Chapter 22 - Cloning and biotechnology Flashcards
vegetative propagation
➜ plants can reproduce asexually using a cutting (meristem - undifferentiated plant cells that form in the vegetative organs of a plant)
Vegetative organs
- root and shoot tips
- axillary buds (where leaves and stem meet)
- Vascular cambium (between xylem and phloem)
Process of vegetative propagation
➜ over time a miniature plant (plantlet) forms at the vegetative organs and is attached to parent
➜ these are clones of parent
➜ at maturity plantlet detaches from parent when it is of photosynthesising by itself
➜ new plant = same phenotype so growing and harvesting easier
➜ some plants have runners or horizontal stems that form over soil surface, pointing sufficiently far away from parent so when ready it will grow far away from parent so it doesn’t compete for water or nutrients or is overshadowed by parent
e.g strawberries, peppermint, spider plant
adventitious roots
➜ roots forming under nodes of runners
➜ The runner dies when the plantlet is self-sustaining
Propagation techniques
➜ dont need seeds
➜ as well as runners, tubers, rhizomes, bulbs, suckers and offsets can be used to propogate
➜ All modes of vegetative propagation contain modified stems that can generate meristematic tissue
Potato tubers
➜ swollen modified roots that form eyes on their surface
➜ eyes can sprout new growth = chitting
➜ starch stored in tuber fuels early growth of new plant
Rhizomes
➜ Ginger forms rhizomes
➜ it is a modified stem that grows horizontally underground
➜ New growth stems from nodes in the rhizome, forming new stems and adventitious roots
➜ section used in cookery is the rhizome
bulbs
➜ onion and garlic
➜ form bulbs that can grow adventitious roots underground and leafy shoots above ground
Suckers
➜ growths thatappear from root system of many trees and shrubs which can provide meristematic tissue for vegetative propagation
➜ poplars, cherries and plums
Offsets
➜ small virtually complete daughter plants that have been asexually produced on the mother plant
➜ tulips and lillies
Practical: Producing Cuttings
➜ Cuttings can grow to full-sized plants much quicker than plants grown from seeds
➜ equipment:
∘ sharp scissors
∘ potting compost
∘ pots to house new plants
∘ hormone rooting powder - contains auxins that promote mitosis and cell differentiation in new root growth
∘ plastic bag to cover cutting
➜ cut below a stem node - small length of stem
➜ dip base of cutting in rooting powder
➜ once new roots have started to grow, it can be planted into soil
Cultivars
➜ gardners etc can harness natural process of vegetative repro to artificially clone
➜ like cutting, layering, grafting, division and budding
➜ this creates cultivars - strains of genetically identical plants for foodstuffs or commercial blooms
➜ methods rely on the formation of meristematic tissue from which plant organs can differentiate
Cauliflower cloning
➜ many plant cells = totipotent
➜ cauliflower used as it is comprised mostly of actively dividing cells and can withstand being handled
➜ small piece of plant cut = explant
➜ explant then disinfected to prevent fungi
➜ used on endangered species
Micropropagation and tissue culture method
➜ wear eye protection for god knows what reason
➜ wipe app surfaces down and soak all apparatus in sterilant (so no fungi contamination)
➜ break off a small floret of cauliflower and use scalpel to cut thin section of floret (5-10mm long) thin section = explant
➜ soak explant in sterilising solution for 15 min and swirl every 5 min = to make sure only cauliflower cells present
➜ take out explant using STERLISISED forceps and add to agar growth medium which has all nutrients needed for plant to grow (agar also sterilised dw)
➜ leave container on sunny window for 3 weeks
RESULTS:
➜ clones a full cauliflower from explant
➜ shows totipotency = explant has capability to produce all diff cel types to make cauli
Advantages of plant cloning
➜ plants produced are free of disease
➜ same genotype and phenotype
➜ GM plants are immune to certain diseases
➜ process is rapid so large yield
➜ small plants = easily transported
➜ pants that are difficult to grow via seeds can be cloned
➜ grown in any country = any season
➜ rare and endangered species can be propogated to save them
➜ whole plants can be created from GM cells
➜ use of cultivars prevent risk of F₁ hybrids
Disadvantages of plant cloning
➜ expensive and labour intensive process
➜ process is susceptible to microbial contamination
➜ no genetic variation = risk of large scale loss due to disease etc
➜ New plants have to be carefully screened for abnormalities that could lead to the new plants being infected
➜ risk of an unexpected secondary metabolic chem reaction causing stunted growth or evend eath in new explants
Asexual reproduction in animals
➜ less common than plants
➜ some reproduce asexually by parthenogenesis - growth and development of embryo clones occur without fertilisation e.g aphids
➜ other naturally occuring cloning = identical twins
Identical twins
uno i always wanted a twin 😔
➜ egg fertilised by sperm in a singleton birth
➜ forms zygote
➜ single zygote undergoes a few mitotic divisions to become embryo
➜ identical twins referred as monozygotic
➜ at embryo stage, embryo split into 2
➜ 2 embroyes formed = identical and both develip in utero
➜ result = identical offspring, identical pheno and genotype
➜ non identical twins formed from diff egg and sperm they ARE NOT clones
Embryo twinning
➜ produces offspring that are genetically identical to each other and not parents
➜ been routine procedure to boost yield of livestock and promote desired allele since 1980’s
➜ key step is the deliberate division of the emrbyo into 2 half embryos
➜ then inserted into surrogate mum for gestation and birth
➜ births identical twins
➜ some cases - emrbyos are split into single identical cells which can be implanted into separate surrogate mummies
➜ guarantees desirable characteristic in offspring but not how many offspring
Reproductive cloning
DOLLY SHEEP IN EDINBURGH, 1996
Somatic Cell Nuclear Transfer (SCNT)
➜ 3 separate animals are required:
∘ animal to be cloned via cell
∘ female to donate egg cell
∘ surrogate mummy
➜ Procedure
∘ animal to be cloned donates a somatic (body) cell
∘ egg cell extracted from donor and enucleated (nucleus is removed by suction and discarded)
∘ nucleus from somatic cell injected into egg cell
∘ hybrid zygote is now treated to encouraged to divide via mitosis
∘ embryo implanted into surrogate mother for gestation and birth
Therapeutic cloning
➜ cloned cells are used to replace dead or damaged cells that cause a loss of function in an individual
➜ embryos go through reproductive cloning (dolly sheep process) but the embryos are removed and subdivided
➜ embryo cell = totitpotent so it is artificially differentiated into any type of specialised cell
➜ high potential but little clinical progress cuz UK dum
For animal cloning
➜ well accepted and non controversial in livestock farming
➜ maximising agricultural output as desirable characteristics can be cloned
➜ can remove less desirable characteristics from gene pool over time
➜ help preserve endangered species
➜ Provide regenerated organs for patients suffering from degenerative disease - organs would be a direct match so no risk of rejection
Against animal cloning
➜ SCNT is hit or miss (100’s of attempts to make dolly bestie)
➜ unknown long term effects of cloning (Subsequent cloning attempts have led to a high number of early deaths and genetic abnormalities in the clones)
➜ some cloned animals tend to grow abonormally large (Large Organ Syndrone - LOS) which can cause breathing and circulatory problems
➜ disrupts normal mechanisms or regulation of gene expression
➜ destroys embryos - ethical?
Biotechnology
➜ harnesses the processes in living organisms to useful products like food/meds
➜ carry out useful services liek sewage treatment, composting and bioremediation(use of microorganisms to degrade contaminants of an environmental accident)
Microorganisms
➜ most useful group of organisms that carry out biotechnological processes because they
- have simple growth requirements
∘ their food is cheap and readily available
∘ occupy very little space
- reproduce quick
- no non-productive tissues and organs
-can be grown on an industrial scale to perform duties useful to large numbers of the human population
Brewing and distilling
➜ brewing uses yeast to respire sugars from barley malt and produce ethanol (primary product) and CO2 (by product)
➜ this is anaerobic and known as fermentation
➜ some alcoholic drinks have a higher alcohol content than fermentation alone can produce, so these drinks are first fermented then distilled to concentrate alc
e.g
∘ whiskey (nasty+it burns) and bourbon are distilled from a barley beer
∘ brandy is distilled from grape wine (yum)
∘ other spirits use generic fermented ethanol distilled through botanicals (berries, herbs etc) to extract flavours
Baking bread
➜ wheat/rye mixed with yeast and other stuff to make doUGH
➜ culture of yeast is in fact a mixture of several different naturally occurring species
➜ commercial bakers = control species of yeast used
➜ artisn bakeries use wild yeast cultures, preserved and cultivated regularly
➜ Yeast enzymes begin by hydrolysing the starch in flour to maltose
➜ Maltose is then hydrolysed to produce monosaccharides which can be used for aerobic respiration
➜ O2 runs our so yeast respires anaerobically
➜ aerobic and anaerobic resp produce CO2 (bubbles) throughout dough causing it to rise
➜ baking kills yeast and causes the gas pockets to expand so bread rises more
Cheesemaking
➜ pasteurised milk = raw material
➜ bacteria are used to digest lactose producin g lactic acid and it lowers pH of milk
➜ low pH=proteins denature in milk leading do separation of curds (solid) and whey (liquids)
➜ curds are pressed and processed into cheese
➜ mould spores from saprotrophic fungi can be artifically added into blue veined cheeses
Yoghurt making
➜ starter culture of Lactobacillus bulgaricus and Streptococcus thermophilus bacteria are introduced to pasteurised milk
➜ bacteria use sugar in milk to respire and produce lactic acid (waste)
➜ lactic acid denatures proteins in milk causing them to coagulate (stick together) producing thick and sour yogurt
➜ flavours added to make it taste yum
Penicillin production
➜ species of mould from the Penicillium genus can be cultured in industrial fermenters
➜ known as deep tank fermentation
➜ extractin & purification of product produces large volumes of the drug for therapeutic use
➜ first wonder drug = produced at large scale
∘ penicillin mould added to culture medium with sugars, AA and minerals
∘ stainless steel tank has a water jacket to maintain a constant fermentation temp outside the cultutre medium
∘ bubbles supply O2 and cause mixing of culture and theres a sterile air injection
Insulin production
➜ previously diabetes were treated with pig insulin (hard to isolate, expensive and not as effective)
➜ Recombinant DNA technology can incorporate the gene for human insulin into the genome of the bacterium, Escheriscia coli
➜ Recombinant bacteria are grown in batch fermenters, and each bacterial cell expresses insulin
➜ insulin is released into the batch medium and can be purified for medicinal use at a later stage
Mycoprotein production
myco means fungus
➜ meat substitute and is low fat and high fibre
➜ microorganism used is Fusarium venenatum, a filamentous fungus
➜ source of glucose is added to the tank
➜ O2 also supplied to allow aerobic resp can occur which yields maximal growth of hyphae
➜ Nitrogen introduced in the form ammonia
➜ product in mycoprotein = fungus, rather than a substance produced by microorganisms
Bioremediation
➜ humans contamnate land/water with toxic substances and it can be remediated by removing pollutants and restoring land
➜ e.g oil spills, industrial accidents, acidic damage from mining and cleanup of crime scenes
➜ biore rely on oxidative digestion of pollutants
➜ bioventing (Naturally occurring microorganisms perform aerobic digestion of the contaminants and release non-polluting products) are sometimes enough
➜ biostimulation - relies on naturally occurring microorganisms but adds nutrients that promote microbial diegestion of pollutants
➜ GM has been trialled to create microorganisms capable of biorem
Emerging uses of biotechnology
➜ Production of biofuels (to replace the use of fossil fuels)
➜ Production of vaccines and antibodies for the treatment of disease
➜ Production of hardy crop plants that can grow in arid conditions
➜ Counteracting threats from bioterrorism or bio-warfare
Advantages of using microorganisms to make food
➜ microorganisms reproduce quickly so selective breeding quicker
➜ microoganisms can be grown on waste substrates (e.g whey - waste product of cheese making)
➜ growth not seasonal
➜ inputs = cheap like glucose, oxygen, ammonia
➜ fermenters can be set up anywhere in world so food can be produced in extreme climates
➜ low fat, high protein products (mycoprotein)
➜ microorganisms efficient at converting energy into biomass
Disadvantages of using microorganisms to make food
➜ sterile technique is expensive
➜ micro reproduce quick so could mutate quick too
➜ perception that food is nutritious but has no flavour etc
➜ bacteria can be infected by viruses
➜ people may not like their food going through microorganisms and wont buy it
➜ fermenter contaminated by pther bacteria could ruin product
➜ fermenter produced products are high in nucleic acids which is harmful in large quantities so it must be processed to be safe to eat
Culturing Microorganisms
➜ work aseptically - keep bunsen lit at yellow as flame creates convection current above bench, preventing contamination from air
➜ hot agar jelly poured into sterile petrie dish and left to cool
➜ inoculating loop passed through flame
➜ use loop to take sample from culture tube (flame before and after removing and replacing plug)
➜ open petri teeeny weeeny bit
➜ and wipe on surface of agar
➜ sterilise loop again
➜ tape at intervals around dish and store upside down
➜ incubate 25°C for 24 hrs
Batch fermentation
➜ microo grown in batches in fermentation vessel
➜ product removed after culture cycle and fermenter is cleaned for new batch
➜ known as closed culture
Continuous fermentation
➜ microo continually grown and the products harvested
➜ nutrients added ans waste is removed through process
optimum conditions in fermenter
➜ pH - for enzymes for max rate for max yield
➜ temp - use a water jacket for max yield
➜ O2 - to max resp
➜ nutrient supply - for max yield
➜ agitation - paddles for even distribution of temp, pH, O2 and nutrients
➜ contamination - steam cleaned in between b atches
➜ waste removal - to prevent negative effects on growth
Measuring populations of microo
Direct counting
includes all cells (both living and dead) and involves taking samples to count these individual microorganisms
Measuring populations of microo
Viable counting
involves culturing samples of microorganisms and counting the colonies that grow (only live cells)
Measuring populations of microo
Turbidity
➜ measure of living and dead microorganisms
➜ measure of the cloudiness of a suspension using a colorimeter
➜ as microo can be grown in broth culture
➜ measuring turbidity can be then used to estimate n.o of cells in broth
➜ as microo reproduce suspension becomes more turbid (cloudy)
➜ can be measured by colorimeter and data logger or light sensor or turbidity meter
➜ can then plot a pop curve
Standard population growth curves
➜ Lag Phase - pop increases slowly as microo pop adjusts to new env and starts to reproduce
➜ Log Phase - pop grows at exponential rate due to high nutrients
➜ Stationary Phase - pop reaches max as it is limited by resources, n.o of death = n.o being reproduced by binary fiss
➜ Decline Phase - due to lack of nutrients and death due to toxic substance build up, death rate exceeds reproduction rate
binary fission
➜ single circular DNA molecule undergoes DNA replication
➜ plasmids present undergo DNA replication
➜ parent cell divides into two cells, with the cytoplasm roughly halved between the two daughter cells
➜ two daughter cells each contain a single copy of the circular DNA molecule and a variable number of plasmids
rate of cell division by binary fission:
N = N₀ x 2ⁿ
N = the final number of bacteria
N₀ = the initial number of bacteria
n = the number of division
Immobilised Enzymes
(IE)
an enzyme that is attached to an insoluble material to prevent mixing with the product
➜ can be immobolised several ways:
∘ attached to an inert substance - by covalent bonding or attachment
∘ enclosed in a capsule - matrix?
∘ contained within partially perm membr
why immobolise enzyme?
➜ for use in industrial processes, enzyme can be reused in future processes rather than being discarded after it has been used once
➜ Reusing the enzyme also avoids the need to separate the enzyme from the product in downstream processing
∘ IE are containted within a column through which substrate is filtered in solution
∘ as substrate runs through column, enzyme substrate complexes are formed
∘ products are produced and these flow out column leaving enzymes behind
Advantages of immobilised enzymes
➜ no enzyme in product (uncontaminated) so no need to further filter end product
➜ IE can be reused multiple times - cost effective and efficient
➜ IE has high temp and pH change tolerance
∘ immobilisation makes enzymes more stable
Disadvantages of immobilised enzymes
➜ expensive equipment
➜ IE is costly to buy so not helpful for small industries
➜ ROR is sometimes lower when using IE as enzymes can’t freely mix with substrate
Lactose free dairy products like milk
∘ enzyme: Lactase
∘ converts lactose to glucose and galactose
Semi-synthetic penicillin
➜ overcomes issues of penicillin resistance
∘ Enzyme: Penicillin acylase
∘ converts penicillin to stronger one
Glucose products used to sweeten and thicken foods
∘ Enzyme: Glucoamylase
∘ starch and other dextrins to glucose
Fructose to sweeten foods that must have low sugar
∘ Enzyme: Glucose isomerase
∘ starch and other dextrins to glucose
Purified samples of L-amino acids used in food production
∘ Enzyme: Aminoacylase
∘ Separates out L-amino acids from D-amino acids
Acrylamide required in disposable nappy production
∘ Enzyme: Nitrilase
∘ Converts acrylonitrile into acrylamide
Using IE to breakdown lactose in milk
➜ there is container of alignate beads bound with lactase
➜ milk is poured in
➜ lactase breaks lactose down into glucose and galactose which can be easily absorbed by the lactose intolerant
➜ lactose = disaccharide
