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Lecture – 33
Other aspects: cryopreservation, seed banks etc.

[FL] Today, we will have a look at two other aspects cryopreservation and seed banks.
(Refer Slide Time: 00:18)

Now seed banks are very important for the purpose of conservation, because if we think about a large sized tree and you have a limited area so, you can only have n number of trees in your area. But if you could collect the seeds of these trees and if you could preserve them for posterity, so we should be able to regenerate these trees using these seeds. So, seed banks become very important as a measure of ex-situ conservation and before we move forward let us have a look at this paper.
Refer Slide Time: 00:51)
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It says, regeneration of whole fertile plants from 30000 year old fruit tissue buried in Siberian permafrost. So, what happens in this case is that, Siberia is a location in Russia; it is on the northern and eastern portions of Russia and this area is very cold. So, this area has some areas, it has some regions that are a permafrost.
Now, permafrost is a region that always remains under sub freezing conditions. So, the temperature here is always less than 0 degree Celsius. So, the water is always frozen. So, it is permanently frozen; permafrost.
Now, in these areas, when some excavations were done, then some fruits were found and when radiocarbon dating of these fruits was done then, it turned out that these fruits were at least 30,000 years old.
The exact age was 3,1800 plus minus 300 years. So, in this case, when the scientist had access to this fruit tissue, they tried to regenerate these plants and when that was done; the plants were again formed.
Refer Slide Time: 01:58)
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So, these are the plants, they are also giving out flowers and if we look at the flowers from the relative species that is present today will observe quite a number of variations from this old sample.
So, what is happening in this case is that, we are having an access to a form of this plant that was existing around 30,000 years back and does not exist today. So, this is a very good example of what preservation can do for us. So, this is a form of biodiversity that is lost from the earth, because it was present quite a long back. But, because it was preserved in the form of this tissue, so we can access this biodiversity even after a huge span of 30,000 years. Now, this was when these tissue were kept in a very cold condition just by nature.
Refer Slide Time: 02:53)
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Now, this is another example; germination, genetics, and growth of an ancient date seed.
Now, date palms are found in a number of areas, speaking in the drier areas. (Refer Slide Time: 03:04)
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And these are 2000 years old date palms seeds that were found in Israel and then, these seeds were germinated back. So, this is one of the plant that came up through this germination, and then these plants are now growing and they can be propagated.

So, here also, the place where these seeds were found, these areas were having a number of date palm trees in antiquity. But close to around 800 years back, we started getting historical records that there is no date palm found in these areas anymore. So, at least for the last 800 years, we did not have any of these varieties that were growing here in the antiquity and the prime reason was climate change. But these date palms seeds were stored in a jar around 2000 years back and then because the whole area is very dry, and the seeds were kept in an area where they were kept cool.
So, even after 2000 years, people have been able to germinate these seeds and get the plants back. So, this tells us the power of preserving of the seeds. So, in this lecture, we are going to have a look at this preservation of seeds in a greater detail. How can we use preservation of seeds as a measure of conservation of these species? So, let us begin with what a seed is. So, seed is an embryonic plant enclosed in an outer protective coating. (Refer Slide Time: 04:25)

(Refer Slide Time: 04:34).

So, if we look at trees in the forest, so here we will have a fruit, when this fruit opens up, it releases the seeds and then the seeds fall off. So, these are seeds; if we cut open a seed then inside we will find something like this. So, we will have a seed coat in the outer area; in most of the cases this is a protective layer so, it will be very thick and very strong. Then, we will have some region that has endosperm. Now endosperm is the food source for the embryonic plant.
(Refer Slide Time: 04:46)

So, when the seed falls on to the ground, it will not be able to perform photosynthesis right away. So, there is some amount of stored food that is kept for the embryonic plant. And then this is the embryonic plant, these are cotyledons or the embryonic leaves, this is hypocotyl or the embryonic stem. So, when this seed falls into the ground and when it gets right conditions, right amount of moisture, right temperature, then this plant will grow out of this seed.
And while doing so, it will utilize its endosperm for the initial period till it is able to throw a leaf out. And once this leaf has come out then it will be able to perform photosynthesis and gain energy from the sun from that point onwards. Now, the important point to note here is that, because seeds have this stored food in the form of endosperm, so they also act as food for a number of organisms including us. So, when we are eating rice, when we are eating wheat, what we are eating is the seed of the rice or the wheat plant. Now, similarly, out there in nature there are a number of organisms that eat these seeds and some organisms even collect these seeds for posterity such as, ants or squirrels.
Now, if we want to conserve these seeds, we will have to protect them from these animals and we will also have to protect them from the microorganisms, because microorganisms also want to eat all of these. So, how do we do that? So, we begin by collection of good seeds.
(Refer Slide Time: 06:34)

So, what do we mean by good seeds? The good seeds are these characters; they are well ripened and healthy. So, you should not collect your seed that is half ripened, because in that case, the embryonic plant inside might not be well formed and it might not be able to germinate later on, which is our prime purpose when we are creating the seed bank.
Then, they should be pure and free from inert materials and weed seeds, because finally, later on we are going to plant these seeds back in to the environment so, we do not want any weeds to come to with it. Then, it is also should be free of inert materials. So, inert materials include things such as soil or things like husk. Now why should we have seeds free from inert materials? Because these inert materials might also be harboring some microorganisms. Then, these seeds should be viable and have good germination capacity; viable means that these seeds should be living.
So, for instance, if you take a seed and if you say, dig the seed, so because of the high temperature the embryonic plant inside would die and when that plant dies then we will not be able to generate this seed any longer. So, when we are collecting good seeds for our seed banking purpose then they should be viable and they should have a good germination capacity. Now, it should be uniform in its structure and appearance.
Why uniform, because in a number of seeds that are infested or infected will observe some amount of structural abnormalities. So, when our seeds are uniform, then it gives us an indication that these seeds are healthy. Then, they should be free from damage and should not be broken and infected by pests and diseases. So, free from damage means that, when we are processing these seeds so, in a number of situations, we will find that we have collected some fruits and then these fruits have seeds inside.
So, these seeds have to be taken out. So, we will have to remove all the pulp that is inside and then when we have this seed out, then we will dry it, may be put it into some machine for some grading purposes. Now when we are doing such activities, there should not be any mechanical damage to the seeds, because if there is a mechanical damage and remember that the seed coat is a protective covering so, if there is a damage to this protective covering then the seed is very much susceptible to further infections or infestations. So, it should not be broken and it should not be infected with pests and diseases.
(Refer Slide Time: 08:59)
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So, when we are collecting the seeds, what are the best days for a seed collection, how do we know that our seed is right enough to be collected? So, there are laboratory methods and then, there are field methods. Now laboratory methods include things like the maximum dry weight. So, for instance, if you look at a fruit so, initially we will have a flower. Now when this flower is converting into a fruit, so there will be a small size fruit, then this fruit will grow bigger and then finally, this fruit will attain the maximum size. (Refer Slide Time: 09:21)
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Now, we would find some small seeds here as well, but only in the larger size fruits will have the seeds that have reached the maturity. So, when we look at the maximum dry weight as a laboratory method, what we are doing is that, we take a sample of the fruits. So, basically, we begin by saying our day and we have the weight. So, in this graph, at day 0, we will have a fruit with a very small weight and then slowly and steadily the weight will increase and then later on, this weight may remain constant and then it will start falling.
Now, this is the weight of fruit, because initially we had a very small fruit, then at this point we have the mature fruit and then at this point, this fruit is drying off or it has split or it has become infested. So, there are some other organisms say, ants or monkeys that have started eating away the fruit. So, in the laboratory method, when we say that our fruit should have reached the maximum dry weight, we want our fruit to be in this range.
So, this is the maximum dry weight and when we are using this laboratory method, we want to ensure that our fruits have reached this weight. Next is the chemical analysis of fat and nitrogen content. Now this is important, because in a seed that is well developed, we will be having an endosperm that is well developed. So, it will be having quite a heavy amount of fat and even nitrogen inside to act as initial starting point for the embryonic plant.
So, we can perform a chemical analysis of the amount of fat and nitrogen that is there in the seeds. Next, we could go for examination of embryo development and endosperm of sample seeds through X-ray radiographs. Now in this case what we are doing is that, we take a sample of the seeds from the fruits and then we perform an x-ray examination of those to understand whether these parts have fully developed or not. If they have fully developed that means that our fruits are now ready to be collected for seeds.
Next you have moisture content of fruits. So, in a number of cases the moisture content will also tell us whether these fruits are ripened fully or not. Then, you have the field methods. So, field methods include density of fruits or also weight of fruits, color of fruits. So, for instance, in a number of fruits will observe a change in color from green to say, yellow or green to red and that will tell us whether these fruits are matured fully or not. And also a visual examination of seed contents after cutting. So, what we are referring to here is that, in a number of seeds such as bamboo seeds, so you will have this husk, but inside there will not be any seed.
(Refer Slide Time: 12:51)

And in certain situations, when this fruit is completely developed, we will have a seed inside. So, you can always take a few samples out and observe what is inside the seed, do you have your embryonic plant inside or not? So, that can also be done. (Refer Slide Time: 13:16)
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Next, which trees should we collect our seeds from? So, we want to develop a seed banking facility. So, which trees should we collect our seeds from? So, the first point is that, you should collect from dominant or co-dominant trees. Now, dominant or co-dominant trees means, trees that are the highest, that are there in that particular stand. So, a dominant tree has come out and is getting sunlight from all the directions. So, you should take seeds from dominant or co-dominant seed, because they are the largest size trees and they are also considered as having the best genotype. Then, you should collect from a minimum of 10 seeds at a time, preferably from 25 to 50 trees..
So, you are not collecting just from one particular tree, because you want to have a diversity of genotypes that is there. You should collect from trees that are far from each other to avoid collecting from half siblings or parents. Now, in this case, what we are referring to, is that if you have a tree and if you try to understand that if there is a fruit or a seed here and when it gets dispersed, what is the maximum distance that it can go out. (Refer Slide Time: 14:11)
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So, for instance, if this is the maximum distance to which you can have the seeds or the fruits of this plant or this tree. So, the next tree that should be collected should be outside of this distance. So, this should be the next tree. Why? Because if you are collecting from a tree that is here when this particular tree; so, let us call this tree 1, tree 2 and this is the third tree.
So, if this is within this distance, so it is quite possible that T 3 is the progeny of T 1. So, essentially, if say, this tree was a female tree and it was getting pollens so, these flowers got pollinated, they formed the seed and then, this seed fell here and this form our T 3.
So, in this case, when we are collecting seeds from T 1 and T 3, the amount of genetic diversity between both of these collections will be less, because they are related to each other. Whereas, when we are collecting from T 1 and T 2 then, because they are very far from each other, there is a reduced possibility that both of these trees are related to each other.
So, in that case, the amount of genetic diversity between both of these will be much greater. So, you should collect from trees that are far from each other to avoid collecting from half siblings or parents. Now, before collecting, mark the individual trees and create a record for these, collect equal numbers of cones, fruits, or seeds per tree. Now, why is this important? Because essentially what we are doing is a sampling procedure. So, for instance, if we collected say, 1000 seeds from here and say, 10 seeds from here, then this will not be representative of what is being found in the nature.
In nature, we are having one tree that is T 1 and one tree that is T 2. So, if we are collecting 1000 seeds from here, we should also be collecting 1000 seeds from the second plant. So, this is what is being said here; collect equal number of cones, fruits, or seeds per tree, whatever your collecting. Next, mixing of seeds can be done for large scale collections. So, it is not necessary that you should keep the seeds of T 1 separate from those of T 2; you can mix all of these depending on the conditions.

Refer Slide Time: 16:45).

Now, when we are specially collecting for seed banking purposes, then we generally increase the number of seeds of fruits that needs to be collected. So, in place of 25 to 50, we generally say let us have even more number of trees per gene pool; 50 to 100 trees. And in this process, dominant and co dominant trees are preferred, but we also might go for a random sampling, including poorer than average trees, because even those poorer trees might be having some genes that are important for biodiversity.
So, it is quite possible that our tall and dominant trees are having less amount of disease resistant, but are shorter trees are having a better disease resistance. So, when we are collecting for conservation purposes only so, in that case, a random sampling is preferred, including those of poorer than average tree. Next, collection in better seed years for better representation of parents. Now what is this saying? So, when we talk about a seed year, so essentially if you have these trees in the forest.
(Refer Slide Time: 17:43)

Now, the seeding can happen in two ways, one is a sporadic seeding. So, in this sporadic seeding, what happens is that, in a particular year this tree might be having fruits and this tree might be having fruits, but the other trees are not having fruits or it is possible that a number of these trees are having fruits, but the number of fruits that are being given out are very less.
So, typically in the forest, we observe that in a period of say, 3 to 5 years there is one year in which we will have the maximum amount of fruiting and seeding. And then, this will remain subsided for another 2, 3 more years and then it will again peak up; so, we will observe a graph such as these.
Refer Slide Time: 18:35)
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So, here we have the number of seeds or number of fruits per tree and here we have the time or let us call it the year. So, here say, we have the year 1990, 1995 say, 2000, 2005, 2010, 2015 and so on. Now, what happens is that, typically we observe that there is a peak and then we will have a break, then another peak and then another break and then another peak and then another break and so on. So, these years, in which we are having the highest number of seeds of fruits per tree, are known as seed years. So, this is one seed year, this is another seed year, this is another seed year, this is another seed year and all of these seed years show a certain periodicity. So, essentially if this period is say, 7 years then this period will also be around 7 years, this period again will be around 7 years.
So, when we are collecting in better seed years, what is happening is that we are having more number of a fruits per tree and also more number of trees that are fruiting. So, essentially we have a larger number of seeds. When we have a larger number of seeds then the amount of diversity is also greater, because in our previous situation here, if we collected in this particular year, we will only have the diversity of these two trees, but when we are collecting from our good seed year, what is happening is that, this tree is also fruiting, this tree is also fruiting, this tree is also fruiting, this tree is also fruiting, this tree is also fruiting, and this tree is also fruiting. So, in that case, we will have the diversity of all the trees together; so which is why, a good seed year is preferred for the collection of seeds. Next, larger quantity of seeds to be collected for each provenance. So, a provenance means a region.
So, for instance, in the case of teak, you can have take from Madhya Pradesh, you can have teak in Kerala, you can have teak in Tamil Nadu, you can have teak in Karnataka. So, all of these are different locations from which you can have the teak seeds. And when we are collecting for the purpose of conservation, when we are collecting to create a seed bank, we want to have as many seeds as possible. So that, for each sample for say, every species like teak, if we have say, around 1000 seeds then it is normally observed that the viability of the seeds goes down with time.
(Refer Slide Time: 21:20)

So, this is viability and this is the time. So, right after collection, you will have the maximum viability, but then later on, it will go on decreasing and finally, it will reach a viability of 0, after say, in this case let us say this is 100 years. So, this is the viability curve. Now, if you are collecting a very less number of seeds then, with this graph you might have a zero viability at the age of say, 30 years. So, we always want to have a larger number of seeds for provenance so that, even after a long period, even after a very long period has elapsed, we still have some seeds that are viable and that we can use to propagate the species.
Refer Slide Time: 22:12)
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Now, when we are doing the collection, we need to organize a number of things; we need to organize collection teams. So, these collecting teams have to be taught about how this collection needs to be done, what are the do’s and do not’s that should be done. So, for instance, if there is a tree and if there is a seed that has fallen down into the ground. So, after it has falling into the ground, it might be covered with dirt, it might be having some fungi together with this dirt. So, this seed should not be collected whereas, seeds that are still there on the trees are better. So, things which is these have to be taught to all of these collecting teams. Then organization of transport, because you do not want to keep seeds out there in the field, we need to bring them to our facility as soon as possible so that, they are preserved in a better manner
So, organization of transport is required, you might require some equipment. So, equipment for climbing of trees, equipment for plucking of branches, equipment for depulping of seed; all these equipments are may be required.
Organization of records; so for every sample you need to know what is this species, where this species was collected, when was this particular sample collected, and so on. Organization of permits; so this is especially important when you are moving your seeds across the international boundaries. So, for instance, if you want to send your seeds to say, Norway, so there might be an number of permits that are required, especially from the biodiversity point of view. And then, organization of seed extraction from the fruits. So, once you have brought your seeds or fruits to the facility, next you need to extract the seeds and process them.
(Refer Slide Time: 23:52)
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Now, what are the ways of collecting the seeds? So, you can collect seeds after they have fallen out naturally. So, which is known as the natural seed fall; the second one is manual shaking. So, you can go to a tree and you can shake that tree, so that all the fruits come down and then you can collect them. Or you can make use of equipment for mechanical shaking of trees. Or you can make use of tree funnels. Now, tree funnel is an interesting concept. So, here what we do is that, if there is this tree that is now giving out the seeds; what we do is that, we construct a funnel around this tree and this funnel is normally make made of cloth.
Refer Slide Time: 24:17)
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So, these are say, some poles. So, this is a pole and this is the funnel made of cloth or a plastic sheet. Now, what would happen in that case is that, if there is any fruit on the tree, let us represent it by a red color. So, these are the fruits and when these fruits break open or when these fruits fall down. So, they will fall on this funnel and then get collected here. Now, this is especially important for those fruits or seeds that are small in size, because in the case of these small sized fruits or seeds, your fruits and seeds might disperse to a very large distance. And then, once they have mixed up with the dirt or the soil that is surrounding these trees, then it become very difficult to collect them back. But then, with this mechanism, your seeds will fall in to the funnel, they are not getting in contact with the soil and so, they are not getting into contact with the microorganisms.
So, once your seeds have collected here, you can take them away. So, this is specially used for conditions such as cones. So, in the case of pine cones, there seeds are very small so, this is one thing that could be used. Another thing is raiding of animal caches like, those of squirrels and ants.
Now these animals, squirrels and ants, they collect a number of fruits and they collect a number of seeds and then they bring them to their own homes and then they collect them there or they store them there for weathers that are not very good.
Now, in these cases, if you raid a squirrel cache so, what we will find is, seeds from a number of different trees surrounding those areas. So, that could also be one-way that can be used. Normally, we do not go for these because the places where these animals store the fruits and seeds are generally damp and in those damp areas these are very high probability that you could have a fungal infestation. But then, talking back about our first example, the Siberian permafrost, there the fruits were collected from one of a squirrel cache. So, that is one example where we collected seeds or fruits from a very old sample. Another is collection by plucking.
So, in the case of plucking you can climb a tree and then you can pluck different fruits once they have matured, or you can have collection by cutting breaking and sawing. So, in this case, you can cut a branch or you can pull a branch to break it or you can saw a branch to break it and once this branch has come down, you can then collect the fruits from these plants.
(Refer Slide Time: 27:16)
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Now, once your fruits have been collected, there are a number of other operation that need to be done. One is de-pulping. So, in this case you have separating the pulp out from the fruit. Now pulp is a soft tissue, it has a lot of moisture and so, there is a larger chance that your seeds might get infested, if they are surrounded by the pulp. So, we remove this pulp; this can be done by hand by soaking the fruit in water or by using a mechanical device. Next after pulping, you could go for a drying under the shade or a sun drying, depending on the species, depending on the seeds you will have to dry the seeds.

Now why is drying done? Because typically, our seeds have a larger moisture content and at larger moisture content, the probability of the seeds getting infected or infested becomes larger. Whereas in the case of dried out seeds, there is hardly any microorganisms that can grow there. Now a case in point here would be the date palm seeds, that we saw earlier. So, even after 2000 years, they were able to retain their viability, because they were stored in a cool and dry place.
Now, in certain situations, if you need to dry these things very fast or when the weather is not supporting, we can even go for drying with artificial heat or using kilns. Now in the case of some seeds, you have wings attached to the seeds. So, in this case, you will have a seed, so this is a seed and then there is a wing attached to it. Now why are these wings attached to the seeds? Because remember, you have this tree and this tree had a fruit and this fruit had a seed.
(Refer Slide Time: 28:38)
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(Refer Slide Time: 28:48)
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Now, if this fruit breaks open, the seed will directly fall down, whereas if this seed has wings attached to it; so, if this seed has these wings, then when there is even a small blow of wind, these seeds will be able to disperse to a farther of place. So, just for facilitating dispersion, there could be a number of seeds that have these wings. Now once you have collected these seeds, there is no purpose of having the wings together.
And besides, if you have a seed with the wings, so the later processing might become difficult. So, for instance, if you want to grade these seeds, for instance, you wanted the bigger size seeds in place of this smaller size seeds, because the larger size seeds are more matured; they are having more amount of endosperm. Now in that situation, when you are putting your seeds through a grader, then these wings may come and block the holes or block the seeds. So, in those situations, we may go for a de-winging operation. So, both of this wings are taken off.
Then you could have, threshing and sieving operation so as to remove all that husk or all the broken parts of the wings or the broken parts of the fruits that are together. So, you can have this threshing and sieving operation or a blowing operation. Then we can go for a sorting and a grading operation. Now in sorting and grading, we do two kinds of things; one is a visual examination, in which you ensure that your seeds are not having any defects, especially they are not broken down mechanically, their seed coats are intact; they are not showing any signs of infestation..
(Refer Slide Time: 30:33)
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So, for instance, if you have a seed, if there is a seed and then there is a black colored spot here and a black colored spot here. So, this might show that there is some amount of disease that is coming up on the seeds. So, there is some amount of microorganisms growth that is going on in the seeds. So, in the grading operations and in the sorting operation, we remove these seeds. The second sort of grading operation is when you sort your seeds by size. So, in this situation, what happens is that, you have a sieve.
(Refer Slide Time: 31:03)
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So, this is a sieve in which you have holes that are there in different gradation. So, this one is a small diameter, this one is a larger diameter, and this one is the largest diameter. Now, when you put all your seeds here and this particular sieve is shaking in this manner.
So now, when all of these seeds fall and when they move down, all the smaller seeds will come out through this hole and collect in one bucket. The larger size seeds will come down from this hole and collect in this bucket, then this bucket, and then this bucket. Now these seeds are the largest sized seeds and these one are very small sized seeds and then these are the medium size seeds.
Now, because these seeds have the largest amount of endosperm and because, these seeds have reached the complete maturity. So, the amount of viability in these seeds and also the amount of regeneration that we will observe from these seeds is much greater, typically than these smaller seeds.