060708090
incubation time (days )
pmol / gFW of Z and Z R
0
1
2
3
4
pmol/gFW of iP
ZZR iP
A
0
50
100
150
200
0 102030405060708090
incubation time (days)
pmol / g FW of ABA
0
1
2
3
4
5
6
7
8
pmol / g FW of IAA
ABA IAA
B
Fig. 6 Hormonal endogenous levels (pmol gFW-1) extracted from flowering 4-month-old plants of
Psygmorchis pusilla. (A) cytokinins Z, ZR and iP, (B) ABA and IAA.
0
1
2
3
4
0 30 60 90 120
incubation time (days )
floral spike/ plant
BA 0 BA 1 BA 2 BA 4
Fig. 5 Hormonal effects on in vitro Psygmorchis pusilla flowering. Average floral spike number in plants
previously incubated during 48 h in basal liquid medium (BA 0) and liquid media with different BAP concentrations (1,
2 and 4 µM), and then shifted to solidified media with the same hormonal composition. Mean separation (n=15) by
Bonferroni’s test at p ≤ 0.05.
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Floriculture, Ornamental and Plant Biotechnology Volume V ©2008 Global Science Books, UK
Vaz and Kerbauy In vitro flowering of Psygomorchis pusilla
Despite the great complexity in-
volved in flowering process, our results
suggest that, in P. pusilla cultivated in
vitro, during the different stages of flower
development (Fig. 8), i.e. resumption of
floral buds, growth of floral spikes and
floral bud anthesis, plants require dif-
ferent media composition, temperatures
and photoperiods. More conspicuous
flowering in these plants showed to be
dependent on the size and number of
leaves, indicating that floral development
is somewhat linked to vigour during vege-
tative growth.
Largely different from other studied
orchids which show ability to form repro-
ductive structures under in vitro condi-
tions, the flower buds of Psygmorchis
pusilla can easily reach the anthesis
stage, producing beautiful small stalk
with various yellow flowers. Its apparent
higher tolerance to ethylene makes this
species an interesting material for basic
and applied research, as well as for com-
mercial proposes (Fig. 9). Further mole-
cular approaches comparing wild and im-
proved flowering genotypes will provide a
better understanding of its flowers deve-
lopment.
ACKNOWLEDGEMENTS
We thank Dr. Rita de Cássia Ribeiro-Figueiredo for carbohydrate analyses. Psygmorchis pusilla studies were supported by Fundação de Amparo à Pesquisa do Estado de
São Paulo (FAPESP, São Paulo, Brazil).
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