Plants are the most important resources of the earth as they play a major role for the survival of human beings. So the earth is considered to be the marvelous space to biodiversity. There are several species that are different in their genetical and physical characters. The earths ecosystem stability is maintained by plants and are
very beneficial to human mankind by utilizing the co2 and releasing the o2 for the food production through the process called photosynthesis. The important materials like pharmaceuticals, shelter, rubber, food, cosmetics, timber, traditional products, clothing etc are obtained from the plants. They help in soil fixation and prevent from soil erosion. As there is increase in human population there is increase in their needs due to this deforestation occurs i.e the cutting of trees and using the space for residential areas for industries of paper, wood etc. the natural calamities like volcano, earth quakes plays a major role in the destruction of plants. CITES (Convention on International Trade in Endangered Species) of wild fauna and flora was invented in July 1975 which is the agreement done by the government that the flora and flora usage for trading internationally is banned.
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Conservation is used to save the species or components of the earth for long term in artificial or natural habitats. Plant diversity is conserved by many protocols(Ayad et al., 1997; Karp et al., 1997). They are
Ex situ conservation
In situ conservation
Ex situ conservation:
It is also known as off-site conservation. In these the plant species are protected from the threat habitat and and are grown in in seed banks, zoos etc under human care (CBD Secretariat, 2003). The species like seeds, tissues, whole plants are conserved in the artificial environment and helps in increasing the plant population . It is very useful but it cannot save plants from the extinction. The plants can be safe guarded in botanical gardens even if the habitats are decimated. It also helps in recovery of some species by studying the threats of endangered species.
In situ conservation:
The In situ conservation is also said to be on- site conservation (Smart et al., 2002). In these the species are grown in the natural environment. The recovery of species takes place in the natural habitat where the adaptations take place naturally. So the conservation of seed to be a better option as it can done in a very small area when compared to conservation of several plants species as it requires large space. But they should be protected from disasters that occur naturally and human activities.
Classification of seeds:
Seeds can be classified in to three categories by differing in their behavior. They are orthodox, intermediate and recalcitrant (Roberts 1973; Ellis et al 1990)
These can survive under low moisture content( < 10%) with out any loss of genetic variation and vitality. From the mother plants they dessicate naturally after maturation and when they are stored under low moisture, poor effects are not observed (Roberts 1973). These are mostly produced in mediterranean regions. Almost 20 to 30% water content is present in these seeds and when 5% of water is present after drying then also viability is seen. Due to this reason they can be stored for longer periods. In orthodox seeds as the temperature is decreased the life span of the seed increases as the water content in the seed increases.
We can tell that the temperature and the longevity of seeds is indirectly proportion. Some seeds can be stored up to 5-25 years under 0-5oc with out any change in their viability and genetic variation so they can be called medium term storage seeds. Some seeds can be stored for 100 years under -10 to -15 o c. The life span of the seeds becomes twice or thrice longer when the temperature is decreased for every 50c and 1% moisture content decrease has been given by koopowitz and kaye in 1990 .
These seeds can be viable only for a shorter period of time unlike the orthodox seeds and hence calles as short lived seeds. After maturation they must collect these seeds and sowed if not they will die immediately as they do not have mechanism of dormancy(Berjak et al 1990). They cannot tolerate if the moisture is 15 -30% . They loose their viability if they are stored under cold conditions. They are mostly produced by coffee, mango that are temperate species. There size is large and moisture content is high when compared to orthodox seeds and they differ in their morphology, structure but the sizes of embryos of orthodox seeds are larger when compared to recalcitarant seeds( Chin et al, 1989). The dessication tolerance and the properties of water are not linked and there fore the models have not been proposed.(pammenter et al 1991,1993). The exact behavior of these seeds are not still known( Vertucci and Farrant, 1995). Embryos are not present in some recalcitrant seeds( Berjak et al 1996). Under saturated salt and cooling conditions the coffee seeds were freezed and it was demonstrated by ( Dussert et al 1997).
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These show the properties of orthodox and recalcitrant seeds. Moisture content of these seeds reduces same like orthodox seeds but they cannot survive in low temperatures. At 10o c they can be stored. The water content can be dried up to 10 -15%. The seeds that are sensitive to chilling and can tolerate low temperatures are called as intermediate seeds. The tropical species like neem seeds are economically important and can tolerate dessication for limited time and cannot be stored for longer time period. ( Gamene et al 1996:hong et al 1996).
The seeds can be stored for longer period and the life span is increased by placing the seeds under glass state (Murthy et al 2003).
All the seeds from different regions are collected based on the nature and the ability to tolerate the conditions. They are cleaned and stored in the seed banks for further use. The temperature is -18o c and humidity is 15% ( Eberhart et al 1997). In order to protect the seeds from contamination, damage they should undergo few steps before storage. Proper cleaning should be done in order to remove the debris and inert material , infected seeds, damaged seeds. They must be cleaned after harvesting. The main advantages of storing the seeds are easy to handle, can be maintained and stored in less space, low labor is required, not an expensive method, can store the seeds for longer time period. The seeds are collected depending on their texture i.e. hard, soft , pulpy so the extraction of seed is the first process in cleaning. If a fruit contains immature seeds it can preserved at 10 ââ‚¬" 15 oc. the hard and dry fruits can be preserved at shady places, the fruits that are moist should be placed in the trays containing holes for the passage of air. Extraction of the seed is done after the fruit gets ripened. Then check the seeds that should be free from infections by placing them on a flat surface. The seeds that are infected or damaged must be taken out and the remaining are dried and to prevent them from further infection or damage or contamination ,they are sealed with silica gel and packed in containers.
Mechanism of lipids in storage of seeds and germination:
The major products of the plants are seeds which contain carbohydrates , lipids etc and play a major role in germination and storage of seeds (Kermode, 1995). The properties of seeds i.e the lipids that are stored in the seeds, the glass state inside the cells helps the sun flower seeds in development (Lehner, 2006). The stored lipids causes lipid peroxidation due to which the aging of the sun flower seeds is observed (Baill et al 1996). Lipids are rich in oily seeds and plays an important role in storage and germination of seeds. They are present in the form of triglycerides in most of the plants. The triglycerides helps the seeds in germination and storage physiology. The lipids physical behavior change is seen during ageing (Vertucci 1992; Walters et al 2005). In ageing reactions the lipids act as reserviors for the substrates (Walters , 1998). The seeds cannot tolerate low temperatures as the triglycerol composition is effected ( Stanwood, 1987). The seeds of Cuphea Carthagenesis when stored at 25oc the viability of seeds is higher but when stored at 5oc the seed viability decreases ( Crane et al 2006). The crystallization of triacylglycerols leads to death of seeds when they are exposed to water (Crane et al 2003). This damage is mainly due to long chain fatty acids as they do not melt at 25oc.
Seed germination depends on the triacylglycerol crystallization. The damage of intermediate seeds is caused by the interactions between the water and triacylglycerols when they are stored for longer period (Sacande et al 2000, Ellis et al 1991) and due to the crystallization the seeds may tend to die ( Crane et al 2003). Crane et al in the year 2003 has invented that the seeds are sensitive if the melting temperature of lipids is more than 27oc and some species can be stored at ââ‚¬" 18o c if they have melting temperature less than 27oc. if the seeds are treated with higher temperature. In order to melt the lipids before imbibitions the seed damage can be prevented and can tolerate the exposure to sub zero temperature. The neem seeds (Azardirachta indica) can be beneficial by treating them at high temperature around 35oc (Sacande et al 1998). The lipids in the seeds are shattered by the oil bodies, these are stabilized by oleosins (Haung, 1992). The cotyledons structure can be disrupted and coalescence when the seeds are imbibed because of the presence of oil bodies with very low quantity of oleosins. The phase behavior, composition and crystallization of the seeds mainly depends on the lipid phase behavior and causes damage. For example in Cuphea Wrightti the damage of cells is caused due to the change in the lipid phase (Volk et al 2006). The hydrophobic and hydrophilic interactions are also caused due to the water effect on the crystallized lipids. Intermediate storage behavior (Ellis et al 1990) is the disease seen in intermediate seeds and is caused due to the interactions between the crystallized lipids and water. ( crane et al 2003). The magnitude of unfreezable water is determined by DSC. Cooling of unfrozen water mechanism is explained by kinetics not by thermodynamics (Bronshteyn & Steponkus, 1993). The unfrozen water content correlates negatively with the lipid content in the seeds (Dusssert et al 2001). Relative humidity of seeds may differ by the water content. Ice crystals are formed in oily seeds when the interaction takes place between lipids and water (Vertucci 1989). Unfrozen water measures the glass state formation in seeds and there was no difference in orthodox and recalcitrant seeds (Berjak et al ; 1992). DSC (Differential scanning calorimetry ) measures the unfrozen water in the seeds .
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DSC ( Differential scanning calorimetry) :
It is discovered by E. S. Watson and M. J. O Neill. It helps in measuring the thermal parameters like glass transition, melting etc. It also helps in protecting the tissues that are cryopreserved.
The heat flow and the transition of water in samples is measured by DSC as the function of time and temperature. A tissue with known weight is placed in the aluminium pan which is sealed tightly and also placed in a reference pan. Then these are cooled at the same temperature at a programmable rate. The heat flow difference is measured by the cooling and heating between the two pans and a graph is plotted between the temperature and time as a result a thermogram is obtained. DSC helps in analyzing the water physical state during the cooling and heating events. By the exothermic peak and endothermic peak during cooling and rewarming the liquid glass state and ice state transitions can be determined by the heat flow.
It helps in measuring physical parameters like glass transitions, melting, freezing (Benson et al 1996). The heat flow and the transition of water state can be determined by DSC by the function of time and temperature (Bachiri et al 2001). In cryobiology the physical phase of water can be estimated by heating and cooling the bio molecules and is considered to be the important application of DSC and by the heat flow data the phase transitions are estimated and are analysed by the exothermic peak during cooling and endothermic peak during rewarming (Mazur, 2004). Thermal profile by using the methods of cryopreservation provides the information on the prevention of ice formation and glass stabilization (Mazur, 2004).
DSC also helps to measure the quality, purity of the molecules or mixtures and also in comparing the kinetic substances, stability, characterization and safety investigation (Black et al 2002). The time, temperature , boiling points and the crystallization percentage can be measured by DSC. The molecules stability that are effected by thermal and oxidation are also measured. The cryoprotectives capability and osmotic changes in water transport in the cells are explained by the various sugar properties of thermodynamics in DSC ( Bachiri et al 200 & Luo et al 2002). During cryopreservation the toxicity on the seeds can be minimized by using DSC and by optimizing the crypprotectents that are added ( Benson et al 1996). The encapsulation, dehydration and vitrification protocols are developed and is said to be the major application of DSC and are also used to store the seeds for longer period ( Bachiri et al 2001). The complex behavior of the storage of seeds and different water phases can be studied by the DSC (Vertucci ,1990). The association of bio molecules with phase transitions and thermo physical properties of them are also studied.
The water content required to preserve the seeds , the optimum dessication rate and the relation between the un frozen water and injury caused due to dessication is determined by DSC (Pritchard et al 1995). The plants and animals cold tolerant can also studied by DSC (Wharton and Block ,1997). The formation of ice inside the cell on freezing and crystallization on warming it again can be determined by DSC, when the seeds are cryopreserved under liquid nitrogen. The freezable water present in many recalcitrant seeds is also determined by DSC and the ice formed inside the cells is also harmful to the tissues and when the seeds are exposed to lower temperatures( Vertucci, 1989).
Lipids properties that are stored inside the cell are also identified by the DSC and these are linked with the germination of seeds, the behavior of the seeds under storage and damaged caused due to imbibition (Crane et al 2003, Walters et al 2005). Thermal transitions that takes place during lipid phase on cooling and in dry seeds on thawing can also be characterized by DSC (Vertucci, 1992). It can tell about the changes in tryacylglycerol and composition of fatty acid chains (Crane et al 2003, Walters et al 2005). By the comparision of thermo grams the thermal properties changes of lipids can be observed by DSC when the seeds are stored under different conditions i.e under dry and cool conditions and when harvested at different times (Vertucci, 1992).
DSC also helped in identification of hydration window limits of coffee species during cryopreservation (Dussert et al 2001). Soyabean seeds and pea moisture levels, tissues thermal transitions are detected and comparision of these seeds with the seeds that are exposed to lower temperature are compared with DSC. The transition state of lipids and water were estimated by DSC by the level of moisture (Vertucci 1989). The phase transitions of water level in fern spores are also determined by DSC (Ballessteros, 2007). It also helps in stabilization and folding of protein. Ultra tight interactions of molecules are measured by DSC. It is used in process development. During manufacturing the biocomparability is assessed. It can also be used to study the antibody domains. It is used in protein engineering. The effects in the change of structure of molecule can also be determined by using this technique. It is also used in rank order binding. It also helps in providing the insights in refolding and unfolding mechanisms. The reversibility of any thermal processes can be monitored. It is easy to study the molecules with out labelling in their native state and the capacity of heat that is excessive can be measured continuously. The proteins conformational energetic can be monitored and also this technique has the ability to monitor biopolymers.
Advantages of DSC:
The main advantage of DSC is convenient to use. In case of eutectic impurities the analysis of DSC from 1-2 mg sample is very impressive globally. The smaller amount of sample is required and analysis is very quick in DSC. It is also useful in chromatographic techniques for assessing the stability in pharmaceutical development.
The quantity of water from the sample that has to be cryopreserved is quantified from the thermo diagram that has been produced by DSC . transitions in lipid phase can also be observed while freezing or when the samples are warmed . it is also used to determine the relationship with the lipid quantity in the seed to the high moistures.
The technique like DSC is commonly used for the quantification of drugs how ever there are many draw backs of this technique like they show slow heating rates and even it has limitations in usage to measure the solubility of drug. The more advanced techniques like HyperDSC (HDSC) which is a replacement of DSC which works on high heating rates and using this technique we have many advantages. Currently there are latest thermal analysis techniques are available which offers great approach towards the cryoprotection study of the plants. The techniques like Hyper DSC can scan at 500oc min-1 cooling and warming rates. This type of equipments possess low mass furnace design because they are dependent on platinum resistant thermometers and thus they are quick to respond. Hyper DSC is more or less similar to the direct plunging method and the rapid re warming cryopreservation methods. How ever this technique shows great stability profiling for Tgs , detection and characterization specially in glassy states and non homogenous mixtures which contains dehydrated germ plasm. The other advantage of hyper DSC is that measurement of small glass transitions can be done on the basis of differential kinetics. Higher precision rates can be explained for melting phenomenon and for detecting small Tgs.