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Module 1: Plant Development

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In-vitro Culture Initiation

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So, todaywe willstudy about different forms of cultures which are used and their applicationsmeans in plant biotechnology and what are the nutritional requirements of plant cellsbefore starting with the different forms of in-vitro cultures.So, then starting with culture initiation we all now know that any part of the plantcan be used as an explant to initiate different forms of cultures.So, these plant parts are used to initiate and establish in-vitro grown cultures.So, why do you think in-vitro plant cell culture or tissue culture can be of use because, theycan act as model systems to study the effect of various environmental parameters on theplant physiology and it is growth and development.So,because as under in-vitro conditions which are controlled environmental conditions meansyou can study each factor at a time, while in whole plants under natural conditions thesethesoil conditions or your environmental parameters are not under control.So, field grown plants we have now we know that they can undergo photosynthesis.So, you do not have to provide carbon sources or nutrients to these plants exogenously theycan themselves procure from the soil and use sunlight to produce the food source.But in case of in-vitro cultures this photosynthetic capacity is impaired they do not carry outas large amount of photosynthesis as the whole plunders.So, they need to be supplemented with exogenous carbon source this is generally sucrose usedas carbon source now what all different parts of the plant which can be used as explants.They can be nodal they can be internodal segments they can be apically apical or axillary meristematicregions petiloles even your petals flower petals ovules ovaries, so any part of theplant can be used effectively as an Explant.Now, how to decide which kind of an explant to be used, it also depends on on what kindof in-vitro culture you would like to carry out.So, we will see when we will be discussing different forms of in-vitro cultures whatare the explants used which can lead to different forms of in-vitro cultures.So, what should be taken care of when you are starting to initiate these in-vitro cultures,if suppose you are bringing an outside plant material to inside to be used as an explantfirst thing is you need to make sure that you bring it in a nice box or very quickly,so that the catabolic activity.As soon as you from the plant the breakdown begins.So, you need to minimize that catabolic activity, so in order to minimize you should bring itin an icebox and as quickly as possible.Now the second thing is there will be contaminants if you are dependent on the outside plantsas explants.So, then you need to do surface sterilization which is one of the key and the initial stepsin initiating any kind of in-vitro cultures.So, how to carry out surface sterilization, when I say surface sterilization which meansthat removing these surface contaminants.So, first thing is that the material which you bring to the lab from outside it shouldget rid of the soil.So, continuous water wash is done with the help ofa surfactant in it or any detergentin it, for example savlon is used sometimes you can use any detergent.Then subsequent washing so that the detergent is lost from the surface.Now then you treat it witha wetting agent generally used wetting agents is up to 70percent of ethanol volume by volume, this is what is used in plant tissue culture labs.So, when I say wetting agents like for example, the 70 percent ethanol is used as wettingagent what does it do from the word itself you can make out wetting agent.it just increase the time period for the detergent or any disinfectant we are using this in increasingthe bymaking it wet for the longer time, because if we use100 percent the ethanol that it canevaporate it easily as compared to 70 percent ethanol.Then why at all to use it through . It also so.Closer to what she said wetting agent is not too wet, but wet in a sense hence that itwould facilitate the penetration of the disinfectant inside the explant.So, as to increase the probability of removing all the contaminants or killing the contaminants.Now when I say it increases the probability or the penetration of the disinfectant, howhow do you think it is increasing the penetration of the.Increasing the . Surface related to surface tension very good,but what does it do to surface tension and off what the liquid in which the wetting agentis there.So, suppose you then provide the disinfectant.So, what it would do is it reduces the surface tension of the liquid, when it when you sayreduces the surface tension of the liquid which means means the attraction between themolecules of the liquid.So, that they can get dispersed and get inside the gaps of the available on the explant surfaceand thereby increasing the probability of penetration and disinfection yeah one morepoint.Now when you are using seeds as explants to generate either in-vitro seedlings or anyforms of cultures, sometimes youwhich we observed in lab also for viola odorata.So, this is one of the medicinal plants the seeds were not able to give rise to the plants,despite the fact that whatever was given in literature was being provided as the mediumfor germination.So, that to avoid seed dormancy or to overcome the seed dormancy then these seeds shouldbe treated with either dilute acids or even heating treatment is given.So, now why do you think and what does the term seed dormancy means to you and why doyou think heating or dilute acid exposure is, how is it helping in overcoming the seeddormancy what it must be doing and of course after the treatment with dilute acid thenit has to be water washed properly.So, that the acid is lost is there a role of dormancy.So, till the surroundings think in nature, till the environmental conditions are conducivefor the plant to grow or arise the metabolic activity it has to be kept viable at the positionit is.So, then the metabolic activity has to be arrested; now for that the seed dormancy isused by nature.Now if it becomes so tough that even when the environment is conducive the seed is notable to come out.So, generally what is needed is you there is a covering around the seed which is calledas seed coat.So, when the seed coat becomes very hard such that the penetration by water is very difficult.So, then it is difficult to rupture the seed coat for the seedling to come out.So, then the acid treatment or your heat treatment can help in removing this coat.So, that it facilitates by the exposure of the environmental conditions conducive andthe media conditions the seed can sprout into the seedling.So, how the explants sterilization is done this is from um a book Google book, wheresimple protocol is given.So, generally what is done is you bring the plant material from the outside you give awater wash continuous and then with two percent up to savlon wash can also be given alongwith water and then after that that you treat it with distilled water.So, as to remove the detergent and then after treatment with distilled water you take itin the laminar hood, now the laminar hood they are are laminar hood is what have youused a LAF.Yeah.So, what is LAF laminar air flow ? So, why do we use that either there is a horizontalflow or there is a vertical flow.So, how is it maintaining the aseptic conditions?So, how is that positive pressure being created ?Hepa filter.There is a hepa filter through which sterile air can be pushed in through the mesh withhigh pressure, so that there is a positive air flow outside the LAF.So, the sterile environment inside and you must have heard while working in the LAF itis always preferred to work behind the flame, you enlighten the flame lighten the flameand then it is preferred to work behind the flame or close to the flame.Why because around the flame again the sterile environment probability is higher becauseof the heat generated.So, then when the explants once have the the surface contaminants have been removed bywater wash now that before you treat it with the disinfectant there is a treatment givenby the wetting agent for 15 to 30 seconds.So, and then after treating it with the wetting agent again the ethanol is removed by waterwash intermittent water washes is given for every treatment and then after you treat afterthat you treat it with disinfectants.Now disinfectants generally used in plant tissue culture technology is is either youuse sodium hypochlorite which is up to 20 percent or you use your mercury chloride 0.01to 0.1 percent.Now, mercuric chloride or sodium hypochlorite are your disinfecting agent.So, the exposure time the concentration of the disinfectant has to be optimized dependingon on the kind of species or whether you are working with the tree species, whether youare working with an old explant whether you are working with a young explant all thatwould have to be optimized.So, if you are generally for in-vitro cultures young explants are preferred, why do you thinkyoung explants are preferred?Young explants means parts of the plant which are younger meristematic regions or youngerplant parts.So, why do you think younger plant parts are preferred, I can agree with the meristem butwhy do you think then another question why less contamination ?are more . Very nice very good so because the cells arestill rapidly dividing and have not differentiated the organogenesis and specially the xylemand the phloem bundles have not which become the throughout connections in the whole plant.So, when it is not there so the chances of the contaminant which can be a virus or abacteria living inside the plant to get transferred is less.So, that is one of the reasons meristematic and why younger plants because as you peoplesaid younger plants the cells are still rapidly dividing it is still a growing region of theplant not yet matured.So, because of the rapid division the ability of differentiating redifferentiating is muchhigher than a matured plant part.So, now what is Callus induction, generally in nature callus is a wound response wheneverthere is a wound created it is a repair mechanism in nature.So, suppose there isa lesion which is made in the plant, now thecells near that damagedregion they start rapidly dividing till they cover that damaged region and once they coverwhy do itthey need to cover it because now the inside of the plant has been exposed.So, it has to be now again protected from the outside environment which has so manyother microbes and may be pathogenic environment.So, then the cells keep on rapidly dividing which are around that lesion till that portiongets covered and then once that covering has happened, further on top of it it is coveredby deposition of lignin wax suberin is one form of wax which gets secreted by these cellsto create a hard coat or a layer.So, that is how callus is formed in nature as a wound response.Now when you need to exploit this in in-vitro cultures what would you like to have, youwould like to have a continuous repair mechanism rather than closing it at any time.So, you would like these cells which have now started rapidly dividing to continue todivide forever till you subculture it and provide medium.Generally you may see that even small when you take leaf explants and if you providethe right kind of medium.So, I the question was what do you think will become critical in that right kind of mediumto reprogram the cells or to bring them to dedifferentiated states it is your .Hormones.Hormones.So, generally auxins and cytokinins we will come to that.So,if you can give the right atmosphere and even the environmental conditions not allspecies will give a callus induction response if you provide it light conditions.Some callus induction responses from some plants also happen when you put them undercomplete dark conditions.So, all the conditions have to be optimized depending on species depending on age of theexplant depending on type of the explant and the genotype.So, how is it induced in-vitro the cells from any plant species can be cultured asepticallyon in a nutrient medium.Now any plant part tissue with living cells can be used as an explant as I said, thenunder the influence of plant growth regulators generally when we say plant hormones theyare endogenously produced plant growth compounds or regulators.But if they are synthetically produced or exogenously provided right, then we call themas growth regulators.So, what happens when there is an excision and there is rapid division it will forcebecause, the cells will continue to divide around that region they will because you areusing a tissue which is an organized culture.So, the epidermis it is a tissue so there will be a dermal tissue also in that it isa complete organ which you are using.So, once the cells continue to divide they will rupture the epidermis the topmost dermaltissue and the cells will come out and after few days you will see that there is a blobof tissue which looks like a callus.Now generally it takes one to five weeks for callus proper complete callus induction totake place.If you see that even after two months or so oh you are not observing complete callus inductionor it has become impaired, then which means that either nutrient conditions or growthregulators have to be relooked and then you should revise the media.Now, there can be different forms of callus depending on the species on which you areworking.Now this hardness of friability of the callus depends on the cell to cell contact, how closelythey are connected with each other how strongly and the water content in the callus.So, which means the fresh weight to drive weight ratio of the callus.So, generally for suspension cultures or in-vitro cultures friable callus is preferred, friablecallus which means the cells of that callus should be able to get dispersed easily whengiven suitable conditions.Because hard and compact callus cannot be will become limited where what limitationdo you think you can face the plant cell cultivation, if your beginning material which is the callusis very hard and compact if you not to exploit plants sensitivity growth you need to massproduce it is not it.Now, mass producing the callus you can do 2 days you continue to derive the callus inthe solidified mediumaround you dispersed the cells and bring them in suspension andthen you continue to the and scale it up.So, but what is the problem if I continue to do it in the solidified medium?There the entire tissue is not exposed to the medium.So, how does the callus afford to multiply or grow it is because of that compactnesson the solid medium.The cells which are in touch with the medium they get the nutrients and then from thenit is subsequently transfer to the other cells which are not very close to the media.Now this is a demerit in one sense what demerit?This also gives heterogeneity to the callus nature because there are physical gradientsnutrient gradients.So, thereby leading to biochemical gradients and maybe their bio synthetic capacity.So, that is why callus can be heterogeneous in nature.Now if you need a synchronous culture and all cells giving you reproducible resultsin terms of productivities then all cells have to be given the same environment.So, if the cells get dispersed in the liquid medium all cells are getting exposed to thesame environment.So, the chances are high that the culture would be synchronous in nature and then themass transfer limitations which you people were referring to will also be overcome tosome extent.Now, because they are on the solidified medium, the callus growth in comparison to cells insuspension is also slower.While if you compare it with bacterial cells, the doubling time is still 24 to 72 hourshere and there in bacterial cells is between 10 to 12 hours or 24 hours.Now, callus inductions seeds are preferred at they can withstand severe sterilizationconditions.So, I would still sayit depends on the kind of culture, you would like to even if yousee the books different books say different things, but it is very species specific.So, generally if you want to end up depending on the objective of your study.If you are if the objective is to produce a particular phytochemical from that cellusing plant cell cultivation, then in the end what do you need?You need a very high yielding cell line.So, for generating a very high yielding cell line product yielding cell line, your startingmaterial if it is a high yielding, the chances are all the cells there are high yielding.So, if you can get a callus from that explant which is high yielding, the probability ishigh that you will end up in plant cell lines which are high yielding.So, ideally what should be done is, you should pick up a plant variety which is high yielding.For example, I will give you an example I work with production.So, there are neem as ubiquitous in our country.It can be found in many regions of the country, but because of the geographic and climaticvariation even in the neem variety which is present in different parts of the countryincluding north and South Indiathe yields are variable.So, you must pick up a variety which is higher yielding average high yields are there andthen you must pick up that explant or that region of the plant, which is highest yieldingin that plant.So, for example, in seeds generally have the highest yield of So, we picked up a plantwhich is inI think a Trivandrum variety which was very high yielding in comparison to therewereplants in IIT Delhi also.But then we made a comparison we picked up the plant from here in South India which wasshowing high yield and from that plant we picked up the seed of that plant because theseed is known to have highest yield and that seed was used to generate the callus.So, what I mean to say here is, it it is not necessary that you need to pick up the seedonly you must pick up that explant a part of the plant which shows highest yield ofthat particular metabolite.So, once you have suppose the callus successful callus induction happens after 2 to 5 weeksthen how do you subculture?Let it grow for some time the initial 2 to 3 subcultures, the entire callus which hasbeen induced should be transferred to the fresh medium.You should not disintegrate it because factors like minimum inoculum density which you musthave heard in your microbial fermentation or fermentation technology that critical inoculumdensity is crucial for the growth subsequent growth of the culture.So, you should not the minute the callus has come out that does not mean that you ruptureit disintegrated into fewer smaller sizes and any size or any small amount of cellswould lead to multiplication.There has to be a critical size a minimum inoculum size which is needed for multiplicationfurther.So, once it has grown to some 10 millimeter cubes or so, then you take that callus after3 subcultures of the entire callus blob tissue then you can divide it into two and furthersubculture it for propagation.Now, coming to the suspension culture.So, now, how fro[m]- suspension culture induced from callus?Once you have got the callus then you bring it in suspension there are different waysin which suspension cultures are created either you can use Buchner funnels I will show youa picture.So, either you can use Buchner funnels and if you want a very synchronous culture orvery fine suspension, then you can even in pipe it out few cells else people have donecallus induction with single cells even and then subsequent cell suspension also withsingle cells or small amount of cells.So, you can pipe it out the cells and then use that as inoculum or you can directly filtertheremove the settled aggregates and use rest of the cells remove the liquid part and removerest of the cells as filtrate to be used for suspension culture initiation.Now, what is key here is that, as you do in fermentation of microbes inoculum age, inoculumsize has a role to play in your growth and production kinetics same is true here.Now it is not that if you directly take the cells they will start dividing it dependson what all conditions?It depends on the production medium.Now if you suddenly bring the cells from your inoculation inoculum preparation medium twoproduction medium which is completely different the cells may not divide or there may be along lag depending on the size of the inoculum which you have used.So, the production media then the conditions which you are using in the environmental orthe media composition which you are using is critical then your minimum size of thecells which you are using is critical.Now, there are techniques how you can reach to synchronous cultures.Now for doing synchronous cultures people you see sieves cell sieve methods.Now this can lead to the filtrate different filtrates which will be obtained from differentmesh sizes can then be used for generation of suspension cultures as with a high probabilitythat they would be all synchronous in nature.Now why do you think these all filtrate?Suppose I use 80 mesh size or 150 mesh size and I use that filtrate to generate a cellsuspension why do you think that this increases the probability of getting a synchronous culture? So, because as she said with the same size of the cells coming in same shape of the cellscoming in and the probability is higher that their metabolic activity or their age wouldalso be same so, thereby leading to synchronous cultures and when I say nurse tissue culturethere is a nurse tissue nursing tissue involved.So, in this technique what is done is, you take a callus small piece of a callus of thesame variety or of a different variety and put it on a sterile environment and a petridish and put a small sterile filter paper on top of it and the the metabolites or theliquid which gets released by this fresh callus which is nursing will wet the tissue on topof it.Now this wet tissue which is now supposedly having all the nutrients and other metaboliteswhich have been released onto that tissue, you then inoculate your single cell usingthe piped that single cell from which you need to generate the callus.Once it gets the nursing from this parent callus tissue underneath the filter paperwhich is wet filter paper, you will see that the cells will begin to divide.The cells as they divide after some amount of division you will find the callus becomevisible and then use that callus for subsequent sub culturing can lead to a real synchronouscallus or cell culture because you have started with single cell leading to it is subsequentclones.So, this is what is called a single cell culture technique.And this is to achieve synchronous culture what I was talking as the cell sieving method.Now, another way of carrying out the single cell culture technique is also micro culturechamber method which used to be used which can be used even now.Our in which you use a sterile petri dish that sterile petri dish you put paraffin oiloil on top of that sterile petri dish and then you make use of sterilecover slips, Ihope you people know what our cover slips.Now these take three cover slips two cover slips when they are put on top of that glassslide it with the oil, oil they will form this gap which is shown here this gap betweenthe two cover slides.Now, because there is oil underneath it will form a small oil boundary so, as to give asterile environment so, that whatever is inside now in this square would not be in touch withthe outside world.Then you inoculate your one drop of liquid nutrient with your cell inside that gap andthen you cover this with the third cover slip on top with the paraffin oil.So, now, it becomes like a square small chamber mini chamber or micro chamber where that singlecell is in the drop of the liquid nutrient and then you can see under the mice microscopelet it divide.The cells would divide and then once you have seen it has reached to some groups of cellsmass then that can be subsequently transferred and can lead to generation even of the wholeplant.So, this is called as micro culture chamber technique.So, before we begin with different forms of culture, it is important for us to know twothings.One is how will we find whether the cells are growing and the second is how will youfind and whether the cells are viable.So, how do we find what parameters do we use to quantify the plant cell growth or planttissue growth there are different ways.One is you do simple dry cell weight estimation in which you dry the biomass in oven at 60degree C, the plant biomass or the callus or the tissue biomass and let it dry untilconstant weight is achieved.So, that is simple you calculate the biomass concentration.Now, the second is you use packed cell volume because plant cells are large in size thisworks appreciably well.So, you can use a centrifuge tube and then let the plant biomass because they form aggregates,they settle you will see that the plant biomass after some time will settle down and thenin comparison to the total volume occupied in that centrifuge, that percentage whichthe biomass occupies is represented as packed cell volume.That can also be an indication a rough indication for growth of the biomass.Now what else?Fresh way to drive it which is called as growth index.Now growth index is another parameter which is used when you measure the growth of thecallus which is final fresh weight after some amount of time, you calculate the fresh weightfinal minus the initial fresh weight which means the amount of fresh weight which hasincreased to the initial amount of fresh weight which was there.So, this ratio is called as growth index and is a parameter which gives you an indicationof how much growth has happened.Now, talking about plant cell viability there are many different ways, but here I have listedon three different ways which are generally used in literature to determine plant cellviability one is the fluorescein diacetate vital stain.So, this particular stain in it is based on the activity of esterase in the viable cells.Now these enzymes can reduce this I to produce a fluorescent product which you can then becausethe cells are viable and then the membrane integrity is in place.So, they can hold on to the fluorescent product which you can see under the UV light and percentageviability is given as the number of fluorescent cells to the total number of cells.So, those which are fluorescent would be viable because it shows integrity of the cell membranesstill in place and it also shows the activity of esterases in the that the esterases arestill active metabolically active cells in the viable cells.So, this is how percentage viability can be calculated.The second way is using tetrazolium salts which you must have heard even in your animalcell cultures and others MTT assay.So, TC is a Tryphenyltetrazolium salt it is used and it generally is dependent on theactivity of dehydrogenases enzymes.Now the dehydrogenases is it isit signifies the the metabolic activity of the cell becausewhether the mitochondria is actively functional or not.So, so what it does is,because it is dependent on dehydrogenases.So, once it reacts with this tetrazolium salt it forms a red Formosan product.This red Formosan product can be measured spectrophotometrically, which can then beused to calculate percentage viability of the cell.Now Evans blue Evans blue any kind of such stains they depend on whether now once thecell membrane is the cell is alive the membrane integrity is in place.If the membrane integrity is in place it is selectively permeable.Now selectively permeable either you can use not taking up a particular stain as a viablemarker or taking up a void particular stain and not letting it go as a viable marker.So, in Evans blue dead cells become?Blue.Blue which means what are you looking at?Because the cell membrane integrity of the non-viable cells is not there.So, the will be able to get in and stain the cell . So, that is why we Evans blue is usedto can be used for determining the viability of the plant cells applications.So, in agriculture in what way production of higher yielding or drought resistant orherbicide resistant or salt resistant species of crops.Then horticulture and forestry if you want to do micro propagation or if you want toproduce synthetic seeds.Then in production of phytopharmaceuticals your callus cell suspension cultures or hairyroot cultures which is organ culture comes to play a role.Then products from transgenic plants you must have heard about plants or GMOS or or plantswith specificnutrient quality like your golden rice or plants with which arewhich can beprotectedfrom certain insect pests.So, for example, your cotton hm ok .