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Module 1: Plant Metabolism

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Somatic Embryogenesis and Protoplast Culture

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Now, in somatic embryogenesis, there are two stages.If you need to produce embryos from somatic cells which is called as somatic embryogenesisthere are two stages.Stage one where there is induction of embryogenesis which means that the cells which are meantto form embryos or or which are prone to become embryogenic in nature you induce them to undergothat program or the cells which are not determined to make embryos are determined then throughreprogramming to make embryos.So, stage one is induction of embryogenesis and stage two is then once the zygote is formedthe different stages it undergoes to produce your in vitro plantlet.So, requirements for these two stages are different in terms of media.It is not necessary generally whatever media composition is working well with callus inductionto induce embryos it may work well, but once for the development after induction of theembryos it might need revision of the media, specific media composition.So, the different stages in the development of embryos differ morphologically.So, we will be looking at there is a picture.There are four different stages before the hypocotyl region appears.So, embryogenic cells generally, what are they?They those which have competence and embryogenic in nature they are generally found to be smallrich in cytoplasm and they are the vacuole size is less.And those cells which are non-embryogenic they will be large vacuolated and less densecytoplasm.So, embryogenic cells will have more dense cytoplasm in comparison to non-embryogeniccells.And generally, it is observed that they are once they are transferred into low auxin containingmedium then it induces pro embryos.Now, once the somatic embryos they are transferred from induction medium to developmental mediumthenwhen it is taking place from a single cell, the induction, the single cell willdivide into a number of cells 4 to 5, then forming of globular structure.Once the globular structure is formed it undergoes further development which means differentiationto become a torpedo shape, and then after torpedo shape it undergoes further vascularbundle formation.Generally, you can see in the hypocotyl regions where the xylem elements start becoming visibleand in the cotyledon is region.So, and then the cotyledons the shoot promidium and the root promidium appear.So, development and maturation, it is similar to the zygotic embryo.Also, there are four stages known globular, then heart shaped, then torpedo and cotyledonarystage.Now, all these are morphologically distinct.You can clearly see once the embryo starts developing the somatic embryos was.And moreover, biochemically also molecularly they are different because differentiationis taking place vascular bundle formation is taking place and thereby leading to fromthe meristem region there will be a root meristem, shoot meristem region appearing.So, what happens?At torpedo stage cell differentiation occurs establishing the root and shoot meristem.Now, what is the torpedoes stage?This third-one which looks like a torpedo.Now, small cytoplasmically rich cells which are embryogenic are observed in these meristemsand the rest of the cells, they are vacuolated and matured cells which will form the bulk.Now, what are the factors which can affect embryogenesis?It is nutrient medium specially sucrose and reduce nitrogen.Then light gases, gases which means dissolved oxygen here, then exogenous hormones yourgrowth regulators.So, what can be the different applications of somatic embryos?You can use them for micro propagation.They are also used as organ cultures for secondary metabolite production.So, when I say secondary metabolite production how do you think somatic embryos can be usedfor large scale secondary metabolite production?Micro propagation last class itself we know, large amount of small plant light can arise.So, then how come secondary metabolite production?Third application is given as they can be used as model system to study growth of theplants.So, that is also quite obvious.How does an embryo developing sites?So, they can act as model systems to embryos the real zygotic embryos.So, but how come they can be used for secondary metabolites?How and why do you think?Somatic embryos are more organized than the cell cultures.Ma'am, is it because all of them are being formed from the somatic cells they are moreconsistent in the production of the secondary metabolite, and no large amount of variationand in the quality of secondary metabolite cannot be seen.So, that is the reason.One of the reasons can be that.Being an organized structure it is less prone to somaclonal variation.So, it is and moreover secondary metabolism is also linked in plant cells with organogenesisand differentiation.It is the higher order function.Secondary metabolism came later, primary metabolism is for growth and development.So, secondary metabolism is a part of higher functions which means much more organizedfor survival.So, that needs cell to cell contacts, organogenesis to happen, and differentiation into organformation is nothing, but getting into those organized functions.Different cells come together to perform a specific function, so it is more defined now.So, these kind of cells when come together they become organized structures, there thesecondary metabolism is found to be more visible in terms of biosynthetic capacity.So, secondary metabolites yields are found to be higher in organized structures.Now, and the other is they are more stable than the callus or cell cultures.In the last class also, they were showing for micro propagation somatic embryos canbe cultivated in reactors.So, once somatic will give rise to another somatic embryos, multiplication should happenedknow.Large scale cultivation means what?That in the reactors multiplication is happening.So, once somatic embryos will form another somatic embryo.So, it has to start from the cells.So, a logically can you give propose a solution to it?What should be the starting material?What kind of cells?All cells, the somatic or much closer which will have higher probability.You remember we had talked about cells with embryogenic which are determined to produceembryos, and those which are induced to produce embryos, pre-determined embryogenic potential.So, the cells which are now you have been able to find if, if you can find those cellsas which have embryogenic potential, you will use those cells bring them in suspension,do large scale cultivation and then give them conditions to form organized structures calledsomatic embryos.Now, meristem culture we were discussing.Meristem culture is nothing, but you make use of the meristematic regions of the plantwhich are lateral bud regions, axillary bud regions or apical meristems and which willbe useful in making virus free plants.We also discussed about the reasons.Now, virus distribution is uneven throughout the plant and is much less in a meristem,the reasons were virus cannot travel quickly enough through the plasmodesmata.These are connections.So, as these meristematic region the cells are rapidly dividing.So, the rate at which they are dividing and informing new cells, the virus to travel fromthrough these connections is the rate is lesser than that.So, one is this, the probability of virus transfer is reduced.The second is as we were talking about the presence of vasculature is missing, vascularbundles in the meristematic regions is missing.They are still to form vascular elements.So, the transfer of therefore, viruses difficult to these regions.So, therefore, meristem culture is preferred to generate virus free plants.Coming onto protoplast culture.Now, what is protoplast?Cells devoid of.Cell wall.Cell wall.So, where do you think, why do you think protoplast culture people do?. It can be of use.Cell cannot be used for hybridization.. Even with whole plants we see in agriculturehybrids are produced, but it is about what is much easier, making the processmuch easier.So, when we say somatic hybridization, it will facilitate because somatic cells caneasily be combined together.What are hybrids?What are hybrids?When I say a hybrid, it is used for hybridization.Means what?So, when two cells can be combined such that the two nuclei combine to make one nuclei,all their cellular content, one cytoplasm and one nucleus.So, the nuclei I have to combine together and the cytoplasm gets mixed.So, that is.So, what is done?The cells we know that the plant cells are attached to each other.They are attached through the pectin rich layer.What is that layer called?Middle lamella.Middle lamella.So, that layer has to be removed before the cell wall is removed, so now, the attachmentis off.Now, the cell wall is removed for the protoplasm to come together.So, now, how the cell wall is removed.There are two ways to remove the cell one is mechanical, the other is enzymatic.So, in mechanical you we use a very sharp blade to cut the cell wall.Now, if the plasma membrane in general will is closer to the cell wall, so there are chancesthat the membrane can get ruptured.So, what should be done to avoid that?To ease out this process, mechanical separation of cells what should be done?Can you give a solution?Everything is done under the microscope.First prerequisite is what?To disintegrate, to separate the cells.So, now, that is operation requires pectineus treatment or, so once suppose the cells havebeen separated you have a single cell.Now, there is a cell wall and the plasma membrane, I need to cut the cell wall.Knowing that the plasma membrane is very close how to avoid that plasma membrane to be ruptured.. .Very nice, very good.So, now, can you just elaborate on this?Plasmolysis will help how? content like both and putting a hypocotyls. Do you agree?Hm.So, that is what is done.So, you change the osmotic pressure by adding osmoticums such that such that the cell membranedistracts contracts such that the cell wall can be easily cut off.Now, what care should be taken in this?. The viability should not be lost.. So, as it is cut the balance has to be maintainedbecause now it is becoming more fragile with the only plasma membrane.So, then you you have to bring back the osmotic pressure such that if it is plasmolysed andonce the cell wall is out the, the cell is out the plasma membrane is the only barrierbetween the outside and the inside now it was plasmolysed.So, now, what will happen?It was plasmolysed because of?. Higher or lower concentration outside of thesolutes of the salts?Hyper means?He said.. Agreed.. So, if it is higher concentration and theplasma membrane or the plasmolysed or your plasma membrane is the only barrier left.It is very high.So, now, what will happen?The viability will reduce.So, now, you would like to balance it.Now, while balancing you would like to reduce the concentration.It is recommended not to suddenly reduce the osmotic pressure.What is; why it is recommended so? pressure water by .And the cell will burst.So, this care has to be taken.The balance of the osmotic pressure when you are creating protoplasm.So, these are functional individual cells with plasma membrane as the only outermostlayer.What can be the different applications of making protoplast or doing protoplast cultures?You can obtain single cells.Why single cells?It is you want to do clonal propagation from single cells you want to then divide pharmacalusand then regenerated into plantlets.So, this is a clonal propagation.Plant cell transformation or animal transformation and is the uptake of extracellular geneticmaterial.So, or even desirable genetic modification in you need to do some updates from extracellularto intracellular.Now, what is what facilitates that uptake?In plant cell, if you want to do transformation what is the barrier, first barrier?Cell wall . Cell wall.So, protoplast is devoid of cell wall.So, now transformation is much easier because the only barrier is plasma membrane, completeplant of single cell origin can be produced, then transformation is facilitated.Now, you can even study.Once you have protoplast depending on the media composition, once the protoplast startsdividing the nutrient conditions of these divided cells may be different from the originalprotoplast.So, the nutrient conditions can even lead.Now, the new cells need not be protoplast because they they are the genetic machinerythere is to reduce cell walls, so the newer cells which will get produced will eventuallydevelop cell wall.So, their nutritional requirements would be different and they can also be used for isolationof different cell organelles if the studies after be done.So, then I was talking about what are the different ways of making protoplast, one ismechanical the other is enzymatic.Now, mechanical involves cutting of plasmolysed cell with a sharp blade to release the protoplast.Now, enzymatic would involve exposing the cells to various kinds of enzymes we know.What kind of enzymes?We need to remove the cell wall.So, all those enzymes which can degrade the cell wall or decompose the cell wall, forexample.. What are then, you should know what is thecomposition of the plant cell wall, cellulose, hemicellulose, pectinase.Hm.So, is nothing, but a mixture of macerozyme, driselase, these are names given by some companiesfortheir origin of organism is generally fungus.So, these class of enzymes in together as a mix are also called as for cell wall degradation,plant cell wall degradation.So, then how it is done?Use sterilize the explant.So, generally you sterilize the leaf explants.The epidermis is removed, and then and the explant is incubated with these enzyme mixturesfor some time.And it is said that flaccid leaves facilitate peeling, flaccid leaves means.Think logically, what can it mean?Is it referring to the dry leaves or something?Right.Drooping leaves.Drooping leaves means what?Lesser.. Turgor pressure.So, when we are using cells then actively growing cells are used which are from theexponential place for isolation of protoplast.Now, conditions which should be optimized if you want to isolate protoplast there isare enzyme concentration, exposure time, enzyme composition, what are the different kindsof enzymes we are using, then osmotic pressure, the presence of osmoticums.So, the osmotic pressure, what kind of solutes, what concentrations we are using for the osmoticums.Now, the enzyme generally used are in the concentration range of 0.5 to 2 percent, wheremacerozyme which is a mixture of these kinds of enzymes or separately you can use cellulose,pectinase, hemicellulose, another enzyme mix like drieselase.So, enzyme solution.And sorbitol, mannitol, what is the purpose?Osmoticbalance.Very nice.So, they are used as a osmoticums to balance out the osmotic pressure.Then calcium chloride, for membrane stability.This, it neutralizes neutralizes the surface charge, reduces the surface charge.So, that.. It is protoplast or hybridization.So, generally, if they have to be brought together for hybridization the surface chargeshould be neutralized, so that they do not repel, the membranes have to fused, is notit.So, therefore, it is treated with calcium chloride.And it is also said that the genotype and the environmental factors can also impactprotoplast isolation.So, the factors which will affect protoplast culture, if you only want to do culture ofprotoplast then obviously, because the only barrier is plasma membrane.So, osmotic pressure has to be balanced out carefully.So, osmotic pressure, osmolarity of the medium is adjusted to the same level as the enzymeor the washing solution to which you are exposing it, otherwise the cell can rupture or getnon-viable.Then, prolonged culture at high osmotic pressure can result in the browning of the cells.The osmolarity browning means necrosis or viability loss.Now, the osmolarity of the medium what is done?It is gradually decreased with cell wall formation and cell division.Gradually decrease we have discuss, the reason being suddenly if you decrease it the watermay gush inside the cell and the cell may burst.So, as the cell wall is getting formed, with the barrier is getting formed the osmoticpressure is reduced.Abrupt decrease therefore, should be avoided in the osmotic pressure.What are the neutral nutritional requirements?As I said generally to begin with they can be similar to cell or callus culture, butonce the cells start dividing in or then you need to the media composition might need tobe revised.Presence of high ammonium concentration can be toxic to protoplast.Why?We were that day discussing that the nitrate and ammonium are the nitrogen source for theplant cells, is not it.And easy assimilation is ammonium.Nitrate also once it gets assimilated taken up, it is converted into ammonium ions tobe integrated into ammonia amino acids or many different other wherever it is needed.So, then why?Generally, it is observed that if you give higher concentrations of ammonium, um thereis viability loss, there is loss in growth of the plant.This is called as ammonium toxicity.Knowing that, is not it interesting.Ammonium ultimately is needed, nitrate is also getting converted to ammonia only beforeit gets into the metabolism.So, why not to give ammonium in directly.If you give ammonium, try to give ammonium directly to the plants it can be toxic tothe plant cells ammonium.Why so?We get . Very nice, very good.So,ammonium assimilation is found to be also related to release a chance because it hasto be taken in, so in order to balance out there are channels.So, even ion transport takes place with something getting in something, there is an reflux andinflux parallel.So, that is called as to maintain the membrane potential.So, once the hydrogen ions are refluxed, then there is acidification in the environmentof the cells and there is the pH of the cell gets disturbed.So, which is said to be one of the reasons why plants then the growth is inhibited.You have usually carbon nitrogen vitamins plant growth regulators, they are manipulatedto facilitate which is case of any form of cultures.Now, growth regulators.Combination of auxin and cytokinin it stimulates growth and division we know that.So, similar combination of cell and protoplast culture can be used, that is why I said combinationwhich we are using for callus or cell culture might work initially for the protoplast cultures,but once the protoplast culture dividing into cells then it might need revision.And environmental factors, light, intensity, very high light intensity can inhibit divisionin protoplast.So, there generally it is preferred that you use dim light or you carry out in dark.So, and what else, pH temperature is generally the same what to use for your plant cell cultures,right.Heat treatment and electric impulse is found to induce division in protoplast.It is said that immobilization sometimes helps in protoplast cultures which is very obvious.Why do you think immobilization would be useful in protoplast cultures?Do not read just think?Think it is support into the cell.Right.Protection.So, because we know that it is very fragile there is only the cell membrane barrier.So, encapsulating it in a gel form is giving it more protection.Now, nurse sculpture and condition media is sometimes found to be useful to protoplastcultures.Nurse tissue culture technique which we had read earlier what was that?Using the callus . Not using another, may be the extracts ofthe other cells can be useful for the growth of their desired culture.So, protoplast fusion of somatic hybridization how is it done and what is it?It is an alternate to conventional breeding.Protoplast fusion can overcome sexual incompatibility and obtaining somatic hybrids, if you wantto use somatic cells to produce hybrid cells.Now, fusion of somatic cells and production of hybrids is known as somatic hybridization.Now, fusion by treatment of somatic.So, what all is needed?You need either sodium ions, so it is treated with sodium nitrate, maybe it will be thencalcium chloride, then peg.Peg is?Polyethylene glycol.Polyethylene glycol.So, it is facilitated it is said you need to fuse, you need to bring the cells together.So, therefore, you will like to treat it with cations like sodium ions or calcium ions andpolyethylene glycol.How is polyethylene glycol?Peg is used in transformation, is not it.Yeah What is it doing?Permeability.So, it is manipulating the permeability of the cell membrane reversibility, reversibly.So, if if it is used, it would facilitate what?The cell membranes have to fuse together, so that there is a transfer of nuclei, organelle,cytoplasm has to become one in hybrids, is not it.So, then transient pores have to be formed and the membranes have to get fused.So, polyethylene glycol is helping in fusion.So, all this is facilitating nothing, but coming together of the cells, fusion of themembranes by changing the permeability of the membranes, fluidity of the membranes.So, what are the steps involved?Isolation of protoplast first, then induction of the membrane fusion, mixing of cytoplasmand organelles would then happen, then formation of sin carrions.Formation of sin carrions after the mixing is happened there will forms sin carrions.Sin carrions means?. Two nuclei is coming together to form onenuclei.Then selection of fusion product.So, once you have obtained you would select the right, the desirable whether the properfusion has happened not under the microscope you will select the fused cells or hybrids.Now, this then you use those cells you give them optimum conditions for those hybridsto multiply, once they multiply then they would form callus and then from callus youcan regenerate into hybrid plants.So, protoplast fusion membrane properties this is what we were discussing.Now, process of protoplast fusion it requires what?It would require direct contact of the protoplast.So, what is facilitated?The addition would be facilitated by increasing divalent cations which means calcium or sodiumions.So, we have already discussed this.This would change the surface properties of the cell membranes, and using polyethyleneglycol would change the fluidity of the membranes, reversibly changing the or reversible permeabilityincrease in changing the permeability of the membranes to take up the extracellular material.Now, direct DNA or macro molecule uptake which is now cell membranes we know are semi-solidstructures.This is what we have learnt and fluid mosaic model.Now, fluidity of this membrane it is a physiological characteristics.So, now, what is required?How it is done?We need to have as I said reversibly increase the pore size.Generally, this is what is happening in the cells also.It is the cells if they want to take up some macromolecules, there are pores which aretransient pores which are getting formed and then even when it gets damaged, there is poreformation, but there is process which is present in the cell to again bring back the membraneto its position and closing the holes.So, the same process is being facilitated by using your polyethylene glycol or any chemicalsthen or even short duration of.You must have heard for transformation heat shock treatment is done or electric pulseselectroporation is done, so same thing can be used here to improve results of somatichybridizations.So, this is one of the protocols which is used for protoplast fusion in which you usehigh calcium ion concentration, electric charge and the pH is also changed because it is saidto have a profound effect on thefusion of these protoplast.So, what is it done?You mix sodium, nitrate peg and sucrose, sucrose being an osmoticumknowing that once the protoplasts are there.So, you need to balance the osmotic pressure.Then youheat it at 35 degree C or thus the the protoplast in this mixture and then youcentrifuge the different whichever cells you want to produces a somatic hybrid those cellsare centrifuged and then again it is left at 30 degree C for some time for the fusionto happen.So, one is you are facilitating diffusion and then you are giving time for the sin carrionsto form, once the sin carrions are formed then you check its do the screening and thenchange the media composition for it to divide and form the cultures .