Conservation of Medicinal Plants

Objectives

At the end of this lecture, student will be able to

• Discuss the objectives and methods of conservation of medicinal plants

• Discuss methods of in situ and ex situ conservation of medicinal plants

Conservation of Medicinal Plants

• All cultures from ancient times to the present day have used plants as a source of medicines

• As per WHO, 80% of the world's population depend on traditional medicine for their primary health care needs

• The  greater  part  of  traditional  therapy  involves  the  use  of  plant extracts or their active principles ignoring the fact that these plants will not be available on a continuing basis

• However, one need to ensure concerted efforts   to make the plants available on a continuing basis in spite of facing the following threats

• Threats posed include increasing demand, a vastly increasing human population and extensive destruction of plant-rich habitats such as the tropical forests, wetlands, Mediterranean ecosystems and parts of the arid zone

• Today many medicinal plants face extinction or severe genetic loss, but detailed information is lacking

• For most of the endangered medicinal plant species no conservation action has been taken to ensure sufficient materials to the gene banks

• Now a days too much emphasis has been put on the potential for discovering new wonder drugs, and too little on the problems involved in the use of traditional medicines by local populations

• In the light of this situation, WHO, IUCN, WRI and WWF in consultation with each other have framed certain guidelines in 1988 to conserve medicinal plants

• The aim of these WHO Guidelines is to provide a framework for the conservation and sustainable use of plants in medicine

Conservation of Medicinal Plants

In situ conservation

Ex situ conservation

CONSERVATION OF POPULATIONS OF MEDICINAL PLANT SPECIES IN NATURAL HABITATS

In situ conservation

• In situ conservation means ensuring that the populations of species of plants and animals continue to grow and evolve in the wild - in their natural habitats

• in situ conservation is achieved by

• setting aside areas as nature reserves and national parks (collectively termed "Protected Areas")

• by ensuring that as many wild species as possible can continue to survive in managed habitats, such as farms and plantation forests

In this regard the policy creation includes

• Identifying which of the protected areas are most important for medicinal plants;

• Targets and techniques for recording and monitoring medicinal plants in protected areas;

• Techniques and procedures for collection of medicinal plants within protected areas;

• A legal mechanism to ensure that benefits reach local people

• Training of park managers about medicinal plants, including their uses;

• Public education about medicinal plants in protected areas

The methods adopted includes the following assessments

• The Parks Department should assess the extent to which the protected areas system covers the medicinal plants of the country

• It should then create new protected areas and extend existing ones to ensure that all the medicinal plants of the country are conserved

• The Parks Department should devise economic and social incentives for maintaining natural habitats and wild species

• Park managers should ensure that the conservation and exploitation of medicinal plants are incorporated into site management plans

• Species that are heavily depleted by over-collection should be re-introduced into areas where they once grew wild

• This can be achieved by park departments extending their help to encourage the process of cultivation of the medicinal plants

• They can also provide seeds and saplings for local people, and can even set up small medicinal plant nurseries in suitable areas

Ex situ conservation

• Ex situ conservation means, conservation of the medicinal plants outside their habitat unlike in situ

• Ex situ conservation has the advantage that it is usually easier to supply plant material for propagation, for re-introduction, for agronomic improvement, for research and for education purposes

• The disadvantages of ex situ conservation are that the sample of the species conserved ex situ may represent a narrower range of genetic variation than that which occurs in the wild

• Species conserved ex situ can also suffer genetic erosion and depend on continued human care

• For this reason, ex situ conservation must not replace, but should complement, in situ conservation

• Most important of all, ex situ conservation should not be used as a reason for failing to safeguard representative samples of the medicinal plants and their habitats in nature

• Priority for ex situ conservation should be given to species whose habitats may have been destroyed or cannot be safeguarded

• It should also be used to bulk up populations of depleted or even locally extinct plants for restocking in nature

• In some countries it may be appropriate to conserve all medicinal plants ex situ

• Relating to ex situ conservation of medicinal plants, it is particularly important to conserve a broad genetic base, to permit improvement in the cultivated material

• When collecting the plant material for ex situ conservation, care should be taken not to put the survival of the wild population at risk

Techniques adopted for conservation

1. Germplasm technique

• Germplasm technique means use of seeds in live gene banks

• The advantage of this in vitro or reduced growth storage method includes

• The need for lesser space in growth rooms for maintaining thousands of genotypes

• Absence of diseases and pest attack in culture vessels

• eliminates the need for long and frustrating quarantine procedures during movement and exchange of germplasm

Disadvantages of Germplasm technique

• Some crops do not produce viable seeds

• Some seeds remain viable for a limited duration only and are recalcitrant to storage

• Seeds of certain species deteriorate rapidly due to seed borne pathogen

• Some seeds are very heterozygous not suitable for maintaining true to type genotypes

2. Cryopreservation technique

• “Cryopreservation” is defined as the viable freezing of biological material and their subsequent storage at ultra-low temperatures (-196C)”using liquid nitrogen

Cryopreservation procedures

• Three different procedures have been used for cryopreservation of plant cells:

A. Two-step freezing

B. Vitrification

C. Encapsulationdehydration.

• Two-step freezing: incubation of cells in a  mixture of cryoprotectants (total concentration of 1–2M), which causes moderate dehydration of the cells, followed by a slow freezing step (for example, 1°C/min down to app –35°C).

• Vitrification: based on severe dehydration at non-freezing temperatures by direct exposure to concentrated cryoprotectants (total concentration ranging from 5–8M), followed by rapid freezing

• Encapsulation-dehydration: Cells are encapsulated in alginate beads, cultured on medium with increased sucrose concentration, air-dried using silica gel or the airflow of a flow cabinet and directly transferred to liquid nitrogen

3. Tissue culture technique

• 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, including:

• The production of exact copies of plants that produce particularly good flowers, fruits, or have other desirable traits.

• To quickly produce mature plants the production of multiples of plants in the absence of seeds or necessary pollinators to produce seeds.

• The regeneration of whole plants from plant cells that have been genetically modified.

• The production of plants in sterile containers that allows them to be moved with greatly reduced chances of transmitting diseases, pests, and pathogens

• The production of plants from seeds that otherwise have very low chances of germinating and growing, i.e.: orchids and nepenthes.

• To clean particular plant of viral and other infections and to quickly multiply these plants as ‘cleaned stock’ for horticulture and agriculture

Tissue culture techniques

A. Cell suspension technique

B. Zygotic embryo technique

C. Adventitious bud technique

D. Gene bank technique

Cell suspension Technique:

• Pre-treatment: The cell suspension is inoculated at high density into standard medium containing 6% mannitol and cultured under standard conditions.

• The suspension is harvested when the cell will be dividing rapidly and chilled on ice.

• Cryopreservation: A double strength cryoprotectants solution (1M dimethylsulfoxide (DMSO) + 1M glycerol + 2M sucrose) was prepared and one volume ofcryoprotectants solution is added to one volume of cell suspension and the mixture is incubated on ice for 1hr.

• The mixture is dispensed as 1 ml aliquots and the ampoules are cooled until they reach - 35oC

• Storage: The ampoules are stored in or over liquid nitrogen.

• Thawing: The ampoules are dropped into sterile water at about 40oC with a ratio of 4 ampoules to 150 ml water

• Re- growth: The cells in suspension are transferred to several layers of 5 cm filter paper on the surface of a 9 cm agar plate containing a growth medium and are incubated under standard conditions.

• The cells and upper layers of filter paper are then transferred to fresh medium, until after 5 – 6 days, the cells alone are transferred to agar medium

Zygotic embryos Technique:

• Pre-growth: The embryos are excised aseptically from the seeds then in an open petridish, are exposed to a sterile airflow in a laminar flow cabinet for 3h

• Cryopreservation: The embryos contained in plastic ampoules are immersed in liquid nitrogen

• Thawing: The plastic ampoules are transferred to a water bath at 37 – 38^oC then placed on moist sterile filter paper in petri dishes for 10 days

• Re growth: After 10 days in the absence of medium the embryos are added to a standard growth medium to stimulate growth into plants

Adventitious buds technique:

• Pre growth: uniformed nodes (5 mm in length) containing an adventitious bud are removed from stem sections of plants

• Nodes are placed on agar medium containing 0.7 M sucrose and incubated under standard conditions or two days then transferred to a nylon membrane contained in a dish.

• Cryopreservation: Nodes are transferred to plastic ampoules then immersed in liquid nitrogen and stored under liquid nitrogen.

• Thawing: Nodes are thawed in a water bath at 25oC

• Re-growth:  Because of their size, nodes can be transferred individually to a standard growth medium and the adventitious bud is stimulated to develop as a shoot

Gene bank technique

• A Gene bank conserves genetic wealth through seeds, vegetative propagules, tissue culture, embryos, gametes or cells, DNA etc

• The importance of gene banks has been recognized since long and they have been in existence in various parts of the world for a long time

• The purpose of the Gene Bank

• To undertake and promote long-term conservation of plant genetic resources employing ex-situ conservation for seeds, in-vitro cultures and cryopreservation techniques and assist in in-situ conservation efforts

• To act as the repository of collected material, elite material and endangered material, as also a regional repository of duplicate collections as a part of the global system

• To monitor and maintain the existing collections, facilitating the organisation of regeneration program

• To ensure availability of exotic and indigenous germplasm through periodic seed increase for evaluation, utilization and conservation

• To conduct research related to medium and longterm conservation of germplasm

• To develop and operate a database and information network system on forest genetic resources

• To  support/assist  in  organization  of  post-graduate  education  and short/medium-term training courses on Forest germplasm activities at national, regional, and international levels.

Summary

• All cultures from ancient times -plants as a source of medicines

• WHO- 80% of the world's population depend on traditional medicine

• Threats -increasing demand, a vastly increasing human population and extensive destruction of plant-rich habitats

• WHO, IUCN, WRI and WWF - guidelines in 1988 to conserve medicinal plants

• In situ and ex situ conservation

• In situ conservation - Natural habitat, protected areas, national parks

• Ex situ conservation

• Germ plasm technique - use of seeds in live gene banks

• Cryopreservation technique – Liquid nitrogen, -196⁰C, two step freezing, vitrification and encapsulation-dehydration

• Tissue culture technique – Cell suspension, zygotic embryo, adventitious bud technique