The effect of exogenous and endogenous phytohormones on the in vitro development of gametophyte and...

ORIGINAL PAPER

The effect of exogenous and endogenous phytohormoneson the in vitro development of gametophyte and sporophytein Asplenium nidus L.

V. Menendez • Y. Abul • B. Bohanec •

F. Lafont • H. Fernandez

Received: 27 January 2011 / Revised: 9 May 2011 / Accepted: 30 May 2011 / Published online: 12 June 2011

� Franciszek Gorski Institute of Plant Physiology, Polish Academy of Sciences, Krakow 2011

Abstract The fern Asplenium nidus L. is in great demand

as an ornamental plant. The aim of this work was to inves-

tigate the influence of phytohormones in promoting a

gametophytic and sporophytic growth in homogenized

sporophytes tissue. Exogenous application of 0.5 and

5 lM N6-benzyladenine, 0.05 and 0.5 lM indole-3-acetic

acid (IAA), and 0.3 and 3 lM gibberellic acid (GA3)

favoured sporophyte regeneration, whereas gametophyte

regeneration took place when plant material was cultured in

a hormone-free liquid MS medium. The endogenous

contents of the auxin IAA, the cytokinins trans-zeatin, trans-

zeatin riboside, dihydrozeatin, dihydrozeatin riboside,

isopentenyladenine and isopentenyladenosine, and the gib-

berellins GA1, GA3, GA4, GA7, GA9 and GA20 in growing

gametophytes and sporophytes were evaluated. Similar

levels of the auxin and cytokinins and qualitative differences

in the gibberellins were found between both generations.

Keywords Alternation of generations � Apospory �Asplenium nidus L. � Auxin � Cytokinin � Fern �Gametophyte � Gibberellin � Morphogenesis � Ornamental

Abbreviations

BAP N6-benzyladenine

CKs Cytokinins

DHZ Dihydrozeatin

DHZR Dihydrozeatin riboside

DW Dry weight

FW Fresh weight

GAs Gibberellins

HPLC-MS/MS High-performance liquid

chromatography-tandem mass

spectrometry

IAA Indole-3-acetic acid

iP Isopentenyladenine

iPR Isopentenyladenosine

MS Murashige and Skoog nutrient medium

(1962)

tZ Trans-zeatin

tZR Trans-zeatin riboside

Introduction

A fern exists in two distinct forms during its life cycle: the

small, simple, haploid gametophyte and the large, mor-

phologically complex, diploid sporophyte, and the two

generations are isolated from each other by meiosis and

fertilization. The heteromorphic alternation of generations

in ferns offers the opportunity to understand the ontogeny

of the alternation. The use of the fern life cycle for

investigation avoids the inherent difficulties of studying the

individual generations when sporophyte and gametophyte

are in intimate association.

The regeneration capacity of sporophytic tissue of

Asplenium nidus L. cultured in liquid media has been

Communicated by P. K. Nagar.

V. Menendez � Y. Abul � H. Fernandez (&)

Lab of Plant Physiology, Department of BOS,

c) Catedratico R Urıa s/n, 33071 Oviedo, Spain

e-mail: [email protected]

B. Bohanec

Biotechnical Faculty, Centre for Plant Biotechnology

and Breeding, Jamnikarjeva 101, 1111 Ljubljana, Slovenia

F. Lafont

Mass Spectrometry Unit, Edificio Ramon y Cajal,

Campus Rabanales, Cordova, Spain

123

Acta Physiol Plant (2011) 33:2493–2500

DOI 10.1007/s11738-011-0794-9

reported (Fernandez et al. 1993, 1997) and two organiza-

tion patterns, sporophyte and gametophyte, were observed

from the explants. The differentiation pattern followed by

gametophyte and sporophyte cells in ferns may be affected

by both nutritional and environmental factors, and includes

the loss of close cell to cell communication and subsequent

washing out of certain bioactive compounds (Lucas et al.

2009; Wang and Fiers 2010), and the content dynamics and

potential functioning of phytohormones affecting several

developmental processes in ferns such as germination, sex

determination, alternation of generations without fertiliza-

tion (apogamy) or sporogenesis (apospory), etc. (Banks

1999; Kazmierczak 2003; Menendez et al. 2006, 2009;

Greer et al. 2009; Abul et al. 2010; Somer et al. 2010).

In an attempt to study the role of phytohormones on the

regeneration pattern of sporophytic tissue of A. nidus

towards gametophyte or sporophyte, the effect of the

addition of auxins, cytokinins and gibberellins to the cul-

ture medium was investigated, and the endogenous content

of the auxin IAA, the cytokinins tZ, tZR, DHZ, DHZR, iP

and iPR, and the gibberellins GA1, GA3, GA4, GA7, GA9

and GA20 in gametophyte and sporophyte generations was

evaluated.

Materials and methods

Spore culture

Spores (5 mg) of A. nidus L. obtained from sporophytes

growing in the greenhouse at the Oviedo University (Spain)

were soaked in water for 2 h and then washed for 10 min

with a solution of NaClO (0.5% w/v) containing Tween 20

(0.1% w/v). Then they were rinsed three times with sterile

distilled water. Spores were centrifuged at 700g for 3 min

between rinses and cultured in 100 ml flasks containing

20 ml Murashige and Skoog (1962) medium (MS) sup-

plemented with 2% (w/v) sucrose and 0.7% (w/v) agar and,

unless otherwise noted, the pH was adjusted to 5.7 with 1

or 0.1 N NaOH. The cultures were maintained at 25�C

under cool-white fluorescent light (40 lmol m-2 s-1) with

a 16:8 h light:dark photoperiod. Once germinated, the

gametophytes were transferred to fresh media and watered

to induce fertilization. Isolated sporophytes were cultured

in MS medium with 2% (w/v) sucrose and 0.7% (w/v) agar.

Homogenized cultures from sporophytes

Six-month-old sporophytes (0.3 g fresh weight), formed by

sexual means from spore-derived gametophytes, were

mechanically fragmented using a Waring blender for 15 s

under aseptic conditions. The homogenized tissue samples

were cultured separately in 250 ml Erlenmeyer flasks that

contained 50 ml of liquid MS medium with 2% (w/v)

sucrose and one of the following phytohormones: IAA at

0.05 or 0.5 lM; BAP at 0.5 or 5 lM and GA3 at 0.3 or

3 lM. The liquid cultures were placed on a gyratory shaker

(75 rpm). After 2 months, the explants were transferred to

transparent Petri dishes divided into 25 squares each with

3 ml MS medium with 2% (w/v) sucrose and 0.7% (w/v)

agar, and cultured for 3 months.

Quantification of endogenous auxin, cytokinins

and gibberellins

Samples of gametophytes and sporophytes were taken for

analyses, according to a modified protocol of Villacorta

et al. (2008). The gametophytes used for the hormone

analyses originated from spores and the sporophytes from

spore-derived gametophytes. Each sample (300 mg DW)

was frozen in liquid nitrogen, lyophilized and stored at

-20�C until analysis. The plant material was homogenized

in 15 ml of cold 80% methanol containing 50 ng of deuterated

[2H5]IAA, [2H5]tZ, [2H5]tZR, [2H5]DHZ, [2H5]9DHZR,

[2H6]iP, [2H6]iPR, [2H2]GA1, [2H2]GA3, [2H2]GA4,

[2H2]GA7, [2H2]GA9 and [2H2]GA20 (OldChemIm�,

Olomouc, Czech Republic). After filtration, the residue was

re-extracted with 5 ml of 80% methanol for 2 h and

re-filtered. The obtained two fractions were pooled and

passed through a C18 solid phase extraction, (SPE),

(500 mg) to remove the pigments. The organic solvents

were evaporated under reduced pressure at 40�C, the pH of

the water phase was adjusted to pH 7 and distilled water

was added to a final volume of 20 ml. The sample was

placed onto a pre-equilibrated DEAE-Sephadex A-25

(Amersham Pharmacia Uppsala, Sweden) with a pre-

equilibrated C18 SPE cartridge (BondElut� Varian, Ham-

burg, Germany) coupled underneath. The DEAE cartridge

was eluted with 10 ml of 2 M NH4HCO3, recovering

cytokinins, whereas the C18 cartridge was eluted with

10 ml of 80% methanol, recovering GAs and IAA. This

eluate was absorbed onto 0.5 g of Celite and then purified

using a column of silica (SiO2). The cytokinin fraction was

then placed onto another C18 SPE cartridge and eluted

with 10 ml of ice cold 80% methanol (-20�C). Methanol

was evaporated under reduced pressure at 40�C. The

remaining water phase had 7 ml of PBS buffer added and

was then purified further using an inmunoaffinity column

for isoprenoid cytokinins, according to the manufacturer’s

instructions (OldChemIm�, Olomuc, Czech Republic).

Cytokinins were eluted with 2 ml ice-cold methanol. The

samples were then evaporated under reduced pressure at

room temperature until dried. Phytohormones were ana-

lysed by HPLC/MS carried out on a Varian 1200l triple

quadrupole, working in positive electrospray ionization

mode (ESI?) with a capillary voltage of 5,500 V and acid

2494 Acta Physiol Plant (2011) 33:2493–2500

123

voltage of 40 V. HPLC/MS analysis was carried out by

MRM (multiple reaction monitoring) ion detection mode

working with three transitions for each compound. Liquid

chromatography (LC) was performed using a Polaris 3 lm,

150 9 2.1 mm I.D. analytical column, maintained at 40�C.

The mobile phases consisted of water/0.1% formic acid

(A) and methanol/acetonitrile 25/75 (B). The flow-rate was

0.2 ml/min. In each case, 20 ll of the sample was injected.

The gradients used for auxin and gibberellins were:

t = 0 min (90% A, 10% B); t = 1 min (75% A, 25% B);

t = 10 min (0% A, 100% B) and t = 15 min (0% A, 100%

B) and the gradients for cytokinins were: t = 0 min (92%

A, 8% B); t = 2 min (65% A, 35% B); t = 9 min (0% A,

100% B) and t = 13 min (0% A, 100% B). The levels of

phytohormones in the plant samples were determined from

the area ratios of endogenous to corresponding deuterated

phytohomones.

A curve was prepared always with the same quantity of

isotope labelled added to samples and with concentrations

from 1 to 250 ppb for the compounds analysed. The min-

imum quantification level was 1 ppb (1 ng per ml) for each

compound.

Light microscopy

Observations of plant materials were conducted when

required using an optical microscope (Nikon Eclipse E600)

and photographic equipment (DS Camera Control Unit

DS-L1), taking samples of fresh material from cultures.

Laser scanning confocal microscopy

Plant material was mounted in VectaShield (VECTA-

SHIELD� Vector Laboratories). The autofluorescence of

each of the samples was captured using a laser confocal

microscope, Leica TCS-SP2-AOBS, using 109 and 209

dry objectives and 409 oil immersion objectives. The

green fluorescence was excited with an argon/krypton ion

laser at a wavelength of 488 nm with a detection range of

500–550 nm. The red fluorescence was excited with a

helium–neon laser at a wavelength of 633 nm with a

detection range of 649–702 nm. A transmission image was

also captured. Z-image stacks of between 8 and 45 optical

sections were collected at intervals of 0.94–2.65 l. Maxi-

mum projections were made for each explant.

Data collection and statistical analyses

Statistical analyses were done using SigmaStat� v3.1

software. Deviation from normality and homogeneity of

variance was tested with Shapiro–Wilk and Barlett-Box

tests, respectively. Non-parametric data were preferably

analysed using the 2 9 2 Chi-square test (v2) of frequency,

but Fisher’s test was used when necessary. Experiments

were repeated twice with similar results. Each sample of

data for quantification of endogenous phytohormones was

analysed three times. Analysis of variance (ANOVA) with

post-hoc Sheffe comparisons was used. The level of sig-

nificance was set at a = 0.05 for all tests (Zar 1998).

Results

Effect of phytohormones in homogenized cultures

of sporophytes

Data on the regeneration pattern observed in homogenized

cultures of sporophytes of A. nidus are shown in Fig. 1.

Initially, the explants became brown (Fig. 1a) and after

1–2 weeks, some cells start to divide and form white-

coloured proliferation centres (Fig. 1b, c). Later, these

developed into sporophytic buds when phytohormones

BAP, GA3 or IAA were added to the medium (Fig. 1d, e)

and into aposporous gametophytes when cultured in a

hormone-free medium (Fig. 1f).

After 90 days, the percentage of explants forming

unrecognized structures (stage I) (Fig. 1c) and explants

forming recognized structures, gametophytes or sporo-

phytes, (stage II) (Fig. 1d–f) was noted and significant dif-

ferences were found between the treatments (v52 = 32.735,

p \ 0.001) (Fig. 2). The addition of GA3 or BAP to the

medium favoured the formation of proliferation centres in

the most part of sporophytic explants that finally did not

evolve to visible sporophytes. The numbers of recognized

structures in MS media without phytohormones (gameto-

phytes) or with IAA (sporophytes) (v12 = 0.0132,

p = 0.908) were higher than in the presence of BAP

(v12 = 16.376, p \ 0.001; Yates correction was used in

calculating this test) or GA3 (at 0.3 lM, p \ 0.049 and at

3 lM, p \ 0.001, respectively, both using Fisher’s test).

The regenerated structures cultured under the above

conditions were observed by laser confocal microscopy.

The incidence of different laser waves emitted green or red

autofluorescence by cells. Cells lacking chlorophyll pig-

ments (initial proliferation centres) were emitted in green

colour and cells with chlorophyll pigments (developing

gametophytes or sporophytes) were emitted in red colour

(Fig. 3).

Endogenous content of auxin, cytokinins

and gibberellins

The auxin IAA, the cytokinins tZ, tZR, DHZ and iP, and

the gibberellins GA1, GA3, GA4 and GA9 were detected in

gametophytes and sporophytes (Fig. 4). No significant

differences in the endogenous content of IAA were found

Acta Physiol Plant (2011) 33:2493–2500 2495

123

among both phases of the life cycle (Fig. 4a). The cyto-

kinins tZR and iP were detected at higher levels than Z and

DHZ, but no significant differences between these

cytokinins were noted among gametophytes and sporo-

phytes (Fig. 4b). The endogenous content of IAA was

250–300 pmol/g DW, which was higher than that of the

cytokinins, where the levels were \50 pmol/g DW. Gib-

berellins GA1, GA3, GA4 and GA9 were detected in the

sporophytic tissue and the GA content was the highest of

all the hormone groups quantified. The content of GA4 was

particularly high in the sporophyte (Fig. 4c). Only gib-

berellin GA9 was detected in gametophytic tissue.

Discussion

This works reports that the hormonal status of both culture

medium and explant influences the morphogenic response

in homogenized sporophytic tissue of A. nidus. In the

homogenate cultures, where the plant tissue was disrupted

by mechanical means, the majority of cells lost their

functions and died. The few surviving cells were able to

Fig. 1 Regeneration processes

in homogenized sporophytes of

A. nidus L. a initial explant,

b recovering of division

capacity by some cells, c white-

coloured proliferation centres

without apparent organization,

d incipient primordial frond,

e frond elongation, f aposporous

gametophytes. In all pictures

bar indicates 1 mm

0

10

2030

40

50

60

7080

90

100

Control GA3 (3) GA3 ( 0.3) BAP (5) IAA (0.5) IAA (0.05)

%

Culture conditions (µM)

I

II

Fig. 2 Percentage of explants without visible regenerated structures

(Stage I) and forming well-defined structures (Stage II), from

homogenized sporophytes of A. nidus L., cultured in liquid MS

medium with GA3 (0.3 or 3 lM), BAP (5 lM) or IAA (0.05 or

0.5 lM). Data after 90 days


123

preserve their capacity for division and organization and

eventually formed complete individuals—either gameto-

phytes or sporophytes depending on the culture conditions.

Homogenization of plant material favours that cellular

connections become lost and the surviving cells might

evolve following the same developmental program as did

before to be cultured or another different. The stress

imposed on cells when they are mechanically separated

from each other has been considered in the terms that a

stress signal may be produced during homogenisation,

which, together intercellular interaction, may affect the

expression of specific genes, driving the morphogenetic

events towards gametophyte or sporophyte regeneration

(Teng and Teng 1997).

In previous studies, it has been reported that the dif-

ferentiation pattern followed by sporophyte cells in ferns

may be affected by many factors, such as the physiological

isolation of cells, the age of plant material, nutrients

(sucrose levels), exogenous phytohormones and the phys-

ical state of the culture medium (Teng and Teng 1997;

Fernandez and Revilla 2003; Somer et al. 2010). In

A. nidus, both the physiological isolation of cells and the

new balance of phytohormones in the cells result in one of

two organization patterns to be activated in the sporophytes

explants. The role of phytohormones triggering gameto-

phyte or sporophyte organization is evident during the

initial steps in the progression of one or a few sporophyte

cells in the culture. Before the mechanical disruption takes

place in the sporophytic tissue, the balance of endogenous

phytohormones into the cells, among other factors, allows

sporophyte organization and represses the gametophyte.

After disruption of cellular connections, these balances

become lost and reprogramming gametophyte development

is possible depending upon the new balance of phytoho-

mones in the cells.

The addition of phytohormones to the culture medium

promoted the formation of proliferation centres in the

sporophytes explants. The capacity of these centres to

regenerate sporophytes varied with the phytohormone: the

higher concentration of the auxin IAA allowed sporophyte

regeneration in round about 70% of explants, but GA3 and

BAP stopped organogenesis and the most part of explants

remain at this stage. Auxins increase the growth rate, thus

existing a strong correlation between auxin accumulation

and organ emergence (Reinhardt et al. 2003; Smith et al.

2006). Concerning both GA3 and BAP, perhaps it would

need necessary to short the period of time explants make

contact with the phytohormones, and continue their culture

Fig. 3 A light photograph

(above) and a laser confocal

photograph (below) taken of

regenerated structures in

homogenized sporophytes of

A. nidus L., a cells lacking

chlorophyll pigments emitted a

green colour and b cells with

chlorophyll pigments emitted a

red colour (colour figure online)


123

in a hormone-free medium to allow organogenesis. In any

case, the addition of phytohormones to the culture medium

promoted apospory in homogenate cultures of sporophyte

of A. nidus.

The effect of phytohormones on gametophyte and spo-

rophyte regeneration from sporophytic cells seems to vary

between species. For example, in homogenized cultures of

sporophytes of Adiantum capillus-veneris, Davallia ca-

nariensis and Polypodium cambricum, addition of the

cytokinin BAP to the medium induced the formation of

aposporous gametophytes and/or dedifferentiated cellular

aggregates, whereas the lack of BAP favoured sporophyte

regeneration. Conversely, in Asplenium adiantum-nigrum,

BAP promoted the regeneration of both aposporous game-

tophytes and sporophytes (Somer et al. 2010). Aposporous

gametophytes are produced in Drymoglossum piloselloides

(L.) in both juvenile and older fronds cultured in a modified

MS medium supplemented with kinetin (Kwa et al. 1988),

while low levels of auxins and cytokinins promoted

apospory and high levels of calli in Pteris vittata L. pinnae

strips (Kwa et al. 1995). Moreover, Kwa et al. (1997)

described different morphogenetic capacities in two types

of callus cultured in a hormone-free medium, despite their

common origin, in the fern Platycerium coronarium.

Recently, Martin et al. (2006) found a synergistic effect

from a combination of the cytokinins BAP and kinetin that

induced apospory in fronds and crozier explants of silver

fern Pityrogramma calomelanos L. A wide range of culture

conditions can affect morphogenesis in sporophytic tissues

of ferns; therefore, each species or part of the plant demands

a singular study of its behaviour when cultured in vitro.

In this study, differences in the light emitted by different

regenerated structures were shown for the first time, using

confocal microscopy, highlighting the differentiation pro-

cess that occurs in the proliferation structures derived from

explants until sporophyte and gametophyte organization

was evident. During the initial steps of regeneration process,

the cells are white-coloured that mostly emitted in green

colour, whereas regenerated gametophyte and sporophyte

structures that are full of chloroplasts emitted in red colour,

due to the presence of chlorophyll. Confocal imaging could

be useful to follow in detail the spatial organization of a new

gametophyte or sporophyte from the sporophytic cells.

Analyses revealed the presence of endogenous levels of

several phytohormones in the growing gametophyte and

sporophyte of A. nidus L. Unfortunately, few studies have

been done on the presence of the major phytohormones in

different tissues of lower plants for comparison with those

of higher plants. The levels of IAA found in the gameto-

phytes and sporophytes are similar to those detected in the

water fern Salvinia molesta (Arthur et al. 2007) and in

sporophytic tissue of Psilotum nudum (Abul et al. 2010).

Also, a certain degree of uniformity is observed in the

cytokinin content. In both generations of A. nidus L. were

found higher levels of the cytokinins tZR and iP than of tZ

and DHZ. The levels of tZ detected were the lowest,

\5 pmol/g DW. Levels particularly high of tZR and iP

were recently found in sporophytic tissue of P. nudum

(Abul et al. 2010). Following with lower plants, Ivanova

et al. (1992) reported that Chlamydomonas reinhardtii had

high amounts of iP-type cytokinins (90%); tZ and DHZ

were also present. For the multicellular green algae

Cladophora capensis and Ulva sp., a prevalence of both iP

and cZ-type cytokinins was found (Stirk et al. 2003). We

did not analyse cZ but the levels of tZ, however, were low.

Finally, Menendez et al. (2009) reported levels especially

high of iP and iPR linked to sex, in female gametophytes of

Blechnum spicant. From our experience, high levels of

iP-type cytokinins seem to be a frequent event in ferns.

The analyses of GAs revealed the presence of gibber-

ellins belonging to both the 13-hydroxylation and non-

hydroxylation pathways in sporophytic tissues of A. nidus,

200

220

240

260

280

300

SG

plant tissue

pm

ol I

AA

/ g D

W

0100200300400500600700

G S

plant tissue

pm

ol/g

DW

GA1

GA3

GA4

GA7

GA9

GA20

0

10

20

30

40

50

60

G S

plant tissue

pm

ol/g

DW

Z

ZR

DHZ

DHZR

iP

a

b

c

Fig. 4 Endogenous content of phytohormones in gametophyte

(G) and sporophyte (S) of A. nidus L., a indole-3-acetic acid,

b cytokinins and c gibberellins


123

with an especially high level of GA4, whereas only GA9

was observed in gametophytes. In sporophytic tissues of

P. nudum, the non-hydroxylation pathway appears to be

preferential, with a strong presence of GA4 (Abul et al.

2010). In other species of ferns such as B. spicant, analyses

of gametophytic and sporophytic tissues have revealed the

presence of a wider range of GAs belonging to either the

early 13-OH pathway or the non-OH pathway (Menendez

et al. 2006). Gibberellins are involved in a wide range of

developmental processes in vascular plants, including the

induction of cell division and elongation. Recently, it has

been suggested that the hormonal use of GAs was central

to the evolution of vascular plants (Hirano et al. 2007;

Yasamura et al. 2007). Ferns are in the middle way

between mosses, in which GAs may not be utilized, and

higher plants (von Schwartzenberg et al. 2007). Some

differences of the endogenous content of GAs observed in

the mentioned reports support the necessity to explore

in the endogenous content of these compounds as well as in

the molecular mechanisms acting in different phylogenetic

branches into this plant group.

The effect of phytohormones on the regeneration pro-

gram followed by sporophyte cells of A. nidus together the

cellular disruption seems to be influential in the initial steps

of recovering division activity by these cells and gene

reprogramming. Analyses of the endogenous content of

phytohormones in growing gametophytes and sporophytes

revealed possible differences in the metabolism of

gibberellins between both generations in this species. The

information gained here might help us in the establishment

of accurate protocols for controlling sporophyte or game-

tophyte regeneration, with important theoretical and prac-

tical repercussions.

Acknowledgments The authors thank to Science and Technologies

Ministry for supporting this research through the project MEC2006

CGL 06578, and also FYCIT Program Severo Ochoa.

References

Abul Y, Menendez V, Gomez-Campo C, Revilla MA, Lafont F,

Fernandez H (2010) Occurrence of phytohormones in Psilotumnudum. J Plant Physiol 167:1211–1213

Arthur GD, Stirk WA, Novak O, Hekera P, van Staden J (2007)

Occurrence of nutrients and plant hormones (cytokinins and

IAA) in the water fern Salvinia molesta during growth and

composting. Environ Exp Bot 61:137–144

Banks JA (1999) Gametophyte development in ferns. Annu Rev Plant

Physiol Plant Mol Biol 50:163–186

Fernandez H, Revilla MA (2003) In vitro culture of ornamental ferns.

Plant Cell Tissue Organ Cult 73:1–13

Fernandez H, Bertrand AM, Sanchez-Tames R (1993) In vitro

regeneration of Asplenium nidus L. from gametophytic and

sporophytic tissue. Sci Hortic 56:71–77

Fernandez H, Bertrand A, Sanchez-Tames R (1997) Plantlet regen-

eration in Asplenium nidus L. and Pteris ensiformis L. by

homogenization of BA treated rhizomes. Sci Hortic 68:243–247

Greer GK, Dietrich MA, Stewart S, Devol J, Rebert A (2009)

Morphological functions of gibberellins in leptosporangiate fern

gametophytes: insights into the evolution of form and gender

expression. Bot J Linn Soc 159:599–615

Hirano K, Nakajima N, Asano K, Nishiyama T, Sakakibara H,

Kojima M, Katoh E, Xiang H, Tanahashi T, Hasaebe M, Banks J,

Ashikari M, Kiatano H, Ueguchi-Takana M, Matsuoka M (2007)

The GID1-mediated gibberellin perception mechanism is con-

served in the lycophyte Selaginella moellendorfii but not in the

bryophyte Physcomitrella patens. Plant Cell 19:3058–3079

Ivanova M, Todoruv I, Pashankov P, Kostova L, Kaminek M (1992)

Estimation of cytokinins in the unicellular green algae Chla-mydomonas reinhardtii Dang. In: Kaminek M, Mok D, Zazi-

malova E (eds) Physiology and biochemistry of cytokinins in

plants. SPB Academic Publishing, The Hague, pp 483–485

Kazmierczak A (2003) Induction of cell division and cell expansion at

the beginning of GA3-induced precocious antheridia formation

in Anemia phyllitidis gametophytes. Plant Sci 165:933–939

Kwa SH, Wee YC, Loh CS (1988) Production of aposporous

gametophytes from Drymoglossum piloselloides (L.) Presl.

fronds strips. Plant Cell Rep 7:142–143

Kwa SH, Wee YC, Lim TM, Kumar PP (1995) IAA-induced

apogamy in Platycerium coronarium (Koening) Desv. Gameto-

phytes cultured in vitro. Plant Cell Rep 14:598–602

Kwa SH, Wee YC, Lim TM, Kumar PP (1997) Morphogenetic

plasticity of callus reinitiated from cell suspension cultures of the

fern Platycerium coronarium. Plant Cell Tissue Organ Cult

48:37–44

Lucas WJ, Ham BK, Kim JY (2009) Plasmodesmata—bridging the

gap between neighboring plant cells. Trends Cell Biol

19:495–503

Martin KP, Sini S, Zhang CL, Slater A, Madhusoodanan PV (2006)

Efficient induction of apospory and apogamy in vitro in silver

fern (Pityrogramma calomelanos L.). Plant Cell Rep

25:1300–1307

Menendez V, Revilla MA, Bernard P, Gotor V, Fernandez H (2006)

Gibberellins and antheridiogen on sex in Blechnum spicant L.

Plant Cell Rep 25:1104–1110

Menendez V, Revilla MA, Fal MA, Fernandez H (2009) The effect of

cytokinins on growth and sexual organ development in the

gametophyte of Blechnum spicant L. Plant Cell Tissue Organ

Cult 96:245–250

Murashige T, Skoog F (1962) A revised medium for rapid growth and

bio-assays with tobacco tissue cultures. Physiol Plant

15:473–497

Reinhardt D, Pesce ER, Stieger P, Mandel T, Baltensperger K,

Bennett M, Traas J, Friml J, Kuhkemeier C (2003) Regulation of

phyllotaxis by polar auxin transport. Nature 426:255–260

Smith RS, Guyomarc’h S, Mandel T, Reinhardt D, Kuhlemeier C,

Prusinkiewicz P (2006) A plausible model of phyllotaxis. Proc

Natl Acad Sci USA 103:1301–1306

Somer M, Arbesu R, Menendez V, Revilla MA, Fernandez H (2010)

Sporophytes induction studies in ferns in vitro. Euphytica

171:203–210

Stirk WA, Novak O, Strnad M, van Staden (2003) Cytokinins in

macroalgae. J Plant Growth Regul 41:13–24

Teng WL, Teng MC (1997) In vitro regeneration patterns of

Platycerium bifurcatum leaf suspension culture. Plant Cell Rep

16:820–824

Villacorta N, Fernandez H, Prinsen E, Bernard P, Revilla A (2008)

Endogenous profile of phytohormones in hop. J Plant Growth

Regul 27:93–98


123

Von Schwartzenberg K, Fernandez M, Blaschke H, Dobrev PI, Novak

O, Motyka V, Strnad M (2007) Cytokinins in the bryophyte

Physcomitrella patens: analysis of activity, distribution and

cytokinin oxidase/dehydrogenase overexpression reveal a role of

extracellular cytokinins. Plant Physiol 145:786–800

Wang GD, Fiers M (2010) CLE peptide signaling during plant

development. Protoplasma 240:33–43

Yasamura Y, Crumpton-Taylor M, Fuentes S, Harberd N (2007) Step-

by-step acquisition of the gibberellin-DELLA growth regulatory

mechanism during land-plant evolution. Curr Issues Biol

17:1225–1230

Zar JH (1998) Biostatistical analysis. Prentice Hall, New Jersey


123

The effect of exogenous and endogenous phytohormones on the in vitro development of gametophyte and...

Documents

Transcript of The effect of exogenous and endogenous phytohormones on the in vitro development of gametophyte and...