CHAPTER 1
GENERAL INTRODUCTION
Aquilaria malaccensis Lam. (synonym –Aquilaria agallocha Roxb.) is among
one of very few economic tree species of tropical and sub tropical rain forest which has
aromatic and medicinal values. This species is commonly known as agarwood and has
various local names such as Aloe wood, Eagle wood, Sashi, Gaharu, Agaru etc. It is an
evergreen tree found growing naturally in forests of north east India, mainly in lowland
and on hillsides between altitudes of 100 m up to 1000 msl under high humid, subtropical climate with rainfall 1800-3500 mm per annum. It is a soft, medium size shadetolerant tree grows up to 15–25 m tall on average and can reach heights as much as up
to 40 m, 60 cm in diameter and adapted to live in various habitats. It regenerate through
seeds and found growing associated with varieties of herb, shrub and tree species in the
forest. There are fifteen species in the genus Aquilaria of which eight are known to
produce agarwood (Ng et al., 1997; Kosmiatin et al., 2005). Two species namely A.
malaccansis Lam. and A. khasiana Hall. are available in the north eastern states of India
and third species, A. macrophylla Miq., occurs in the Nicobar Islands (Giri, 2003).
Others species like A. yunnanensis and A. sinensis are common in China (Wu et al.,
2007). A. malaccensis is the main species of commerce and chiefly distributed in 10
countries: Bangladesh, Bhutan, India, Indonesia, Iran, Malaysia, Myanmar, Philippines,
Singapore and Thailand (Oldfield et al, 1998). In India it is confined to the north east
region of India and mostly distributed in the foot-hills of Arunachal Pradesh, Assam,
Manipur, Meghalaya, Mizoram, Nagaland, Tripura and West Bengal (Asian regional
workshop, 1997). Recently, natural population of the species has been reported existing
in pockets in Assam and Arunachal Pradesh (Tabin et al., 2012). Earlier, this species
was mainly reported from the hills and forests of Arunachal Pradesh, Nagaland,
Manipur, Mizoram, and Tripura as well as in West Bengal (Palit, 1996). In Arunachal
Pradesh, it is found under natural conditions and distributed in the rocky terrain forests
of Changlang, Lower Dibang Valley, Lohit and Papum Pare district (Tabin, 2012).
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A. malaccensis is known to be one of the most important species of commerce
and valued for its production of impregnated resinous heart wood that gives fragrance
(Anonymous, 1948). Agarwood is highly demanded for medicine, incense and perfumes
across Asia and the Middle East. It is medicinally used as stimulant tonic and diuretic.
The plant is being used to treat small pox, rheumatism, illness during and after
childbirth; to relieve spasms especially in the digestive and respiratory systems,
abdominal pain, asthma, cancer (used as a general tonic), colic, chest congestion,
diarrhea, hiccups, nausea, nerves and treatment of regurgitation (Burkill, 1966;
Chakrabarty et al., 1994; Kim et al., 1997; Barden et al., 2000; Bhuiyan et al., 2009).
The plant is also reported to possess remarkable anti-cancer activity (Gupta et al.,
1994/1995). In Egypt, Arabia and throughout the northeast part of Bangladesh,
agarwood is described as a stimulant, cardiac tonic and carminative (Bhuiyan et al.,
2009). It is used as a traditional sedative, analgesic and digestive medicine in traditional
Chinese and Japanese medicine (Abdullah et al., 2007; Liu et al., 2008). It bears acrid,
bitter, warm, aromatic properties and plays an important role in incense and perfume
industries too. It has been used for centuries as incense in Buddhist, Hindu and Islamic
ceremonies (Naef, 2011). The term ‘Agarwood’ refers to its resinous heartwood which
turns aromatic and highly valuable as a result of fungal pathogenesis (Shrivastava et al.,
2008). The formation of resin is a natural process of a tree in response to fungal attack,
which requires several years to form. The resin is formed inside the wood when a larva
of stem borer insect (Zeuzera conferta Walker) bore the stems of standing trees carrying
fungi of aerobiological origin along its body or make congenial for other organism to
spread during infestation or enter through injuries/wound. The ecosystem, productivity
and sustainability of any plant species depend on the diversity of other plant species.
Due to over exploitation of A. malaccensis for its wood and oil, this tree species is now
rarely found in wild habitat. Consequently, it is included in IUCN red data list of the
year 2013 as vulnerable and considered critically endangered in the North east India. It
is at the verge of extinction from the natural forests hence listed on the Appendix II of
the Convention on International Trade in Endangered Species of Wild Fauna and Flora
(CITES); hence there is an urgent need to apply modern technologies for the
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conservation and existence of this particular species as well as for preservation of its
germplasm. The application of biotechnological methods such as plant tissue culture
technique for its regeneration and help of microbial inoculants for their field
establishment may be the best alternatives for the above purposes.
"Tissue culture" generally refers to the growth of eukaryotic cells in-vitro. It is
often used interchangeably with cell culture however; "tissue culture" can also be used
to refer to the culturing of tissue pieces, i. e. explant culture or whole organs. Plant
tissue culture is a practice used to propagate plants under sterile conditions, often to
produce clones of a plant. Different techniques in plant tissue culture may offer certain
advantages over traditional methods of propagation, such as, production of exact copies
of plants that produce particularly good flowers, fruits, or have other desirable traits,
quick production of mature plants, production of multiples of plants in the absence of
seeds or necessary pollinators to produce seeds, production of plants from seeds that
otherwise have very low chances of germinating and growing, production of secondary
metabolites,
genotype
modification,
germplasm
conservation,
etc.
Basically
the
technique consists of taking a piece of a plant (such as a stem tip, node, meristem,
embryo, or even a seed) and placing it in a sterile, (usually gel -based) nutrient medium
where it multiplies. The formulation of the growth medium is changed depending on
trial and error basis to get the plant that produce undifferentiated callus, multiplication
of
the number of plantlets, development of roots, or multiplication of
embryos for
artificial seed. German scientist Gottleib Haberlandt in 1902 first originated this
technique to demonstrate the totipotency of plant cell. Later on this technique has
further been improved in several ways for plant propagation, storage and their
improvement.
Considerable progress has been made in this field and many medicinal,
ornamental and fruit cultivars and forest trees are being micro propagated on
commercial scale (Bajaj, 1991). Tissue culture also facilitates the exchange of
germplasm within and across the countries). This technique is being increasingly
exploited
for clonal multiplication (micro
propagation),
in-vitro conservation of
valuable indigenous germplasm threatened with extinction (Arora and Bhojwani, 1989),
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production of secondary metabolites (Bajaj et al., 1988) and establishment of host
pathogen interaction in-vitro in cellular level (vidhyasekaran, 1993). Micropropagation
systems based upon the production of multiple shoots directly from explants allow one
to maintain clonal material in a juvenile state. A modification of this approach is the
production of undifferentiated callus masses from which either roots or shoots can be
regenerated in vitro. Given the proper stimulus, almost any part of a plant can be
induced to form callus, and it is often possible to regenerate roots and shoots from this
callus. This method of vegetative propagation is often termed as organogenesis. Plant
tissue culture has been widely employed as an experimental technique for studying
somatic
embryogenesis
and
organogenesis.
Embryogenesis is the developmental
pathway of numerous well-organized, small embroids resembling the zygotic embryos
from the embryogenically potential somatic plant cells of the callus tissue or cells of
suspension culture.
On the other hand, Organogenesis is the development of
adventitious roots and shoots from meristematic cells of undifferentiated callus mass.
Plant regeneration via somatic embryogenesis can be of immense commercial interest
for developing automated clonal mass propagation system, provides nil or considerably
low somaclonal variation among regenerates and may be exploited for cryopreservation
(Bajaj, 1983). Callus mediated somatic embryos with dependable growth characteristics
and plant regeneration are beneficial for raising improved varieties through in-vitro
selection from high yielding callus lines and possibly also for genetic transformation.
Organogenesis is a method of rapid clonal propagation of genetically uniform plants.
For improvement of crops, tissue culture techniques provide a rich source of variability.
Increasing the genetic variability already available has enormous potentials from the
point of crop improvement (Bajaj and Gosal, 1982). The use of in-vitro genetic
variation often termed as somaclonal variation, results in regeneration with genetic
variation either by pre-existing as various heterogeneous cells or through culture
techniques.
Plant Tissue culture is also important for the species which has very short
dormancy period and dependent on any particular reproductive period as in case of
traditional method of sexual reproduction. Even improvement of characters is possible
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through tissue culture method by adopting biotechnological inputs. A. malaccensis Lam.
have restricted period of seed viability (Saikia, 2011; Tabin, 2012; Shankar, 2012). The
plant is generally propagated through seeds which germinate readily after maturity;
however their rate of germination sharply reduces with the increase in the period of
storage. This limitation may also be conquered by regenerating the plants through plant
tissue culture technique. The in-vitro propagated plants furnish a ready source of
uniform, sterile, and compatible plant material for mass multiplication and germplasm
conservation of rare, endangered and threatened medicinal plants (Anis and Faisal,
2005; Uppendra et al., 2005). The tissue culture raised plants may also provide raw
materials with uniform active principles of importance to perfumery and medicine
industries. Taking these factors into consideration, techniques of plant tissue culture was
adopted for propagation of Aquilaria seedlings and to supplement the conventional
methods of regeneration by enhancing the rate of multiplication in a short time and
space. Thus it is possible to produce plants in large numbers starting from a single
individual. By using tissue culture raised agarwood plantlets, growers may obtain more
oleoresin than if they grow from random seeds. The uniform growth with tissue culture
plantlets may make the process of inducing oleoresin formation easier and volume of
oleoresin more predictable. The plantlets, generated in-vitro under aseptic conditions,
eliminate all microbes including naturally occurring symbionts hence unable to make
relationship with other plants when grown outside the laboratory. Among naturally
occurring beneficial microorganisms, mycorrhizal fungi occupy most important position
as they develop symbiotic relationship with more than 90% of land plants. The first
report that root fungi may be beneficial to plant was observed in Indian pipe
(Kamienski, 1881). The symbiosis between fungi and root was named by Frank (1885)
as ‘Mykorrhizen’ from Greek meaning ‘fungus-root’. Seven types of mycorrhizal
associations have been recognized namely ectomycorrhizae, arbuscular mycorrhizae,
ecto-endo
mycorrhizae,
arbutoid
mycorrhizae,
monotropoid
mycorrhizae,
ericoid
mycorrhizae and orchidaceous mycorrhizae are known (Smith and Read, 1997; Mukerji
et al., 2002). Amongst them, the arbuscular mycorrhhizae (AM) association was the
most common one. AM fungi forms endo-mycorrhizal symbiosis with roots of higher
5
plants. Members of more than 80% of existing vascular plant families are capable of
forming the AM associations. Arbuscular mycorrhizal (AM) fungi are ancient and
widespread group among mycorrhizae which form symbiotic associations with roots of
many land plant species (Smith and Read, 1997). The fungus-plant relationships are
usually described as mutually beneficial. The AM fungi obtain their energy through an
obligate symbiosis with vascular plants; the AM, although non-vascular plants also are
reported to form the AM (Russell and Bulman, 2005). The AM fungi are named by their
formation of highly branched intra-cellular fungal structures ‘arbuscules’ which are the
site of phosphate exchange between fungus and plants. Vesicles, which contain lipids
and
are carbon storage structures,
are found
commonly in most genera of
Glomeromycota although this depends on environmental conditions (Smith and Read,
1997). AM fungi are obligatory biotrophs, have very broad host range which makes
them definitely different from the biotrophic fungal plant pathogens as well as other
root symbionts. A clear interdependency has been observed between two partners,
because fungi supply mineral nutrients and water to their host plants in return receives
photo synthetically derived carbon compounds from them (Smith and Read, 1997).
Depending on the individual AM fungi and soil conditions, Aquilaria species show
large positive growth responses to AM colonization (Tabin et al., 2009). The AM
colonization in the roots help in improved growth through increase in phosphorus (P)
uptake despite low P availability in the soil (Moyersoen et al., 1998). Most land plants
form associations with mycorrhizal fungi and have mutualistic associations between
fungi and plant roots which help to rejuvenate the species in the forest under natural
condition. In addition to the nutrient uptake activity, the extra radical mycelium of AMF
also releases substances that cause the soil and its organic components to aggregate
(Bearden and Petersen, 2000), and protect from pathogens through their antibiosis
(Bouamri et al., 2006). Once the symbiotic relationships is established between AM
fungi and the roots of host plant (Bever et al., 2002), they use growth stimulators and
bioremediation agents of degraded or polluted lands (Raman and Sambandan, 1998) to
act as bio-fertilizers which favors plant growth and development (Abbott and Robson,
1984) thus help in reducing toxic chemicals from soil. AM fungi belong to fungal
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phylum Glomeromycota (Schuessler et al., 2001). The Glomeromycota is divided into 4
orders, 8 families and 10 genera namely Acaulospora, Entrophospora, Glomus,
Paraglomus, Gigaspora, Sclerocystis, Scutellospora, Diversispora, Geosiphon and
Archaeospora. They were classified on the basis of spore development and morphology
(Daniell et al., 2001; Vandenkoornhuyse et al., 2002); however, with the development
of molecular biology techniques, AM fungi could increase as many as 10 families with
18 AM genera on date (Redecker et al., 2013). All members of the AM fungi are
asexual. The vegetative mycelium and intra radical structures are aseptate and
multinucleate. Most spores are between 50 and 500 μm in diameter depending on the
species. The AM fungi are not yet successfully cultured in the absence of plant root.
AM fungi are common in tropical forests of Asia but are uncommon in many forests in
Africa and South America (Ganesan et al., 1991). They have the potentials to influence
the diversity and distribution of host species (Kiers et al., 2000), rather distribution of
host plant is dependent on its mycorrhizal partner (Soni and Vyas, 2007). The increase
in the seedling growth and development of Dyera polyphylla and Aquilaria filaria tree
species were reported in Indonesia (Turjaman, et al., 2006). AM fungi also improved
the growth of the Brazilian pine Araucaria angustifolia (Zandavalli et al., 2004). Phytohormonal (gibberellins; auxins) changes throughout the entire plant under the influence
of AM symbiosis have also been described (Keinan et al., 2002). Other impacts of AM
fungi on the plant are their ability to increase the tolerance against drought (Davies et
al., 1992; Karaki et al., 2004), to overcome water deficiency (Robert, 2001), to decrease
negative effects of heavy metals in plants (Tonin et al., 2001; Becerril et al., 2002), to
colonize root over wide range (5-8) of soil pH (Read et al., 1976) and to increase
disease resistance (Zeng, 2006). AMF also protect plants against environmental stress
such as soil salinity (Giri, 2004), drought (Guissou et al., 2001), reduced disease
occurrence in host (Caron, 1989) and reduce damage caused by soil-borne plant
pathogens (Bouamri et al., 2006; Tabin et al., 2009).
Although plant tissue culture technique has got several advantages and
applications, there are still some obstacles which limit its widespread use. For example,
the success rate of transplantation of in-vitro raised plantlets to soil is very low in many
7
cases (Pierik, 1988). Weak root system is one of the major difficulties in the successful
establishment of micropropagated plantlets in field conditions (Pierik, 1988). The
arbuscular mycorrhizae (AM) in particular play a significant role in ensuring the health
of tissue culture raised plantlets (Gianinazzi and Gianinazzi-Pearson, 1989). In general,
the inoculation of AM fungi increases the survival rate and biomass of tree species
(Guadarrama et al., 2004). Mycorrhization of in-vitro propagated plantlets have a
‘positive impact’ on their post transplanting performance (Rai, 2001). AM fungi
improve
rooting
and
survival and
overall growth
(Wang et
al.,
1993) of
micropropagated plants. AM has also been reported to cause increased nutrient uptake
(Pearson and Jakobsen 1993), reduced transplant mortality and injury (Biermann and
Linderman, 1983), improved water relations (Gianinazzi et al., 1990), deposit of more
chlorophyll contents (Thaker and Jasrai, 2002) and increased contents of DNA and
RNA (Senthilkumar et al., 2000) in micropropagated plants. Mycorrhizae not only help
the establishment and growth of tissue culture raised herbaceous plants (Chavez and
Ferrera-Cerrato, 1990; Ravolanirina et al., 1989) but also economically important tree
species (Azcon-Aguilar et al., 1992). In general, mycorrhizal fungi help in the
development of a stronger root system (Ponton et al., 1990). These potentials of
symbiotic association between AM fungal species and plant roots strengthen the belief
of its significance in averting the transplantation shock brought about by unfavorable
environmental conditions due to alteration in humidity and nutritional conditions. The
studies on Leucaena leucocephala convincingly demonstrate that the mycorrhizal
association helped in successful establishment of tissue culture raised plantlets in the
field conditions by alleviating the transplantation shock. A survey of literature reveals
that inoculation of AMF into the roots of micropropagated plantlets plays a beneficial
role (Puthur et al., 1998; Vosatka et al., 1999) Even though methods for
micropropagation protocols of Aquilaria malaccensis has been standardized during the
present studies, the success rate of transplantation to the field is not known. To the best
of our knowledge, no reports exist on the influence of AM fungi in increasing the
resistance of micropropagated A. malaccensis to the transplantation shock. Therefore,
the present studies were undertaken with the major objectives of assessing the effect of
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AM fungal species in averting transplantation shock of micropropagated plantlets of A.
malaccensis. The present investigations propose to develop standard, fast and
reproducible protocols for in-vitro regeneration of A. malaccensis using tissue culture
techniques and their enhanced establishment in the field using endo-mycorrhizal
symbiosis.
To through the light on above aspects, the present research work was designed with
the following specific objectives:
1. To develop and standardize a rapid protocol for raising A. malaccensis seedlings
through in-vitro regeneration.
2. To induce somatic embryogenesis and organogenesis from callus culture.
3. To establish micropropagated plantlets of A. malaccensis in natural environment
using suitable endo-mycorrhizal symbiosis.
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