Conservation of Medicinal Plants: Techniques and Applications, Lecture notes of Pharmacognosy

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Conservation of

Medicinal Plants

To.

Dr. Prince Ahad Mir.

By.

Keshav Kumar.

Introduction

 Medicinal plants and traditional medicine play an important role in the health care system of most developing countries. ‘Plant Conservation is a broad group of activities which aims to prevent plants from becoming extinct. It includes the direct conservation of wild populations, collections of plants with gardens, education programmes, invasive species control, recovery and restoration work, research programmes, training... for which the Institute of Biodiversity Conservation (IBC) has initiated the development of a project on Conservation and Sustainable Use of Medicinal Plants (CSMPP).

Traditional Methods of conserving medicinal plants.  The rural people who obtain fuel wood and medicine from the vegetation around them as they cannot meet the expense of alternative fuels and modern medicine. This results in vegetation loss and environmental damage. For such damage major steps have been taken towards conserving the medicinal plants. They include:  Discouraging cutting down indigenous trees  Encouraging the local people to plant fast-growing exotic and indigenous trees for domestic use.  The inauguration of a national tree planting day and the creation of nature reserves.

Germplasm Technique for Conservation  Germplasm conservation of vegetative propagated crops, forest species especially those with recalcitrant seeds in live gene banks, in fields poses tremendous problems in terms of required land space and labor input during annual or perennial replacing, testing and documentation.  The advantages of in vitro or reduced growth storage include little space necessary in growth rooms for maintaining thousands of genotypes and the absence of diseases and pest attack in culture vessels.  In vitro storage eliminates the need for long and frustrating quarantine procedures during movement and exchange of germ plasma

Cryopreservation technique for conservation of plants.  Cryopreservation is defined as the viable freezing of biological material and their subsequent storage at ultra low temperatures (- 196 °C) using liquid nitrogen.  The use of liquid nitrogen, either by itself or as a source of nitrogen gas, is based on the following unique combination of features:

1. Chemically inert
2. Relatively low cost
3. Non-toxic
4. Non-flammable
5. Readily available

Cryopreservation procedures

 Three different procedures have been used for cryopreservation of plant cells: two-step freezing, vitrification and encapsulation- dehydration.  Two-step freezing: This procedure includes an incubation of cells in a mixture of cryoprotectants (total concentration of 1 – 2 M), which causes moderate dehydration of the cells, followed by a slow freezing step (for example, 1 °C/min down to app – 35 °C).  Vitrification: This procedure is based on severe dehydration at non-freezing temperatures by direct exposure to concentrated cryoprotectants (total concentration ranging from 5 – 8 M), 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.

Fundamental aspects of cryopreservation

➢ Freezing behavior of plant tissue, ultra structural aspects of freezing adaptation. ➢ Physical and biochemical studies of cryopreservation, induced damage, molecular change, molecular mechanisms of freezing tolerance.

2. Freezing:- cells are variably sensitive to low temperature, and this depends on the plant species. Given below are four types of freezing methods:-  Slow freezing method.  Rapid freezing method.  Stepwise freezing method.  Dry freezing method. 4. Storage :- After freezing, storage of the frozen cultures at a specific temperature is an important step. The frozen cell pr tissue are stored at temperature ranging from - 70 ºC to - 196 ºC; but above - 130 ºC, formation of ice crystals occur within the cells which reduce cell viability. The frozen cultures are stored in liquid nitrogen refrigerator at - 150 ºC in the vapor phase or at - 196 ºC at liquid phase.  Taxonomic classification of the material  Culture history  Morphogenic potential  Any genetic manipulation  Somaclonal variation  Culture medium and  Growth kinetics

5. Thawing :- It is carried out by placing the frozen samples in ampoules, which are then immersed in warm water bath maintained at 37 - 45 ºC temperature with vigorous swirling. In this way, rapid thawing occurs at 500 - 750 ºC/min rate, formation of intracellular ice crystals is prevented, thus protecting the cells form the damage. 6. Re-culture :- Cryoprotectants are removed from the thawed germ plasma by washing it several times. The obtained material is re-cultured in a fresh medium by following standard procedures. Some workers do not wash the thawed material and culture them directly, because certain vital substances which are released from the cells during freezing are essential for vitro cultures.

Cold storage

 Cold storage refer to the conservation of germ plasma at a low temperature ranging from 1 - 9 ºC. Since the plant material growth in cold storage is delayed in comparison to complete stoppage in cryopreservation, it is a slow growth germ plasma conservation technique. Cold storage has an advantages that the plant cell or tissue are protected from the damaging effect of cryoprotectants. Long-term cold storage method is simple, economical, and yields germ plasma having better survival rate. Many in vitro developed shoots or plants of fruit tree species have been stored in this manner.

Low-pressure and Low-Oxygen Storage

 Low-pressure Storage and Low-Oxygen Storage have been developed as alternative to cryopreservation and cold storage techniques of germ plasma conservation. Graphically represents tissue culture storage under conditions of normal atmospheric pressure, low pressure, and low oxygen

Low Pressure Storage (LPS)  In this technique, the surrounding atmospheric pressure of the surrounding atmospheric pressure of the plant material is lowered, which in turn partially reduces the pressure exerted by the surrounding gases of the germ plasma. This reduced partial pressure lowers the in vitro growth of plants. LPS systems are suitable for storing plant materials for short and long-term  Storage for short-term increases the shelf-life of some plant materials. For examples:- fruits, vegetables, cut flowers, and plant cuttings.

Low-Oxygen Storage (LOS).

 In this technique, the oxygen level is reduced, but the atmospheric pressure is maintained at 260 mmHg by adding inert gases (mainly nitrogen). Partial pressure of oxygen below 50 mmHg reduces the growth of plants (of organized and unorganized tissues), because in the presence of low oxygen less carbon dioxide is produced. Thus, the photosynthetic activity is reduced, which further inhibits the growth and dimension of plant tissue. LOS system has a drawback that conserving plant materials for a long-term in the presence of low oxygen inhibits the plant growth after reaching certain dimensions.