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Module 1: Plant Metabolism

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Synthetic Seed Technology

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So, an extension of Somatic Embryogenesis, we will be talking about Synthetic Seed Technology.Now synthetic seed technology involves somatic embryogenesis, for which is needed for largescale propagation of plant material.Now, even somatic embryos can be used for clonal propagation of plants and they canbe genetically stable, depending on whether they are indirectly produced or directly producedfrom the explant.Now large-scale propagation requires what; planting material of suitable age, and ofhigh germination rate.So, a large number of somatic, the advantage of somatic embryogenesis is that,with thesame genetic composition you can produce a large number of plantlets in vitro by scalingup somatic embryos.So, either you can have embryogenic cell lines; and then you scale up the embryonic cell culture;and then in a two stage cultivation, convert it into a somatic embryo form . So, the technologyis , now in synthetic seeds what is you cannot directly use; in vitro conditions you candirectly use the somatic embryos, then regenerate into plantlets.But if you use to , if you want to use it outside as seeds for planting material, thengenerallyit is encapsulated.It now encapsulated, there are four ways in which synthetic seeds are produced; one isuncoated, desiccated somatic embryos this is one way of producing synthetic seeds; thencoated, desiccated somatic embryos.Now desiccated means what, dried; then encapsulated, hydrated somatic embryos which means, theencapsulation is a hydrogel or which can provide water content; then uncoated hydrated embryos.Now,there are advantages merits and demerits associated with all these four types dependingon the hydration, dehydration, tolerance of the species of the somatic embryo; it canbe decided whether to encapsulated, not encapsulated.So,you need to make sure whether the encapsulation there will be a balance out between it isvigorousness to convert the time it takes to regenerate into plantlets and the protectionfrom the environment, such that it does not become non viable.So, let us talk about the coatings.Uncoated, desiccated somatic embryos what are they, somatic embryos they are dried toreduce the moisture content up to 8 to 15 percent.So, the moisture content is reduced such that it is observed, that they can remain viableup to a period of 12 months; which means, they can be stored for a year long period.Now somatic embryos they are, in comparison to seeds they are less vigourous.So, the vigorousness means what, the capacity of survival or remaining healthy in orderto regenerate into a new plantlet.Now, the survival rates of the embryos it also depends upon the desiccation tolerance;which means, how much that species is tolerant towards dehydration or loss of water.Now in that case, if suppose a species or the somatic embryos is sensitive, then hydratedcoatings are used.Hydrated coatings they are generally for example, made of hydrogels; what are hydrogels forexample, very commonly used, when you make your beads.Calcium.What kind of beads do you make?Calcium alginate.You use sodium alginate mixed with calcium chloride to make calcium alginate and whichis a gel bead.So, hydrogel means what, hydrophilic.So, which means a lot of water they can swell, because of the absorption of water.Now this water is nothing, but a dispersed phase in the cross linking that crosslinkmatrix of the polymer and the cation which you are using.So, desiccated embryos they have, please remember they have poor conversion while hydrated encapsulatesomatic embryos they have higher conversion frequencies, sometime similar to that of theuncoated somatic embryos.So, what all can be used to generate more number of somatic embryos or to increase thevigorousness of the somatic embryos; people have used amino acids, organic acids.Organic acids like aspartic acid, malic acid; so in the form of salts potassium malate orpotassium aspartate these are called as even succinate, so these are called as organicacids.So, supplementing the media with this organic acid as also helped in increasing the numberof embryo generation and even the conversion of embryos.Now abscisic acid, which is ABA; abscisic acid has been found to improve the developmentof embryos.There are four different stages, we learnt in somatic embryos.So, the rapidness through which the somatic embryo converts it into a cotyledonary stage;where rootand shoot primordium is formed and which leads to regeneration of plantlets.Now what all other factors which can affect is, oxygen concentration; because if the seedbecomes encapsulated, there is high chance that there will be mass transfer limitations;and which factor becomes mass transfer limited is oxygen, because oxygen is sparingly soluble.So, steep oxygen gradients have also form, have been found to be limiting the somaticembryo regeneration.So, in that case as you used in fermentation technology, oxygen vectors are used; theseare nothing but organic compounds like your,for example, you will hear dodecane or these arenothing but hydrocarbons of fluoro hydrofluorocarbons.So, why are they used as a oxygen vectors, even in fermentations you will find that oxygenvectors are added in the medium to improve the oxygen transfer capacity.Now how does it improve?Because these have higher solubility of oxygen in comparison to water; like in water howmuch at ambient temperature and pressure?8 mg per liter around.So, but for example, in dodecane it is around 55 mg per liter; then in hydrofluorocarbonsit can go even up to 100 more than 100 mg per liter.So, depending on the size of these hydro fluorocarbons or hydrocarbons you disperse them into theaqueous solution, they will form a film around a gas bubble.So, what are all different phases are now involved; the gas bubble to this oxygen vectorfilm, from the oxygen vector film to the aqueous layer, from the aqueous layer to the cell.So, now because what will improve, the driving force improves, is not it; because there ishigher oxygen transferred from the gas to the oxygen vector film, so thereby leadingto better oxygen transfer rates.So, these are also sometimes preferred, but depending on what is the droplet size, howit is dispersed in the medium that is also a critical think.So, agarose, what other these hydrogels are used materials agarose, sodium alginate, polyoxyethylene,so they form a filmwhich has a non damaging effect on the embryo.So, hydrogels I have told you what are hydrogels, water loving polymeric matrix.So, which absorb water, so water is the dispersed phase and these metrics polymeric matrix whichthen swells up.Now, let us talk about germplasm conservation; somatic embryogenesis or synthetic seeds canalso be used for germplasm conservation.Generally seeds are used for germplasm conservation, but there are demerits associated with it,we will talk about it.So, first thing what are the different ways in germplasm conservation is done; one iscalled as in-situ conservation, the other is ex-situ conservation.Now, in situ preservation of the plant material is using your biological national parks, orreserves, biosphere reserves you will find, then gene sanctuaries are where the originalplant material in it is natural habitat is maintained.Now there are limitations to this kind of conservation process, the limitations logicallywhat is it? space.One space, but more than that, if we are talking about conservation what will be the impactingfactor on this strategy.Any geographical or an environmental calamity, so you cannot save it.Then ex situ preservation is what, that you provide suitable conditions in the gene bankto preserve this material; for example, you preserve them either in the form of seeds,synthetic seeds, or different forms of in vitro cultures like even shoots, meristems,or somatic embryos.But then conditions have to be provided; which means, the media, how do you cryopreserveit, or how do you preserve it matters.So, that the viability is not lost during the prolong storage period, we will talk aboutthat.So, I said there is a demerit associatedif you are conserving the genetic material inthe form of seeds.The demerits are, there it can lead to seed dormancy which is; for example, we workingwith viola odorata that benafsha plant, if you remember for cyclotides production I wastalking about and we found it very hard.We got the seeds from one batch, we got from UK and the other we were trying to procurefrom Australia ah; but we did our level best, but those seeds could not regenerate intoplants.And the seeds and it was known that viola suffers from this demerit, the seed generallypropagation of viola odorata is not preferred through seeds, because of the seed dormancyproblem.So, it is generally propagated vegetatively through runners, which is a part of the stem,lower part of the stem.So, that is a problem, seed dormancy can be a problem, seed viability can be a problem;then seed destruction by indigenous, pathogens can be a problem, so where you can lose thegenetic material.Then they are also confined to, now seed if you are using a seed for conservation, thenit is confined only two plants which propagate through seeds.Plants which do not propagate through seeds which are vegetatively propagated, then therehas to be a way for conservation of such plant material.So, vegetatively propagating plants they are generally conserved using in vitro conservationtechniques , where I said you used different parts like shoots, meristem, somatic embryoskind of cultures for preservation.Now the advantage is, that you can conserve as she was saying one of the demerits largespace is required; but if you are using in vitro cultures in small amount of space youcan conserve a store a large amount of material.Then it overcomes the problems of climatic dependency or environmental dependency, thenprovides large amount of material for culturing, for regeneration later.So, Cryopreservation; now once we are talking about conservation, for storage you need tostore it under right conditions and properly, so that the material remains viable duringthe storage periods.So, for that cryopreservation techniques are important.So, how do we cryopreserved plant materials or any kind of cells cell cultures.Now cryopreservation is what, preservation under subzero temperatures, freezing temperature,subfreezing I would say.So, generally you will find whether it is animal cells, plant cells, microbesliquidnitrogen.So, for plant cell cultures, animal cell cultures the biological material is stored under liquidnitrogen or in the vapours of nitrogen.You can , why are we storing the material at such low temperatures; because it has beenobserved that temperatures in the liquid nitrogen which is so low, subzero or entire metabolicactivity of the cell can be brought to stand still, can be arrested.So, what is preferred, you gradually first reduce the metabolic activity and then bringit to stand still.Now then, what is the right way?You take the biological material and put it in liquid nitrogen.So, it can be stored, but there is a demerit; this is not how it is stored, specially thecells where viability is important.Now why do you think, it is not stored as it is, bringing it in and storing it in liquidnitrogen; cell viability is adversely affected under subzero temperatures, the first questionwhich arises is why?Water in the cell . Very nice.So, there will be crystallization happening.So, what will happen, it will form crystals the crystals will grow an intern as the crystallizationof water might happen, many things happen; one is they can be direct damage effects tothe organelles, they can pierce the cell membrane, rupture the cell membranes, or the organellemembranes; thereby reducing the viability, what else there can be dehydration.What else dehydration in what different ways?Internal crystallization is more related to internal damage.So, how does the freezing begins?Suddenly everything all together, so outside there is water getting frozen, then the cell.So, what happens?There is a water.There is a water yeah, there is a water loss causing dehydration and as you said some effectsdue to the internal water also freezing, not been available; so then that also causes dehydrationand loss in viability.So, what is done?Cryo protection is done, before cryopreservation.So, what is cryo protection?We use cryoprotectants agents which can reduce the damage because of cryopreservation.Now what are cryoprotectants?Cryoprotectants, now in nature also plants must be, because plants cannot move is notit.So,extreme in cold countries, how do plants manage that is called as.Very good that that process is called as cold acclimation.So, what does it mean?It means that entire genetic program is switched on, which leads to up regulation down regulationof many proteins, and also which in turn leads to change in the metabolism, change in thegrowth, reducing the growth, balancing out the energy levels, theamount of PR proteinswhich means, specially antioxidants; then the cell membrane composition in terms ofunsaturation of your lipid bilayer changes; then PR proteins antifreeze proteins are producedsugars, poly , alcohols, polyols; then proteins which can act as or substances which can actas osmoticum's, the cell starts producing.So, similar thing we try to embibe in vitro.So, we add cryoprotectants which include glycerol, thenyou must have heard about the glycerolstocks; why do we prepare glycerol stocks?That is the very reason; the glycerol stock composition also vary, sometimes you willsee in literature people use 45 percent glycerol stock, some people use 20 percent glycerolstock depending on what species you are working; whether you want to cryopreserved bacterialcells, whether you want to cryopreserve fungal cultures, whether you want to cryopreserveplant cells; the glycerol percentage keeps wearing.So, these are sometimes DMSO.Now these are called as cryoprotectants depending on the different nature in which they areuse; some are cell penetrating agents, some are cell dispersing agents which can easilyget mixed with water and balance out the osmotic pressure.So, what is cryo protection?So, I will repeat cryo protection is necessary for cryopreservation of the plant materialunless they are naturally dehydrated.So, generally when we say cold acclimation whichplants use, it is nothing but a slowdehydration process; you will find that suddenly the spring comes and you will find new budscoming in, new leave starts coming in.So, how is it happening?Because these axillary buds or these buds which will regenerate into new plant materialare cryopreserve or are cold acclimized, so through this process which I was talking about.So, chemicals such as DMSO which we use chemicals in vitro conditions, glycerols, various sugars,sugar alcohols they help by protecting the living cells against the damage under subfreezingconditions.Now the question arises, how?So, how do they protect?These compounds they do what; you increase, you must have heard during your 10th, 12thstandard, lowering of freezing temperatures.How does it happen?You increase the solute concentration; the freezing point reduces of the solution orthe solvent.So, the same thing happens here , now you lower the freezing point of water by addingthese certain agents, like sugars.So, it lowers the temperature at which the freezing would first occur and can alter thecrystal structure of ice even.So, the colligative properties of the cryoprotectants it minimizes the harmful effect by the crystallizationof ice.So, what all properties of cryoprotectants are essential?They have high solubility in aqueous phase they should have; then they should be highlypenetrating, so that they can reach inside the cell; then they should be low toxic, shouldnot be very toxic to the cell.So, the toxicity has to be taken into account; which means, the amount of cryoprotectantyou are using, the concentration it matters has to be optimized.So, what are the different ways through which these cryoprotectants work?The phenomena which can cause damage to the cells during cryopreservation; so what isthe phenomena?Mainly occurs during the freezing stage and includes solution effects, extracellular iceformation, dehydration, and intracellular ice formation.So, what do we mean by all these four things, what is solution effect; an ice crystal whichgrows in freezing water, solutes are exuded, causing them to become concentrated in theliquid water.Once as you people said the ice is forming, so rest of the cytoplasm is getting concentrated.So, this high concentration of some of the solutes can be very damaging.Now extracellular ice formation how is it happening?When tissues are cooled slowly, water migrates out of the cell, we know because of the osmosis;now as the water migrates it will cause ? Dehydration.Dehydration.Now when tissues are cold slowly water migrates out of the cell and the ice forms in the extracellularspace.So, sometimes the ice which is forming in the extracellular space will also cause damageby pressing the cell wall and the cell membrane.So, generally you will find that when extracellular ice formation startstaking place, the intercellularspaces they get widened; there is cell to cell contact.So, now, you will find there is more gap and the water there gets crystallized and thecells are then; the cell walls and the cell membranes may get damaged because of the crystalformation outside.Now, dehydration, now you understand how dehydration would occur; migration of the water causingextracellular ice formation, can also cause cellular damaged, because inside now thingsare more concentrated.So, everything is interrelated even the solution effects, the extracellular everything is happeningsimultaneously.So, then intracellular ice formation; if suppose intracellular ice formation happens, thenthe crystal is growing, it can directly cause damage to the intracellular organelles.So, if I ask you in what different ways the cell damage, or subzero freezing, or cryopreservationmay damage the viability of the cell, you should be able to tellSo, what is the procedure?The different cryoprotectants which are used are one is called as permeating cryoprotectant;for example, DMSO, methonal, glycerol there are highly soluble in water.Then non-permeating can be: sugars, sugar alcohol, high polymeric substances, molecularweight polymers.Now you can use the best strategy is to use a combination of these cryoprotectants, becausethe mode of action is different; some are cell permeating, some can causereduce thedehydration process by reducing the osmotic pressure, so can have cosmetic effect.So, better the proposed solution is, you use a mixture of these cryoprotectants.So, what is the general protocol for freezing, it is not when you are freezing, it is recommendedto do slow freezing not sudden; it is not recommended to bring the biological materialdip it in your cryoprotectant and suddenly you put it in minus 176.So, that adaptation is what, when you are doing a slow freezing versus very fast freezing.If I give you a hint, then when you do slow coolingit will give the cell the time to equilibrate;this equilibrium, vapour pressure equilibrium will intern facilitate the internal waternot to freeze, but remain at sub cool temperatures.The idea is, to prevent internal crystallization.So, how does it happen, once you lower the temperature the concentration of the extracellularliquid increases, as more water is converted to ice.Now, since the vapour pressure of the frozen solution is lower than the intra cellularsuper cooled liquid, the vapour pressure of the slowly cooled cells reaches equilibriumwith external ice by a fluxing water.So, you are giving time for the cells what you are saying to adapt.So, thus slow freezing can prevent the intracellular ice formation and consequently the freezinginjury by the crystal formation; and then subsequently the crystal growing inside thecell.So, in order to avoid this crystallization recrystallization, so we try to thaw it.Which means what are you doing, to improve the heat transfer rate there has to be a biggerdriving force.So, you will be suggested to put it in a water bath, 35 to 40 degree C or you thaw usingyour thumb from subzero which is minus 76 to , what is the palm temperature approximately.So, that is what is recommended to prevent this recrystallization of ice.So, generally for plant cells what do we at a constant rate of 0.5 to 2 degree C, we areat this rate it is slowly brought down to some intermediate temperature before it isimmersed in the liquid nitrogen, and then rapid thawing is recommended ok.So, the applications of this preservation or conservation is, you can have clonal propagationor same genetic material propagated later and you can conserve the genetic material.Preservation of rare genomes can be done using this method and free storage of cell culturesor even cell lines, for even secondary metabolite production or any other commercial applicationcan be done .