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Module 1: Plant Cell Bioreactors

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Yesterday wespoke about genetic transformations in plant cell. Now I said genetic transformationsin plant cells in vitro it is generally carried out by using vectors.Now,in nature genetic transformation is happening in plant cells via a agrobacterium transformations.So, what happens in agrobacterium transformation? It has a plasmid which is called the tumorinducing plasmid or a route inducing plasmid. Now, these plasmids have certain gene whichcan get integrated into the plant chromosome thereby causing transformation in plant cells.Now this can be exploited in vitro conditions by removing some of the genes which are notnecessary for the transfer and putting up your polylinker sites or your multiple cloningsite which you know as NCS with unique restriction site so, that you can insert your desiredgene along with your marker system into the T-DNA.Generally the selective agent inbecause these are all binary vector we were also talkingabout their are conjugative plasmid and there are non transmissible plasmid. So, now, fornon transmissible plasmid which are non conjugate; if for them the facilitation is done usingconjugate plasmid. So, in that case we use binary vectors.Now, sometimes because this Ti and Ri plasmids are quite large big in size 200 kbp, Ri isfurther larger it is 250kbp. So, now, beingvery big plasmids and low poppy numbers for multiplicationand agrobacterium the success of transformation might be less. So, generally what is doneis to remove, the bulkiness the virulence region this particular Ti plasmid which isinherently present is deassamed which means the oncogenes which are present which in natureget transmitted as a part of infection into the plant chromosome.Now, oncogenes are what oncogenes are your auxin and cytokinin synthesizing which leadto this rapid multiplication and morphogenetic events change in the biochemical makeup speciallywith respect to the growth hormones. Now these are removed and your this is called as disarmedplasmid and your virulence gene is in tak. This virulence genes region in the disarmedplasmid is kept separately from another artificial plasmid which you prepare which is much smallerin size which is called as a binary vector like pcambiya which we hear in plant transformationthese are called as binary vectors because they have an origin of replication for ecolias well as for agrobacterium. So, that they can multiply and get maintainedan ecoli and also in agrobacterium and they will have your T-DNA. Now it is a deassmedplasmid the inherent Ti plasmid, but this vector will have the T-DNA with your polylinkersite and CS with your marker system with your promoter and your 3 prime utr polyer signal.So, there by when these two are their they work in trans and this virulence genes whichis present in the deassmed plasmid will help in packaging the this T-DNA and processingthis T-DNA helping for its integration into the plant chromosome and thereby the expressionof the desired gene which you have inserted in this area.So, what happens in nature? Crown gall disease is caused by the infection of agrobacteriumtumefaciens and leads to a neoplastic growth. It utilizes opines produced by the host cellsapart from your oxions and synthesis your cytokinin genes that are opine genes alsopresent in this T-DNA which once gets integrated the plant start producing these certaincompoundswhich are which in carbon and nitrogen and this Ti plasmid which is there in the agrobacteriumnot everything is getting integrated. So, in the rest of the plasmid, there are opinecatabolism genes. Now, once the plant produces these opinesthe catball, they induced the opine catabolism gene present in the agrobacterium Ti plasmidsuch that the agrobacterium can use it. So, in nature agrobacterium is a parasite forthe plant. Transformation is associated with an accomplished by transfer of stable replicatingportion of Ti plasmid DNA to the plant cell. So, the replicating portion of the Ti plasmidis your T-DNA. We will see how it happens. Precise mechanism is unknown, but molecularanalysis has shown that 25 nucleotides which are called as direct repeats; direct repeatme some of the base pairs which are after a gap continuously found to be present inmore than one pairs. So, more than one time they are continuouslyrepeated. Repeated sequencing flanking the boundaries of the integration sites left andright border it reads from the right to the left. The genes that are to be introducedin the plant cells must be inserted between the left and therefore, the right border.So, what happens in the structure of the Ti plasmid, there are four regions; one is yourtDNA region this is the main transfer DNA responsible for the tumour induction it hasall those genest. The second reason in this Ti plasmid is responsible for replication,the third region it is responsible for conjugation. So, formation such that the transfer can takeplace the forth region responsible for virulence where genes are there. So, it is importantfor processing and the transfer of T-DNA. Now, structure of T-DNA it is broken downinto three parts the transfer of T-DNA into plant chromosome brings about physiologicaland morphological changes in the tissue due to expression of genes located in the T-DNA.Onc region which is oncogenic region tms one. Now these are different locus or positionsof the genes tms 1 and tms 2, it represents shooty locus which means they represent orthey express to produce cytokinins. So, that is why they are called as shooty locus. Whatkind of cytokinin? generally it is ipa; ipa is your iso pentile adolescence.Then the tmr region, the rooty locus is responsible for auxin synthesis generally what kind ofauxin IAA, Indole Acetic Acid. Then OS region is your Opine Synthesis region responsiblefor synthesis of amino acids and sugar derivatives opines are nothing, but amino acids and sugarderivatives collectively called as opines. These are low molecular weight nitrogen containingcompounds organic compounds. There are many different types of opine some of them areoctopine menopine nopaline and sometimes agrobacterium themselves. They are classed depending onthe type of opine, they are producing, some are called as mono monopinetype of agrobacterium,some are octoppine. So, in terms of the class ofor the types of opines produced agrobacteriumare also classified. Now, 25 base pair repeat sequence which iscalled as a left border and the right border is essential for the T-DNA transfer. Now howdoes it take place? These are some of the links which you can go through help you tounderstand. So, this is a just a pictorial representationof the entire Ti plasmid. Now you will have have oncogenic genes, you will have cytokininproducing genes, you will have opine synthesizing genes, then this entire is called as oncogenicregion. Then you will have t d r region; r regionwill be including your left border the right border and all these genes then conjugativetransfer I told you. So, this is the reason which are responsible for conjugative transfer,then opine catabolism genes are also present, origin of replication is present, the relianceregion is present. So, T-DNA does not contain the outside the other genes neither the catabolismgenes neither of origin of replication. The virulence gene that is I when we say deassmedplasmid which means the T-DNA region has been taken out replaced.Now, virulence region it is generally 35 kbp long, it isorganised in 6 operons virulenceA, B, C, D, E and G and each of these regions play a different role during this transferprocess. It is not only transfer which is happening from the time itinduces there isthe signals like yourdefence or some molecules which like poly phenol compounds which getreleased by the plants injured plant cells, they act as cuse they induce some of thesevirulence regions in the agrobacterium. And these different proteins will be responsiblefor first induction of subsequent other regions of this virulence gene which will some ofthe proteins will be utilised in packaging, some of the proteins will be utilised in creatinga some of the proteins are utilised in protecting the T-DNA as it enters the plant cells someof the proteins are responsible for conjugation. So, virulence a is a product specific innermembrane protein that recognises and is responsive to the plant phenolic compounds.So, this is what is responsible to recognise plant phenolic compounds. So, it acts as areceptor. Virulence G as acts as a transcription activator of other loci of the virulence region.The product of Vir C and D are involved in generation and processing of T-DNA which meanscreating a nig and then replicating and then packaging it which means means the five primeand is gapped. Virulence B and R are involved in follow forming structural components whichfacilitate movement of T-DNA. Virulence H allows bacteria to survive in the presenceof bactericidal compounds from plant during the infection .So, how does it happen? The early event is the nicking of the Ti plasmid between thethird and the forth bases of the bottom strand of the 25 base per repeat the virulence Doperon encodes and endonuclease that produces nick in the border sequence. Then the initiationof DNA synthesis begins in the 5 prime to 3 prime end.Now, it is accompanied by synthesis and secretion of certain other molecules like glucose , celluloseor surface proteins which have pathogenic characteristics. The T strand now which ismade replicated. Now it is a single strand. Now it as to enter the plant chromosome andthen the nuclear membrane through the nuclear membrane into the plant chromosome.Now, the T strand in order to transverse through the bacterial membrane plant cell wall thenplant cell membrane and then the nuclear membrane during this entire process. It must avoiddegradation with nucleases the T-DNA exist as DNA protein complex.Therefore, I talkedabout capping which protects it and mediates its travels the T-DNA is converted to thedouble strand DNA prior to . So, this double strand DNA is happening prior to integrationin the plant chromosome. Placing the foreign genes into T-DNA regionof the Ti plasmid is possible to clone many make copies clone means making copies. Theintroduced genes with the multiplication of the residing plasmid within the bacterialwith the bacterial growth. So, this is what we were talking about thisis how it happens the various events which you are talking about the various differentkinds of. So, you can read that and you can correlate with the figure.So, they have demonstrated the various virulence proteins means which are responsible one isacting as a receptor , then it further induces other regions of the virulence gene afterinduction induction involves production of where D where E where E is involved. Onceit enters the plant chromosome to protect it from the nucleases, then where B is involvedin conjugations. So, you can correlate and remember how the events take place.So,. Now the Ti plasmids cannot be used directly because of as I was saying, generally we havebinary vectors and these vectors are inserted into agrobacteriums which are disarmed.Now, disarmed I told you the inherent plasmid T-DNA has been replaced and only virulencegene is kept . The reason being they are large in size absence of unique restriction site.So, it is difficult to insert your gene of interest. Then tumor induction, why do youthink tumor induction has to be avoided? If you do not disarm there will be oncogenicgenes present. So, the morphogenetic event would be causing tumors or causing root adventitiousroots that was not the purpose if that happens what what will happen ? It depends on theobjective of the study. You are trying to do a targeted transformationinto the plant cell. So, more the number of genetic transformation; more metabolic burden.So, any integration there is leading to removal of certain endogenous genes and replacement,there is a high chance. So, more the removal more there is a chance that some of the crucialwho knows if any crucial endogenous genes get silent or gets replaced and this is adisease. Only if desired, you would like to have it otherwise why would you like to haveunnecessary growth on your plastic growth in the plant. Depending on if you are workingfor whole plants then; obviously, you are not wanting to have new plastic growth inthe whole plant. But what if the purpose of secondary metabolite production using plantcell cultures? If suppose is for secondary metabolite productionusing plant cell cultures, is this useful or not?Yes yes useful. Why?Becausethe grow . They will grow very fast , they will not needhormones in the medium generally this is what is observed. If oncogenic genes; this is howthey are exploited for secondary metabolic production because if you take a wild argobacteriumstrands with their wild Ti plasmid natural Ti or Ri plasmid which we call as herry roots.So, their what is happening? We are not having any specific mca for specific gene which wehave inserted this is natural infection which we exploit in lab. So, it would lead to what?Integration of the inherent T-DNA incident T-DNA thereby leading to expression of oncogenes. This is what leads to hormone free growth in transform cultures. And once you have hormonefree growth faster growth more branching and because it is stress associated.So, the possibility is higher that the secondary metabolite yield increases, then the untransformedis it useful or not? And it is advantages, but then still there is a selection neededafter transformation selection of the cell line why? You understand what I am tryingto ask, I infect the agrobacterium and I will have more than one cell lines I say stillthere is a selection needed .Even in if it is a targeted transformation and used in minus still there is a selectionneeded in agrobacterium transformations. Selection of because you will end up you mayend up in many different cell lines isn't it as you end up in untransformed callus induction.So, why why now selection is needed? We have got once I have confirmed that this particularcell line is transformed generally using pcr, we will talk about those methods once I haveconfirmed why cannot I stick to just that line.It might not be regenerated in all the generations the plasmids .It as it is a stable transformation event because it gets , it is not transient expressionit as got integrated into plant chromosome. I will give a hint we were talking about stress.It has been found in literature that agrobacterium infections are stress to the plant cells.So, where will compromise? Some cells might got resistant to that stresssome might not be effected by that stress. Depends on; they act like transposable elements.So, sometimesthe copy number and the position is not in your hand. So, if the copy numberis not in your hand and the position is not in your hand, the cell lines which come outwill vary in growth as well as yield and being a stress, there will be a compromise and growth.So, there is a need for another step of selection even after transformation to reach to thehighest yielding and highest or with better growth the in comparison to others.So, among what has been transformed, generally you select based on your secondary metaboliteyield and growth index. So, then the tumor cells cannot develop into normal shoots. Disarmingof Ti plasmid or removal of tumor inducing property is an essential steps. Generallyit is observed that once you do agrobactirium infection even if you have ended up in celllines their regeneration capacity is hampered because of the stress into the whole plants.So, when you are working for transgenic plants all the more the tumor regions or the oncoregions have to be taken out . This is done by replacement of tumor inducing genes intransfer T-DNA by selectable marker providing resistance against antibiotics like kanamycin.Now going back to my question, I said it is recommended that the desired gene has to beinserted near the 5 prime end. Whether it five prime end is scabbed in protected.So, our inserted DNA is more safe there. And your marker systems your resistance antibioticresistance gene is on the other end. So, that if it is nicked once the transformation isdone, you would not like to have the antibiotic resistance gene in the plant because any extragene insertion is a metabolic burden. Promoters and polyadenylation signals isolated fromnopaline synthase genes. So, because in nature already these opines are getting expressed.So, people have utilise the same expression promoter like CaMV 35S promoter; CaMV 35Spromoter was used then here nopaline synthesis 3 prime utr is used because it has been foundto express very well in the plumps. So, what are binary vectors these are basedon the principle that virulence genes may be located on a helper Ti plasmid having thewhole of the T-DNA located. So, this disamed Ti plasmid is called as the helper Ti plasmid.In this case T-DNA is found on a separate vector which is binary vector design to replicatein both E.coli and agrobacterium. So, it has the origin of replication of both the E.coliand the agrobacterium and capable of. Now why do you think there is a need for E. coliorigin of replication, why unnecessarily having this E. coli step in between?To rep to transfer to it to be able to grow it in bacterias because it having multiplycopies. Hm.Because this is conjugate figure plasmid. So, the copy numbers cannot multiply here.So, you need to have more copies and then you transform those into the agrobacteriumand then this agrobacterium is used transform; agrobacterium is used to transform the plantcells. I will stop here. There the other kind of vectors also present called as intermediateand helper vectors, but generally you will find in literature the success rate is veryhigh with binary vector. So, most of these transformations are done using binary vectors.