CHAPTER 13 – PHOTOSYNTHESIS IN HIGHER PLANTS

CHAPTER 13

PHOTOSYNTHESIS IN HIGHER PLANTS

  • Green plants carry out ‘photosynthesis’, a physico-chemical process by which they use light energy to drive the synthesis of organic compounds.
  • Ultimately, all living forms on earth depend on sunlight for energy.
  • The use of energy from sunlight by plants doing photosynthesis is the basis of life on earth. Photosynthesis is important due to two reasons:

(a) it is the primary source of all food on earth.

(b) It is also responsible for the release of oxygen into the atmosphere by green plants.

  • chlorophyll (green pigment of the leaf), light and CO2 are required for photosynthesis to occur.

 

  • Two leaves experiment for importance of chlorophyll for starch formation – a variegated leaf or a leaf that was partially covered with black paper, and one that was exposed to light. On testing these leaves for starch it was clear that photosynthesis occurred only in the green parts of the leaves in the presence of light.
  • Half-leaf experiment for importance of CO2 for starch formation – a part of a leaf is enclosed in a test tube containing some KOH soaked cotton (which absorbs CO2), while the other half is exposed to air. The setup is then placed in light for some time. On testing for starch later in the two halves of the leaf the exposed part of the leaf tested positive for starch while the portion that was in the tube, tested negative.

 

Early Experiments

Joseph Priestley (1770)               – revealed the essential role of air in the growth of green plants

– discovered oxygen in 1774.

– Bell jar experiment

– hypothesised that Plants restore to the air whatever breathing animals and burning candles remove.

1

 

Jan Ingenhousz (1730-1799)      – showed that sunlight is essential to the photosynthesis.

Julius von Sachs (1854)              – provided evidence for production of glucose when plants grow.

                                                      – Glucose is usually stored as starch.

– showed that the green substance in plants is located in special bodies (later called chloroplasts) within plant cells.

T.W Engelmann (1843 – 1909)   – Used a prism to split light into its spectral components and then illuminated a green alga, Cladophora, placed in a suspension of aerobic bacteria. The bacteria were used to detect the sites of O2 evolution.

observed that the bacteria accumulated mainly in the region of blue and red light of the split spectrum.

described first action spectrum of photosynthesis, which resembles roughly the absorption spectra of chlorophyll a and b.

The empirical equation representing the total process of photosynthesis for oxygen evolving organisms was then understood as:

CO2 + H2O —Light –> [CH2O] + O2

where [CH2O] represented a carbohydrate (e.g., glucose, a six-carbon sugar).

Cornelius van Niel (1897-1985)  – a microbiologist

– studies of purple and green bacteria,

– demonstrated that photosynthesis is essentially a light-dependent   reaction in which hydrogen from a suitable oxidisable compound reduces carbon dioxide to carbohydrates.

2H2A + CO2Light –> 2A + CH2O + H2O

– In green plants H2O is the hydrogen donor and is oxidised to O2.

– When H2S is the hydrogen donor for purple and green sulphur bacteria, the ‘oxidation’ product is sulphur or sulphate and not O2.

– Hence, he inferred that the O2 evolved by the green plant comes from H2O, not from carbon dioxide.

– This was later proved by using radioisotopic techniques.

The correct equation, that would represent the overall process of photosynthesis is therefore:

6CO2 +12H2O —Light –> C6H12O6 + 6H2O + 6O2

 

SITE OF PHOTOSYNTHESIS

  • Photosynthesis occurs in green leaf in the chloroplasts.
  • mesophyll cells in the leaves, have a large number of chloroplasts. Usually the chloroplasts align themselves along the walls of the mesophyll cells, such that they get the optimum quantity of the incident light.
  • Within the chloroplast there is the membranous system consisting of grana, the stroma lamellae, and the fluid stroma. There is a clear division of labour within the chloroplast. The membrane system is responsible for trapping the light energy and also for the synthesis of ATP and NADPH. In stroma, enzymatic reactions incorporate CO2 into the plant leading to the synthesis of sugar, which in turn forms starch.
  • The former set of reactions, since they are directly light driven are called light reactions. The latter are not directly light driven but are dependent on the products of light reactions (ATP and NADPH). Hence, to distinguish the latter they are called, by convention, as dark reactions. However, this should not be construed to mean that they occur in darkness or that they are not light- dependent.

Screenshot (42)  HOW MANY PIGMENTS ARE INVOLVED IN PHOTOSYNTHESIS?

  • A chromatographic separation of the leaf pigments shows that the colour that we see in leaves is not due to a single pigment but due to four pigments:

Chlorophyll a (bright or blue green in the chromatogram),

chlorophyll b (yellow green),

xanthophylls (yellow) and

carotenoids (yellow to yellow-orange).

  • Pigments are substances that have an ability to absorb light, at specific wavelengths.
  • wavelengths at which there is maximum absorption by chlorophyll a, is in the blue and the red regions, also shows higher rate of photosynthesis. Hence, we can conclude that chlorophyll a is the chief pigment associated with photosynthesis.
  • These graphs, together, show that most of the photosynthesis takes place in the blue and red regions of the spectrum; some photosynthesis does take place at the other wavelengths of the visible spectrum.
  • Though chlorophyll is the major pigment responsible for trapping light, other thylakoid pigments like chlorophyll b, xanthophylls and carotenoids, which are called accessory pigments, also absorb light and transfer the energy to chlorophyll a. Indeed, they not only enable a wider range of wavelength of incoming light to be utilised for photosyntesis but also protect chlorophyll a from photo-oxidation.

Screenshot (43)

WHAT IS LIGHT REACTION?

  • Light reactions or the ‘Photochemical’ phase include light absorption, water splitting, oxygen release, and the formation of high-energy chemical intermediates, ATP and NADPH.
  • several complexes are involved in the process.
  • The pigments are organised into two discrete photochemical light harvesting complexes (LHC) within the Photosystem I (PS I) and Photosystem II (PS II). These are named in the sequence of their discovery, and not in the sequence in which they function during the light reaction.
  • The LHC are made up of hundreds of pigment molecules bound to proteins.
  • Each photosystem has all the pigments (except one molecule of chlorophyll a) forming a light harvesting system also called antennae.
  • These pigments help to make photosynthesis more efficient by absorbing different wavelengths of light. The single chlorophyll a molecule forms the reaction centre.
  • The reaction centre is different in both the photosystems.
  • In PS I the reaction centre chlorophyll a has an absorption peak at 700 nm, hence is called P700, while in PS II it has absorption maxima at 680 nm, and is called P680

Screenshot (44) THE ELECTRON TRANSPORT

  • In photosystem II the reaction centre chlorophyll a absorbs 680 nm wavelength of red light causing electrons to become excited and jump into an orbit farther from the atomic nucleus. These electrons are picked up by an electron acceptor which passes them to an electrons transport system consisting of cytochromes.
  • This movement of electrons is downhill, in terms of an oxidation-reduction or redox potential scale.
  • The electrons are not used up as they pass through the electron transport chain, but are passed on to the pigments of photosystem PS I.
  • Simultaneously, electrons in the reaction centre of PS I are also excited when they receive red light of wavelength 700 nm and are transferred to another accepter molecule that has a greater redox potential.
  • These electrons then are moved downhill again, this time to a molecule of energy-rich NADP+.
  • The addition of these electrons reduces NADP+ to NADPH + H+.
  • This whole scheme of transfer of electrons, starting from the PS II, uphill to the accepter, down the electron transport chain to PS I, excitation of electrons,transfer to another accepter, and finally down hill to NADP+ causing it to be reduced to NADPH + H+ is called the Z scheme, due to its characterstic shape. This shape is formed when all the carriers are placed in a sequence on a redox potential scale.

Screenshot (45)  Splitting of Water

  • The electrons that were moved from photosystem II must be replaced.
  • This is achieved by electrons available due to splitting of water.
  • The splitting of water is associated with the PS II; water is split into H+, [O] and electrons.
  • This creates oxygen, one of the net products of photosynthesis.
  • The electrons needed to replace those removed from photosystem I are provided by photosystem II.
  • 2H2O ——-> 4H+ + O2 + 4e
  • water splitting complex is associated with the PS II, which itself is physically located on the inner side of the membrane of the thylakoid.

Cyclic and Non-cyclic Photo-phosphorylation

  • Living organisms have the capability of extracting energy from oxidisable substances and store this in the form of bond energy.
  • Special substances like ATP, carry this energy in their chemical bonds.
  • The process of whichATP is synthesised by cells (in mitochondria and chloroplasts) is named phosphorylation.
  • Photo- phosphorylation is the synthesis of ATP from ADP and inorganic phosphate in the presence of light.
  • When the two photosystems work in a series, first PS II and then the PS I, a process called non-cyclic photo-phosphorylation occurs.
  • The two photosystems are connected through an electron transport chain, as seen earlier – in the Z scheme.
  • Both ATP and NADPH + H+ are synthesised by this kind of electron flow.
  • When only PS I is functional, the electron is circulated within the photosystem and the phosphorylation occurs due to cyclic flow of electrons.
  • A possible location where this could be happening is in the stroma lamellae.
  • While the membrane or lamellae of the grana have both PS I and PS II the stroma lamellae membranes lack PS II as well as NADP reductase enzyme.
  • The excited electron does not pass on to NADP+ but is cycled back to the PS I complex through the electron transport chain. The cyclic flow hence, results only in the synthesis of ATP, but not of NADPH + H+.
  • Cyclic photophosphorylation also occurs when only light of wavelengths beyond 680 nm are available for excitation.

Chemiosmotic Hypothesis

  • The chemiosmotic hypothesis has been put forward to explain the mechanism of synthesis of ATP.
  • Like in respiration, in photosynthesis too, ATP synthesis is linked to development of a proton gradient across a membrane.
  • This time these are membranes of the thylakoid.
  • There is one difference though, here the proton accumulation is towards the inside of the membrane, i.e., in the lumen.
  • In respiration, protons accumulate in the intermembrane space of the mitochondria when electrons move through the ETS.
  • Processes that take place during the activation of electrons and their transport to determine the steps that cause a proton gradient to develop are –

(a) Since splitting of the water molecule takes place on the inner side of the membrane, the protons or hydrogen ions that are produced by the splitting of water accumulate within the lumen of the thylakoids.

(b) As electrons move through the photosystems, protons are transported across the membrane. This happens because the primary accepter of electron which is located towards the outer side of the membrane transfers its electron not to an electron carrier but to an H carrier. Hence, this molecule removes a proton from the stroma while transporting an electron. When this molecule passes on its electron to the electron carrier on the inner side of the membrane, the proton is released into the inner side or the lumen side of the membrane.

(c) The NADP reductase enzyme is located on the stroma side of the membrane. Along with electrons that come from the accepter of electrons of PS I, protons are necessary for the reduction of NADP+ to NADPH+ H+. These protons are also removed from the stroma.

  • Hence, within the chloroplast, protons in the stroma decrease in number, while in the lumen there is accumulation of protons.
  • This creates a proton gradient across the thylakoid membrane as well as a measurable decrease in pH in the lumen.
  • This proton gradient is important because it is the breakdown of this gradient that leads to release of energy.
  • The gradient is broken down due to the movement of protons across the membrane to the stroma through the transmembrane channel of the F0 of the ATPase.
  • The ATPase enzyme consists of two parts:

one called the F0 is embedded in the membrane and forms a transmembrane channel that carries out facilitated diffusion of protons across the membrane.

The other portion is called F1 and protrudes on the outer surface of the thylakoid membrane on the side that faces the stroma. The breakdown of the gradient provides enough energy to cause a conformational change in the F1 particle of the ATPase, which makes the enzyme synthesise several molecules of energy-packed ATP.

  • Chemiosmosis requires a membrane, a proton pump, a proton gradient and ATPase.
  • Energy is used to pump protons across a membrane, to create a gradient or a high concentration of protons within the thylakoid lumen.
  • ATPase has a channel that allows diffusion of protons back across the membrane; this releases enough energy to activate ATPase enzyme that catalyses the formation of ATP.
  • Along with the NADPH produced by the movement of electrons, the ATP will be used immediately in the biosynthetic reaction taking place in the stroma, responsible for fixing CO2, and synthesis of sugars.

Screenshot (46)

WHERE ARE THE ATP AND NADPH USED?

  • The products of light reaction are ATP, NADPH and O2.
  • Of these O2 diffuses out of the chloroplast while ATP and NADPH are used to drive the processes leading to the synthesis of food, more accurately, sugars.
  • This is the biosynthetic phase of photosynthesis.
  • This process does not directly depend on the presence of light but is dependent on the products of the light reaction, i.e., ATP and NADPH, besides CO2 and H2
  • Immediately after light becomes unavailable, the biosynthetic process continues for some time, and then stops. If then, light is made available, the synthesis starts again.
  • CO2 is combined with H2O to produce (CH2O)n or sugars.
  • Melvin Calvin used radioactive 14C in algal photosynthesis studies which led to the discovery that the first CO2 fixation product was a 3-carbon organic acid or 3-phosphoglyceric acid (PGA).
  • He also contributed to working out the complete biosynthetic pathway; hence it was called Calvin cycle after him.
  • In another group of plants, first stable product of CO2 fixation is 4 carbon organic acid oxaloacetic acid or OAA.
  • The Primary Acceptor of CO2 is a 5-carbon ketose sugar – ribulose bisphosphate (RuBP).

THE CALVIN CYCLE

  • Calvin and his co-workers then worked out the whole pathway and showed that the pathway operated in a cyclic manner; the RuBP was regenerated.
  • The Calvin pathway occurs in all photosynthetic plants; it does not matter whether they have C3 or C4 (or any other) pathways.
  • The Calvin cycle can be described under three stages: carboxylation, reduction and regeneration.
  1. Carboxylation –

Carboxylation is the fixation of CO2 into a stable organic intermediate.

Carboxylation is the most crucial step of the Calvin cycle where CO2 is utilised for the carboxylation of RuBP.

This reaction is catalysed by the enzyme RuBP carboxylase which results in the formation of two molecules of 3-PGA.

Since this enzyme also has an oxygenation activity it would be more correct to call it RuBP carboxylase-oxygenase or RuBisCO.

  1. Reduction –

These are a series of reactions that lead to the formation of glucose.

The steps involve utilisation of 2 molecules of ATP for phosphorylation and two of NADPH for reduction per CO2 molecule fixed.

The fixation of six molecules of CO2 and 6 turns of the cycle are required for the removal of one molecule of glucose from the pathway.

  1. Regeneration –

Regeneration of the CO2 acceptor molecule RuBP is crucial if the cycle is to continue uninterrupted.

The regeneration steps require one ATP for phosphorylation to form RuBP.

  • Hence for every CO2 molecule entering the Calvin cycle, 3 molecules of ATP and 2 of NADPH are required.
  • It is probably to meet this difference in number of ATP and NADPH used in the dark reaction that the cyclic phosphorylation takes place.
  • To make one molecule of glucose 6 turns of the cycle are required.
  • Total 18 ATP, 12 NADPH, 12 CO2 are used synthesis of for 1 molecule of glucose.

 Screenshot (47)

THE C4 PATHWAY

  • Plants that are adapted to dry tropical regions have the C4 pathway
  • Though these plants have the C4 oxaloacetic acid as the first CO2 fixation product they use the C3 pathway or the Calvin cycle as the main biosynthetic pathway.
  • C4 plants are special: They have a special type of leaf anatomy, they tolerate higher temperatures, they show a response to highlight intensities, they lack a process called photorespiration and have greater productivity of biomass.
  • The particularly large cells around the vascular bundles of the C4 pathway plants are called bundle sheath cells, and the leaves which have such anatomy are said to have ‘Kranz’ anatomy.
  • ‘Kranz’ means ‘wreath’ and is a reflection of the arrangement of cells.
  • The bundle sheath cells may form several layers around the vascular bundles; they are characterised by having a large number of chloroplasts, thick walls impervious to gaseous exchange and no intercellular spaces.
  • This pathway also known as Hatch and Slack Pathway, is a cyclic process.
  • The primary CO2 acceptor is a 3-carbon molecule phosphoenol pyruvate (PEP) and is present in the mesophyll cells. The enzyme responsible for this fixation is PEP carboxylase or PEPcase.
  • The mesophyll cells lack RuBisCO enzyme.
  • The C4 acid OAA is formed in the mesophyll cells.
  • It then forms other 4-carbon compounds like malic acid or aspartic acid in the mesophyll cells itself, which are transported to the bundle sheath cells.
  • In the bundle sheath cells these C4 acids are broken down to release CO2 and a 3-carbon molecule.
  • The 3-carbon molecule is transported back to the mesophyll where it is converted to PEP again, thus, completing the cycle.
  • The CO2 released in the bundle sheath cells enters the C3 or the Calvin pathway, a pathway common to all plants. The bundle sheath cells are rich in an enzyme Ribulose bisphosphate carboxylase-oxygenase (RuBisCO), but lack PEPcase.
  • Thus, the basic pathway that results in the formation of the sugars, the Calvin pathway, is common to the C3 and C4
  • the Calvin pathway occurs in all the mesophyll cells of the C3 plants while in the C4 plants it does not take place in the mesophyll cells but does so only in the bundle sheath cells.

Screenshot (48)

PHOTORESPIRATION

  • RuBisCO is the most abundant enzyme in the world.
  • It is characterised by the fact that its active site can bind to both CO2 and O2 – hence the name.
  • RuBisCO has a much greater affinity for CO2 than for O2.
  • This binding is competitive. It is the relative concentration of O2 and CO2 that determines which of the two will bind to the enzyme.
  • In C3 plants some O2 does bind to RuBisCO, and hence CO2 fixation is decreased. Here the RuBP instead of being converted to 2 molecules of PGA binds with O2 to form one molecule and phosphoglycolate in a pathway called photorespiration.
  • In the photorespiratory pathway, there is neither synthesis of sugars, nor of ATP. Rather it results in the release of CO2 with the utilisation of ATP.
  • In the photorespiratory pathway there is no synthesis of ATP or NADPH. Therefore, photorespiration is a wasteful process.
  • In C4 plants photorespiration does not occur. This is because they have a mechanism that increases the concentration of CO2 at the enzyme site.
  • This takes place when the C4 acid from the mesophyll is broken down in the bundle cells to release CO2 – this results in increasing the intracellular concentration of CO2.
  • In turn, this ensures that the RuBisCO functions as a carboxylase minimising the oxygenase activity.
  • Because of absence of photorespiration in C4 plants, productivity and yields are better in these plants.
  • These plants show tolerance to higher temperatures.
Characteristics C3 Plants C4 Plants
Cell type in which the Calvin cycle takes place Both Bundle sheath
Cell type in which the initial carboxylation reaction occurs Both Mesophyll
How many cell types does the leaf have that fix Co2. Three: Bundle sheath, palisade, spongy mesophyll Two: Bundle sheath and

Mesophyll

Which is the primary Co2 acceptor RuBP PEP
Number of carbons in the primary Co2 acceptor 5 3
Which is the primary Co2 fixation product PGA OAA
No. of carbons in the primary Co2 fixation product 3 4
Does the plant have RuBisCo? Yes Yes
Does the plant have PEP Case? No Yes
Which cells in the plant have Rubisco? Mesophyll/Bundle sheath/none Mesophyll/Bundle sheath/none
Co2 fixation rate under high light conditions Medium High
Whether photorespiration is present at low light intensities Sometimes Negligible
Whether photorespiration is present at high light intensities Sometimes Negligible
Whether photorespiration would be present at low CO2 concentrations High Negligible
Whether photorespiration would be present at high CO2 concentrations Sometimes Negligible
Temperature optimum 20-25C 30-40 C

FACTORS AFFECTING PHOTOSYNTHESIS

  • The rate of photosynthesis is very important in determining the yield of plants including crop plants.
  • Photosynthesis is under the influence of several factors, both internal (plant) and external.
  • The plant factors include the number, size, age and orientation of leaves, mesophyll cells and chloroplasts, internal CO2 concentration and the amount of chlorophyll. The plant or internal factors are dependent on the genetic predisposition and the growth of the plant.
  • The external factors include the availability of sunlight, temperature, CO2 concentration and water.
  • As a plant photosynthesises, all these factors will simultaneously affect its rate. Hence, though several factors interact and simultaneously affect photosynthesis or CO2 fixation, usually one factor is the major cause or is the one that limits the rate. Hence, at any point the rate will be determined by the factor available at sub-optimal levels.
  • Blackman’s (1905) Law of Limiting Factors – If a chemical process is affected by more than one factor, then its rate will be determined by the factor which is nearest to its minimal value: it is the factor which directly affects the process if its quantity is changed
  • For example, despite the presence of a green leaf and optimal light and CO2 conditions, the plant may not photosynthesise if the temperature is very low. This leaf, if given the optimal temperature, will start photosynthesising.

Light

  • It includes light quality, light intensity and the duration of exposure to light.
  • There is a linear relationship between incident light and CO2 fixation rates at low light intensities.
  • At higher light intensities, gradually the rate does not show further increase as other factors become limiting.
  • Light saturation occurs at 10 per cent of the full sunlight.
  • Hence, except for plants in shade or in dense forests, light is rarely a limiting factor in nature.
  • Increase in incident light beyond a point causes the breakdown of chlorophyll and a decrease in photosynthesis.

Screenshot (50)

Carbon dioxide Concentration

  • Carbon dioxide is the major limiting factor for photosynthesis.
  • The concentration of CO2 is very low in the atmosphere (between 0.03 and 0.04 per cent).
  • Increase in concentration upto 0.05 per cent can cause an increase in CO2 fixation rates; beyond this the levels can become damaging over longer periods.
  • The C3 and C4 plants respond differently to CO2 At low light conditions neither group responds to high Co2 conditions. At high light intensities, both C3 and C4 plants show increase in the rates of photosynthesis.
  • C4 plants show saturation at about 360 µlL-1 while in C3 plants saturation is seen only beyond 450 µlL-1. Thus, current availability of CO2 levels is limiting to the C3
  • The fact that C3 plants respond to higher CO2 concentration by showing increased rates of photosynthesis leading to higher productivity has been used for some greenhouse crops such as tomatoes and bell pepper. They are allowed to grow in carbon dioxide enriched atmosphere that leads to higher yields.

Temperature

  • The dark reactions being enzymatic are temperature controlled.
  • Though the light reactions are also temperature sensitive they are affected to a much lesser extent. The C4 plants respond to higher temperatures and show higher rate of photosynthesis while C3 plants have a much lower temperature optimum.
  • The temperature optimum for photosynthesis of different plants also depends on the habitat that they are adapted to.
  • Tropical plants have a higher temperature optimum than the plants adapted to temperate climates.

Water

  • Even though water is one of the reactants in the light reaction, the effect of water as a factor is more through its effect on the plant, rather than directly on photosynthesis.
  • Water stress causes the stomata to close hence reducing the CO2 Besides, water stress also makes leaves wilt, reducing the surface area of the leaves and their metabolic activity as well.

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CHAPTER 13 – PHOTOSYNTHESIS IN HIGHER PLANTS

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CHAPTER 11 – TRANSPORT IN PLANTS

Chapter 11

Transport in Plants

  • Plants need to move molecules over very long distances, much more than animals do; they also do not have a circulatory system in place.
  • Water taken up by the roots has to reach all parts of the plant, up to the very tip of the growing stem. The photosynthates or food synthesised by the leaves have also to be moved to all parts including the root tips embedded deep inside the soil.
  • Movement across short distances, say within the cell, across the membranes and from cell to cell within the tissue has also to take place.
  • In a flowering plant the substances that would need to be transported are water, mineral nutrients, organic nutrients and plant growth regulators.
  • Small distance transport – by diffusion and by cytoplasmic streaming supplemented by active transport.
  • Long distance Transport – through the vascular system (the xylem and the phloem) and is called translocation.
  • In rooted plants, transport in xylem (of water and minerals) is essentially unidirectional, from roots to the stems.
  • Organic and mineral nutrients however, undergo multidirectional transport.
  • Organic compounds synthesised in the photosynthetic leaves are exported to all other parts of the plant Including storage organs. From the storage organs they are later re-exported.
  • The mineral nutrients are taken up by the roots and transported upwards into the stem, leaves and the growing regions. When any plant part undergoes senescence, nutrients may be withdrawn from such regions and moved to the growing parts.
  • Hormones or plant growth regulators and other chemical stimuli are also transported, though in very small amounts, sometimes in a strictly polarised or unidirectional manner from where they are synthesised to other parts.

Means of Transport

Diffusion

  • Passive movement and may be from one part of the cell to the other, or from cell to cell, or over short distances, say, from the intercellular spaces of the leaf to the outside.
  • No energy expenditure takes place.
  • Molecules move in a random fashion, the net result being substances moving from regions of higher concentration to regions of lower concentration.
  • Diffusion is a slow process and is not dependent on a ‘living system’.
  • Diffusion is obvious in gases and liquids, but diffusion in solids rather than of solids is more likely.
  • Diffusion is very important to plants since it the only means for gaseous movement within the plant body.
  • Diffusion rates are affected by the gradient of concentration, the permeability of the membrane separating them, size of the substances, temperature and pressure.

Facilitated Diffusion

  • The diffusion of any substance across a membrane also depends on its solubility in lipids, the major constituent of the membrane.
  • Substances soluble in lipids diffuse through the membrane faster.
  • Substances that have a hydrophilic moiety, find it difficult to pass through the membrane; their movement has to be facilitated. Membrane proteins provide sites at which such molecules cross the membrane.
  • They do not set up a concentration gradient: a concentration gradient must already be present for molecules to diffuse even if facilitated by the proteins. This process is called facilitated diffusion.
  • In facilitated diffusion special proteins help move substances across membranes without expenditure of ATP energy.
  • Facilitated diffusion cannot cause net transport of molecules from a low to a high concentration – this would require input of energy.
  • Transport rate reaches a maximum when all of the protein transporters are being used (saturation).
  • Facilitated diffusion is very specific: it allows cell to select substances for uptake.
  • It is sensitive to inhibitors which react with protein side chains.
  • The proteins form channels in the membrane for molecules to pass through. Some channels are always open; others can be controlled. Some are large, allowing a variety of molecules to cross.
  • The porins are proteins that form huge pores in the outer membranes of the plastids, mitochondria and some bacteria allowing molecules up to the size of small proteins to pass through.
  • When an extracellular molecule bound to the transport protein; the transport protein then rotates and releases the molecule inside the cell, e.g., water channels (made up of eight different types of aquaporins.)

Passive symports and antiports

  • Some carrier or transport proteins allow diffusion only if two types of molecules move together.
  • In a symport, both molecules cross the membrane in the same direction; in an antiport, they move in opposite directions.
  • When a molecule moves across a membrane independent of other molecules, the process is called uniport.

Active Transport

  • Active transport uses energy to pump molecules against a concentration gradient.
  • Active transport is carried out by membrane-proteins. Hence different proteins in the membrane play a major role in both active as well as passive transport.
  • Pumps are proteins that use energy to carry substances across the cell membrane. These pumps can transport substances from a low concentration to a high concentration (‘uphill’ transport).
  • Transport rate reaches a maximum when all the protein transporters are being used or are saturated.
  • Like enzymes the carrier protein is very specific in what it carries across the membrane.
  • These proteins are sensitive to inhibitors that react with protein side chains.

Comparison of Different Transport Processes

  • Proteins in the membrane are responsible for facilitated diffusion and active transport and hence show common characterstics of being highly selective; they are liable to saturate, respond to inhibitors and are under hormonal regulation.
  • But diffusion whether facilitated or not – take place only along a gradient and do not use energy.

Plant-Water Relations

  • Water is essential for all physiological activities of the plant and plays a very important role in all living organisms.
  • It provides the medium in which most substances are dissolved. The protoplasm of the cells is nothing but water in which different molecules are dissolved and (several particles) suspended. A watermelon has over 92 per cent water; most herbaceous plants have only about 10 to 15 per cent of its fresh weight as dry matter.
  • Of course, distribution of water within a plant varies – woody parts have relatively very little water, while soft parts mostly contain water. A seed may appear dry but it still has water – otherwise it would not be alive and respiring!
  • Terrestrial plants take up huge amount water daily but most of it is lost to the air through evaporation from the leaves, i.e., transpiration. A mature corn plant absorbs almost three litres of water in a day, while a mustard plant absorbs water equal to its own weight in about 5 hours. Because of this high demand for water, it is not surprising that water is often the limiting factor for plant growth and productivity in both agricultural and natural environments.

Water Potential

  • Water potential (Psi w) is a concept fundamental to understanding water movement. Solute potential (Psi s) and pressure potential (Psi p) are the two main components that determine water potential.
  • Water molecules possess kinetic energy. In liquid and gaseous form they are in random motion that is both rapid and constant. The greater the concentration of water in a system, the greater is its kinetic energy or ‘water potential’. Hence, it is obvious that pure water will have the greatest water potential.
  • If two systems containing water are in contact, random movement of water molecules will result in net movement of water molecules from the system with higher energy to the one with lower energy. Thus water will move from the system containing water at higher water potential to the one having low water potential.
  • This process of movement of substances down a gradient of free energy is called diffusion.
  • Water potential is denoted by the Greek symbol Psi and is expressed in pressure units such as pascals (Pa).
  • By convention, the water potential of pure water at standard temperatures, which is not under any pressure, is taken to be zero.
  • If some solute is dissolved in pure water, the solution has fewer free water and the concentration of water decreases, reducing its water potential. Hence, all solutions have a lower water potential than pure water; the magnitude of this lowering due to dissolution of a solute is called solute potential or Psi s. Psi s is always negative. The more the solute molecules, the lower (more negative) is the Psi s.
  • For a solution at atmospheric pressure (water potential) Psi w = (solute potential) Psi s.
  • If a pressure greater than atmospheric pressure is applied to pure water or a solution, its water potential increases. It is equivalent to pumping water from one place to another.
  • Pressure can build up in a plant system when water enters a plant cell due to diffusion causing a pressure built up against the cell wall, it makes the cell turgid, this increases the pressure potential. Pressure potential is usually positive.
  • Though in plants negative potential or tension in the water column in the xylem plays a major role in water transport up a stem. Pressure potential is denoted as (p.
  • Water potential of a cell is affected by both solute and pressure potential. The relationship between them is as follows:

Osmosis

  • The plant cell is surrounded by a cell membrane and a cell wall. The cell wall is freely permeable to water and substances in solution hence is not a barrier to movement.
  • In plants the cells usually contain a large central vacuole, whose contents, the vacuolar sap, contribute to the solute potential of the cell.
  • In plant cells, the cell membrane and the membrane of the vacuole, the tonoplast together are important determinants of movement of molecules in or out of the cell.
  • Osmosis is the term used to refer specifically to the diffusion of water across a differentially- or semi-permeable membrane. Osmosis occurs spontaneously in response to a driving force.
  • The net direction and rate of osmosis depends on both the pressure gradient and concentration gradient.
  • Water will move from its region of higher chemical potential (or concentration) to its region of lower chemical potential until equilibrium is reached. At equilibrium the two chambers should have the same water potential.


In above Fig two chambers, A and B, containing solutions are separated by a semi-permeable membrane.

  • Solution of which chamber has a lower water potential? – B
  • Solution of which chamber has a lower solute potential? – B
  • In which direction will osmosis occur? – A(B
  • Which solution has a higher solute potential?- A
  • At equilibrium which chamber will have lower water potential?- Equal
  • If one chamber has a psi of -2000 kPa, and the other -1000 kPa, which is the chamber that has the higher psi ?- second with -1000kP.

  • Experiment – a solution of sucrose in water taken in a funnel is separated from pure water in a beaker through a semi-permeable membrane (Egg membrane – Can be obtained by removing the yolk and albumin through a small hole at one end of the egg, and placing the shell in dilute solution of hydrochloric acid for a few hours. The egg shell dissolves leaving the membrane intact). Water will move into the funnel, resulting in rise in the level of the solution in the funnel. This will continue till the equilibrium is reached. External pressure can be applied from the upper part of the funnel such that no water diffuses into the funnel through the membrane. This pressure required to prevent water from diffusing is the osmotic pressure and this is the function of the solute concentration; more the solute concentration, greater will be the pressure required to prevent water from diffusing in. Numerically osmotic pressure is equivalent to the osmotic potential, but the sign is opposite. Osmotic pressure is the positive pressure applied, while osmotic potential is negative.

Plasmolysis

    • The behaviour of the plant cells (or tissues) with regard to water movement depends on the surrounding solution.
    • If the external solution balances the osmotic pressure of the cytoplasm, it is said to be isotonic.
    • If the external solution is more dilute than the cytoplasm, it is hypotonic and if the external solution is more concentrated, it is hypertonic.
    • Cells swell in hypotonic solutions and shrink in hypertonic ones.
    • Plasmolysis occurs when water moves out of the cell and the cell membrane of a plant cell shrinks away from its cell wall. This occurs when the cell (or tissue) is placed in a solution that is hypertonic (has more solutes) to the protoplasm. Water moves out; it is first lost from the cytoplasm and then from the vacuole.
    • The water when drawn out of the cell through diffusion into the extracellular (outside cell) fluid causes the protoplast to shrink away from the walls. The cell is said to be plasmolysed.
    • The movement of water occurred across the membrane moving from an area of high water potential (i.e., the cell) to an area of lower water potential outside the cell.
    • The process of plamolysis is usually reversible.
    • When the cells are placed in a hypotonic solution (higher water potential or dilute solution as compared to the cytoplasm), water diffuses into the cell causing the cytoplasm to build up a pressure against the wall, that is called turgor pressure. The pressure exerted by the protoplasts due to entry of water against the rigid walls is called pressure potential. Because of the rigidity of the cell wall, the cell does not rupture.
    • This turgor pressure is ultimately responsible for enlargement and extension growth of cells.

Imbibition

  • Imbibition is a special type of diffusion when water is absorbed by solids – colloids – causing them to enormously increase in volume. e.g., absorption of water by seeds and dry wood.
  • The pressure that is produced by the swelling of wood had been used by prehistoric man to split rocks and boulders.
  • If it were not for the pressure due to imbibition, seedlings would not have been able to emerge out of the soil into the open.
  • Imbibition is also diffusion since water movement is along a concentration gradient; the seeds and other such materials have almost no water hence they absorb water easily.
  • Water potential gradient between the absorbent and the liquid imbibed is essential for imbibition.
  • In addition, for any substance to imbibe any liquid, affinity between the adsorbant and the liquid is also a pre-requisite.

Long Distance Transport of Water

  • Long distance transport of substances within a plant cannot be by diffusion alone. Diffusion is a slow process. It can account for only short distance movement of molecules. For example, the movement of a molecule across a typical plant cell (about 50µm) takes approximately 2.5 s.
  • In large and complex organisms, often substances have to be moved across very large distances. sometimes the sites of production or absorption and sites of storage are too far from each other; diffusion or active transport would not suffice. Special long distance transport systems become necessary so as to move substances across long distances and at a much faster rate.
  • Water and minerals, and food are generally moved by a mass or bulk flow system.
  • Mass flow is the movement of substances in bulk or en masse from one point to another as a result of pressure differences between the two points. It is a characteristic of mass flow that substances, whether in solution or in suspension, are swept along at the same pace, as in a flowing river.
  • This is unlike diffusion where different substances move independently depending on their concentration gradients.
  • Bulk flow can be achieved either through a positive hydrostatic pressure gradient (e.g., a garden hose) or a negative hydrostatic pressure gradient (e.g., suction through a straw).
  • The bulk movement of substances through the conducting or vascular tissues of plants is called translocation.
  • The higher plants have highly specialised vascular tissues – xylem and phloem.
  • Xylem is associated with translocation of mainly water, mineral salts, some organic nitrogen and hormones, from roots to the aerial parts of the plants.
  • The phloem translocates a variety of organic and inorganic solutes, mainly from the leaves to other parts of the plants.

How do Plants Absorb Water?

  • The responsibility of absorption of water and minerals is of the root hairs.
  • Root hairs are thin-walled slender extensions of root epidermal cells that greatly increase the surface area for absorption.
  • Water is absorbed along with mineral solutes, by the root hairs, purely by diffusion.
  • once water is absorbed by the root hairs, it can move deeper into root layers by two distinct pathways: Apoplast pathway, Symplast pathway.
  • Apoplast is the system of adjacent cell walls that is continuous throughout the plant, except at the casparian strips of the endodermis in the roots.
  • The apoplastic movement of water occurs exclusively through the intercellular spaces and the walls of the cells.
  • Movement through the apoplast does not involve crossing the cell membrane.
  • This movement is dependent on the gradient.
  • The apoplast does not provide any barrier to water movement and water movement is through mass flow.
  • As water evaporates into the intercellular spaces or the atmosphere, tension develop in the continuous stream of water in the apoplast, hence mass flow of water occurs due to the adhesive and cohesive properties of water.
  • The symplastic system is the system of interconnected protoplasts. Neighbouring cells are connected through cytoplasmic strands that extend through plasmodesmata.
  • During symplastic movement, the water travels through the cells – their cytoplasm; intercellular movement is through the plasmodesmata.
  • Water has to enter the cells through the cell membrane, hence the movement is relatively slower.
  • Movement is again down a potential gradient.
  • symplastic movement may be aided by cytoplasmic streaming.

  • Most of the water flow in the roots occurs via the apoplast since the cortical cells are loosely packed, and hence offer no resistance to water movement. However, the inner boundary of the cortex, the endodermis, is impervious to water because of a band of suberised matrix called the casparian strip.
  • Water molecules are unable to penetrate the layer, so they are directed to wall regions that are not suberised, into the cells proper through the membranes.
  • The water then moves through the symplast and again crosses a membrane to reach the cells of the xylem.
  • The movement of water through the root layers is ultimately symplastic in the endodermis. This is the only way water and other solutes can enter the vascular cylinder.
  • Once inside the xylem, water is again free to move between cells as well as through them. In young roots, water enters directly into the xylem vessels and/or tracheids. These are non-living conduits and so are parts of the apoplast.

  • Some plants have additional structures associated with them that help in water (and mineral) absorption. A mycorrhiza is a symbiotic association of a fungus with a root system.
  • The fungal filaments form a network around the young root or they penetrate the root cells. The hyphae have a very large surface area that absorb mineral ions and water from the soil from a much larger volume of soil that perhaps a root cannot do.
  • The fungus provides minerals and water to the roots, in turn the roots provide sugars and N-containing compounds to the mycorrhizae.
  • Some plants have an obligate association with the mycorrhizae. e.g., Pinus seeds cannot germinate and establish without the presence of mycorrhizae.

Water Movement up a Plant

Root Pressure

  • As various ions from the soil are actively transported into the vascular tissues of the roots, water follows (its potential gradient) and increases the pressure inside the xylem. This positive pressure is called root pressure, and can be responsible for pushing up water to small heights in the stem.
  • Effects of root pressure is also observable at night and early morning when evaporation is low, and excess water collects in the form of droplets around special openings of veins near the tip of grass blades, and leaves of many herbaceous parts. Such water loss in its liquid phase is known as guttation.
  • Root pressure can, at best, only provide a modest push in the overall process of water transport. They obviously do not play a major role in water movement up tall trees.
  • The greatest contribution of root pressure may be to re-establish the continuous chains of water molecules in the xylem which often break under the enormous tensions created by transpiration.
  • Root pressure does not account for the majority of water transport; most plants meet their need by transpiratory pull.

Transpiration pull

  • The flow of water upward through the xylem in plants can achieve fairly high rates, up to 15 metres per hour.
  • Most researchers agree that water is mainly ‘pulled’ through the plant, and that the driving force for this process is transpiration from the leaves. This is referred to as the cohesion-tension-transpiration pull model of water transport. But, what generates this transpirational pull?
  • Water is transient in plants. Less than 1 per cent of the water reaching the leaves is used in photosynthesis and plant growth. Most of it is lost through the stomata in the leaves. This water loss is known as transpiration.

TRANSPIRATION

  • Transpiration is the evaporative loss of water by plants. It occurs mainly through the stomata in the leaves.
  • Besides the loss of water vapour in transpiration, exchange of oxygen and carbon dioxide in the leaf also occurs through pores called stomata (sing.: stoma).
  • Normally stomata are open in the day time and close during the night.
  • The immediate cause of the opening or closing of the stomata is a change in the turgidity of the guard cells.
  • The inner wall of each guard cell, towards the pore or stomatal aperture, is thick and elastic. When turgidity increases within the two guard cells flanking each stomatal aperture or pore, the thin outer walls bulge out and force the inner walls into a crescent shape.
  • The opening of the stoma is also aided due to the orientation of the microfibrils in the cell walls of the guard cells. Cellulose microfibrils are oriented radially rather than longitudinally making it easier for the stoma to open.
  • When the guard cells lose turgor, due to water loss (or water stress) the elastic inner walls regain their original shape, the guard cells become flaccid and the stoma closes.

  • Usually the lower surface of a dorsiventral (often dicotyledonous) leaf has a greater number of stomata while in an isobilateral (often monocotyledonous) leaf they are about equal on both surfaces.
  • Transpiration is affected by several external factors: temperature, light, humidity, wind speed. Plant factors that affect transpiration include number and distribution of stomata, number of stomata open, per cent, water status of the plant, canopy structure etc.
  • The transpiration driven ascent of xylem sap depends mainly on the following physical properties of water:
  • Cohesion – mutual attraction between water molecules.
  • Adhesion – attraction of water molecules to polar surfaces (such as the surface of tracheary elements).
  • Surface Tension – water molecules are attracted to each other in the liquid phase more than to water in the gas phase.
  • These properties give water high tensile strength, i.e., an ability to resist a pulling force, and high capillarity, i.e., the ability to rise in thin tubes. In plants capillarity is aided by the small diameter of the tracheary elements – the tracheids and vessel elements.
  • The process of photosynthesis requires water. The system of xylem vessels from the root to the leaf vein can supply the needed water.
  • As water evaporates through the stomata, since the thin film of water over the cells is continuous, it results in pulling of water, molecule by molecule, into the leaf from the xylem.
  • Also, because of lower concentration of water vapour in the atmosphere as compared to the substomatal cavity and intercellular spaces, water diffuses into the surrounding air. This creates a ‘pull’.
  • Measurements reveal that the forces generated by transpiration can create pressures sufficient to lift a xylem sized column of water over 130 metres high.

Transpiration and Photosynthesis – a Compromise

  • Transpiration has more than one purpose; it
    • createstranspiration pull for absorption and transport of plants
    • supplieswater for photosynthesis
    • transportsminerals from the soil to all parts of the plant
    • cools leaf surfaces, sometimes 10 to 15 degrees, by evaporative cooling
    • maintainsthe shape and structure of the plants by keeping cells turgid
  • An actively photosynthesising plant has an insatiable need for water. Photosynthesis is limited by available water which can be swiftly depleted by transpiration.
  • The humidity of rainforests is largely due to this vast cycling of water from root to leaf to atmosphere and back to the soil.
  • The evolution of the C4 photosynthetic system is probably one of the strategies for maximising the availability of CO2 while minimising water loss.
  • C4 plants are twice as efficient as C3 plants in terms of fixing carbon (making sugar). However, a C4 plant loses only half as much water as a C3 plant for the same amount of CO2 fixed.

Uptake and Transport of Mineral Nutrients

  • Plants obtain their carbon and most of their oxygen from CO2 in the atmosphere. However, their remaining nutritional requirements are obtained from minerals and water for hydrogen in the soil.

Uptake of Mineral Ions

  • Unlike water, all minerals cannot be passively absorbed by the roots.
  • Two factors account for this:

(i) minerals are present in the soil as charged particles (ions) which cannot move across cell membranes and
(ii) the concentration of minerals in the soil is usually lower than the concentration of minerals in the root.

  • Therefore, most minerals must enter the root by active absorption into the cytoplasm of epidermal cells.
  • This needs energy in the form of ATP.
  • The active uptake of ions is partly responsible for the water potential gradient in roots, and therefore for the uptake of water by osmosis. Some ions also move into the epidermal cells passively.
  • Ions are absorbed from the soil by both passive and active transport.
  • Specific proteins in the membranes of root hair cells actively pump ions from the soil into the cytoplasms of the epidermal cells.
  • Like all cells, the endodermal cells have many transport proteins embedded in their plasma membrane; they let some solutes cross the membrane, but not others.
  • Transport proteins of endodermal cells are control points, where a plant adjusts the quantity and types of solutes that reach the xylem.
  • the root endodermis because of the layer of suberin has the ability to actively transport ions in one direction only.

Translocation of Mineral Ions

  • After the ions have reached xylem through active or passive uptake, or a combination of the two, their further transport up the stem to all parts of the plant is through the transpiration stream.
  • The chief sinks for the mineral elements are the growing regions of the plant, such as the apical and lateral meristems, young leaves, developing flowers, fruits and seeds, and the storage organs.
  • Unloading of mineral ions occurs at the fine vein endings through diffusion and active uptake by these cells.
  • Mineral ions are frequently remobilised, particularly from older, senescing parts.
  • Older dying leaves export much of their mineral content to younger leaves.
  • Similarly, before leaf fall in decidous plants, minerals are removed to other parts.
  • Elements most readily mobilised are phosphorus, sulphur, nitrogen and potassium.
  • Some elements that are structural components like calcium are not remobilised.
  • An analysis of the xylem exudates shows that though some of the nitrogen travels as inorganic ions, much of it is carried in the organic form as amino acids and related compounds.
  • Similarly, small amounts of P and S are carried as organic compounds.
  • In addition, small amount of exchange of materials does take place between xylem and phloem.
  • Hence, it is not that we can clearly make a distinction and say categorically that xylem transports only inorganic nutrients while phloem transports only organic materials.

Phloem Transport: Flow from Source to Sink

  • Food, primarily sucrose, is transported by the vascular tissue phloem from a source to a sink.
  • Usually the source is understood to be that part of the plant which synthesises the food, i.e., the leaf, and sink, the part that needs or stores the food.
  • But, the source and sink may be reversed depending on the season, or the plant’s needs.
  • Sugar stored in roots may be mobilised to become a source of food in the early spring when the buds of trees, act as sink; they need energy for growth and development of the photosynthetic apparatus.
  • Since the source-sink relationship is variable, the direction of movement in the phloem can be upwards or downwards, i.e., bi-directional. This contrasts with that of the xylem where the movement is always unidirectional, i.e., upwards.
  • Hence, unlike one-way flow of water in transpiration, food in phloem sap can be transported in any required direction so long as there is a source of sugar and a sink able to use, store or remove the sugar.
  • Phloem sap is mainly water and sucrose, but other sugars, hormones and amino acids are also transported or translocated through phloem.

The Pressure Flow or Mass Flow Hypothesis

  • The accepted mechanism used for the translocation of sugars from source to sink is called the pressure flow hypothesis.
  • As glucose is prepared at the source (by photosynthesis) it is converted to sucrose (a dissacharide). The sugar is then moved in the form of sucrose into the companion cells and then into the living phloem sieve tube cells by active transport.
  • This process of loading at the source produces a hypertonic condition in the phloem.
  • Water in the adjacent xylem moves into the phloem by osmosis. As osmotic pressure builds up the phloem sap will move to areas of lower pressure.
  • At the sink osmotic pressure must be reduced. Again active transport is necessary to move the sucrose out of the phloem sap and into the cells which will use the sugar – converting it into energy, starch, or cellulose.
  • As sugars are removed, the osmotic pressure decreases and water moves out of the phloem.
  • the movement of sugars in the phloem begins at the source, where sugars are loaded (actively transported) into a sieve tube. Loading of the phloem sets up a water potential gradient that facilitates the mass movement in the phloem.
  • Phloem tissue is composed of sieve tube cells, which form long columns with holes in their end walls called sieve plates.
  • Cytoplasmic strands pass through the holes in the sieve plates, so forming continuous filaments.
  • As hydrostatic pressure in the phloem sieve tube increases, pressure flow begins, and the sap moves through the phloem.
  • Meanwhile, at the sink, incoming sugars are actively transported out of the phloem and removed as complex carbohydrates.
  • The loss of solute produces a high water potential in the phloem, and water passes out, returning eventually to xylem.
  • A simple experiment, called girdling, was used to identify the tissues through which food is transported.
  • On the trunk of a tree a ring of bark up to a depth of the phloem layer, can be carefully removed.
  • In the absence of downward movement of food, the portion of the bark above the ring on the stem becomes swollen after a few weeks.
  • This simple experiment shows that phloem is the tissue responsible for translocation of food; and that transport takes place in one direction, i.e., towards the roots.

 

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CHAPTER 11- TRANSPORT IN PLANTS

CHAPTER 12 : BIOTECHNOLOGY AND ITS APPLICATIONS

CHAPTER 12

BIOTECHNOLOGY AND ITS APPLICATIONS

  • Biotechnology essentially deals with industrial scale production of biopharmaceuticals and biologicals using genetically modified microbes, fungi, plants and animals.
  • The applications of biotechnology include therapeutics, diagnostics, and genetically
    modified crops for agriculture, processed food, bioremediation, waste treatment, and
    energy production.
  • Three critical research areas of biotechnology are:
    (i) Providing the best catalyst in the form of improved organism usually a microbe or pure enzyme.
    (ii) Creating optimal conditions through engineering for a catalyst to act, and
    (iii) Downstream processing technologies to purify the protein/organic compound.

BIOTECHNOLOGICAL APPLICATIONS IN AGRICULTURE

There are three options that can be thought for increasing food production
(i) agro-chemical based agriculture;
(ii) organic agriculture; and
(iii) Genetically engineered crop-based agriculture.

  •  We have succeeded in tripling the food supply by Green Revolution but yet it was not
    enough to feed the growing human population.
  • Increased yields have partly been due to the use of improved crop varieties, but mainly due to the use of better management practices and use of agrochemicals (fertilisers and pesticides).
  • However, for farmers in the developing world, agrochemicals are often too expensive, and further increases in yield with existing varieties are not possible using conventional breeding.
  • So there is a need to find alternative path that our understanding of genetics can show so that farmers may obtain maximum yield from their fields and to minimise the use of fertilisers and chemicals so that their harmful effects on the environment can be reduced. Use of genetically modified crops is a possible solution.
  • Plants, bacteria, fungi and animals whose genes have been altered by manipulation are called Genetically Modified Organisms (GMO).
  • Genetic modification has:
    (i) Made crops more tolerant to abiotic stresses (cold, drought, salt, heat).
    (ii) Reduced reliance on chemical pesticides (pest-resistant crops).
    (iii) Helped to reduce post-harvest losses.
    (iv) Increased efficiency of mineral usage by plants (this prevents early exhaustion of
    fertility of soil).
    (v) Enhanced nutritional value of food, e.g., Vitamin ‘A’ enriched rice.
    In addition to these uses, GM has been used to create tailor-made plants to supply
    alternative resources to industries, in the form of starches, fuels and pharmaceuticals.
  • By applications of biotechnology in agriculture, pest resistant plants are produced,
    which could decrease the amount of pesticide used.
  • Bt toxin is produced by a bacterium called Bacillus thuringiensis (Bt for short).
  • Bt toxin gene has been cloned from the bacteria and been expressed in plants to
    provide resistance to insects without the need for insecticides; in effect created a
    bio-pesticide. Examples are Bt cotton, Bt corn, rice, tomato, potato and soyabean etc.

Bt Cotton:

  • Some strains of Bacillus thuringiensis produce proteins that kill certain insects such as lepidopterans (tobacco budworm, armyworm), coleopterans (beetles) and dipterans (flies, mosquitoes).
  • B. thuringiensis forms protein crystals during a particular phase of their growth. These crystals contain a toxic insecticidal protein.
  • This toxin does not kill the Bacillus because this protein exists as inactive protoxins but once an insect ingest the inactive toxin, it is converted into an active form of toxin due to the alkaline pH of the gut which solubilise the crystals. The activated toxin binds to the surface of midgut epithelial cells and create pores that cause cell swelling and lysis and eventually cause death of the insect.
  • Specific Bt toxin genes were isolated from Bacillus thuringiensis and incorporated into the several crop plants such as cotton. The choice of genes depends upon the crop and the targeted pest, as most Bt toxins are insect-group specific.
  • The toxin is coded by a gene named cry. There are a number of them, for example, the proteins encoded by the genes crylAc and cryllAb control the cotton bollworms, that of crylAb controls corn borer.

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Pest Resistant Plants:

  • Several nematodes parasitise a wide variety of plants and animals including human beings.
  • A nematode Meloidegyne incognitia infects the roots of tobacco plants and causes a
    great reduction in yield.
  • A novel strategy was adopted to prevent this infestation which was based on the
    process of RNA interference (RNAi).
  • RNAi takes place in all eukaryotic organisms as a method of cellular defense.
  • This method involves silencing of a specific mRNA due to a complementary dsRNA
    molecule that binds to and prevents translation of the mRNA (silencing).
  • The source of this complementary RNA could be from an infection by viruses having
    RNA genomes or mobile genetic elements (transposons) that replicate via an RNA
    intermediate.
  • Using Agrobacterium vectors, nematode-specific genes were introduced into the host
    plant.
  • The introduction of DNA was such that it produced both sense and anti-sense RNA in
    the host cells. These two RNA’s being complementary to each other formed a double
    stranded (dsRNA) that initiated RNAi and thus, silenced the specific mRNA of the
    nematode.
  • The consequence was that the parasite could not survive in a transgenic host
    expressing specific interfering RNA. The transgenic plant therefore got itself protected from the parasite.

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BIOTECHNOLOGICAL APPLICATIONS IN MEDICINE

  • By enabling mass production of safe and more effective therapeutic drugs.
  • Further, the recombinant therapeutics do not induce unwanted immunological
    responses as is common in case of similar products isolated from non-human sources.
  • At present, about 30 recombinant therapeutics have been approved for human-use the world over. In India, 12 of these are presently being marketed.

    Genetically Engineered Insulin

  • Management of adult-onset diabetes is possible by taking insulin at regular time
    intervals.
  • if enough human-insulin was not available, that one would have to isolate and use
    insulin from other animals.
  • Insulin used for diabetes was earlier extracted from pancreas of slaughtered cattle and
    pigs.
  • Insulin from an animal source, though caused some patients to develop allergy or other types of reactions to the foreign protein.
  • Insulin consists of two short polypeptide chains: chain A and chain B, which are linked together by disulphide bridges.
  • In mammals, including humans, insulin is synthesised as a prohormone (like a
    pro-enzyme, the pro-hormone also needs to be processed before it becomes a fully
    mature and functional hormone) which contains an extra stretch called the C peptide.
  • This C peptide is not present in the mature insulin and is removed during maturation
    into insulin.
  • The main challenge for production of insulin using rDNA techniques was getting insulin assembled into a mature form.
  • In 1983, Eli Lilly an American company prepared two DNA sequences corresponding to A and B, chains of human insulin and introduced them in plasmids of E. coli to produce insulin chains. Chains A and B were produced separately, extracted and combined by creating disulfide bonds to form human insulin.

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Gene Therapy

  • Gene therapy is the corrective therapy for hereditary disease.
    Gene therapy is a collection of methods that allows correction of a gene defect that has
    been diagnosed in a child/embryo. Here genes are inserted into a person’s cells and
    tissues to treat a disease.
  • Correction of a genetic defect involves delivery of a normal gene into the individual or
    embryo to take over the function of and compensate for the non-functional gene.
  • The first clinical gene therapy was given in 1990 to a 4-year old girl with adenosine
    deaminase (ADA) deficiency. This enzyme is crucial for the immune system to function.
  • The disorder is caused due to the deletion of the gene for adenosine deaminase.
  • ADA deficiency can be cured by bone marrow transplantation or by enzyme
    replacement therapy, in which functional ADA is given to the patient by injection.
    But the problem with both of these approaches that they are not completely curative.
  • In gene therapy, lymphocytes from the blood of the patient are grown in a culture
    outside the body. A functional ADA cDNA (using a retroviral vector) is then introduced
    into these lymphocytes, which are subsequently returned to the patient. However, as
    these cells are not immortal, the patient requires periodic infusion of such genetically
    engineered lymphocytes. However, if the gene isolate from marrow cells producing
    ADA is introduced into cells at early embryonic stages, it could be a permanent cure.

Molecular Diagnosis

  • For effective treatment of a disease, early diagnosis and understanding its
    pathophysiology is very important but using conventional methods of diagnosis (serum and urine analysis, etc.) early detection is not possible.
  • Recombinant DNA technology, Polymerase Chain Reaction (PCR) and Enzyme Linked
    Immuno-sorbent Assay (ELISA) are some of the techniques that serve the purpose of
    early diagnosis.
  • Presence of a pathogen (bacteria, viruses, etc.) is normally suspected only when the
    pathogen has produced a disease symptom. By this time the concentration of pathogen is already very high in the body. However, very low concentration of a bacteria or virus (at a time when the symptoms of the disease are not yet visible) can be detected by amplification of their nucleic acid by PCR.
  • PCR is now routinely used to detect HIV in suspected AIDS patients. It is being used to
    detect mutations in genes in suspected cancer patients too. It is a powerful techqnique
    to identify many other genetic disorders.
  • PCR –
    A single stranded DNA or RNA, tagged with a radioactive molecule (probe) is allowed to hybridise to its complementary DNA in a clone of cells followed by detection using
    autoradiography. The clone having the mutated gene will hence not appear on the
    photographic film, because the probe will not have complimentarity with the mutated
    gene.
  • ELISA is based on the principle of antigen-antibody interaction. Infection by pathogen can be detected by the presence of antigens (proteins, glycoproteins, etc.) or by detecting the antibodies synthesised against the pathogen.

TRANSGENIC ANIMALS

  • Animals that have had their DNA manipulated to possess and express an extra (foreign) gene are known as transgenic animals.
  • Transgenic rats, rabbits, pigs, sheep, cows and fish have been produced, although over 95 per cent of all existing transgenic animals are mice.
  • common reasons to produce transgenic animals:
    (i) Normal physiology and development:
    Transgenic animals can be specifically designed to allow the study of how genes are
    regulated, and how they affect the normal functions of the body and its
    development, e.g., study of complex factors involved in growth such as insulin-like
    growth factor.
    By introducing genes from other species that alter the formation of this factor and
    studying the biological effects that result, information is obtained about the
    biological role of the factor in the body.
    (ii) Study of disease:
    Many transgenic animals are designed to increase our understanding of how genes
    contribute to the development of disease. These are specially made to serve as
    models for human diseases so that Investigation of new treatments for diseases is
    made possible.
    Today transgenic models exist for many human diseases such as cancer, cystic
    fibrosis, rheumatoid arthritis and Alzheimer’s.
    (iii) Biological products:
    Medicines required to treat certain human diseases can contain biological products,
    but such products are often expensive to make.
    Transgenic animals that produce useful biological products can be created by the
    introduction of the portion of DNA (or genes) which codes for a particular product
    such as human protein (α-1-antitrypsin) used to treat emphysema.
    Similar attempts are being made for treatment of phenylketonuria (PKU) and cystic
    fibrosis.
    In 1997, the first transgenic cow, Rosie, produced human protein-enriched milk (2.4
    grams per litre). The milk contained the human alpha-lactalbumin and was
    nutritionally a more balanced product for human babies than natural cow-milk.
    (iv) Vaccine safety:
    Transgenic mice are being developed for use in testing the safety of vaccines before
    they are used on humans.
    Transgenic mice are being used to test the safety of the polio vaccine. If successful
    and found to be reliable, they could replace the use of monkeys to test the safety of
    batches of the vaccine.
    (v) Chemical safety testing:
    This is known as toxicity/safety testing. The procedure is the same as that used for
    testing toxicity of drugs.
    Transgenic animals are made that carry genes which make them more sensitive to
    toxic substances than non-transgenic animals. They are then exposed to the toxic
    substances and the effects studied. Toxicity testing in such animals will allow us to
    obtain results in less time.

    ETHICAL ISSUES

    The manipulation of living organisms by the human race cannot go on any further, without
    regulation. Some ethical standards are required to evaluate the morality of all human
    activities that might help or harm living organisms.
    Going beyond the morality of such issues, the biological significance of such things is also
    important. Genetic modification of organisms can have unpredicatable results when such
    organisms are introduced into the ecosystem.
    Therefore, the Indian Government has set up organisations such as GEAC (Genetic
    Engineering Approval Committee), which will make decisions regarding the validity of GM
    research and the safety of introducing GM-organisms for public services.

Bio-patent:

  • The modification/usage of living organisms for public services (as food and medicine
    sources, for example) has also created problems with patents granted for the same.
  • There is growing public anger that certain companies are being granted patents for
    products and technologies that make use of the genetic materials, plants and other
    biological resources that have long been identified, developed and used by farmers and
    indigenous people of a specific region/country.
  • Rice is an important food grain, the presence of which goes back thousands of years in
    Asia’s agricultural history. There are an estimated 200,000 varieties of rice in India
    alone. The diversity of rice in India is one of the richest in the world.
  • Basmati rice is distinct for its unique aroma and flavour and 27 documented varieties of Basmati are grown in India. There is reference to Basmati in ancient texts, folklore and poetry, as it has been grown for centuries.
  • In 1997, an American company got patent rights on Basmati rice through the US Patent and Trademark Office. This allowed the company to sell a ‘new’ variety of Basmati, in the US and abroad.
  • This ‘new’ variety of Basmati had actually been derived from Indian farmer’s varieties.
    Indian Basmati was crossed with semi-dwarf varieties and claimed as an invention or a novelty.
  • The patent extends to functional equivalents, implying that other people selling
    Basmati rice could be restricted by the patent.
  • Several attempts have also been made to patent uses, products and processes based
    on Indian traditional herbal medicines, e.g., turmeric neem.
  • If we are not vigilant and we do not immediately counter these patent applications,
    other countries/individuals may encash on our rich legacy and we may not be able to
    do anything about it.

Biopiracy

  • It is the term used to refer to the use of bio-resources by multinational companies and
    other organisations without proper authorisation from the countries and people
    concerned without compensatory payment.
  • Most of the industrialised nations are rich financially but poor in biodiversity and
    traditional knowledge. In contrast the developing and the underdeveloped world is rich in biodiversity and traditional knowledge related to bio-resources. Traditional
    knowledge related to bio-resources can be exploited to develop modern applications
    and can also be used to save time, effort and expenditure during their
    commercialisation.
  • There has been growing realisation of the injustice, inadequate compensation and
    benefit sharing between developed and developing countries. Therefore, some nations
    are developing laws to prevent such unauthorised exploitation of their bio-resources
    and traditional knowledge.
  • The Indian Parliament has recently cleared the second amendment of the Indian
    Patents Bill, that takes such issues into consideration, including patent terms
    emergency provisions and research and development initiative.

 

To download notes in pdf format please click on the following link.

biotechnology and its applications

How to prepare for NEET in last 6 months

How to prepare for NEET in last 6 months

“Winter is coming” but now winter has finally arrived and as the exam days are nearing, anxiety level is increasing. Now preparation is entering into final phase and mind is clouding with fear of unknown. New questions are arising in mind, so in this article I will try to tackle some of these questions –

What should be my strategy for last six months? Should I solve the questions or read the theory?

Regarding strategy every individual has a different way of study, so you should decide your own strategy. But there are certain things you should follow.

  1. Read NCERT at least 4-5 times thoroughly. Remember most of the paper will be from NCERT itself, so if you must complete NCERT 4-5 times and also take note of examples, terms, diagrams and labelling.

Don’t stop if you have done so because, you will find new things in NCERT even if you are reading it carefully 7th/8th/9th time. So read NCERT daily.

  1. Solve papers of AIPMT/NEET (2007-17), AIIMS (2007-17), AIEEE/JEE mains (2011-17 phy and chem) at least twice.
  2. Give attention to each subject equally.
  3. Prepare a notebook in which only write those things, formulas, terms, examples, facts which you are forgetting regularly. Keep revising this notebook. Read this notebook on the day before exam and on the day of exam. It will help you much as it contains all those things which you do not remember.
  4. Stay positive!!! Keep away the negative factors like mobile (Facebook, WhatsApp etc.), TV, internet, movies, party and gossiping friends.
  5. Some friends always talk like how will we get selected, it’s too tough and other negative things. Stay away from these.

Some students ask me about my favoured strategy… so here it is!!!

According to me your syllabus should be completed or nearing completion. If it is not, then try to finish it as soon as possible.

Now here is the strategy to solve papers… you should start solving past year papers regardless of your syllabus condition. Don’t wait for syllabus to be completed. Believe me it’s never gonna happen, as some part of your syllabus will be left here and there.

Now there is a method of solving past papers.

First starts with recent papers (2016-17). Starts with one paper a week. It’s easy and practical to do.

Let’s assume… you solved paper on Sunday. It will take 3-4 hours to solve and 1 hr to check and analyse. Don’t worry about time, take as much time you needed as it’s the first paper and our focus is more on solving more questions for now. Try to attempt as many questions you can. Try to take some calculated risk too. Now check the paper and see how many questions are right/wrong/left.

Suppose you solved only 10 questions of physics and 15 of chemistry and 40 of biology.

Now divide your remaining 6 days of week accordingly. Like

Monday – 20 Qs of physics (around 60% of wrong+left); solve these questions by going through the theory not by seeing the solution. First read the theory of these question and try to solve by yourself. If you can’t then only take help of solution, teacher or friend. Now suppose 10 qs you solved yourself and 10 with the help.

Tuesday – remaining 15 Qs of physics and 10 Qs of Monday which you didn’t solve yourselves. Again same procedure.

Wednesday, Thursday – same schedule for chemistry.

Friday, Saturday – same for biology.

2nd week – start 2nd paper and you will be able to solve more questions than last week.

Gradually you will be able to solve more and more questions. And then you can solve more papers per week.

It will take 2-3 hrs daily. In remaining time, you can read according to your way.

Look, paper solving is must and it should start now as I have mentioned 33 papers and you will solve them atleast twice so total 66 papers. Now around 170 days are remaining in exams and so u have around 2-3 days for each paper only.

How many questions will be from NCERT and what about those which are not from NCERT?

As syllabus for whole paper is syllabus of CBSE, so paper will be based on NCERT. Paper of biology and organic, inorganic chemistry will be mostly directly from NCERT.

But again, why should you believe me? What about your friends who are saying “NCERT to sabhi padte h, selection lana h to kuch alag padho”.

Well, I will say don’t believe on anyone. Solve past papers and find out yourself. You must be done with NCERT so you will be able to find the questions from NCERT.

Now in above mentioned strategy of solving papers, you will find some questions which are not from NCERT. Take note of these and read the theory about these from books or internet. It will help to cover the subject better.

What will be cutoff?

            To have an idea of previous year cutoff is very impotent. NEETcutoff is generally around 75%.

Now to get 75%, you must solve 85-90% questions (150-160 Qs). So you have to take risk in exam.

I prefer studying in night, should I prefer early morning study instead?

Although morning study is better, but I will suggest that you should study at your suitable time, when your efficacy is best.

So, in the end I will suggest you to study in a smartly planned way.

“work hard, work smart”. REVISION is the key. All that you have read is a waste if you don’t revise it. so, make sure you keep revising everything you have read.

All the best for your exams.

for any further information..

please whatsapp me on +918401546940

Dr. Anurag mittal

CHAPTER 11 : BIOTECHNOLOGY: PRINCIPLES AND PROCESSES

Chapter 11

Biotechnology : Principles and Processes

[you can download the notes from the link given at the end of theory]

Biotechnology deals with techniques of using live organisms or enzymes from organisms to produce products and processes useful to humans.

  • Traditional form – based on natural capabilities of microorganisms. making curd, bread or wine, which are all microbe-mediated processes, could also be thought as a form of biotechnology. However, it is used in a restricted sense today,
  • Modern form – it uses genetically modified organisms to achieve the same on a larger scale. Further, many other processes/techniques are also included under biotechnology. For example, in vitro fertilisation leading to a ‘test-tube’ baby, synthesising a gene and using it, developing a DNA vaccine or correcting a defective gene, are all part of biotechnology.
  • The European Federation of Biotechnology (EFB) has given a definition of biotechnology that encompasses both traditional view and modern molecular biotechnology. The definition given by EFB is as follows:

‘The integration of natural science and organisms, cells, parts thereof, and molecular analogues for products and services’.

PRINCIPLES OF BIOTECHNOLOGY

  • Among many, the two core techniques that enabled birth of modern biotechnology are :
    • Genetic engineering: Techniques to alter the chemistry of genetic material (DNA and RNA),to introduce these into host organisms and thus change the phenotype of the host organism.
    • Maintenance of sterile (microbial contamination-free) ambience in chemical engineering processes to enable growth of only the desired microbe/eukaryotic cell in large quantities for the manufacture of biotechnological products like antibiotics, vaccines, enzymes, etc.
  • Sexual reproduction has many advantages over asexual reproduction. The former provides opportunities for variations and formulation of unique combinations of genetic setup, some of which may be beneficial to the organism as well as the population. Asexual reproduction preserves the genetic information, while sexual reproduction permits variation.
  • Traditional hybridisation procedures used in plant and animal breeding, very often lead to inclusion and multiplication of undesirable genes along with the desired genes. The techniques of genetic engineering which include creation of recombinant DNA, use of gene cloning and gene transfer, overcome this limitation and allow us to isolate and introduce only one or a set of desirable genes without introducing undesirable genes into the target organism.
  • A piece of DNA, which is somehow transferred into an alien organism, most likely would not be able to multiply itself in the progeny cells of the organism. But, when it gets integrated into the genome of the recipient, it may multiply and be inherited along with the host DNA. This is because the alien piece of DNA has become part of a chromosome, which has the ability to replicate.
  • In a chromosome there is a specific DNA sequence called the origin of replication, which is responsible for initiating replication. Therefore, for the multiplication of any alien piece of DNA in an organism it needs to be a part of a chromosome(s) which has a specific sequence known as ‘origin of replication’. Thus, an alien DNA is linked with the origin of replication, so that, this alien piece of DNA can replicate and multiply itself in the host organism. This can also be called as cloning or making multiple identical copies of any template DNA.
  • The construction of the first recombinant DNA emerged from the possibility of linking a gene encoding antibiotic resistance with a native plasmid (autonomously replicating circular extra-chromosomal DNA) of  Salmonella typhimurium.
  • Stanley Cohen and Herbert Boyer accomplished this in 1972 by isolating the antibiotic resistance gene by cutting out a piece of DNA from a plasmid which was responsible for conferring antibiotic resistance.
  • The cutting of DNA at specific locations became possible with the discovery of the so-called ‘molecular scissors’- restriction enzymes.
  • The cut piece of DNA was then linked with the plasmid DNA. These plasmid DNA act as vectors to transfer the piece of DNA attached to it. A plasmid can be used as vector to deliver an alien piece of DNA into the host organism.
  • The linking of antibiotic resistance gene with the plasmid vector became possible with the enzyme DNA ligase, which acts on cut DNA molecules and joins their ends. This makes a new combination of circular autonomously replicating DNA created in vitro and is known as recombinant DNA.
  • When this DNA is transferred into Escherichia coli, a bacterium closely related to Salmonella, it could replicate using the new host’s DNA polymerase enzyme and make multiple copies. The ability to multiply copies of antibiotic resistance gene in coli was called cloning of antibiotic resistance gene in E. coli.
  • there are three basic steps in genetically modifying an organism
    • identification of DNA with desirable genes;
    • introduction of the identified DNA into the host;
    • maintenance of introduced DNA in the host and transfer of the DNA to its progeny.

TOOLS OF RECOMBINANT DNA TECHNOLOGY

Key tools of Recombinant DNA technology are – restriction enzymes, polymerase enzymes, ligases, vectors and the host organism.

  1. Restriction Enzymes

  • In 1963, the two enzymes responsible for restricting the growth of bacteriophage in Escherichia coli were isolated. One of these added methyl groups to DNA, while the other cut DNA. The later was called restriction endonuclease.
  • The first restriction endonuclease isolated – Hind II.
  • Restriction endonuclease cut DNA molecules at a particular point by recognising a specific sequence of base pairs. This specific base sequence is known as the recognition sequence.(For Hind II – sequence of 6 base pairs).
  • Today we know more than 900 restriction enzymes that have been isolated from over 230 strains of bacteria each of which recognise different recognition sequences.

Naming of enzymes –

  • First letter of the name comes from the genes
  • The second two letters come from the species of the prokaryotic cell from which they were isolated, e.g., EcoRI comes from Escherichia coli RY 13.
  • Next letter derived from the name of strain.
  • Roman numbers following the names indicate the order in which the enzymes were isolated from that strain of bacteria.

Action of enzyme –

  • Restriction enzymes belong to a larger class of enzymes called nucleases. These are of two kinds; exonucleasesand endonucleases.
  • Exonucleases remove nucleotides from the ends of the DNA whereas, endonucleases make cuts at specific positions within the DNA.
  • Each restriction endonuclease functions by ‘inspecting’ the length of a DNA sequence. Once it finds its specific recognition sequence, it will bind to the DNA and cut each of the two strands of the double helix at specific points in their sugar -phosphate backbones.
  • Each restriction endonuclease recognises a specific palindromic nucleotide sequences in the DNA.
  • The palindrome in DNA is a sequence of base pairs that reads same on the two strands when orientation of reading is kept the same. For example, the following sequences reads the same on the two strands in 5→3 This is also true if read in the 3→5direction.

5—— GAATTC —— 3

3—— CTTAAG —— 5

  • Restriction enzymes cut the strand of DNA a little away from the centre of the palindrome sites, but between the same two bases on the opposite strands. This leaves single stranded portions at the ends. There are overhanging stretches called sticky ends on each strand.
  • These are named so because they form hydrogen bonds with their complementary cut counterparts. This stickiness of the ends facilitates the action of the enzyme DNA ligase.
  • Restriction endonucleases are used in genetic engineering to form ‘recombinant’ molecules of DNA, which are composed of DNA from different sources/genomes.
  • When cut by the same restriction enzyme, the resultant DNA fragments have the same kind of ‘sticky-ends’ and, these can be joined together (end-to-end) using DNA ligases .
  • Normally, unless one cuts the vector and the source DNA with the same restriction enzyme, the recombinant vector molecule cannot be created.

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Fig: Steps in formation of recombinant DNA by action of restriction endonuclease enzyme – EcoRI

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Fig: Diagrammatic representation of recombinant DNA technology

 

Separation and isolation of DNA fragments :

  • The cutting of DNA by restriction endonucleases results in the fragmentes of DNA. These fragments can be separated by a technique known as gel electrophoresis.
  • Since DNA fragments are negatively charged molecules they can be separated by forcing them to move towards the anode under an electric field through a medium/matrix. Nowadays the most commonly used matrix is agarose which is a natural polymer extracted from sea weeds.
  • The DNA fragments separate (resolve) according to their size through sieving effect provided by the agarose gel. Hence, the smaller the fragment size, the farther it moves.
  • The separated DNA fragments can be visualised only after staining the DNA with a compound known as ethidium bromide followed by exposure to UV radiation.
  • We can see bright orange coloured bands of DNA in aethidium bromide stained gel exposed to UV light.
  • The separated bands of DNA are cut out from the agarose gel and extracted from the gel piece. This step is known as elution. The DNA fragments purified in this way are used in constructing recombinant DNA by joining them with cloning vectors.

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Fig: A typical agarose gel electrophoresis showing migration of undigested (lane 1) and digested set of DNA fragments (lane 2 to 4)
  1. Cloning Vectors

  • Plasmids and bacteriophages have the ability to replicate within bacterial cells independent of the control of chromosomal DNA.
  • Bacteriophages because of their high number per cell, have very high copy numbers of their genome within the bacterial cells.
  • If we are able to link an alien piece of DNA with bacteriophage or plasmid DNA, we can multiply its numbers equal to the copy number of the plasmid or bacteriophage.
  • Vectors used at present, are engineered in such way that they help easy linking of foreign DNA and selection of recombinants from non-recombinants.

Features required to facilitate cloning into a vector.

Origin of replication (ori):

  • This is a sequence from where replication starts and any piece of DNA when linked to this sequence can be made to replicate within the host cells.
  • This sequence is also responsible for controlling the copy number of the linked DNA.
  • So, if one wants to recover many copies of the target DNA it should be cloned in a vector whose origin support high copy number.

    Selectable marker :

  • In addition to ‘ori’, the vector requires a selectable marker, which helps in identifying and eliminating nontransformants and selectively permitting the growth of the transformants.
  • Transformation is a procedure through which a piece of DNA is introduced in a host bacterium.
  • Normally, the genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, tetracycline or kanamycin, etc., are considered useful selectable markers for coli. The normal E. coli cells do not carry resistance against any of these antibiotics.

    Cloning sites:

  • In order to link the alien DNA, the vector needs to have very few, preferably single, recognition sites for the commonly used restriction enzymes.
  • Presence of more than one recognition sites within the vector will generate several fragments, which will complicate the gene cloning.
  • The ligation of alien DNA is carried out at a restriction site present in one of the two antibiotic resistance
  • For example, you can ligate a foreign DNA at the Bam H I site of tetracycline resistance gene in the vector pBR322. The recombinant plasmids will lose tetracycline resistance due to insertion of foreign DNA but can still be selected out from non-recombinant ones by plating the transformants on ampicillin containing medium. The transformants growing on ampicillin containing medium are then transferred on a medium containing tetracycline. The recombinants will grow in ampicillin containing medium but not on that containing tetracycline. But, nonrecombinants will grow on the medium containing both the antibiotics. In this case, one antibiotic resistance gene helps in selecting the transformants, whereas the other antibiotic resistance gene gets ‘inactivated due to insertion’ of alien DNA, and helps in selection of recombinants.
  • Selection of recombinants due to inactivation of antibiotics is a cumbersome procedure because it requires simultaneous plating on two plates having different antibiotics. Therefore, alternative selectable markers have been developed which differentiate recombinants from non-recombinants on the basis of their ability to produce colour in the presence of a chromogenic substrate.
  • In this, a recombinant DNA is inserted within the coding sequence of an enzyme, a-galactosidase. This results into inactivation of the enzyme, which is referred to as insertional inactivation. The presence of a chromogenic substrate gives blue coloured colonies if the plasmid in the bacteria does not have an insert. Presence of insert results into insertional inactivation of the a-galactosidase and the colonies do not produce any colour, these are identified as recombinant colonies.

    Vectors for cloning genes in plants and animals :

  • Viruses and bacteria are used to transfer genes into plants and animals which transform eukaryotic cells and force them to do what the bacteria or viruses want.
  • For example, Agrobacterioumtumifaciens, a pathogen of several dicot plants is able to deliver a piece of DNA known as ‘T-DNA’ to transform normal plant cells into a tumor and direct these tumor cells to produce the chemicals required by the pathogen.
  • Similarly, retroviruses in animals have the ability to transform normal cells into cancerous
  • A better understanding of the art of delivering genes by pathogens in their eukaryotic hosts has generated knowledge to transform these tools of pathogens into useful vectors for delivering genes of interest to humans.
  • The tumor inducing (Ti) plasmid of Agrobacterium tumifacienshas now been modified into a cloning vector which is no more pathogenic to the plants but is still able to use the mechanisms to deliver genes of our interest into a variety of plants. Similarly, retroviruses have also been disarmed and are now used to deliver desirable genes into animal cells. So, once a gene or a DNA fragment has been ligated into a suitable vector it is transferred into a bacterial, plant or animal host (where it multiplies).
    1.jpg
Fig: E. coli cloning vector pBR322 showing restriction sites (Hind III, EcoR I, BamH I, Sal I, PvuII, PstI, ClaI), ori and antibiotic resistance genes (ampR and tetR). Rop codes for the proteins involved in the replication of the plasmid.
  1. Competent Host (For Transformation with Recombinant DNA)

  • Since DNA is a hydrophilic molecule, it cannot pass through cell membranes. In order to force bacteria to take up the plasmid, the bacterial cells must first be made ‘competent’ to take up DNA.
  • This is done by treating them with a specific concentration of a divalent cation, such as calcium, which increases the efficiency with which DNA enters the bacterium through pores in its cell wall.
  • Recombinant DNA can then be forced into such cells by incubating the cells with recombinant DNA on ice, followed by placing them briefly at 420oC (heat shock), and then putting them back on ice. This enables the bacteria to take up the recombinant DNA.
  • In micro-injection method, recombinant DNA is directly injected into the nucleus of an animal cell.
  • In another method, suitable for plants, cells are bombarded with high velocity micro-particles of gold or tungsten coated with DNA in a method known as biolisticsor gene gun.
  • And the last method uses ‘disarmed pathogen’ vectors, which when allowed to infect the cell, transfer the recombinant DNA into the host.

PROCESSES OF RECOMBINANT DNA TECHNOLOGY

Recombinant DNA technology involves several steps in specific sequence such as –

  • isolation of DNA,
  • fragmentation of DNA by restriction endonucleases,
  • isolation of a desired DNA fragment,
  • ligation of the DNA fragment into a vector,
  • transferring the recombinant DNA into the host,
  • culturing the host cells in a medium at large scale and
  • extraction of the desired product.
  1. Isolation of the Genetic Material (DNA)

  • Nucleic acid is the genetic material of all organisms without exception. In majority of organisms this is deoxyribonucleic acid or DNA.
  • In order to cut the DNA with restriction enzymes, it needs to be in pure form, free from other macro-molecules. Since the DNA is enclosed within the membranes, we have to break the cell open to release DNA along with other macromolecules such as RNA, proteins, polysaccharides and also lipids. This can be achieved by treating the bacterial cells/plant or animal tissue with enzymes such as lysozyme (bacteria), cellulase(plant cells), chitinase(fungus).
  • genes are located on long molecules of DNA interwined with proteins such as histones.
  • RNA can be removed by treatment with ribonuclease whereas proteins can be removed by treatment with protease. Other molecules can be removed by appropriate treatments and purified DNA ultimately precipitates out after the addition of chilled ethanol. This can be seen as collection of fine threads in the suspension.

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Fig: DNA thatseparates out can beremoved by spooling
  1. Cutting of DNA at Specific Locations

  • Restriction enzyme digestions are performed by incubating purified DNA molecules with the restriction enzyme, at the optimal conditions for that specific enzyme.
  • Agarose gel electrophoresis is employed to check the progression of a restriction enzyme digestion. DNA is a negatively charged molecule, hence it moves towards the positive electrode (anode).
  • The process is repeated with the vector DNA also.
  • The joining of DNA involves several processes. After having cut the source DNA as well as the vector DNA with a specific restriction enzyme, the cut out ‘gene of interest’ from the source DNA and the cut vector with space are mixed and ligase is added. This results in the preparation of recombinant DNA.
  1. Amplification of Gene of Interest using PCR (Polymerase Chain Reaction)

  • In this reaction, multiple copies of the gene (or DNA) of interest is synthesisedin vitro using two sets of primers (small chemically synthesised oligonucleotides that are complementary to the regions of DNA) and the enzyme DNA polymerase.
  • The enzyme extends the primers using the nucleotides provided in the reaction and the genomic DNA as template.
  • If the process of replication of DNA is repeated many times, the segment of DNA can be amplified to approximately billion times.
  • Such repeated amplification is achieved by the use of a thermostable DNA polymerase (isolated from a bacterium, Thermusaquaticus), which remain active during the high temperature induced denaturation of double stranded DNA.
  • The amplified fragment if desired can now be used to ligate with a vector for further cloning.

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Fig: Polymerase chain reaction (PCR) : Each cycle has three steps: (i) Denaturation;

(ii) Primer annealing; and (iii) Extension of primers

  1. Insertion of Recombinant DNA into the Host Cell/Organism

  • There are several methods of introducing the ligated DNA into recipient cells. Recipient cells after making them ‘competent’ to receive, take up DNA present in its surrounding.
  • So, if a recombinant DNA bearing gene for resistance to an antibiotic (e.g., ampicillin) is transferred into coli cells, the host cells become transformed into ampicillin-resistant cells. If we spread the transformed cells on agar plates containing ampicillin, only transformants will grow, untransformed recipient cells will die. Since, due to ampicillin resistance gene, one is able to select a transformed cell in the presence of ampicillin. The ampicillin resistance gene in this case is called a selectable marker.
  1. Obtaining the Foreign Gene Product

  • When you insert a piece of alien DNA into a cloning vector and transfer it into a bacterial, plant or animal cell, the alien DNA gets multiplied.
  • In almost all recominant technologies, the ultimate aim is to produce a desirable protein. Hence, there is a need for the recombinant DNA to be expressed.
  • The foreign gene gets expressed under appropriate conditions. The expression of foreign genes in host cells involve understanding many technical details.
  • After having cloned the gene of interest and having optimised the conditions to induce the expression of the target protein, one has to consider producing it on a large scale.
  • If any protein encoding gene is expressed in a heterologous host, is called a recombinant protein.
  • The cells harbouring cloned genes of interest may be grown on a small scale in the laboratory. The cultures may be used for extracting the desired protein and then purifying it by using different separation techniques.
  • The cells can also be multiplied in a continuous culture system wherein the used medium is drained out from one side while fresh medium is added from the other to maintain the cells in their physiologically most active log/exponential phase. This type of culturing method produces a larger biomass leading to higher yields of desired protein.
  • Small volume cultures cannot yield appreciable quantities of products. To produce in large quantities, the development of bioreactors, where large volumes (100-1000 litres) of culture can be processed, was required. Thus, bioreactors can be thought of as vessels in which raw materials are biologically converted into specific products, individual enzymes, etc., using microbial plant, animal or human cells. A bioreactor provides the optimal conditions for achieving the desired product by providing optimum growth conditions (temperature, pH, substrate, salts, vitamins, oxygen).
  • A stirred-tank reactor is usually cylindrical or with a curved base to facilitate the mixing of the reactor contents. The stirrer facilitates even mixing and oxygen availability throughout the bioreactor. Alternatively air can be bubbled through the reactor.
  • The bioreactor has an agitator system, an oxygen delivery system and a foam control system, a temperature control system, pH control system and sampling ports so that small volumes of the culture can be withdrawn periodically.

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Fig: (a) Simple stirred-tank bioreactor; (b) Sparged stirred-tank bioreactor through whichsterile air bubbles are sparged

 

  1. Downstream Processing
  • After completion of the biosynthetic stage, the product has to be subjected through a series of processes before it is ready for marketing as a finished The processes include separation and purification, which are collectively referred to as downstream processing.
  • The product has to be formulated with suitable preservatives. Such formulation has to undergo thorough clinical trials as in case of drugs. Strict quality control testing for each product is also required. The downstream processing and quality control testing vary from product to product.

To download notes in pdf format please click on the following link.

principles and processes in biotechnology

NEET- 2016 All India Quota Allotment (Round-2)

Neet2016 2nd Counseling results ….
Cutoff rank
MBBS (UR) 5849
(OBC) 5878
(SC) 37095
(ST) 63909
MBBS (UR-PH) 206738
(OBC-PH) 211340
(SC-PH) 375154
(ST-PH) 366952
BDS     (UR) 8413
(OBC) 8448
(SC) 41971
(ST) 68730

to download result file..please click on the following link.

All India Quota Under Graduate Allotment -2016 (Round-2)

NEET-2016 allotment list (after 1st Counseling)

Neet2016 1st Counseling results are out..
Cutoff rank
MBBS (UR) 4733
(OBC) 4781
(SC) 30020
(ST) 53031
MBBS (UR-PH) 160789
(SC-PH) 362248
(ST-PH) 366239
BDS (UR) 7579
(OBC) 7589
(SC) 38204
(ST) 67190
BDS. (UR-PH) 180514
(SC-PH) 347251
(ST-PH) 329022

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Round 1 Allotment