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In Mauritius, orchids have not been well studied and little is known about their reproductive biology. 98 species have been described of which 9 are endemic: however, 24 species have been extirpated or have become extinct (Strahm & Bosser, 1996).
These figures will undoubtedly change as we learn more about these orchids. Recent studies have been conducted and suggest that there are other taxa to be described from Mauritius and a number of species are in decline. The main focus of my studies is on the reproductive biology and conservation of Mauritian orchids.
The rarest of the orchids on Mauritius is the epiphytic Angraecum cadetii, which is reduced to only thirteen known individuals, of which four are flowering plants, none of which have produced seed in the last two seasons. Angraecum cadetii are endemic to the Mascarene Islands. It was described in 1987 and named after the Reunion botanist Th.Â Cadet, having been confused with the Reunion endemic A.Â bracteosum for many years. (Brian Moris, 1970)
Fortunately much of what little remains are found within the boundaries of the Black River Gorges National Park. However, the region has been invaded by aggressive exotic plant species, particularly the Strawberry Guava Psidium cattleianum, which smothers out the native vegetation and prevents regeneration, resulting in monotypic stands of guava. As a result of this habitat destruction and degradation, only 13 individuals of A.Â cadetii are now known, making it probably the rarest of the Mauritian orchids.
Fruit success has been non-existent but there is some hope that the species can reproduce without human intervention. However, flower and plant density is so low that the probability of the pollinator removing pollen and then depositing them successfully is very slim. Studies on artificial pollination and micro-propagation have begun. Collecting of native orchids is cornparatively rare.
Since A.Â cadetii is not showy, poaching is not a threat to its survival. Clearly however, without some urgent intervention this species could well become extinct on Mauritius in a few years.
The Aim of this project is to experiment some aspects of in vitro culture of Orchid in view of the developing it on large scale production.
The specific objectives are as follows:
To initiate leaf and root culture of orchids and observe their development. Orchids used is Angraecum Cadetii
Use of different concentration of plant growth regulators on the development of Orchids
To observe and monitor the development of explants
CHAPTER 1-LITERATURE REVIEW
Orchids are the most beautiful flowers that god has ever created. They fit into the Orchidaceae family. Orchids exhibit an incredible range of diversity in size, shape and colour of their flowers. These flowers are both botanically and economically very important. Because of their long lasting and wonderful flowers, they have a very high market value. The number of different kinds of orchids around the world cannot be determined exactly but there are around 600-800 genera and 25,000-35,000 species. Thus orchids represent the most highly evolved family. (Hawkers, 1965)
MORPHOLOGY OF THE ORCHID
Kingdom - Plantae
Division - Magnoliophyta
Class - Liliopsida
Order - Asparagales
Family - Orchidaceae
Tribe - Vandaeae
Botany of the Plant
Among the plant families, orchids are the largest groups. Orchids have the most complicated flowers among the monocotyledons. They have varying growth habits. Most of them grow on trees and a few of them are semi-aquatic that is they grow in water. Their leaves occur in all dimensions with bract like structures and broad structures. Their roots are fibre- like structures or they can be fleshy.
The orchid flower
The Orchid plants are grown mainly for their flowers. They have got a remarkable beauty due to their tremendous differentiation. Orchid flowers are zygomorphic and perfect (Pool, 1929). Orchids have perianth having normally six petal-like segments. The inner ones are called petals and the outer sepals. In agraecoids, most of them are similar, but in Bulbophyllum, there are some differences. The capsule is three valved containing numerous small seeds resembling sawdust. (Brian Moris 1970).
Vegetative Growth of orchids
There are two types of growth in Orchids namely the monopodial and sympodial growth.
This Type of growth consists of the main stem growing from the center of the plant. No pseudobulbs are formed. Growth continues in one direction and is apical.
Sympodial mean multiple stem. At the end of the flowering season, new axis replaces old ones. These orchids have pseudobulbs. Food and water are stored in the pseudobulbs. They function very much like rhizomes. There will be growth more than one at a time.
Habit of growth of orchids
Orchids can be terrestrial or epiphytic when they are in their natural environment. The terrestrial orchids rise from the ground and the epiphytic orchids grow on trees to maximize their opportunity for expansion.
VEGETATIVE PROPAGATION OF ORCHIDS
Most of the orchids are propagated either sexually or asexually. The main disadvantage of orchids is that they are highly heterozygous. Therefore they cannot be raised through seeds and the main way of cultivating orchids is by asexual vegetative propagation
1.2.1 Sexual reproduction
Sexual reproduction method in orchids helps the seed to be cultured on a special culture medium to germinate. Young orchids plantlets are transplanted to separate pots (one only) so as it grow individually. There are two kinds of drawbacks while using the sexual reproduction, such as the impurity of rare species occurred as long as hybrid might occur and this lead to the extinction of the original. Secondly the shortage and importance of you're the only remaining species may lessen once it is allowed to be reproduced on a large scale. So it is favourable to preserve the rare species than to let others contribute in its extinction.Â (C.H.laws et al.1995)
Asexual reproduction produces new plantlets without the fusion of gametes; it resembles the parent plants, except when there for mutations. The offspring can be used as a protective seed and held as an agent for dispersal in seed plant.
Nowadays, tissue culture is modern method used. Tissues are taken from various part of a parent plant and it can be leaf, root or shoot. This tissue is cut into very tiny pieces and place in a culture medium where it become a tiny plantlets. As the plant become adult, it is then transplanted into other pots. Appropriate equipments, aseptic conditions and high level of technology are the source of good tissue culture system and eventually this lead to the profitability of species.
Operative propagation is a type of asexual reproduction used mostly in propagation of phalaenopsis. It is a secure and reliable method. According to C.H.laws , "I have performed this kind of operations for so many times and the success rate has been 100%. When I first started using this method, I had difficulty finding a suitable plant for such an operation, but after much thinking and experimenting with various operating techniques, I can now easily and confidently operate on almost any full-grown phalaenopsis for propagation purposes."
1.2.3 Orchid production in the world
Orchid production is considered one of the main important growing businesses in the world. They have a very high product value in the international market. There are two types of production of orchids, the cut flower production and the potted plant production. About the cut flower, more importance is given on the floral traits. The world's biggest orchid producers are Taiwan and Singapore.
Orchid production in Mauritius
In Mauritius, the production of orchids is done by small growers. Some do collections of the different orchid plants and as a hobby. It is produced for the local market only.
ORCHID TISSUE CULTURE
Vegetative propagation of orchids is a very fast way technique as compared to seeds but it is not as rapid enough to meet the demands and yield of the world markets. Tissue culture is very valuable when referring to mass propagation. The first plant that contributed to the tissue culture of ornamentals is the Orchids and the in vitro technique has boost up the profitability of various industries. (Arditti and Ernst, 1993).
Brief history of orchids culture in Vitro
In 1908, Bernard was the scientist who discovered the first application of biotechnology to the culture of orchids. The fungus mycorrhizal nourish in the Orchid's embryo. The orchid together with the fungus was inoculated in vitro (Arditti and Ernst, 1993).
Moreover, new research works led to the development and identification of the nutrients by the fungi. (knudson, 1992). Seed was observed to be germinating in the media which formulated these nutrients.
Afterwards, it has been discovered that explants other than seed such as buds of inflorescence stalk, could be used in vitro of orchid. The best example that demonstrated it is the phalaenopsis plantlets. (Rotor, 1949)
In 1960, there has been a great impact of tissue culture of orchids. It was introduced that various thousands of uniform seedling plants could be formed from a single bud of Cymbidium duration of 1 year (Morel, 1960). This method helps in the economic and commercials used of orchids. The technique prepared by Morel was then tested on the micropropagation of Miltonia and Cattleya. Odontoglossum and the related genera were a successful experiment. ( Lecoufle, 1967).
While using in vitro cultured, we need both the chemical and physical of the plant cells put culture medium. The growth medium and the external environment (light, temperature, humidity, etc.), play an essential role in tissue culture of orchids. Furthermore the growth medium supply essential mineral ions, additional supplements such as amino acids and vitamins, and addition of a fixed carbon source in the dorm of sugar, mostly sucrose ( as the plant cells are not photosynthesis) are very crucial for orchid propagation. Moreover, water is of vital component (principal biological solvent), physical components such as pH, temperature, light duration and osmotic pressure which need to be maintain in the respective limits.
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As is implied by the name, the stock solution supplies those elements required
in large amounts for plant growth and development. Nitrogen, phosphorus,
potassium, magnesium, calcium and sulphur (and carbon, which is added separately)
are usually regarded as macroelements. These elements usually comprise
at least 0.1% of the dry weight of plants. Nitrogen is most commonly supplied as a mixture of nitrate ions (from the KNO3) and ammonium ions (from the NH4NO3). Theoretically, there is an advantage in supplying nitrogen in the form of ammonium ions, as nitrogen must be in the reduced form to be incorporated into macromolecules. Nitrate
ions therefore need to be reduced before incorporation. However, at high concentrations,ammonium ions can be toxic to plant cell cultures and uptake of
ammonium ions from the medium causes acidification of the medium. In
order to use ammonium ions as the sole nitrogen source, the medium needs to
be buffered. High concentrations of ammonium ions can also cause culture
These elements are required in trace amounts for plant growth and development,
and have many and diverse roles. Manganese, iodine, copper, cobalt,
boron, molybdenum, iron and zinc usually comprise the microelements, although
other elements such as nickel and aluminium are frequently found in
some formulations. Iron is usually added as iron sulphate, although iron citrate can also be
used. Ethylenediaminetetraacetic acid (EDTA) is usually used in conjunction
with the iron sulphate. The EDTA complexes with the iron so as to allow the
slow and continuous release of iron into the medium. Uncomplexed iron can
precipitate out of the medium as ferric oxide.
Only two vitamins, thiamine (vitamin B1) and myoinositol (considered a B
vitamin) are considered essential for the culture of plant cells in vitro. However,
other vitamins are often added to plant cell culture media for historical
Amino acids are also commonly included in the organic supplement. The
most frequently used is glycine (arginine, asparagine, aspartic acid, alanine,
glutamic acid, glutamine and proline are also used), but in many cases its inclusion
is not essential. Amino acids provide a source of reduced nitrogen and,
like ammonium ions, uptake causes acidification of the medium. Casein
hydrolysate can be used as a relatively cheap source of a mix of amino acids.
220.127.116.11 Carbon source
Sucrose is cheap, easily available, readily assimilated and relatively stable and
is therefore the most commonly used carbon source. Other carbohydrates
(such as glucose, maltose, galactose and sorbitol) can also be used
and in specialised circumstances may prove superior to sucrose.
18.104.22.168 Gelling agents
Media for plant cell culture in vitro can be used in either liquid or 'solid'
forms, depending on the type of culture being grown. For any culture types
that require the plant cells or tissues to be grown on the surface of the
medium, it must be solidified (more correctly termed 'gelled'). Agar, produced
from seaweed, is the most common type of gelling agent, and is ideal for routine
applications. However, because it is a natural product, the agar quality
can vary from supplier to supplier and from batch to batch. For more
demanding applications (see, for instance, the section on microspore culture
below and Chapter 3), a range of purer (and in some cases, considerably more
expensive) gelling agents are available. Purified agar or agarose can be used,
as can a variety of gellan gums.
1.3.3 The essential of Clonal propagation
The most essential contributions of in vitro techniques to orchid industry is the used of Mass Rapid Clonal Propagation (MRCP), it was initiated by Morel. The use of clones is of vital constituent in the economic development and production of orchids. Furthermore, the developments of various methodologies are used for fast large-scale multiplication. Development of vegetative propagation can also produce clones but one cutting can produce only plantlets whereas by the use of in vitro techniques, several homogeneous plantlets can be obtained from explants such as root or bud. It is an important aspect for the production of orchids for large scale market value.
TYPES OF PROPAGATION IN VITRO
Axillary bud (Mosich el al. 1974) and shoot tip culture (kip et al. 1970, 1972, Groth 1973, Intuwong and Sagawa 1974, Sing 1976, Lim_ho 1982, Hasegawa and Goi 1987, Choi et al, 1980) play an important role in vitro technique of orchids. Moreover, different explants that can be used are root tip; leaves base (Loh et al. 1975) and off shoot segment (Sing et al.1998)
Axillary Bud Culture
The simplest technique of propagation in tissue culture is the culture of axillary bud. Axillary buds are located on the plant where shoots are likely to form. Furthermore, buds culture developed an ontogeny route (Philips and Hubstenberger, 1995). Bud propagation increase in the shoot number and thus increase the production of some orchids. Moreover, Axillary bud is one of the most popular ways of propagation (Sagawa, 1990). Dendrodium is a sympodial type of orchids where there are many shoots and production of bud is not a problem whereas in orchids such as monopodial orchids such as phalaenopsis and Vanda, stem are short and buds can be mostly obtained from flower stalks.
Shoot tip/Meristem culture
The most widely used methodology in commercialized tissue culture system of orchids is meristem/shoot tip culture. The shoot meristem is characterized by a set of totipotent cells which have a great morphologenic potential in culture and is situated at the tip of the shoot. For instance, in Phalaenopsis orchid, it is believed that shoot tip removal can destroy the mother plant thus it is not a widely used technique.
Using in vitro from plant, root cultures can be recognized. Primary or lateral roots can be used in simple media. The growth of root in culture is potential limitless as the roots are indistinct organs. Root culture was one of the first achievements of the up-to-date plant tissue culture but this is not extensively used for plant transformation studies.
Protocorm like bodies are known as PLBs can be describing as stages that form before plantlets. It is found in almost all Orchids explants. PLB is depicted as a greenish mass of tissue with tiny root hairs. The term protcorm like bodies was quoted by Champagnat in 1977(Pierik, 1987), to describe the structure formed from the shoot tip in vitro, as it resembled to the protocorms formed by orchid seed as they started to germinate. The bulblets has the ability to cell division and it increase in culture by forming clumps. PLBs and sub-culturing are separated, this lead to the continuous division of the cell.
The process of Somatic embryogenesis can be considered as the early stage f PLBs by some investigators of orchids. (Chen et al, 1994) but it has not yet been recognized in many orchids.
Orchid tissue culture pathway
The regeneration pathways of orchids culture can be followed the various stages:
Selection of the explants such as root or leaves or shoot
Development of PLB from the explants
Proliferation of these PLBs
The growth of the PLB into organ such as shoots and roots.
NUTRIENTS REQUIRED BY ORCHIDS IN CULTURE
Complex Additives used in the media
A very large variety of various complex additives have been implemented in the Orchid In vitro culture (Arditti, 1977). Moreover, the tissue culture media contained definite constituents of elements such as minor elements, major elements, vitamins, plant growth regulator. Natural extracts also are used and this proved to be a successful attempt. Furthermore, it is discovered that when adding organic matter in basal medium, the growth of orchid's plantlets are boosted up.
Murashige & Skoog Medium (MS)
Murashige & Skoog Medium (MS) is used for micropropagation, organ culture, Callus culture and cell suspension culture.Murashige & Skoog Medium (MS) was established by Murashige & Skoog (1962) for in vitro callus culture of Nicotiana tabacum (family- Solanaceae).
Murashige & Skoog Medium (MS) provides all essential macroelements, microelements, & Vitamins for the growth of plant cell, tissue and organ culture in vitro. This medium with high concentration of salts is used for cultivating plant cell, tissue and organ culture.
Coconut water is the clear liquid inside young coconuts, which is effectively used in the tissue culture since past half century. It was firstly used for the clonal propagation of carrot (Caplin and steward, 1948). The use of coconut water help in increasing the PLB's proliferation, but how it does so, it is very not clear.
Coconut water helps also in the germination of seeds to induce vigorous root formation in stem cutting and promote rapid growth of plants. Moreover coconut water contains a very wide range of biochemical, some of which act as growth regulators. These include, 1-3 dephenylurea which depicted the cytokinin activity (shartz and steward, 1995) and zeatin described as an adenine (Arditti and Ernst, 1993). The development of the orchids changes the composition of water but in general, it is high in vitamins, amino acids, sugars and certain salt.
PLANT GROWTH REGULATORS AND TISSUE CULTURE
The main point as far as plant concern is that of plasticity and totipotency. Thus particular media component can be used in the development of plant cells in culture. Plant growth regulators are the important features for determining the development pathway of plant cells. Plant growth regulators used mostly are plant growth hormones or their synthetic analogues.
Development of the plant growth regulators and their used in plant culture were done due to the initial observation made in 1950s. There are difficulties in the predicting of the plant growth regulators, this is due to the great impact cultures have between species, cultivars. However, some main beliefs do hold accurate and have become the exemplar on which plant tissue regimes are based. There are five main classes of plant groth regulators such as auxins, cytokinins, Gibberellins, Absicisic acid and ethylene. The most widely used plant growth regulators are auxins and cytokinins and this is usually used together. The ratio used for Auxin to cytokinin determines the type of culture need to be established. Root culture is favoured when a high level of auxin to cytokinin ration is used; shoot culture favoured high level of cytokinin to auxin ratio and callus formation is due to the middle ratio.
Auxins promote both cell division and cell growth