Ex situ conservation of two threatened ferns of the Western Ghats through in vitro spore culture
Johnson
Marimuthu 1 & Visuvasam Soosai Manickam 2
1 Department
of Plant Biology and Biotechnology, 2 Centre for Biodiversity and
Biotechnology,
St. Xavier’s
college (Autonomous), Palayamkottai, Tamil Nadu 627002, India
Email: 1ptcjohnson@gmail.com
(corresponding author), 2 vsmanickam@gmail.com
Date of publication (online): 26
July 2011
Date of publication (print): 26
July 2011
ISSN 0974-7907 (online) |
0974-7893 (print)
Editor: V. Irudayaraj
Manuscript details:
Ms # o2687
Received 28 January 2011
Final received 08 June 2011
Finally accepted 27 June 2011
Citation: Marimuthu, J. & V.S. Manickam (2011). Ex situ
conservation of two threatened ferns of the Western Ghats through in vitro
spore culture. Journal of Threatened Taxa 3(7): 1919–1928.
Copyright: © Johnson Marimuthu &
Visuvassam Soosai Manickam 2011. Creative Commons Attribution 3.0 Unported
License. JoTT allows unrestricted use of this article in any medium for
non-profit purposes, reproduction and distribution by providing adequate credit
to the authors and the source of publication.
Author Details: Dr. M. Johnson standardized
the large scale multiplication protocol for thirteen rare and endangered ferns
of the Western Ghats under the Ministry of Environment and Forest, New Delhi
sponsored project. He has also published about 66 papers in national and
international journals on large scale propagation, phytochemical, antimicrobial
activity and isozymic profile of Indian medicinally important plants.
Rev. Dr. V.S. Manickam carried out
several projects sponsored by DST, DBT, Moen, UGC. During the last twenty five years he has surveyed
Pteridophytes of the Western Ghats and Angiosperms of Tirunelveli Hills. He
coordinated the All India Coordinated Project on Taxonomy for Pteridophytes and
Gymnosperms. He has completed several projects on ex situ conservation of
several rare and endangered ferns and angiopserms. He has published six books
and more than 300 research papers.
Author Contribution: JM executed the work and contributed for
paper writing; VSM was the
prinicpal investigator of the project and he only suggested the problem, given
guidelines and contributed for paper writing.
Acknowledgements: The authors
acknowledge the financial assistance from Ministry of Environment and Forests,
Government of India, New Delhi, India.
Abstract: The present study was intended
to produce a protocol for the conservation of two endangered ferns of southern
Western Ghats of India using in vitro spore culture. In addition this study
reports spore germination, gametophyte development, changes in the reproductive
phases and sporophytes formation of the medicinally important ferns Pronephrium
triphyllum (Sw.) Holttum and Sphaerostephanos unitus (L.)
(Holttum). Matured spores of the two selected ferns were harvested, filtered
through 40μM nylon membrane and sterilized with 0.1% mercuric chloride for
3 to 5 min and rinsed with sterile distilled water for 15 min showed less
frequency of mortality and a high percentage of spore germinations. For Pronephrium
triphyllum, the spores sown on the Knop’s basal agar medium showed
the highest percentage (38.3±1.13) of germination. Highest percentage (52.3±1.43)
of sporophyte formation was observed in Knop’s liquid medium. For Sphaerostephanos
unitus, the highest percentage (36.8±1.31) of spore germination was observed
in the Knop’s basal agar. The highest percentage of sporophyte formation was
observed only in Knop’s medium (76.8±1.41), other media failed to induce
sporophyte formation. The in vitro raised plantlets were hardened and
established in the natural habitat and distributed to various botanic gardens
as a part of ex situ conservation. Cytological and isoperoxidase analysis confirmed the genetic uniformity
between mother plants and in vitro raised sporophytes / plants. The established
protocol of the present study will be useful for the multiplication and
conservation of the two threatened ferns of the Western Ghats. The same
protocol may also be applicable to similar threatened ferns.
Keywords: Conservation, ex situ, ferns, isoperoxidase, Pronephrium
triphyllum, Sphaerostephanos unitus, spore.
For images, tables -- click here
Introduction
The Western Ghats is
one of the hotspots of the world and also one of the significant geographical
regions. Around 233 species of
ferns occur in southern India (Manickam & Irudayaraj 1992). In China, South Africa, USA, Europe and
Canada, the ferns are used as medicines to cure diseases such as chest
complaints, cancer, rheumatism, bowel disorder, ulcer, cough, fever and
Alzheimer disease. In China alone,
401 kinds of pteridophytic medicines have been used for various ailments (Luo
1998). The economic value of the ferns has been enumerated by various authors
from time to time (Kaur 1989). Today, the diversity of plant life is facing
serious threats, largely due to habitat loss, habitat degradation and
increasing exploitation of natural resources. It has been estimated that
globally 30% of the flora is threatened (Raven 1999). The decline in the number
and quality of the habitats is attributed to encroaching urbanization, growing
industrialization, intensive farming and unsustainable harvesting of wild
species. According to the World Resource Institute, India figures among 28
countries that are facing severe effects of increasing ecological imbalance if
preservation is not taken on a war footing. The IUCN report says that in India
7.7% of the plants are under threat. In Western Ghats, a number of epiphytic
and lithophytic ferns are destroyed due to various deforestation activities. In
the Western Ghats, 44 threatened ferns are facing extinction and the
conservation of these species is a major concern of biologists (Manickam 1995).
The establishment of plantations of cash crops like cardamom, coffee, rubber
and tea is the main reason for the destruction of the evergreen forests and
consequent demise of the ferns in the Western Ghats. A reduction in the
anthropogenic pressure on natural populations would contribute to their
conservation in nature. Among the various biotechnological options, also
reported in other agri–horticultural crops, micropropagation through
tissue culture and in vitro spore germination are best applied and commercially
exploited in fern species (Fay 1994). Application of this technology (in vitro
spore germination) for large–scale multiplication of certain species of
ferns from the Western Ghats has been demonstrated (Sara et al. 1998; Manickam
et al. 2003; Johnson et al. 2005; Sara & Manickam 2005; Johnson &
Manickam 2006; Sara & Manickam 2007; Johnson & Manickam 2007; Johnson
et al. 2008). The plant tissue culture as an effective tool to conserve plant
genes and guarantee the survival of the endemic, endangered and over exploited
genotypes is derived from the fact that it makes use of small units (cells and
tissues) without losing the mother plant, takes pressure off the waning wild
populations and makes available large numbers of plants for reintroduction and
commercial delivery. Endangered ferns such as Diplazium cognatum, Histiopteris incisa, Hypodematium
crenatum, Thelypteris confluens, Athyrium nigripes, Pteris vittata, Metathelypteris flaccida, Pteris gongalensis, Pteris confusa, Cyathea
crinita, Cheilanthes viridis, Pronephrium articulatum, and Nephrolepis
multiflora have been multiplied
through in vitro spore culture as a part of ex situ conservation (Sara 2001;
Johnson 2003; Manickam et al. 2003; Irudayaraj et al. 2003; Vallinayagam 2003).
Based on this background, the present investigation was initiated to extend the
good work already done in our laboratory to a few other equally endangered
species. In the present study in vitro spore culture has been attempted as part
of our continued efforts to conserve species of conservation importance and
prospective economic value. Reintroduction of the plants so multiplied through
spore culture in selected forest habitats hitherto untested in our centre has
also been attempted.
Materials and Methods
Two rare and endangered ferns from the
Western Ghats were selected for the present study viz., Pronephrium triphyllum (Sw.) Holttum
(Thelypteridaceae) and Sphaerostephanos
unitus (L.) (Holttum) (Theylpteridaceae).
Matured fertile fronds of the selected species were collected from the wild of
the Western Ghats and established in the green house attached to the Centre for
Biodiversity and Biotechnology, St. Xavier’s College, Palayamkottai, India. The
fronds were washed in running tap water for a few minutes. The fronds were cut
into small pieces and dried over white absorbent paper at room temperature (250C).
After drying the fronds over the absorbent paper at room temperature for 24hr,
the liberated spores were passed through 40mm nylon mesh to remove the
sporangial wall materials and the clean spores were collected and stored in a
refrigerator at 50C (Images 1a & 2a). The spores were surface
sterilized with 0.1% HgCl2 solution for 5min and washed with sterile
distilled water for 15min. The surface sterilized spores were inoculated onto
different media viz., Knops (1906), Knudson (1946), Mitra et al. (1976), Moore’s
(1903), and Murashige & Skoog’s medium (1962) devoid of sugar and plant
growth regulators using sterile Pasteur pipettes and incubated at 250C
± 20C under 12hr photoperiod (1500 lux). The pH of the media was
adjusted to 5.8 before adding agar 0.5% (w/v) and autoclaved at 1210C
for 15min. Both liquid and agar
nutrient media were used for spore germination and sporophyte formation.
Gametophytes regenerated from spores were sub–cultured on different basal
media (Knops, Knudson C, Mitra et al., Moore’s and Murashige and Skoog’s
medium) for sporophyte formation. Germination percentage of the spores, growth
area of the prothalli, and their development pattern were analyzed.
Photomicrographs were taken with a labotriumph microscope. The culture tubes
containing spore raised micropropagated plants of the two selected species were kept at room temperature (30–320C) for a week before transplantation. For acclimatization, the
plants with well developed roots (5–8 cm) were removed from culture
tubes, washed in running tap water to remove the remnants of agar and each
group was planted separately onto a 10cm diameter polycup filled with different
potting mixtures: river sand, garden soil and farm yard manures (1:1:1) and
sand and soil (2:1). The plants were kept in a mist chamber with a relative
humidity of 70%. Plants were irrigated at 8hr intervals for 3–4 weeks and
establishment rate was recorded. The plantlets established in community pots
were transferred to a shade net house for 3–4 weeks and then repotted in
larger pots (20cm diameter) with one plant in each pot.
For cytological analysis, the in vitro
raised young sporophytes (croziers) and immature sporangia in fertile fronds
were squashed in acetocarmine after being fixed in a 1:3:6 mixture of glacial acetic
acid, chloroform and 100% ethyl alcohol for 24 hours and then preserved in 95%
ethyl alcohol. Mitotic and Meiotic chromosomes were observed in several cells
for establishing the correct counts.
For Peroxidase analysis, the explants were
ground well in a mortar and pestle with phosphate buffer (pH 7.0) under ice
cool condition. The resultant slurry was centrifuged at 10,000rpm for 10min at
40C in a mikro 22R centrifuge and the supernatant was used as the
enzyme source and stored in a 700C deep freezer. Vertical
discontinuous poly acrylamide gel electrophoresis (PAGE) was carried out for
separation of isozyme. After the gel running, the gels were incubated in the
dark with acetate buffer (pH 4.6) 85ml + Ethanol (10ml), O–Dianisidine
(100mg) + 3% H2O2 (1ml) + 4ml distilled water for 30min
for staining, 7% acetic acid was used to stop the reaction and fixed the gel
(Smila et al. 2007). The Isoperoxidase profiles were documented using the
Vilber Loubermet Gel Documentation system and the similarity between the mother
plants and in vitro spore derived plants were calculated using the Biogene
software (Vilber Loubermet, Germany).
Results
Pronephrium triphyllum
Spores collected from the mature fronds
showed a variety of contamination and survival rates. On treatment with 0.1%
(w/v) HgCl2 for 5min followed by washing with sterile distilled
water for 15min showed 75–80 % of the spores free from the microbial
contamination. The young spores showed a high percentage of mortality, even
with a short duration of exposure to the sterilants. Spores were cultured in
liquid and solid basal media (Knudson, Knop’s, Mitra, Moore’s and Murashige and
Skoog’s). Microbial contamination was more in the liquid media compared to the
solid media. Spore germination time and germination percentages were dependent
on the composition of the media. The spores sown on the Knop’s basal agar
medium showed the highest percentage (38.3±1.13) of germination, followed in
order, by the Moore’s, Mitra and Knudson C media respectively (22.3±0.81, 21.3±0.83
and 15.3±1.21). The time taken for spore germination also varied. In Knop’s
medium spores germinated after 38 days, while in other media they took a much
longer time for germination. The pattern of germination is of Vittaria type.
After 3–4 weeks, repeated longitudinal and transverse division of the
anterior cells of protonema and expansion of the resultant daughter cells
formed the prothallial plate. The prothallus was cordate type. The prothallus
development was Drynaria type. Highest percentage of prothallus (81.3±1.34)
formation was observed in Mitra medium. The thallus was dioecious,
dorsiventrally flattened which developed a midrib region with a cushion like
structure and notched apical region.
The glandular hairs were present along the
margin and the midrib regions. The sex organs and rhizoids originated from the
midrib region. After 120 days, the male sex organs, antheridias were formed on
the posterior end. After 160 days, the female sex organs, archegonias were
formed on the anterior end (Images 1b–h). For sporophyte proliferation,
the 180 day – old gametophytes were transferred to Knudson C, Knop’s and
Mitra liquid media. After 30 days, the sporophyte emerged from the midrib
region on Knops liquid medium. After 15 days, the sporophytes were transferred
to agar medium for sporophyte elongation. The highest percentage (52.3±1.43) of
sporophyte formation was observed in Knop’s liquid medium compared to the other
two media [Knudson C (16.5±1.31) and Mitra (11.3±0.81)] (Table 1). After 15
days of rooting, the in vitro derived plantlets were washed thoroughly in
running tap water to remove the pieces of agar adhering to the roots and
implanted in the pots containing a mixture of (1:2:1) sterile soil: sand:
farmyard manure irrigated with 10 x diluted Murashige and Skoog’s / Knudson C
liquid medium once a week. The pots were covered with poly bags to maintain the
humidity. The plantlets were kept in a culture room for 15 days. After that,
they were transferred to a green house (R.H. 80%) under constant misting. After
three weeks the plants were transferred to the field. The micropropagated
plants showed 73.5±1.24% establishment during hardening and 72.3±1.24%
establishment in the field at KBG. Subsequently the micropropagated plants were
distributed to various botanic gardens for ex situ conservation. (Table 1)
(Image 4c,d).
Sphaerostephanos unitus
Matured spores were used for culture
initiation. The percentage of microbial contamination was less when the spores
were treated with 0.1% HgCl2 (w/v) for 5min and washed thoroughly
using sterile distilled water for 15min. The survival of explants depended on
the duration of the treatment with sterilants and the prolonged exposure (6–10
min) to 0.1% HgCl2 resulted in high percentage mortality. The spores
were cultured in hormone free liquid and solid media (Knudson C, Knop’s,
Murahige & Skoog’s and Mitra). In the liquid media, the inoculated spores
failed to germinate due to the high incidence of microbial contamination. After
35 days, the spores started to germinate in knops agar medium. The germination
pattern was Vittaria type. The prothallial plate was formed after 30 days of
culture, due to the repeated divisions of the cells. The prothallus development
was Drynaria type. The thallus was dorsiventrally flat with an apical notch.
The prothalli were cordate type. A high percentage of prothalli formation (74.8±1.21)
was observed in Knudson C medium. Glandular hairs were present on the margin
and central areas of the gametophyte. The male and female sex organs formed on
the midrib region. The male and female sex organs formed after 120 and 150 days
respectively. The sporophyte emergence was noticed in the midrib regions after
180 days with the formation of sporophyte and root initials (Image 2b–h).
The highest percentage (36.8±1.31) of spore germination was observed in the
Knop’s basal agar. Germination was not observed on the Murashige and Skoog’s
basal medium. The highest frequency of gametophyte formation and multiplication
(74.8±1.28) were observed in Knudson C basal medium. The sporophyte formation
was observed only in Knop’s medium, that too at a high (76.8±1.41) percentage;
there was no formation of sporophyte in other media (Table 2). Knop’s medium
also promoted the formation and elongation of rhizoids. After 30 days of
rooting, rooted plants were hardened in polycups containing a mixture (1:2:1)
of sand: garden soil: farmyard manure, covered with unperforated poly bags and
irrigated with 10 x diluted MS liquid medium once a week. The plants were kept
in the culture room for 15 days. Seventy eight percentage of the plants were
successfully established in poly cups. After 15 days, the hardened plants were
transferred to 15cm diameter pots and kept in the green house. Eighty–five
percentage of plants were well established in the green house. After six months, the plants, 34cm in
height, having 10 to 12 croziers were repotted, for distribution to various
Botanic Gardens, such as the Calicut University Botanic Garden, Kozhikode;
TBGRI, Trivandrum; Gurukula Botanic Garden, Wyanad and Genepool Garden, Gudalur
for ex situ conservation. Many plants were also transferred to their natural
settings in the Kodaikanal Botanic Garden, Kodaikanal for field establishment
(Table 3) (Images 4a–d).
Isoperoxidase analysis revealed the
genetic uniformity between the mother plants and in vitro spore raised
sporophytes. The MW – Rf values and banding positions confirmed 100%
genetic uniformity. In addition they provided the biochemical marker for the
two selected species. P. triphyllum(Image 3b) showed three different bands in three different active regions
(0.510, 0.664 and 0.764). S. unitus(Image 3c) showed only two bands in two different active regions (0.478 and
0.536). Cytological studies on root tips of ten randomly selected plants
established in KBG revealed the presence of 144 chromosomes in P. triphyllum (Image 3a) and 72 chromosomes in S. unitus (Image 3d) confirming the mother plants
chromosomes.
Discussions
Spores
are tiny objects which are used liberally by ferns for reproduction. A spore contains only half the normal
chromosome number and no embryo. The single celled spores are excellent
experimental material on par with pollen grains and isolated cells of higher
plant species. Observations over the past twenty years reveal that spores are
produced in huge numbers by nature, but the percentage of spore germination and
their developmental physiology rate is very poor due to unfavourable
conditions. Each and every species requires their unique environment for their
growth and development; most of the rare and endangered ferns failed to obtain
the optimal growth condition for their development. The present study also
confirmed the previous observations. However, there are a few published reports
on successful germination of spores in vivo (Theuerkauf 1994). Under natural
conditions, the percentage of spore germination is low due to the prevalence of
unfavourable factors, both biotic and abiotic. It is not unusual that the
spores are dispersed by wind to places unfavourable for their germination. The
spores otherwise having little stored food materials, seldom germinate in the
wild. The spores can germinate under in vitro conditions easily. The in vitro
spore culture methods have advantages over soil based conventional methods. The
in vitro culture techniques have been used to study different aspects of spore
germination, growth and development of gametophytes and sporophytes in ferns
(Nester & Coolbaugh 1986; Hickok et al. 1987; Miller & Wagner 1987; Melan
& Whittier 1990). However there are number of factors such as temperature, humidity, light, and nutrient
compositions (Raghavan 1989) which need to be addressed for successful in vitro
spore culture. Tissue culture which tends to be more sophisticated than spore
culture is also advocated and successfully explored for horticulture
propagation of selected ferns (Hennen & Sheehan 1978; Padhya & Mehta
1981; Higuchi et al. 1986). Successful culture initiation, be it spore culture
or tissue culture, depends on a number of physical and chemical factors. A
number of workers have studied spore germination under the influence of various
physiological and chemical parameters (Mehra & Palta 1971; Sharma &
Vangani 1988; Sharma & Sharma 1991). In the present investigation also, the spores of all the selected
species were cultured under varied conditions with the object of developing
viable protocols for mass multiplication and conservation. In a comparative
perspective, spore cultures are more desirable for rare species conservation
than tissue culture as it retains the genetic variability inherent in the
genetic make up of a species. Nowadays, accurate recordings of the genetic
uniformity and chemical characterization of the plants are needed to be
verified before conservation. Analysis of isoenzyme banding profile is considered to be one of the
best and cheapest system for the analysis of population structure, genetic
uniformity and developmental pattern, due to its role in the metabolic pathway.
It functions in harmony with other enzymes within the organizational framework
of cells and Isoenzyme often exhibits tissue or cell specificity (Zeidler
2000). These banding profiles are useful in differentiating the selected
species and the induced variants. In the present study also, Isoperoxidase
studies revealed the biochemical uniformity between the mother plants and the
in vitro spore raised plants. The earlier reports are directly consonant with
the present study and strengthen the role of isoperoxidase in the study of
genetic uniformity (Nair 2000; Johnson 2003; Nikhat 2004; Sonali 2004). Since
the 1960s, Electrophoresis coupled with isoenzyme has been the tool of choice
for studies of heritable variation by geneticist, systematist and population
biologist (Zeidler 2000). The isoperoxidase profiles will be used as a
taxonomic tool for the characterization of the two important ferns in the
future. The present study completely demonstrated the life cycle and
reproductive biology of the two rare and endangered ferns of the Western Ghats,
India. The established protocol of the present study will be useful for the
multiplication and conservation of the two threatened ferns of the Western
Ghats. The same protocol may also be applied to similarly threatened conserved
ferns.
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