Requirements of a Plant Tissue Culture Lab

1) Lot of good quality water (distilled & double distilled water) which does not contain salts or any

contaminants.

2) Waste water should be properly processed before it is discarded to the environment.

3) Laminar air flow is required to perform aseptic operations.

4) Facilities like A/C, light source, Humidity regulator should be available for optimal growth of

plant tissues /plants

5) Shelves or racks of suitable sizes have to be installed to store the tissue cultures, and fans should

be installed near the racks to remove heat produced during the metabolic activities of growing

cells.

6) Humidity in the air should be maintained at 30% to ensure that the culture tubes neither do not

have excess humidity which may cause contamination problems nor low humidity which may

dry up the culture medium.

7) Sterilization facility for the equipments, glassware, media, explants, work space etc. should be

available.

8) Glassware of all sizes is required for storage, sterilization, culture of the explants.

9) Media formulations / chemicals required for the culture of all kinds of plants/plant materials

should be readily available.

Plant Physiology

Plant physiology deals with the studies of life processes that are similar in many organisms. Thousands

of chemical reactions are underway in every living cell transferring water, mineral salts and gases from

the environment into organized plant tissue. Plants are also endowed with the property of

photosynthesizing, a unique feature that is inevitable for life on earth. Plant function can ultimately be

understood on the basis of the principles of physics and chemistry. Studies in plant physiology depend

strongly upon plant anatomy, cell biology and structural & functional chemistry. At the same time, the

structural sciences of plant anatomy and cell biology become more meaningful because of plant

physiology. It deals with the various processes seen in plants throughout its life from the moment of

conception when it begins as a zygote until death.

Mechanism of Photosynthesis

It is the only mechanism of energy input in the living world. Like energy-yielding oxidation reactions

upon which all life depends, photosynthesis involves oxidation and reduction. The overall process is an

oxidation of water (removal of electrons with the release of oxygen as a by-product) and a reduction of

CO2 to form organic compounds such as carbohydrates.

Photosynthesis is a two component reaction system: Photosynthesis consists of two types of reactions: a light dependent one and a light independent one.

The light dependent reaction is a photochemical reaction, culminating in the generation of NADPH + H+,

ATP are energy rich and form the assimilatory power, the source of energy utilized for CO2 fixation can

go apace irrespective of whether light is present or absent &therefore are collectively designated, dark

reactions. But the driving force for the events of dark reaction is provided by the products of light

reaction. It was Blackman, who first recognized that photosynthesis is a two component reaction

system. Here are some evidences, experimental and otherwise, that confirms this.

Photosynthetic rate in continuous and intermittent light: Warburg (1919) found that Chlorella registered a higher rate of photosynthesis, when exposed to

intermittent light than when exposed to continuous light. The explanation for this observation is that

during the short intervals of dark period, the products of light reaction could be utilized. But, when the

algal cells were exposed to continuous light, the products of the light reaction accumulated and not

enough of them could be utilized since, no dark reaction followed. This accounts for the shortfall in the

products of photosynthesis

1) Employment of inhibitors: Warburg (1920) showed that cyanide added to a

photosynthetic system reduced the rate of photosynthesis. This depression in the rat of photosynthesis

was much greater under intense light than in weak light, indicating that it is the dark reaction which is

affected.

2) Temperature coefficient: The temperature coefficient of any chemical reaction is the

ratio between its rates at two temperatures, 10o C apart from one another (Q10). Almost all chemical

reactions have a Q10 value of two, i.e., the rate of chemical reaction doubles up, if the temperature is

raised by 10oC. Within a certain range of temperature, this is true of photosynthesis also. This means

that part of the photosynthesis reactions are regulated wholly by temperature, unaffected by light.

3) Tracer Technique: Green plants kept in light for some time and shifted to a dark

chamber thereafter, continued to produce the end product of photosynthesis. Radioactive isotopes

supplied under such conditions always showed out in an array of products, wholly considered

photosynthesis. These experiments provide direct proof of theory that photosynthesis consists of a light

reaction and dark reaction.

4) Work of Arnon’s group: This group of workers successfully separated the lamellar and

stroma parts of chloroplasts. The lamellar fragments, on exposure to light produced NADPH2, ATP and

molecular oxygen, the last of which escaped from the system. The assimilatory power, consisting of

NADPH2 and ATP, when fed to the stroma fragment of the chloroplast yielded products of

photosynthesis is made up of two sets of reactions, one light dependent and another light-independent,

but they also identified the physical locations where those reactions occur.

Light Harvesting Complexes

Sun is the source of an incessant flow of energy which forms a range of electromagnetic spectrum, of a

portion of which is perceived by us as a light. A small portion of this light between the wavelength 39-

700 nm is absorbed by the chlorophylls and other photosynthetic pigments and becomes available for

photosynthesis, as evidenced by their absorption spectra.

Light may be thought as made up of waves of particles, called photons. Wavelength and energy are

inversely proportional; light of shorter wavelength carries more energy and of longer wavelength carries

less energy. Usually in any calculation regarding energy carried by light, it is done for an Einstein, an

Einstein being 6.02 X 1023 photons and the energy which carries has been termed as a quantum.

When light is absorbed by chlorophyll, it gets excited which means that an electron is shifted to an outer

orbital, making the chlorophyll molecule charged. The electron that moved to higher orbital tends to

regain its original level to bring the chlorophyll molecule to its ground state. As it does so, energy in the

form of heat is produced and the chlorophyll fluoresces. Since some energy was lost as heat, the

fluorescing chlorophyll molecule emits light lesser energy and so of longer wavelength.

The frequency of light of a given wavelength is defined as the number of waves that hits the surface

each second. At the speed of light travels, the frequency of any light wave is bound to be very high. If

the light of too short wavelength and therefore of very high frequency and high energy hits the surface,

the dislodgement of the electron is bound to be permanent and the damage is irreparable. If on other

hand, as red and blue light waves hit chlorophyll , they are absorbed (as evidenced by the absorption

spectrum of chlorophyll) and energy so become available causes an electron to move into an outer

(higher) orbital, without any danger of its being lost altogether. As the dislodged electron is getting back

taking the chlorophyll molecule to its ground state, energy is released and this is put to use in

photosynthesis. Incidentally in the latter event the chlorophyll‘s electron-hunger is satisfied from

another source, allowing it to get back to its ground state. When we say that energy released in the

process of dislodgement of electron from chlorophyll and return of the chlorophyll to its ground state is

used in photosynthesis, we mean that the light reactions of photosynthesis are on.

We know that photosynthesis in its essence, consists in the reduction of CO2 to CH2O and the source of

hydrogen is water. Otto Warburg, wanted to determine at the figure of 4 quanta per molecule of oxygen

released, which means that 30 kcals of energy per Einstein is spent. This means that photosynthesis has

about 75% efficiency, for the quantum energy carried by an Einstein of red light is about 40 kcals.

Emerson and his co-workers did not get the same results when they repeated the experiments of

Warburg. They found that an average, 10 quanta are required for every molecule of CO2 reduced or O2

evolved. This value certainly does not lend to photosynthesis the same degree of energy efficiency of

Warburg’s value suggests. But this is nearer the calculated value of 8, since each O2 released or CO2

reduced required for 4electron transfers, each of which requires 2 quanta of energy.

We may arrive at the same conclusion from other angle. For the reduction of CO2 to CH2O about 118

Kcals, are required. One Einstein of red light of 660 nm, calculation shows, carries 43 kcals of energy. It

means that at least three photons are required for the purpose. Even if the blue light of 420 nm carrying

70 kcals is considered, it will be still low for one single photon to effect the reduction.

Furthermore calculations showed that under the most intensive light conditions when the highest rate

of photosynthesis was recorded the alga would absorb enough number of photons before the first

molecule of oxygen was released, but in reality, the evolution of oxygen begins almost immediately

upon illumination. Against this background, the idea of a photosynthetic unit (PSU) has been suggested.

It is not one molecule of chlorophyll, in its individual entity that subserves the purposes of

photosynthesis but rather a collection of them.

The experiments of Emerson and Arnold (1932) on Chlorella led them to think that about 2500

molecules of chlorophyll constituted a PSU. They envisaged special reaction sites among the 2500

chlorophylls to which the light harvested was transferred, allowing a photoact, i.e. absorption and

transfer of a light quantum to a trapping centre where it promotes release of an electron.

Some experimental evidence was obtained towards the confirmation of the existence of PSU. Broken

chloroplasts with more than a thousand and more chlorophylls alone were able to show Hill reaction

activity. Photosynthesis inhibitors such as DCMU (Dichloromethyl urea) were effective only when

applied in concentrations of one molecule or more per 2000 chlorophylls, in lesser concentrations, they

were ineffective.

Later studies showed that a PSU needs to consist of 250 chlorophyll molecules, a figure obtained by

dividing the number of 2500 chlorophylls by quantum requirement, i.e., 10. This appears to be more

realistic as the size of the 2500 chlorophylls would be too unwieldy for effective physiological activity.

Other components of light reaction like cytochrome-f, ferredoxin and P-700 occur in the ration of 1

molecule each for 300 chlorophylls. All this points to the possibility of a PSU, made up of 250-300

chlorophylls and their accessory pigments and electron carriers.

The occurrences of a PSU as a distinct morphological entity were obtained by Park and his co-workers

and they named it quantasome. Now, it is believed that the quantasome and PSU are respectively

biophysical and biochemical aspects of the same entity.

Herbicide Resistance in Plants

A weed in a general sense a plant that is considered as unwanted plants in farm fields, gardens, parks

etc., and generally the term is often used to describe the plants that grow & reproduce aggressively.

They are one of the major problems in crop management as they compete with crops for water and

nutrients and as a result decrease farming yields and crop production.

Until the emergence of genetically modified crops, selective herbicides ie., herbicides that only kill a

specific weed and these herbicides come into contact with a planted crop of different species without

harming them. The major drawback in using selective herbicides is each wed requires a different

herbicide, which in large volumes is very costly.

One of the most common herbicide is Glyphosate or ((N-(Phosphonomethyl) Glycine) is a broad

spectrum herbicide used to kill weeds. Glyphosate kills plants by interfering with the synthesis of amino

acids, Phenyl alanine, tyrosine, and tryptophan. It does this by inhibiting the enzyme 5-enol pyruvyl

shikimate-3-phosphate synthatase (EPSPS), which catalyzes the reaction of shikimate-3-phosphate &

phosphoenol Pyruvate to form 5-enol pyruvyl-shikimate-3-phosphate (ESP). ESP is subsequently

dephosphorylated to chorismate an essential precursor in plants for the aromatic acids, i.e.,

phenylalanine, tyrosine, and tryptophan. These amino acids are used as building blocks in peptides and

to produce secondary metabolites such as foliates, ubiquinones and napthoquinones.

Glyphosate has several dissociable hydrogen atoms and it tends to exist as zwitterion where phosphonic

hydrogen dissociates ad joins the amine group and is soluble in water to 12g/l at room temperature.

Some micro organisms have a different version of 5-enolpyruvyl-shikimate-3-phosphate synthatase that

is resistant to Glyphosate inhibition. The version mostly used in genetically modified groups is isolated

from Agrobacterium strain CP4. The CP4 EPSPS gene is engineered for plant expression by fusing the 5’

end of the gene to a chloroplast transit peptide from Petunia EPSPS gene. Transit peptides have an N-

terminal presequence which directs them to an organelle such as chloroplast, mitochondrion etc., The

transit peptide is required for their transport across the relevant membranes from their transport across

relevant membranes from their site of synthesis

This transit peptide is used because it has the ability to deliver bacterial EPSPS to the chloroplasts

because chloroplasts synthesize the amino acids Phenyl alanine, Tyrosine & Tryptophan. The naturally

occurring EPSPS enzyme from Agrobacterium CP4 can be identified from a screen of micro organism cell

extract having very high Glyphosate tolerance kinetic parameters.

After thepre-CP4 EPSPS protein (i.e., the one which contains chloroplast transit peptide amino terminal

extension) reaches the chloroplast, and then the transit peptide is cleaved and degraded in the

cytoplasm like any other nuclear encoded chloroplast targeted protein. This leaves mature CP4 EPSPS

with no chloroplast transit peptide attached.

Internal structure of Chloroplast

Electron microscopy shows the chloroplast to consist of an envelope enclosing a complex of

membranes, the thylakoid system often joined or stacked into grana; the lipid membranes, contrast with

the background when stained with lipophilic electron dense osmium. The space between the envelope

and thylakoid membranes is the chloroplast stroma. The envelope is composed of two membranes each

about 5.6 nm thick separated by the intra envelope space (10 nm) with areas of high electron density

which are possibly contact points between the membranes; they may be involved in transport, i.e., of

proteins between cytosol and stroma. The membranes are lipid bilayers, of galactosyl glycerides and

phosphatidyl choline, containing carotenoids but no chlorophyll.

The stroma contains indistinct granules and particles, mainly of proteins; the enzyme ribulose

bisphosphate carboxylase (Rubisco) is the major soluble protein and may crystallize in unfavorable

conditions such as water stress or air pollution. Other inclusions are products of the photosynthetic

processes; for example, starch granules upto 2µm long accumulate in stroma and disturb the thylakoid

membranes, and globules of lipids and plasto quinine accumulate; RNAs and DNA occur in chloroplasts

which synthesize many of their constituent proteins.

The most noticeable feature of chloroplasts in electron micrographs is the thylakoid. Thylakoid

membranes frequently associate into granal stacks, interconnected by pairs of membranes, called

stromal thylakoids (or alternatively intergranal connections or frets), which are in connect with the

stroma on both the sides. The interface between the appressed membranes is the partition region. In C3

plants over 60 percent of the thylakoid surface is typically in the grana. The end membranes of stacked

thylakoids and the ends of the grana, but not the partition regions, have direct contact with the stroma.

Thylakoid membranes vesicles in the grana are stacked and flattened, but not closed, sacs inter

connected with the other membranes. The vesicles join the stromal lamellae at different points around

the periphery of the granum. The structure derives from the folding and joining of separate sheets of

lamellae which are interconnected and probably originate from a single point, the prolamella body, in

the developing chloroplast. The thylakoid system appears to be a single interconnecting giant closed

vesicle with continuous lumen, a feature of great importance in electron transport and ATP generation.

Composition of the lumen is not known; but proteins, of the water-splitting complex and the light-

harvesting complex for example, may occupy part of the volume and it is unlikely to be homogeneous

aqueous solution of small molecules. Grana differ in extent and size between species, and with

conditions during growth, for example with bright illumination, there is less granal stacking. Grana in

isolated thylakoids stack and unstuck, according to the ionic concentration and light quality.

On the outer surface of stromal and of granal thylakoids in contact with the stroma, are particles of

Rubisco loosely attached and easily removed.

Thylakoid membranes are ‘sided’ in construction, with the water-splitting complexes in the lumen, a PS

II chlorophyll-protein complex, a cytochrome b-f complex and light harvesting complex spanning the

membrane interspersed with the PSI chlorophyll-protein complex on the outer side, and finally enzymes

of carbon metabolism and ATP synthesis on the outer surface. This sidedness allows thylakoids to

transport electrons to the stroma from water in the lumen and accumulate protons in the lumen.

Chlorophyll- a & b occur only in thylakoid membranes and may form 5 percent of their total mass.

Chlorophyll is complexed with, but not covalently bonded to proteins; the hydrophobic phytyl groups of

chlorophyll may be between the membrane proteins and lipids, and the hydrophilic parts of the

porphyrin ring in the protein. This would orientate pigment molecules for efficient energy capture.&

transfer.

Although there is uncertainty about the chlorophyll-protein complexes in the membrane and their

correspondence to their membrane particles, three main complexes contain 90 percent of the

chlorophyll. One corresponds to PSI and its antenna chlorophyll-a; it is called P700 chlorophyll a complex

or chlorophyll-protein complex I, CPI for short. A second is light-harvesting chlorophyll a/b-protein

complex, now called light harvesting complex, LHC, which has only antenna function and no

photochemical activity. The third is less well resolved but contains PSII and its antenna chlorophyll-a. It is

called CPa and is the chlorophyll-protein complex serving as the internal chlorophyll-a antenna of PSII

Electron Transport in Photosynthesis

Photosynthesis of prokaryotic cyanobacteria as well as that of eukaryotic algae and higher plants

produces oxygen and the basic process is similar in them all.

A form of the Hill and Bendall ‘Z’ scheme of the sequence of processes and electron transport leading

from water-splitting through NADP+ reduction is explained below.

Photon capture by the photosystem antennae and excitation transfer to PSII & I provide the energy for

oxidation of water and electron movement to acceptors, which donate e- to biochemical processes, and

for passage of protons into thylakoid lumen, for synthesis of ATP. The electron transport system may be

considered in five parts:

a) A water-splitting complex

b) A photosystem complex

c) An electron carrier chain

d) A PS I complex

e) A group of e- carriers which reduce electron acceptors (NADP+, O2)

Electron transport starts with the capture of photons by chlorophylls and accessory pigments. Transfer

of the energy to reaction centers of PS I & II excites the dimer chlorophylls and causes ejection of

electrons to acceptors, starting e- transport along the chain of redox components. Excitation of P-680 of

PS II results in an oxidized reaction centre P-680* PSII is defined as that part of oxygenic photosynthesis

catalyzing photo induced transfer of e- from water to plastoqiunone (PQ) Passage of electrons along the

electron transport chain moves the protons from the stroma into the thylakoid space and thus creates a

proton gradient. This is used to drive the synthesis of ATP. Plastoquinone, plastocyanin and ferredoxin

are mobile and can transport electrons between the complexes.

With the transfer of H+ from the stromal to lumen side of the thylakoid membrane. This oxidized PS II is

reduced by e- from a water-splitting complex via intermediate states M & Z which are components of

the water splitting complex and electron carrier system between it and the reaction centre. The

energized e- passes, from more to less negative potential, to the primary acceptor pheophytin and then

sequence to the quinine acceptors Qa, Qb, and PQ. Quinones are important carriers of e- and H+ in many

biological processes. From PQ the electron passes to cytochrome f and plastocyanin before reducing an

oxidized PS I reaction centre. Here it is energized again by excitation energy derived from photon energy

trapped in the chlorophyll matrix, and passed via intermediate states AD, A and B to oxidized ferredoxin

(Fd) and NADPH+, which are reduced and are able to enter into biochemical reactions in chloroplast

stroma.

Electron transport chains bridge the thylakoid membranes, allowing electron removed from water held

in the water-splitting complex of proteins, manganese ions and other components inside thylakoid

lumen to pass across the membrane to ferredoxin on the stromal side. Plastoqiunone in the membrane

is reduced by the electrons; the H+ from the stroma attaches to the reduced plastoqiunone and is

carried to the lumen, where it is released oxidized. Thus the electron transport is coupled to plasto

quinine cycle which carries which carries (‘pumps’) H+ from stroma to the thylakoid lumen in reverse

direction to electron transport, increasing H+ concentration in the thylakoid lumen and forming the

protein concentration gradient, the energy of which derives ATP synthesis.