Lesson 1

TISSUE CULTURE

Tissue culture is the science propagation of plants in an in vitro environment to get seedling that are:

Free from diseases.
Have increased genetic viability.
Need for rapid clonal propagation.

This started many years ago but production in masses can be traced around 100 years ago. This was first done on non flowering plants that were needed in large numbers. This was influenced also by the number of diseases that affect fruit crops especially bananas.

Objectives

By the end of this lesson the learner should be able to

Describe the steps involved in Tissue culture.
Indentify chemicals and thier correct concetration used in tissue culture.
Describe how to get explant that are pests and disease free

Preparatory stage

Suckers used in micro propagation are acquired from healthy true to type mother plant with minimum or no damage at all. You should know the genotype in order to give the farmer the correct crop that will cope with the climatic conditions that it will be later introduced to. Its also worthy noting the correct use of the banana i.e. whether you need plantain or exclusively ripening type.

Lesson 2

Stages in tissues preparation

Stage O Acquisation of seedlings from the field and planting them in the shadenet
Stage 1 Acquisation of explants from shade net and cleaning them.
Stage 2 Surface sterlization.
Stage 3 Hood sterlization and inoculation.

*Stage 0

This involves acquiring the selected genotype and planting it in a controlled environment where any possible disease or pest will be controlled. This is preferably done under the shade net. The ex plants are treated against any pathogens that might have attacked itwhile it was in the field. In the growth shed the plants are sprayed regularly with appropriate pesticides to control pests and diseases.

*Stage 1

This is the stage of getting the meristem from the growth shed. It should bedone when the plants are actively growing because this is the time the cells are dividing more rapidly. For this case we shall deal with banana (musa spp). Musa spp plant is bulky and grows completely under ground. Only the pseudo stems emerge from the soil. Since the desired explants are the apical bud of the pseudo stem or a strong sucker the pseudo stem can only give the meristem before flowering, though a young inflorescence can also be used, most researchers recommend the use of young sucker. The sucker should be dug with great care by uprooted at a minimum or no damage. After the successful removal of the sucker it is the rinsed water to remove excess soil then roughly with plenty of water with Sodium hypochlorite (NaOCl). A drop of teepol is added to help in breaking surface tension of water. The explants are subsequently trimmed by removing the excess corm tissues and successive leaf sheaths until the diameter of the base is approximately 7-10 centimeters.

* Stage 2

The surface sterilization is done to kill all microorganisms and systemic pathogen that might be in the tissues. Several compounds are used at different concentrations and varying duration of time. In the preparation room of tissue culture laboratory the meristems are introduced into a solution of citric acid at a rate of 7g/l for a period of 30 minutes. Sodium hypochlorite in form of bleach detergent commercially called Jik and a drop of teepol introduced at a rate of 0.2 to 0.4% active ingredients for a period of 12 minutes. It’s important to note that exceeding time limit damages the delicate cells.

*Stage 3

After a period of 12 minutes the explant are move to the transfer room and NaOCl is poured out and pour in 60% alcohol for 20 minutes. The explants are then rinsed with sterile, distilled water for about 2 to 3 times and allow them to stay in water as trim the size to about 5 to7 cm. Briefly immerse the explants into a solution of cystein hydrochloride (50 mg/l).This should last for less than 3 minutes then you immerse in sterile distilled water. Reduce the size once more to 4cm, while removing the browning spots due to oxidation of phenols and polyphenols compounds. Immerse in a solution of ascorbic acid 15g/l as you transfer them into the culture bottle with the culture media. N.B. Note any time you handle the explants it should be under the laminar flow with forceps that should always be flame sterilized.

Assignment

Discuss chemical compounds used to sterilize explants in tissue culture.
Suggest how to make a solution of 60% ethanol.
Briefly discuss the importance of growing crops in the growth shed.
Visit any tissue culture facility around your area and familiarize with the equipments used and write a report.'

Glossary

Sterile distilled water- water with no dissolved chemicals and autoclaved.
Laminar flow- Inoculating area that gives filtered air with no microorganisms.
In vitro- In a controlled environment.
Inflorescence– Flowering part of a banana.

summary

plants have different ways of sterlizing and the chemical concetration varies from plant to another.for instance,while we use 60% ethanol for 20 minutes on bananas, cassava requires 40% for 5 minutes. Therefore you will be required to work out the protocal to use for different crops.To prepare the solutions e.g. 60% ethanol you are required to pick 60 ml absolute ethanol and top to 100 ml with distiled water.The same is done for all other solutions that are expressed in percentage and remember you must always use distilled water to prepare these solutions.The next lesson will with growth room and aclamatization of young seedlings.

Ref.tissue culture for imropving yeilds.CAIT 1989.

lesson 3

PHOTOSYNTHESIS

Study the general equation for photosynthesis and be able to indicate in which process each reactant is used and each product is produced.
List the two major processes of photosynthesis and state what occurs in those sets of reactions.
Distinguish between organisms known as autotrophs and those known as heterotrophs as pertains to their modes of nutrition.
Explain the significance of the ATP/ADP cycle.
Describe the nature of light and how it is associated with the release of electrons from a photosystem.
Describe how the pigments found on thylakoid membranes are organized into photosystems and how they relate to photon light energy.
Describe the role that chlorophylls and the other pigments found in chloroplasts play to initiate the light-dependent reactions.
Describe the function of electron transport systems in the thylakoid membrane.
Explain the role of the two energy-carrying molecules produced in the light-dependent reactions (ATP and NADPH) in the light-independent reactions.
Describe the Calvin-Benson cycle in terms of its reactants and products.
Explain how C-4 photosynthesis provides an advantage for plants in certain environments.
Describe the phenomenon of acid rain, and how photosynthesis relates to acid rain and the carbon cycle.

What is Photosynthesis?

Photosynthesis is the process by which plants, some bacteria, and some protistans use the energy from sunlight to produce sugar, which cellular respiration converts into ATP, the "fuel" used by all living things. The conversion of unusable sunlight energy into usable chemical energy, is associated with the actions of the green pigment chlorophyll. Most of the time, the photosynthetic process uses water and releases the oxygen. overall reactio equation: 6H2O + 6CO2 ----------> C6H12O6+ 6O2 Chemical equation translation: Six molecules of water plus six molecules of carbon dioxide produce one molecule of sugar plus six molecules of oxygen.Plants are the only photosynthetic organisms to have leaves (and not all plants have leaves). A leaf may be viewed as a solar collector crammed full of photosynthetic cells.The raw materials of photosynthesis, water and carbon dioxide, enter the cells of the leaf, and the products of photosynthesis, sugar and oxygen, leave the leaf.Water enters the root and is transported up to the leaves through specialized plant cells known as xylem (pronounces zigh-lem). Land plants must guard against drying out (desiccation) and so have evolved specialized structures known as stomata to allow gas to enter and leave the leaf. Carbon dioxide cannot pass through the protective waxy layer covering the leaf (cuticle), but it can enter the leaf through an opening (the stoma; plural = stomata; Greek for hole) flanked by two guard cells. Likewise, oxygen produced during photosynthesis can only pass out of the leaf through the opened stomata. Unfortunately for the plant, while these gases are moving between the inside and outside of the leaf, a great deal water is also lost. Cottonwood trees, for example, will lose 100 gallons of water per hour during hot desert days. Carbon dioxide enters single-celled and aquatic autotrophs through no specialized structures.

Nature of light

White light is separated into the different colors (wavelengths) of light by passing it through a prism. Wavelength is defined as the distance from peak to peak (or trough to trough). The energy of is inversely porportional to the wavelength: longer wavelengths have less energy than do shorter ones.The order of colors is determined by the wavelength of light. Visible light is one small part of the electromagnetic spectrum. The longer the wavelength of visible light, the more red the color. Likewise the shorter wavelengths are towards the violet side of the spectrum. Wavelengths longer than red are referred to as infrared, while those shorter than violet are ultraviolet. Light behaves both as a wave and a particle. Wave properties of light include the bending of the wave path when passing from one material (medium) into another (i.e. the prism, rainbows, pencil in a glass-of-water, etc.). The particle properties are demonstrated by the photoelectric effect. Zinc exposed to ultraviolet light becomes positively charged because light energy forces electrons from the zinc. These electrons can create an electrical current. Sodium, potassium and selenium have critical wavelengths in the visible light range. The critical wavelength is the maximum wavelength of light (visible or invisible) that creates a photoelectric effect.

Chlorophyll pigment

A pigment is any substance that absorbs light. The color of the pigment comes from the wavelengths of light reflected (in other words, those not absorbed). Chlorophyll, the green pigment common to all photosynthetic cells, absorbs all wavelengths of visible light except green, which it reflects to be detected by our eyes. Black pigments absorb all of the wavelengths that strike them. White pigments/lighter colors reflect all or almost all of the energy striking them. Pigments have their own characteristic absorption spectra, the absorption pattern of a given pigment.

Chlorophyll is a complex molecule. Several modifications of chlorophyll occur among plants and other photosynthetic organisms. All photosynthetic organisms (plants, certain protistans, prochlorobacteria, and cyanobacteria) have chlorophyll a. Accessory pigments absorb energy that chlorophyll a does not absorb. Accessory pigments include chlorophyll b (also c, d, and e in algae and protistans), xanthophylls, and carotenoids (such as beta-carotene). Chlorophyll a absorbs its energy from the Violet-Blue and Reddish orange-Red wavelengths, and little from the intermediate (Green-Yellow-Orange) wavelengths.

Structure of chlorophyll

Carotenoids and chlorophyll absorb some of the energy in the green wavelength. Why not so much in the orange and yellow wavelengths? Both chlorophylls also absorb in the orange-red end of the spectrum (with longer wavelengths and lower energy). The origins of photosynthetic organisms in the sea may account for this. Shorter wavelengths (with more energy) do not penetrate much below 5 meters deep in sea water. The ability to absorb some energy from the longer (hence more penetrating) wavelengths might have been an advantage to early photosynthetic algae that were not able to be in the upper (photic) zone of the sea all the time.

The action spectrum of photosynthesis is the relative effectiveness of different wavelengths of light at generating electrons. If a pigment absorbs light energy, one of three things will occur. Energy is dissipated as heat. The energy may be emitted immediately as a longer wavelength, a phenomenon known as fluorescence. Energy may trigger a chemical reaction, as in photosynthesis. Chlorophyll only triggers a chemical reaction when it is associated with proteins embedded in a membrane (as in a chloroplast) or the membrane infoldings found in photosynthetic prokaryotes such as cyanobacteria and prochlorobacteria.

The thylakoid is the structural unit of photosynthesis. Both photosynthetic prokaryotes and eukaryotes have these flattened sacs/vesicles containing photosynthetic chemicals. Only eukaryotes have chloroplasts with a surrounding membrane.Thylakoids are stacked like pancakes in stacks known collectively as grana. The areas between grana are referred to as stroma. While the mitochondrion has two membrane systems, the chloroplast has three, forming three compartments.

Stages of photosythesis

Photosynthesis is a two stage process. The first process is the Light Dependent Process (Light Reactions), requires the direct energy of light to make energy carrier molecules that are used in the second process. The Light Independent Process (or Dark Reactions) occurs when the products of the Light Reaction are used to form C-C covalent bonds of carbohydrates. The Dark Reactions can usually occur in the dark, if the energy carriers from the light process are present. Recent evidence suggests that a major enzyme of the Dark Reaction is indirectly stimulated by light, thus the term Dark Reaction is somewhat of a misnomer. The Light Reactions occur in the grana and the Dark Reactions take place in the stroma of the chloroplasts.

Nature of light and dark reaction

In the Light Dependent Processes (Light Reactions) light strikes chlorophyll a in such a way as to excite electrons to a higher energy state. In a series of reactions the energy is converted (along an electron transport process) into ATP and NADPH. Water is split in the process, releasing oxygen as a by-product of the reaction. The ATP and NADPH are used to make C-C bonds in the Light Independent Process (Dark Reactions).

In the Light Independent Process, carbon dioxide from the atmosphere (or water for aquatic/marine organisms) is captured and modified by the addition of Hydrogen to form carbohydrates (general formula of carbohydrates is [CH2O]n). The incorporation of carbon dioxide into organic compounds is known as carbon fixation. The energy for this comes from the first phase of the photosynthetic process. Living systems cannot directly utilize light energy, but can, through a complicated series of reactions, convert it into C-C bond energy that can be released by glycolysis and other metabolic processes.

Halobacteria, which grow in extremely salty water, are facultative aerobes, they can grow when oxygen is absent. Purple pigments, known as retinal (a pigment also found in the human eye) act similar to chlorophyll. The complex of retinal and membrane proteins is known as bacteriorhodopsin, which generates electrons which establish a proton gradient that powers an ADP-ATP pump, generating ATP from sunlight without chlorophyll. This supports the theory that chemiosmotic processes are universal in their ability to generate ATP.

Carbon fixation

Carbon-Fixing Reactions are also known as the Dark Reactions (or Light Independent Reactions). Carbon dioxide enters single-celled and aquatic autotrophs through no specialized structures, diffusing into the cells. Land plants must guard against drying out (desiccation) and so have evolved specialized structures known as stomata to allow gas to enter and leave the leaf. The Calvin Cycle occurs in the stroma of chloroplasts (where would it occur in a prokaryote?). Carbon dioxide is captured by the chemical ribulose biphosphate (RuBP). RuBP is a 5-C chemical. Six molecules of carbon dioxide enter the Calvin Cycle, eventually producing one molecule of glucose.

Some plants have developed a preliminary step to the Calvin Cycle (which is also referred to as a C-3 pathway), this preamble step is known as C-4. While most C-fixation begins with RuBP, C-4 begins with a new molecule, phosphoenolpyruvate (PEP), a 3-C chemical that is converted into oxaloacetic acid (OAA, a 4-C chemical) when carbon dioxide is combined with PEP. The OAA is converted to Malic Acid and then transported from the mesophyll cell into the bundle-sheath cell, where OAA is broken down into PEP plus carbon dioxide. The carbon dioxide then enters the Calvin Cycle, with PEP returning to the mesophyll cell. The resulting sugars are now adjacent to the leaf veins and can readily be transported throughout the plant.

C-4 photosynthsis involves the separation of carbon fixation and carbohydrate systhesis in space and time. Image from Purves et al., Life: The Science of Biology, 4th Edition, by Sinauer Associates. The capture of carbon dioxide by PEP is mediated by the enzyme PEP carboxylase, which has a stronger affinity for carbon dioxide than does RuBP carboxylase When carbon dioxide levels decline below the threshold for RuBP carboxylase, RuBP is catalyzed with oxygen instead of carbon dioxide. The product of that reaction forms glycolic acid, a chemical that can be broken down by photorespiration, producing neither NADH nor ATP, in effect dismantling the Calvin Cycle. C-4 plants, which often grow close together, have had to adjust to decreased levels of carbon dioxide by artificially raising the carbon dioxide concentration in certain cells to prevent photorespiration. C-4 plants evolved in the tropics and are adapted to higher temperatures than are the C-3 plants found at higher latitudes. Common C-4 plants include crabgrass, corn, and sugar cane. Note that OAA and Malic Acid also have functions in other processes, thus the chemicals would have been present in all plants, leading scientists to hypothesize that C-4 mechanisms evolved several times independently in response to a similar environmental condition, a type of evolution known as convergent evolution.

Plants may be viewed as carbon sinks, removing carbon dioxide from the atmosphere and oceans by fixing it into organic chemicals. Plants also produce some carbon dioxide by their respiration, but this is quickly used by photosynthesis. Plants also convert energy from light into chemical energy of C-C covalent bonds. Animals are carbon dioxide producers that derive their energy from carbohydrates and other chemicals produced by plants by the process of photosynthesis.

The balance between the plant carbon dioxide removal and animal carbon dioxide generation is equalized also by the formation of carbonates in the oceans. This removes excess carbon dioxide from the air and water (both of which are in equilibrium with regard to carbon dioxide). Fossil fuels, such as petroleum and coal, as well as more recent fuels such as peat and wood generate carbon dioxide when burned. Fossil fuels are formed ultimately by organic processes, and represent also a tremendous carbon sink. Human activity has greatly increased the concentration of carbon dioxide in air. This increase has led to global warming, an increase in temperatures around the world, the Greenhouse Effect. The increase in carbon dioxide and other pollutants in the air has also led to acid rain, where water falls through polluted air and chemically combines with carbon dioxide, nitrous oxides, and sulfur oxides, producing rainfall with pH as low as 4. This results in fish kills and changes in soil pH which can alter the natural vegetation and uses of the land. The Global Warming problem can lead to melting of the ice caps in Greenland and Antarctica, raising sea-level as much as 120 meters. Changes in sea-level and temperature would affect climate changes, altering belts of grain production and rainfall pattern.

Assignment

Review Questions

1. The organic molecule produced directly by photosynthesis is: a) lipids; b) sugar; c) amino acids; d) DNA

2. The photosynthetic process removes ___ from the environment. a) water; b) sugar; c) oxygen; d) chlorophyll; e) carbon dioxide

3. The process of splitting water to release hydrogens and electrons occurs during the _____ process. a) light dependent; b) light independent; c) carbon fixation; d) carbon photophosphorylation; e) glycolysis

4. The process of fixing carbon dioxide into carbohydrates occurs in the ____ process. a) light dependent; b) light independent; c) ATP synthesis; d) carbon photophosphorylation; e) glycolysis

5. Carbon dioxide enters the leaf through ____. a) chloroplasts; b) stomata: c) cuticle; d) mesophyll cells; e) leaf veins

6. The cellular transport process by which carbon dioxide enters a leaf (and by which water vapor and oxygen exit) is ___. a) osmosis; b) active transport; c. co- transport; d) diffusion; e) bulk flow

7. Which of the following creatures would not be an autotroph? a) cactus; b) cyanobacteria; c) fish; d) palm tree; e) phytoplankton

8. The process by which most of the world's autotrophs make their food is known as ____. a) glycolysis; b) photosynthesis; c) chemosynthesis; d) herbivory; e) C-4 cycle

9. The process of ___ is how ADP + P are converted into ATP during the Light dependent process. a) glycolysis; b) Calvin Cycle; c) chemiosmosis; d) substrate-level phosphorylation; e) Kreb's Cycle

10. Once ATP is converted into ADP + P, it must be ____. a) disassembled into components (sugar, base, phosphates) and then ressembled; b) recharged by chemiosmosis; c) converted into NADPH; d) processed by the glycolysis process; e) converted from matter into energy.

11. Generally speaking, the longer the wavelenght of light, the ___ the available energy of that light. a) smaller; b) greater; c) same

12. The section of the electromagnetic spectrum used for photosynthesis is ___. a) infrared; b) ultraviolet; c) x-ray; d) visible light; e) none of the above

13. The colors of light in the visible range (from longest wavelength to shortest) is ___. a) ROYGBIV; b) VIBGYOR; c) GRBIYV; d) ROYROGERS; e) EBGDF

14. The photosynthetic pigment that is essential for the process to occur is ___. a) chlorophyll a; b) chlorophyll b; c) beta carotene; d) xanthocyanin; e) fucoxanthin

15. When a pigment reflects red light, _____. a) all colors of light are absorbed; b) all col;ors of light are reflected; c) green light is reflected, all others are absorbed; d) red light is reflected, all others are absorbed; e) red light is absorbed after it is reflected into the internal pigment molecules.

16. Chlorophyll a absorbs light energy in the ____color range. a) yellow-green; b) red-organge; c) blue violet; d) a and b; e) b and c.

17. A photosystem is ___. a) a collection of hydrogen-pumping proteins; b) a collection of photosynthetic pigments arranged in a thylakjoid membrane; c) a series of electron-accepting proteins arranged in the thylakoid membrane; d. found only in prokaryotic organisms; e) multiple copies of chlorophyll a located in the stroma of the chloroplast.

18. The individual flattened stacks of membrane material inside the chloroplast are known as ___. a) grana; b) stroma; c) thylakoids; d) cristae; e) matrix

19. The fluid-filled area of the chloroplast is the ___. a) grana; b) stroma; c) thylakoids; d) cristae; e) matrix

20. The chloroplast contains all of these except ___. a) grana; b) stroma; c) DNA; d) membranes; e) endoplasmic reticulum

User:Ngugi mm