So, last class what did wediscuss, we were discussing about the various genetic transformationsin plant cells, what are the different ways in which plant cells can be transformed.So, we were talking about that generally in literature you will find that agrobacteriummediated transformations are used.Agrobacteriums themselves are natural engineers to trans, they have the machinery to successfullytransform the plant cells through a section of DNA present in them plasmid DNA which iscalled as T-DNA which gets stably integrated.So, how we have exploited is that T-DNA inherent T-DNA present in the agrobacterium that isdisarmed which is taken away, and another vectors as which have this segment of T-DNA,a which agrobacterium has the machinery to integrate this plasmid vector which has beenexogenously added into the agrobacterium to integrate into the plant cells.So, now what are the different kinds of vectors which we were discussing last time, the mostcommonly used vectors are binary vectors.Now, binary vectors why are they called binary origin of replications, they can replicatein E. coli as well as in agrobacterium.And then what else the conjugal transfer can take place between E. coli to agrobacterium,and from agrobacterium to plant cells.Now, I was also talking about other vectors which can be used for genetic transformationin plant cells.These are intermediate vectors what are these like for example, pBR3 double two, you willbe hearing if you work in this area.These are smaller size vectors in E. coli a, which contained the T-DNA and they areused to overcome the problems because of the large size of the tthe Ti plasmid.So, as I was saying there is a helper plasmid present in theagrobacterium which is disarmed.Now, this is called as helper plasmid, which will have virulence regions.Now, this small plasmid it can multiply in the E. coli I and then through conjugal transfersit can be brought in the agrobacterium.Now, these plasmids in agrobacterium, they cannot multiply, but then they can be transformedthey can be used to transform the plant cells.Now,co-integrative, sometimes what happens that because of there is homologous recombination.Now, in theagrobacterium, some of the T-DNA regions which have been disarmed, but theother regions if there is a homologous if there is a similarity between that DNA componentto the DNA which is present in these vectors, then transformation would take place, recombinationwould take place, and there will be an exchange genetic material, exchange of genetic material.And this T-DNA where your MCS region is there, where this gets integrated into the entireone single plasmid which can then act as a complete Ti plasmid for the transformationto happen in the plant cells, so then it is called as co-integrative plasmid.Then helper vectors, helper vectors either you have your inherent Ti plasmid which hasbeen disarmed, disarmed means the oncogenic regions have been the T-DNA has been takenout . So, or you have separate plasmids which help in the transformation event which isthey will have regions which are transfer regions which will help in the transport ofthis T-DNA of your small integrative vectors.Now, these are small plasmids maintained in E. coli, these are helper plasmids for E.coli to agrobacterium transform transfer.They contains transfer regions and mobilization regions, which allow the transfer of the conjugationdeficient intermediate vectors into agro.Suppose if there is conjugation which is absent, not all vectors are conjugal vectors . So,if conjugation is absent, then you need helper plasmid which can help in the transfer ofthese vectors into the agrobacterium.So, this is what I was saying co-integrative vectors where homologous recombination entireone co-integrative plasmid inside the agrobacterium can be obtained.Now, what are the different transformation techniques, either what is well-known whichis being used in laboratories is co-culture technique one is that, and the other is directtechniques in planta transformation.Direct the plant is transformed now co-culture with tissue explants meets, it begins within vitro cultures and from the in vitro cultures regeneration is done to get the transformedplant; otherwise directly the plant parts can themselves will get transformed eitherthe seed itself or the zygote [ot/or]- or at the time of floralpollination, just beforepollination when the flowers its come out the there is a flower dip method or floraldip method through which agrobacterium can get into the plant.And after pollination can transform the zygote, thereby in the progeny you will see transformationhappening.Then direct gene transfer or physical delivery methods.So, direct gene transfer either we are using agrobacterium as a tool to transfer or indirectgene transfer methods for example, gene gun which is which I think you must have heardabout . And there are other methods which we will see.So, agrobacterium mediated methods, what it is what is done is the prerequisites for agrobacteriummediated gene transfer, it involves the explants, explants must produce acetosyringone.What was acetosyringone?. So, this was used to induce the virulencegenes present in the agrobacterium.So, acetosyringone, so the type of explants, the age of the explant also sometimes mattersfor the successful transformations.So, there has to be production of these signalling molecules which can attract the agrobacteriumand the reduce theincrease the proximity between the agrobacterium and the plant cells.Now, the explant must produce acetosyringone or other active compounds other phenolic compoundsin order to induce the virulence genes.Then the agrobacterium should have access to cells that are competent for transformations.Sometimes it is observed although transformation is done, but regeneration does not happen.For regeneration to happen, you need competent cells for transformation, and then totipotencyto be maintained in the transformed cells so as to reach into the regenerant plant,so which is a difficult task not that easy.So, nowfor agrobacterium successful transformation in access of the competent cells is necessarynot all cells will be competent in nature.Sometimes you will see that similar method, but you will keep on repeating only some willbe successful, the others may not because of all these regions, not all cells in thecallus will be of the same biochemical nature . Then explants used for transformation thetype of explant the age of explants.Now, orit is generally preferred that they should be actively dividing cells when transformationshould be done.Why, young explants are preferred, not old, why do you think?. Even for transformation, regeneration is thesecond step.For successful transformation to happen it is preferred that you should work with youngerexplants with rapidly dividing cells if you are working with cell cultures.. So, but let us.. Let us do a comparison between matured cellsin suspension and the secondary cell wall comes when organogenesis and it is in thetissue present is not it.. There can be the probability can be higherwhat else.So, matured cells is there one major difference between matured cells andimmature cells.Ifyou remember I was telling in the earlier classes thatthe younger cells the vacuolespresent are small, the cytoplasm is more dense, so the probability is higher . In mature cells,the vacuoles become large the cytoplasm is less dense.So, denser is the cytoplasm higher is the probability for transformation . So, and moreovervacuoles contain enzymes which can rapidly degrade the extracellular DNA . So, it ispreferred that you use younger cells in comparison to matured cells.In literature, you will findthe cells which are used or the tissues used they vary fromcallus, all kinds of in vitro cultures can be transformed, callus, suspension cells,protoplasts, then tissue whole organ entire plant so if there is a wide range.Now, after explants have been co-cultured with agrobacterium, how to detect that whichcells are successfully transformed or not.We have already read now that based on the selection marker or the marker system present,whether it is histochemically based asses which can be used or antibiotic selectionmedium or herbicide selection medium can be used to increase the probability that youwill end up in successfully transformed cells with subsequent sub culturing.It apart from the marker or the selective agent,agrobacterium inhibiting growth inhibitingcompounds other antibiotics like carbon saline throughagrobacterium is sensitive towardsit.So,is also used.Why do you think and when is this used or when should it be used?For transformation, what are you doing you are bringing the agrobacterium in close contactwith the cells, but ultimately what do you want transformed plant cells, you cannot keepgrowing agrobacterium because it will over grow which will subsequently lead to lossin the viability of the cells and the growth.So, after successful transformation, successive culture has to be taken away from the agrobacteriumhas to be removed.So, after 48 hours, generally 48 hoursis the timeline for exposure with the agrobacterium,and then it is kept on the high concentration of carbon saline or any antibiotic where agrobacteriumis sensitive towards . So, then subsequently with reduced concentration of the antibioticyou keep sub culturing.Andafter subsequent sub cultures the antibiotic is removed from the medium, because antibioticitself can be inhibitory to the plant cell growth which you would not like to have forthe transformed cells.In general transformed cells, because we have genetically manipulated them, so their growthrates are compromised.So, the selection marker systems the toxic levels, so what has to be optimized the toxiclevel of the selection agent it is vary species.So, it a species specific the time of exposure is species specific, the antibiotic concentrationis species specific, so that has to be optimized.So, this is the entire protocol you can go through it.This is for your reference the theoretical protocol how it is done.So, what is done is you choose an appropriate explants, then you surface sterilize the explants,the explants are brought inyou growthe agrobacterium in suspension for 24 hours.You look for the growth phase because then the agrobacterium cultures are active.So, generally log phase cultures are preferred.Once the agrobacterium has grown, own there are different strains of agrobacterium whichare present and which are virulentto infect the plant cells, but it is very highly speciesdependent.So, if one agrobacterium like for example, agrobacterium LBA 4404 or 9402.So, these are different strains which are known to be highly virulent, but it is notnecessary that because it has worked with one plant species, it will work equally withthe other.So, you need to choose the agrobacterium strain grow it active phase, log phase cells areused.Then even for exposing the cells you can use different techniques.What is needed either you wound create a wound in the explant and then expose the suspensionto the dip the that explant wounded explant into the suspension.Now, the time of exposure also varies you cannot keep it formore than 15 or 10 minutes.So, generally 10 to 15 minutes, you keep it exposed, then you need to wash it, so thatexcess growth of agrobacterium does not happen when you are keeping it in thefor incubationfor 48 hours.So, you need to you can vary the time there of the exposure the exposure time, you canvary the agrobacterium concentration the self suspension concentration . You can have differentodieswhen you are using the culture to co cultivate with the plant cells.Then you can vary the type of explant that can be varied.Then I was talking about the wounding now wounding itself can be created through differentways.So, people have found that using a syringe filled up with the agrobacterium suspension,you make injuries on the midrib of the leaf explants and that has given rise to successfulincreased efficiency in transformation.Then people use simple dip method, where they expose the explant wounded by a bladeor bycreating holes in the leaves or explants and then exposing it.So, there are different factors which need to be optimized.It looks easy, but there are number of factors which need to be optimized to get a successfullytransformed culture.Then in planta transformation generally uses the floral dip method in which the floweris dipped in the agrobacterium suspension, and then kept for incubation for some timesuch that it resides it is said that either or the seeds themselves can be surface sterilizedand dipped in the suspension such that the agrobacterium cantransform the seeds.And once they regenerate or when the seedling comes out, theyinfect thetissues, the aerialparts or the ground part regions . Or when the seeds, when the flowers are dipped withagrobacterium or brought in contact with the agrobacterium just before pollination, duringpollination or after pollination they the agrobacterium present inside may infect thezygote or may infect firsthand the germ cells which are going to getah involved in the zygoteformation.Thereby leading to progenies which are transformed.Now, talking about direct gene transformtransfer methods, electroporation is a well known techniquewhich is used even for bacterial transformations.Then what are the two different types of electroporation which are frequently used.These are low voltage long pulse methods means time duration isincreased, but lower frequenciesare used.High voltage short pulse method, generally it has been found that high voltage shortpulse methods are found to work better than the low voltage long pulse methods becausethe damage to the DNA is lesser in that case.Now, what is done the optimal voltage, so what can be optimized the voltage which weare using the time for which it is being applied is optimized.Then transformation frequencies are increased several folds by adding certain other techniqueslike heat shock or addingyour peg.Why do you think it helps?Have you heard about competent cells?How are they made competent?Competent cells means they become more susceptible to uptake of the extracellular DNA.How are they made susceptible?Ha, any divalent even magnesium can be used.How is it making it susceptible?So, thereby increasing the proximity between the extracellular DNA and reducing the repulsion,because both are negatively charged so that is the way.And what about PEG, so what it does?. So, what is it actually doing, everythingis now facilitating easy uptake.Polyethylene glycol is also very frequently used.. DMSO, polyethylene glycol yeah.It reduces the . Then, so what is done is in chemical methodsas we were talking about the cells are first made competent.So, divalent ions can be used to reduce the charge repulsion such as to increase or bringincreasethe proximity between the two DNA.The other thing is that there is a usage ofpeople manipulate pH, people addpolyethylene glycolin the medium and the concentration of the DNA is also manipulated it to enhance theefficiency of the transformation.Now, some of these methods you need to go back and check what they do is, they willtry to condense the DNA or concentrate the DNA.And concentrating the DNA reducing the proximity between the extracellular DNA and the plantcell membrane would increase the probability of success, so that is what these things do.Now, lipo infectionthey are alsothe extra cellular uptake of the DNA can be improvedby encapsulating them in the form of liposomes.Now,liposome intake is a well known phenomena in cells.So, liposome they not only will increase the uptake of the DNA, but they will also protectthe DNA from the nucleases, so that way also it helps.Now, what are liposomes, I hope you know what are liposomes, they are similar they willhave both hydrophilic hydrophobic ends.They have a double layer . Like phospholipid bilayer.And they will encapsulate the DNA within.Now, they can be induced to fuse with the cells or the protoplast using PEG and cantherefore, be used for gene transfer.Then there are several advantages what all different advantages with lipo infection,low cell toxicity, stability and storage of nucleic acid can be improved by encapsulation,then high degree of reproducibility and endocytosis is a well-known phenomena already presentin the cell, so that can be easily taken up by the cells.Now, calcium phosphate precipitation another method calcium chloride anyways is being usedif the phosphates are present, then it condenses the DNA precipitates, the DNA such that becauseit is condensed, it reduces or allows the cell bring its much closer to the cell membrane,thereby increasing the probability of uptake of the extracellular DNA.Microinjection this is directgene transfer in which a micro injection assembly is used.It is not that easy, uh under the microscope you hold the cell well using or by freezingit onto a slide, using poly lysine.Poly lysinebecause of the charges presentit will try to stick it at one place, and thensuction pressure is created such that it does not move.And then through that micro injection assembly, the DNA is tried to either directly put inthrough the cell membrane into the cytoplasm or into the nucleus of the plant cell.Now, what other direct methods can be used is in literature, you will see that peoplehave used fiber mediated DNA delivery in which very small size nano fibers are used, siliconfibers are used with high speed, so that they can impregnate the plasma membrane in thecell valve and the DNA is delivered into the cytoplasm.Then laser induced DNA delivery is also reported.Ultrasonication, then macro injection is your hypodermic syringe which we were talking about,where it can be directly injected into the cytoplasm or into the nucleus.Then micro projectile which is your gene gun method in whichat a very high speed, it canbe delivered to the plant cells or the explants cap under an inert atmosphere with heliumgas.And the water vapor using theelectrical charge under high voltage, the water droplets highspeed is created, and it carries the extracellular DNA coated with the inert material metalslike tungsten or gold nanoparticles, which can be injected under high speed directlyinto the explants.I stop here.And then next class we will begin with the plant cell bio reactors that is the last partof the course.