Indian Journal of Experimental Biology
Vol.52, August 2014, pp. 820-824
A simple technique for tracking individual spore and gametophyte development in
Adiantum lunulatum Burm. f. using modified extra thin alginate film technique†
Bhuvnesh Sareen, Amita Bhattacharya, Madhu Sharma*, Anil Sood & Paramvir Singh Ahuja
Division of Biotechnology, CSIR-Institute of Himalayan Bioresource Technology (IHBT)
Palampur 176 061, India
Received 18 September 2013; revised 13 March 2014
A new technique was developed for accurate calculation of percent germination and tracking of individual spores from
germination to gametophyte development in Adiantum lunulatum. High percentage of ETAF immobilized spore germination
(72.4%) was followed by development of gametophytic clumps. The ETAF immobilized clumps were cut into pieces and
multiplied en masse. Apomictic sporophytes developed from the gametophytes. This indicated the potential of ETAF for
mass propagation of A. lunulatum without the need to start from spores. Since individual spores can be tracked from
germination to gametophyte development, the ETAF technique has the potential to be used for (i) harvesting uniformly
developed plants of similar age for extensive experimentations and commercial utilization and (ii) detailed study on
developmental and reproductive biology of different ferns and fern allies.
Keywords: ETAF, Fern spores, Immobilization, In vitro propagation, Percent germination
Ferns have a great ornamental value due to their
foliage. They are used as medicine and food, and also
play an important role in phytoremediation1-3. In
nature, the most prevalent mode of propagation in
ferns is through spores leading to two alternating
generations i.e., gametophytic (haploid) and
sporophytic (diploid). The sporophytes develop
either through fertilization or apogamy4. In vitro
propagation of ferns via spore germination and
subsequent gametophyte multiplication has also been
reported5-7. The in vitro culture through spores can be
used for the mass multiplication and ex situ
conservation of valuable, ornamental, endangered and
medicinal species for their future use8.
Adiantum lunulatum Burm. f. is an important
medicinal fern. Its roots and rhizomes are used for the
treatment of glandular swellings, muscular pain,
rabies, fever, strangury and elephantiasis9-12. Its leaves
are used in treating sprains, balding, bronchitis; and
the leaf juice in dysentery, ulcers, burning sensations,
erysipelas etc13. The spores are used for curing
leprosy, skin and chest diseases, and opthalmia10,11,13-15.
——————
*Correspondent author
Telephone: 91-1894-233339
Fax: 91-1894-230433
E-mail: [email protected]; [email protected]
†
CSIR-IHBT publication number 3530
The plant is also reported to have antifungal and
antimicrobial properties16,17. Mass multiplication of
characterized germplasm of A. lunulatum shall ensure
utilization of these properties for future use.
In this regard, a simple, reproducible and nondestructive extra thin alginate film (ETAF) technique
was developed for the inoculation of A. lunulatum
spores. The technique is a modification of the method
developed by Pati et al.18 for tracking individual
protoplasts. The aim of employing this technique for
fern spores inoculation was to (i) minimize the loss of
spores during the calculation of exact percent
germination and (ii) track individual spores from
germination to gametophyte development.
Materials and Methods
Plant material—Plants of Adiantum lunulatum,
procured from Palampur (1292 m amsl, 32.6°N and
78.19°E), Himachal Pradesh, India, located at
foothills of western Himalaya were maintained in the
fernery of the Institute. Mature sporophylls from
6 month old healthy plants were either kept in
(i) specially designed brown paper packets in a
desiccator containing anhydrous CaCl2 or (ii) in butter
paper packets. These were stored under weight. In
both the cases, the fronds were stored at room
temperature until the dehiscence of microscopic
spores as indicated by dust like impression.
SAREEN et al.: SPORE & GAMETOPHYTE DEVELOPMENT BY ALGINATE FILM IN ADIANTUM
Initiation of aseptic cultures—The spores were
taken in 1 mL of sterile de-ionized water. The spore
suspension was filtered through a folded, sterile filter
paper and surface sterilized using 0.01% mercuric
chloride (w/v) containing a drop of Tween-20 for 2-3
min followed by washing with sterile de-ionized
water for 5 min. After surface sterilization, the spores
were inoculated using extra thin alginate film (ETAF)
technique in petriplate (90 mm) each containing
25 mL Knop medium19 supplemented with 1% (w/v)
sucrose and 0.8% (w/v) agar. The pH of the medium
was adjusted to 5.75 prior to autoclaving at 121 °C
at 15 psi for 20 min.
For preparation of ETAF, the sterilized spores were
suspended in 5% (w/v) solution of sodium alginate in
liquid Knop medium. Fifty microlitre of 75 mM
CaCl2 solution was placed on the surface of 0.8%
(w/v) agar gelled medium (Fig. 1a). A drop of sodium
alginate (prepared as above) containing spores was
then placed over the drop of 75 mM CaCl2 using 1 mL
tips with cut ends (Fig. 1b). This was then
immediately covered with a sterile and clean cover
glass (Fig. 1c-d). Following this, 100 µL of 75 mM
CaCl2 was added to the sides of the cover glass and
allowed to gel for 10 min (Fig. 1e). The cover glass
was then removed with the help of forceps. The Petri
dishes were sealed with parafilm (Fig. 1f).
Petriplates (90 mm) were taken in triplicate and the
experiment was repeated twice. The cultures were
Fig. 1—Modified ETAF technique for spore inoculation
(a) A drop of 75 mM CaCl2 over Knop medium (b) A drop of
sodium alginate containing spores over CaCl2 (c) Spores
embedded in alginate (d) Alginate film covered with cover glass
(e) Gelation with calcium chloride (f) Thin alginate film with
immobilized spores.
821
incubated under culture lab conditions (16 h light
(70±5 µmole m-2s-1): 8 h dark at 25±2 °C). Data on
the time taken for spore germination were recorded.
Three petriplates with four ETAF each were viewed
under the stereozoom microscope (Nikon, SMZ 1500)
for the calculation of percent germination without
random sampling. The percent germination of the
spores was calculated using the formula (number of
spores germinated/total number of spores inoculated
per ETAF) x 100. The different developmental stages
viz., filamentous, spatulate and chordate were tracked
under stereozoom microscope and photographs
were captured using a digital camera (Nikon, DS L15M, Japan).
Gametophyte multiplication and sporophyte
development—The thallus (chordate stage of the
gametophyte) was allowed to develop into clumps
(2.0 cm dia). After 45 days, the gametophyte clumps
(immobilized in ETAF) were cut into four pieces (0.5
cm approx.) and inoculated on same medium in 250
mL Erlenmeyer flask with the help of forceps. The
pieces were allowed to multiply and develop into
sporophytes. Observations were recorded visually at
10 day interval and photographs were taken using the
digital camera (Canon, A2200, USA). Three
replicates of 250 mL Erlenmeyer flasks (Borosil,
India) containing 4 pieces each of gametophyte
clumps were taken and the experiment was repeated
twice. The clumps were sub-cultured on the same
medium at 45 day interval.
The culture conditions remained the same
throughout the study.
Results
The spores were clearly visible through the extra
thin, translucent film of calcium alginate when
viewed under the microscope (Fig. 2d). Thus, each
stage of individual spore germination and
gametophyte development were tracked easily. The
spores increased considerably in size after 10 days of
inoculation and showed asynchronous germination
(Fig. 2a). An exact percent germination of 72.4% was
recorded as the spores were immobilized in ETAF
and did not show agglomeration.
The spore-coat ruptured and a uniseriate
filamentous gametophyte of 2 or 3 cells protruded
after 16 days of inoculation (Fig. 2b-c). The spatulate
and chordate stages developed after 24 and 31 days,
respectively (Fig. 2d-e). Thallus with an apical notch
developed after 45 days. The antheridia developed
at the mid rib region of the dorsal surface of
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INDIAN J EXP BIOL, AUGUST 2014
Fig. 2—Spore germination and gametophyte development using modified ETAF technique (a) Spores immobilized in alginate
film (b, c) Filamentous stage (d) Percent germination, Note - 17 and 13 non-germinated spores in sectors i & ii. shown in white circles
(e) spatulate stage (f) Chordate stage.
the thallus. However, no archegonia development was
observed.
After 35 days of inoculation, the gametophytes
developed in clumps (Fig. 3g). Sporophyte
development was initiated after a total of 56 days
from the gametophytic clumps and complete plant
developed after another 30 to 45 days (Fig. 3h).
Discussion
The study reports a simple, reproducible and
non-destructive technique for the inoculation and
tracking of spore germination and gametophyte
development in A. lunulatum. This technique was
earlier used by Pati et al.18 to track protoplast
development in a highly reproducible manner. In the
present study, however, the technique was further
simplified for the inoculation of fern spores.
Generally, percent germination of spores is
calculated by random sampling20,21. However, in the
modified ETAF technique employed in the present
study, the translucent thin film of alginate facilitated
easy and direct recording of percent spore
germination (Fig. 2d). Following germination, the
gametophytic development of individual spores was
also easily tracked through ETAF (Fig. 3). This can
prove useful in fundamental studies on reproductive
SAREEN et al.: SPORE & GAMETOPHYTE DEVELOPMENT BY ALGINATE FILM IN ADIANTUM
823
Fig. 3—Tracking of fern spore (no. 1-6) germination in ETAF (a) Spores immobilized in alginate film (b) Filamentous stage
shown in red circle (c) Initiation of spatulate stage (d) Spatulate stage (e-f) Chordate stage and mature gametophyte of spore no 4.
(g) Differentiation of gametophytic tissue into clumps in ETAF (h) Complete sporophyte.
biology of ferns and fern allies because a single
population may be comprised of sporophytes
developed either by intra or inter-gametophytic
fertilization or by apogamy. Random sampling on the
other hand, does not allow the tracking of
gametophyte development from individual spores.
The ETAF technique did not hamper the normal
multiplication of the gametophytic clumps
immobilized on alginate. Therefore, ETAF has a
good potential to be used for mass propagation of
A. lunulatum, thereby alleviating the need to start
from spores. Continuous gametophyte multiplication
was accompanied by normal and healthy sporophyte
development as reported earlier in Woodwardia
virginica22. Immobilization in ETAF had no influence
on sporophyte development. As no archegonia
formation was recorded, the sporophyte development
was apomictic. A. lunulatum from western Himalaya
has been reported to be diploid apomict23.
In conclusion, in vitro cultures of A. lunulatum
were raised and multiplied using the ETAF technique,
wherein accurate calculation of percent germination
and tracking of individual gametophyte development
are possible in a reproducible manner. The technique
can also be used for a detailed study of the
morphogenetic, physiological and developmental
changes during individual spore germination and
gametophyte development. These studies in turn can
pave the way for understanding the molecular
mechanism(s) that governs the preference for
apogamy or sexual mode of reproduction in ferns
and fern allies. Moreover, this technique can be
used to harvest uniformly developed plants of
similar age for extensive experimentations and
commercial utilization. Thus, the ETAF technique
has the potential to be extended to a wide variety
of ferns.
Acknowledgement
Thanks are due to the Department of
Biotechnology, Govt. of India and the Council of
Scientific and Industrial Research, New Delhi for
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INDIAN J EXP BIOL, AUGUST 2014
financial assistance and to Dr. Alka Kumari and
Dr. Brij Lal for the plant material.
References
1
Mannan M M, Maridass M & Victor B, A review
on the potential uses of ferns, Ethnobot leaflets, 12
(2008) 281.
2
Salido A L, Hasty K L, Lim J M & Butcher D J,
Phytoremediation of arsenic and lead in contaminated soil
using Chinese brake ferns (Pteris vittata) and Indian mustard
(Brassica juncea), Int J Phytorem, 5 (2003) 89.
3
Tu S, Ma L Q, Fayiga A O & Zillioux E J, Phytoremediation
of arsenic-contaminated groundwater by the arsenic
hyperaccumulating
fern
Pteris
vittata
L,
Int
J Phytorem, 6 (2004) 35.
4
Huang Y M, Hsu S Y, Hsieh T H, Chou H M & Chiou W L,
Three Pteris species (Pteridaceae: Pteridophyta) reproduce
by apogamy, Bot Stud, 52 (2011) 79.
5
Chang H C, Agrawal D C, Kuo C L, Wen J L, Chen C C &
Tsay H S, In vitro culture of Drynaria fortunei, a fern species
source of Chinese medicine “Gu-Sui-Bu, In Vitro Cell Dev
Biol—Plant, 43 (2007) 133.
6
Johnson M, Manickam V S, Benniamin A & Irudayaraj V,
Conservation of endangered ferns of western ghats through
micropropagation, in: Perspective in pteridophytes, edited by
Verma S C, Khuller S P, Cheema H K (Bishen Singh
Mahendra Pal Singh, Dehradun, India) 1984, 183.
7
Mazumder P B, Sharma G D, Chaudhury M D, Mazumder
B & Nath D, In vitro propagation of Helminthostachys
zeylanica (L.) Hook — A rare medicinal fern, Assam
University Journal of Science & Technology: Biological and
Environmental Sciences, 5 (2010) 129.
8
Pennisi E, Tending the global garden, Science, 329 (2010)
1274.
9
Reddy K N, Reddy C S & Trimurthulu G, Ethnobotanical
survey on respiratory disorders in eastern ghats of Andhra
Pradesh, India, Ethnobot Leaflets, 10 (2006) 139.
10 Karthik V, Raju K, Ayyanar M, Gowrishankar K & Sekar T,
Ethnomedicinal uses of pteridophytes in Kolli hills, eastern
ghats, J Nat Prod Plant Resour, 1 (2011) 50.
11 Chopra R N, Chopra K, Handa K L & Kapur L D,
Indigenous Drugs of India, Published by U.N. Dhar and Sons
Private Limited, Calcutta, (1958) 493, 603, 648.
12 Anis M, Sharma M P & Iqbal M, Herbal ethnomedicine of
the Gwalior forest division in Madhya Pradesh, India, Pharm
Biol, 38 (2000) 241.
13 Kumari P, Otaghvari A M, Govindapyari H, Bahuguna Y M
& Uniyal P L, Some ethno-medically important
pteridophytes of India, Int J Med Arom Plants, 1 (2011) 18.
14 Rout S D, Panda T & Mishra N, Ethnomedicinal studies on
some pteridophytes of Similipal Biosphere Reserve, Orissa,
India, Int J of Medicine and Medical Sciences,1 (2009) 192.
15 Pallavi G. An ethno-pharmaco-botanical review of
hamsapadi - Adiantum lunulatum Burm. f. (Adiantum
philippense Linn) Int J Pharma Biol Arch 2 (2011)1627.
16 Guha P, Mukhopadhyay R & Gupta K, Antifungal activity of
the crude extracts and extracted phenols from gametophytes
and sporophytes of two species of Adiantum, Taiwania,
50 (2005) 272.
17 Reddy V L, Ravikanth V, Rao T P, Diwan P V &
Venkateswarlu Y, A new triterpenoid from the fern
Adiantum lunulatum and evaluation of antibacterial activity,
Phytochem, 56 (2001) 173.
18 Pati P K, Sharma M & Ahuja P S, Extra thin alginate film: an
efficient technique for protoplast culture, Protoplasma,
226 (2005) 217.
19 Knop
W,
Quantitative
untersuchung
über
die
ernährungsprozesse der pflanzen, Landwirtsch Vers Stn,
30 (1865) 292.
20 Gomes G S, Randi A M, Puchalski A, Santos D D & Dos
Reis M S, Variability in the germination of spores among
and within natural populations of the endangered tree fern
Dicksonia sellowiana Hook. (Xaxim), Braz Arch Biol Techn,
49 (2006) 1.
21 Quintanilla L G, Amigo J, Pangua E & Pajaroân S, Effect of
storage method on spore viability in five globally threatened
fern species, Ann Bot, 90 (2002) 461.
22 Ferna´ndez H & Revilla M A, In vitro culture of ornamental
ferns, Plant Cell Tissue Org Cult, 73 (2003) 1.
23 Mehra P N & Khullar S P, Biosystematics of Adiantum
lunulatum Burm. F. complex in India with special reference
to western Himalayan Taxa, Cytologia, 42 (1977) 501.