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Module 1: Plant Cell Technology

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Introduction to Plant Cell Technology

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Let us see what is the relevance to industry of plant cell technology.So, talking about some success stories, seethis is one of the plant Silene stenophylla plantwhich wasfound the seeds were found in thepermafrost conditions in Siberian region.So, in the fossils of one squirrel burrow, so these seeds could be then lead to throughin-vitro plant cell cultivation techniques could be revived and that is the plant whichyou can see.So, thats the application of plant cell and tissue culture that such and theage old was30,000 years old permafrost conditions.So, in this region, the seeds could be obtained and then the seeds could be revived to leadto a whole plant.So, now, it is a live plant which is giving rise to new progenies .Similarly, it is plant cell and tissue culture can also be used for conservation of endangeredplants and rare plant species.So, some of the plants which are very rare and are under endangered list, those can berevived and can be conserved using plant cell and tissue culture techniques.So, this isIlex khasiana that is a critically endangered plant in north eastern region ofIndia.So, that was able to be conserved using synthetic seeds and plant tissue culture technology.In order to whocatered to the increasing demand in the market whether it is for agriculturalcrops, whether it is from medicinal compounds, owns, you needartificial seeds.So, plant cell and tissue culture can also be used for production of artificial seedsif you want to do large scale shoot multiplication.So, this is how it is done.Cell immobilization techniques where the embryo which is generated using somatic cells willbe talking about it in detail is encapsulated in gel beads, and these can be used as seedsto lead to plants.So, rapidly multiplying stocks of the plant, so large number of plants can be regeneratedthrough these techniques.Now, you all know about GM crops genetically modified crops in order to improve the eitherthe biomass productivity, the growth, or theproduction of that medicinal compounds, genetic modificationis done.And so one of the most successful example is the BT-cotton.So, almost 60 percent increase was found in the productivities is by bringing in the BT-cotton.So, have you heard about bioplastics?. So, up till more than 50 percent dry cellweight basis, people have been able to express phbs - polyhydroxyalkanoates in plants.So, there is an opportunity to produce bioplastics and plants itself.So, where the plants like switch grass which are rapidly multiplying plants or weeds canbe used to produce at stocks for feed stocks for bioplastics, so that is again throughplant cell technology.Anticancer drugs one of the most popular drugs of vincristine, vinblastine and taxol.So, that is this taxol, it is an anticancer drug which is produced from yew tree.So, and we are in the labor also working with camptothecin, camptothecin is another leadmolecule in India we use it for aeronautic and topotecan for lung cancer, cervical cancer,it is marketed drug, so that is obtained from, in India from nothapodytes nimmoniana thatis again an endangered plant now in western ghats because of rapid uprooting by the industries.So, plants again are valuable stocks for high value medicinal compounds.So, now as I said when you use natural plants for extraction of such high value molecules,what are the disadvantages, what can be the disadvantages?hm Extension of that species . You may lead to endanger plant, then plantgetting endangered.What are the disadvantage? yield isIt may not be enough to sufficient.. For to cater to the demand.Demand Ok, what else?What else, Do you think can be a demerit in natural plant extraction?Hard in the sense, if I would put it in a sense that the downstream processing is moredifficult why?Because there are so many other . Very true, because you are expecting it fromthe whole plant, the plant will have n number of molecules ok.. What else?If there is seasonal variation.hm.And along with seasonal there can be geographic variation.See our cells when we were working withah one of the projects which is on cyclic peptidescalled cyclotides in the lab.So, these are small many proteins from plants which have plethora of therapeutic activitiesso and potential.So, when we were looking at it we collected plants from one from the Himalayan region,one from the Ooty region, and some of the plants we brought and wecultivated them inthe horticulture at IIT madras.For 3 years we cultivated them; and after 3 years we began the study ofcharacterizingthe different types of cyclotides, these mini proteins and the kind of cyclotides whichare present in the plant material.Interestingly what we found was that the plants material which was brought from Himalaya thesame variety, the one which was brought from Ooty, and the one which was cultivated broughtfrom Ooty, but cultivated for 3 years had very different array of cyclotides.Now, the plants were the same which were being cultivated in Ooty and we brought them toIIT madras, so that shows that there is high impact of climatic and geographic variability.And these are a class of secondary metabolites.So, they are defense related compounds.So, even the micro flora which is present around in the soil impacts the kind of secondarymetabolite or the expression of secondary metabolites in the plant.So, that showed us us, there was the difference in the aerial parts to ground parts, therewas a difference in different parts of the plant, there was a difference in the kindof certain novel cyclotides came out which were not present in the other varieties inChennai.And the plants which are those there in the Ooty could not show those novel cyclotideswhich we could detect when we cultivated them in Chennai for 3 years.So, that shows that now an industry cannot work at this pattern, it depends on productquality product.Quality means that has to be consistent product quality and quantity.So, now, for consistent product quality and quantity natural plant extraction has a demeritassociated, because there can be variation as she said with season.Some of the plants for example, it is a bio pesticide it is obtained from neem.Now, from and majorly from the part which has its maximum content which is the seed.So, now all throughout the year seeds are not available, there are only available twicein a year.So, now, for twice in a year or once in a year, one will have to wait for that yearlong and then collect the seed.So, there is demand and supply gap which can crop up.So, and in-vitro cultivation technique will have therefore higher productivity becausewith respect to time and moreover less space and time requirement.So, that is what we were talking about.Then the available land because of the population increase, there has to be a priority drawnbetween agricultural crops, medicinal plants.So, there is a gap in the available land.Then low concentration of the product, because as you people were saying downstream processingis difficult, because there are n number of molecules present in the plant.Now, imagine the same carbon flux is being diverted towards array of different metabolites.Under in-vitro conditions you have the plant cells are amenable to process optimization.Now, process optimization which means that you can divert desirably the metabolic fluxtowards your desired compound.Then limited and non uniform supply this is because of the climatic and geographical variabilityin nature.So, therefore, what can be an alternative strategy, an alternative strategy is likeusing plant cell technology where under controlled environment if you grow the plant cells andyou force them to produce your desired metabolite that is the advantage of plant cell and tissueculture.So, what is plant cell technology?Plant cell technology, there are different forms of in vitro cultures which come underplant cell technology, you can do cell culture, you can do organ culture.Organ culture can include your somatic embryos or your roots, simple roots growing multiplyingthe roots only, or multiply shoots just continue to multiply shoot, or hairy roots agro bacteriumtransformed cultures.So, these can be different forms of cultures which can be used for large scale productionof the high value chemicals . So, asdefinition what does it mean, the cultureof plant seeds organ tissue cells or protoplasts.Now, what are protoplasts?. Cell wall, cells devoid the.Devoid of the cell wall.So, sometimes protoplasts are required they becomes it becomes easier to manipulate anddo hybridization with protoplasts, because cell wall has been removed.So, what is so different about plant cells which has been exploited in plant cell technologyand peculiar about plant cells, a very good advantage which is totipotency.Now, what is totipotency?Totipotency comes from the theory a cell from cell . So, cell theory everybody knows.So, all the genetic information is present in the cell.So, totipotency is the ability of the plant cell, any plant cell, to reverse even if itis organized and performing a specialized function it can reverse back to its multiplicationstage.And again reprogram its genetic information to reorganize itself into some other specializedfunction that is what is totipotency.So, the potential or the inherent capacity of the plant cell to develop into an entireplant.So, even from single cell you can regenerate by rapid multiplication and reprogrammingof the genetic machinery into an entire plant.Then it implies that all the information which is required to generate into a plant is presentin the single cell.It is just that not all information is being used at a given time point.The cell has all its information, but some cells are performing one function some cellsare performing the other function.It does not mean that it does not have the genetic machinery, it is about expression,whether the cell is expressing all the genetic information or not, this is what we also foundwe are coming back to the giving you an example on cyclotides.Why were we able to find novel cyclotides in the same plant variety, although it wasthe same plant which was cultivated from Ooty transferred to IIT madras, suddenly from whereis the novel cyclotide coming which means that the genetic machinery must have beenthere, but must have been encrypted . It is the epigenetic factors or the environmentalconditions which might have been able to induce the expression of these silent genes.So, now how some idea about the history, it began in parallel around the world war timesboth in Europe, in US, they could continuously culture the carrot cells in the medium andkeep it live that was the first breakthrough.And then later they also found they were using coconut milk, and they also found that thereis a compound in the coconut milk the first auxin to be discovered was IAA - Indole AceticAcid which was set to be playing a huge role in expression and keeping the cells life.But everybody then started thinking that IAA is the compound used for in vitro cultures,but when coconut milk also was being used witha this advent of your microscopy and youryour analytical instruments, ands it was also found that apart from auxin, hm there is someother compound which is not IAA auxin present in the coconut milk which is critically affectingsignificantly affecting the in vitro cultures.So, that then came the discovery of kinetins.So, that is how auxins and kinetins came into the picture.So, the initial discovery and all the inventions which took place in plant cell technologydealt withthe nutrient requirements of plant cells, then the different forms of cultures,what can lead to roots, what can lead to shoots which means auxins and cytokinins.So, there was a lot of discovery of hormones and importance of hormones which took place.Then later people came out with nutrient requirements, people also came out with predefined mediumcompositions MS media than gamborgs medium.So, these are are varying in nutrients sometimes in hormones presents.Nitrogen source in some of the media it is present, in some media the nitrogen is notpresent.So, depending on what kind of culture in vitro cultures, you would like to induce some ofthese free defined media work well irrespective of the species on which you are using.So, MS, gamborgs these are very popularly or whites medium these are very popularlyused predefined nutrient media for in vitro cultures depending on the objective, whetheryou want to do root culture, shoot culture or callus, these are used .I was talking about the hormones.So, basically the important there are the hormones also gibberellins, than acetic acid,so, but two major hormones which impact the in-vitro cultivations are auxins and cytokinins.Auxins are said to be root promoting hormones, and cytokinins are shoot promoting hormones.So, generally this is the general trend, but sometimes may vary.So, general trend is that if you vary and keep the auxins and cytokinins concentrationhigh, it may lead to de differentiation of the explant which may lead to callus formation.So, what all things which will impact the plant cell and tissue culture, the explantsource.What kind of explants, use not all explants may lead to the in vitro cultures which youwould like to.So, type of explant which you use also matters.So, which can include any plant plants specially young plants parts are generally used forin vitro cultures, why do you think? to energy .They are more amenable to changes, reprogramming... Medium composition carbon and nitrogen source,then aseptic conditions would be essential, environmental conditions, plant cells, solight plays a major role, and rest as any other fomentation.Now, genetics, genetics in the sense that if you think that using this particular auxinand that and a particular kinin - cytokinin is leading to callus generation or root generationin sunflower may not work with . So, it is species dependent what kind of hormones therefore,everytime that optimization of what kind of hormones and their concentration is important.So, this is a small flowchart of how plant cell fermentation is done.You start with the plant either in vitro inside developed shoots which I showed you like invitro or outside.When outside explants you need to do surface sterilization which means from the surface,the contaminants will have to be removed.And then you bring it to a de differentiated form which is called as callus.After you have callus, you put it in suspension, so that you disperse the cells and it becomesa fine suspension you need to subculture and filter, so that the aggregates are removed.Now, as why did I say that you filter and then subculture, there can be number of subculturecycles required in inoculum preparation or in suspension culture this will facilitatewhat filtering and bringing the cells uniform .. This uniformity is with respect to in termsof.Cell synchronization.Very nice.So, this leads to synchronous culture.If you arepass through cell seeds of uniform some size continuously, you can assume thatwhatever passes through will be uniform in size, and also therefore, maybe more uniformand more synchronous even in their metabolic activity . So, gradually you may lead to synchronouscultures, shape and size.And then you bring it to cell suspension cultures.Once you have cell suspension cultures, then we implement all optimization strategies.What kind of optimization strategies, I will just brief you, and then you bring it to thereactor level where alls scale up strategies are designed before you do the downstream.So, as I was talking about how do you begin you take the plant parts, and then on thesolidified medium with predefined hormonal conditions, it will de differentiate intoa callus form.This callus is transferred into a liquid medium of desired composition with hormones and youget the suspension culture.So, this is also one of the endangered plants for whosefor whom the callus could be generated,this is from a north eastern region.So, let us talk about brief you on the different strategies which we will be learning to optimizethe plant cell bioprocess.Once which is same as bacterial fermentations or microbial fermentations will be optimizingthe media.What techniques generally we use to optimize the media?If you are given to optimize the medium composition, how would you optimize it, how would you optimize?The objective is hm.What else?We can change the concentration of each of the components at a time like carbon sourcewe can supply like 1 percent, 2 percent and accordingly to understand how much of thecarbon sources required by the plant.Ok.So, that we can do for the nitrogen source and sum upSo, there will be some 16 to 18 nutrients . So, each nutrient we will vary in a range1 percent 2 percent up to what?hm Up to some 10, 20, 30.So, how many number of experiments then, 18 nutrients with the.So, how many permutations and combinations we have to imagine .So, my own Ph.D will go . hmOnly optimizing the media is not it? hmAnd plant cells grow slow.hm One single experiment we will take around15 days minimum.Yeah mam. 15 days.So, imagine the number of experiments and the amount of time you will be spending inoptimizing the media.Any other better idea?This is doable.I am not saying this is wrong.ok Yes statistical technique so also .response surface . How will that help?No, the mention will be need to check it and compositions, and those statistical teqhniquesmay give us different compositions, and we canknow that.How, how some more details statistical technique only run that.How?Prachi, she is right, she is right that statistically techniques can be used, buthow are they useful?In what way will they help single factor?We can identify the most . Wonderful, very nice.So, first thing is out of these 16smart person would be to first identify where should Ispend my time and money.I need not even optimize something which is not impacting the result so much . So, firstI will try to identify out of the n number of parameters which I think are present arecritically or most significantly impacting.So, I need the ranking to be done.So, statistical optimization, it helps in reducing the number of experiments.And in those minimum number of experiments, it can help you to identify the most significantones.And then after I have identified the most significant one, then what should I do?. .Again the statistical optimizer design of experiments can be used, it will helping whatyou were talking about response of his methodology.So, what is the response of his methodology?So, we will do with a high high value and high level of that composition.We will same bound a combination the combined in fix.So, response of his methodology is one of the ways these are nothing, butah factorialdesigns.What are factorial experiments?Nowfull factorial orthey are partial sum of the only is there is an entire design space.Once for every parameter there will be from where the single factor experiments will beuseful is if you want to know the range.You must widen your range as far as possible, so that you know the minus and the plus levelwhere you can be sure that there will be an inflection point.There is no point in a in selecting a range where there is no inflection point, how willit maximize the objective function is not it.So, your single factor experiments are useful to identify the inflection thoughrange whereyou are definite to find an inflection point . Now,range where you are definite to findan inflection point is what, you must have a small value and a high value such that oneworks positively and the other is less.Is less So, some three point such that, so that youcan identify that yes there was a hill, some single factor experiments should be selectedin that manner.Once you have identified the positive and the negative end, then you use that in yourstatistical design of experiments.Now, this there can be n number permutation combinations even in this plus minus rangefor all 18 suppose we have selected four more significantly affecting.Even in those fours, selecting minus A and plus A, you will have n number of permutationsand combinations.Now, even in that these design of experiments, they based on statistics they will selectonly few data points recipe, and they will give yourecipe of your experiments .That suppose A, B, C, D were four factors.So, this is how these four factors you carry out the experiment, then they help you toidentify how A, B, C, D is interacting with each other and how is it impacting the youare objective function which may be biomass or your product yield.Once you have identified that interactive effect taken into account, it will help youto optimize and give you predict the most optimum A, B, C, D value in the range whichyou have given which will lead to maximum growth or maximum product formation dependingon what objective function you have used.So that is how media optimization is generally done which in less amount of time with minimumnumber of experiments can give you more can give you better results.Now, optimization of environmental factors also because single factor optimizations youallmust be knowing, they do not take into account the interactive effect.But your statistical optimizations can take into account the interactive effect, thereforecan give you the real optimized value . Addition of precursors.Now, this is another well known strategy precursors are intermediates in the biosynthetic pathway.For example let us take camptothecin or any taxol.So, there arebiosynthetic pathways involved where there will be n number of intermediatesconnecting to the central metabolism that secondarymetabolism.Now, there will be n number of intermediates in that pathway which intermediate to add,or adding any intermediates there which is close to your desired product can lead toincrease in the rate of forward reaction to the desired product?So, that is what is done you select or intermediate which can be after exogenous not all intermediatesif added exogenously will be readily taken up by the cell may become toxic also.So, those intermediates which can be made available to the cell to be taken up insidethe cell and get utilized will lead to enhancement in the rate of forward reaction, and therefore,leading to increased product formation that is what is precursor addition.Now, permeability enhancers one was precursor addition.Then you can even enhance your yield product yield by adding permeability enhancers . So,how do you think permeability enhancers will increase the product yield.Permeability enhancers are what they are compounds which can reversibly permeabilize the cell,reversibly permeabilizing means that the cell viability is not getting impacted such thatthey become non-viable.The increased mass transfers So, because you are disturbing the permeability,so there would be more to and fro motion across the cell membrane which can increase the masstransfer across the cell membrane.What else?So, increasing the mass transfer how will it improve the product yield?The biomass will increase.. hm.So, the product will be more . The required component.that will . Immediate defense.more available now.For product formation, one is the nutrients will can come and cause, but what else whichcan have an immediate impact.What can drive the rate of forward reaction, if you can remove the product?hm yeah.Can make a sink for the product?hm So, by permeabilizing the cell the productis being let out, so which can form a sink for the product and thereby driving the reactionforward, so that is how permeability enhancers given impact.Now, elicitors this is the most promising strategy which is used to improve the productyield in plant cell technology.Elicitors these are molecules which stimulate defense or stress-related responses in plants.Now, majority of these high value compounds as I said are phytoalexins are defense relatedcompounds.So,if you can exogenously add such molecules which can elicit or induce defense responsesin the plant, be it as a signaling molecule which can lead to a cascade effect therebyleading to an enhancement in the secondary metabolism or orsomething for which the plantis alreadyah immune for to increase its defense response, may be fungal components or anypathogenic component which can give the signal to the plant cell and induced the defensemachinery.Plant cell immobilization is also used to improve the productivity.So, these are some advantages . So, one was we were talking about if suppose the speciesis shear sensitive, it forms a aggregates, then it is useful.We have immobilize the cells.So, the encapsulation helps it to overcome the shear forces.