Plant Biotechnology and Tissue Culture
1. What is "totipotency"?
. Fig: Cell Suspension Culture
Fig: Growth curve and different phases of growth during cell suspension culture
Fig: Schematic flowsheet of Agrobacterium mediated gene-transformation.
The ability of a plant cell to perform all the activities of development like a zygote to develop into a complete plant is termed as totipotency. Haberlandt ,1902 first demonstrated totipotency by in vitro technique.
2. How do haploid plants produced?
Haploid (n) plants are obtained by induction of embryogenesis during Anther and Pollen Culture.
3. How are haploid plants made Homozygous diploid ? 2/2020 BotH,TU
The chromosome set of haploid pollen grains are first doubled by mutagenic chemicals like colchine (0.5% for 24-48 hr.). Then homokaryotic fusion resulted into production of homozygous diploid plants ( Vasil and Nitsch,1975).
4. What is Anther Culture?Describe the process with factors.
The culture of anther , containing microspores or immature pollen grains , on a tissue culture nutrient medium to obtain haploid (n) is termed as anther culture.
Steps of anther culture:
(1) At the first step desired plant with flower buds are selected and flower buds plucked.
(2) Surface sterilization:
Next all the collected flower buds sterilized by 70% sodium hypochlorite. .
(3) Then surface sterilized flower buds are washed with sterilized water.
(4) Next the flower buds are cut and stamens are removed by a sterilized knife/ scalpel.
(5) After discarding filaments only the anthers are incubated on nutrient medium at 24° C - 26°C in dark for 3-4 weeks.
(6) Next step may follow any one of the following:
(i) Direct Androgenesis: Here organs are generated directly on the incubated anthers.
Or,
(ii) Indirect method via callus formation: For this strong auxins are added to obtain callus first then organogenesis from the callus e.g., when wheat anthers cultured on a medium having 2,4-D callus produced.
(7) At last the cultured anthers are first transferred on shooting medium and when shoots become 3-5 cm long , then on rooting medium to obtain new plantlets .In case of tobacco a higher ratio of cytokinin (kinetin)led to shoot regeneration, while a higher ratio of auxin promoted root regeneration(Skoog et.al.)
(8) Finally, they are transferred to sterilized soil first, then to the garden .It is called hardening.
Factors :
(1)Genotype of the plant.
(2) The plant must be of pesticide or any type of chemical free.
(3) Stage of pollen :
(i)For Hordeum vulgare and Hyocyamus pre-mitotic stage is suitable.
(ii) Mitotic stage is suitable for plants like Nicotiana tabacum and Datura.
(iii)Post -Mitotic stage is suitable for plants like Atropa belladona .
(4) Pretreatment:
(i) Cold treatment at 3°C-5°C for 2 days or at 7-8°C for 12 days for tobacco ,prior to removel of anther is essential for good result.
(ii) Hot treatment: Brassica campestris requires a brief exposure of anthers at 35°C for 24 hr. or for B. napus it is 32°C for 8 hr.
(ii) Chemicals like Ethrel can enhances haploid production in various species.
(5) Culture media: Addition of 2-3% Sucrose, Chelated iron, nitrate, ammonium salts , amino acids , activated charcoal and growth regulators at different times also play a vital role in anther culture.
(6) Culture Environment: Generally anther culture is maintained in alternating light (12-18 hr. 5000-10000 Lux Per Mt square) at 28°C and darkness (12-16 hr )
at 22°C .
5. State the uses/importance of haploid plants.
Haploid plants are useful in :
(1) direct screening of recessive mutation (as screening of recessive mutation is not possible in diploid or polyploids), and
(2) development of homozygous diploid plants through chromosome doubling of haploid plants.
Cell suspension culture
Courtesy:Lab Associate
6. Describe Cell Suspension Culture.
Multiplication of single cells or small aggregate of cells at higher rate in a continuously agitating liquid culture medium on an orbital shaker is called cell suspension culture.
W.H.Muir(1953) cultured Tagetes erecta and Nicotiana tabacum , L.Nickel (1956) grow Phaselus vulgaris and F.C.Steward and E.M.Stanz (1956) described carrot Explant culture by cell suspension culture method.Suspension culture of higher plant cells in synthetic media were demonstrated by Torrey and Earle in 1965 and cultured cells of Convolvulus.Later on several media were defined by different scholars.
Procedure/Protocol:
Fig: Flowsheet of cell suspension culture technique.
(1) Explant is grown on MS solid medium with 1 gm/lit 2,4-D.
(2) After 5 weeks callus developed.The newly formed callus is transferred to MS liquid medium.
(3) 2 gm of callus is transferred to a 250 ml conical flask with 50 ml liquid medium.
(4) After flaming the neck of the flask,the mouth is closed by aluminium foil or a cotton plug. Then the closure is covered with brown paper.
(5) The flask is then placed on a rotary shaker at 100- 150 rpm to isolate the cell from the callus.
(6) After a week the content of this flask is filtered by sterilized sieve of 200 micro mt. The filtrate contains free cells and cell aggregates.
(7) The filtrate is centrifuged at 500-1000 rpm , the supernatant is poured off.
(8) The filtrate is transferred to new medium and again placed on a rotary shaker.
(9) The subculturing is repeated for 3-4 times and then transfer the content in a new medium in the ratio of 1:3 ( 10 ml cell suspension and 30 ml new medium).
(10)Next the immature embryos are transferred to liduid medium without auxine and glutamine.
(11) After 2 weeks embryos matured and then transferred in petri dishes containing agar medium with BAP orZeatin (0.1 mg /lit).
(12) At last mature embryos transferred on filter paper bridges in tubes wet with MS and sucrose medium for incubation in light.
(13) Finally transferred to pots containing peat, vermiculture and soil for hardening.
7. Briefly explain the two types of Cell Suspension Culture. 2+2/2020 BotH/TU
Types of Cell Suspension Culture:
It is of two types: Batch Culture and Continuous Culture.
(I) Batch Culture: The cell suspension culture which is practised in a fixed volume of culture medium or mass.of cells is termed as Batch Culture. During the culture process , no additions are made to the media, except acid or alkali to control pH. The culture is maintained in conical flasks on orbital shakers at 80--120 rpm.
Types of Batch Culture:
(A) Slow rotatory culture:
(i) It is performed in 250 ml nipple flask with eight nipple like projections.
(ii) Ten such flasks are loaded on the large flat disc of vertical shaker in circular manner.
(iii) During disc rotation, cells within each nipple are alternately bathed in liquid medium and exposed to air.
(B) Shaker Culture:
(i) In this case single cells or cell aggregates are cultured in flxed volume in conical flasks .
(ii) These flasks are mounted by clips on a horizontal large square plate on orbital platform shaker.
(iii) The plate moves by a circular motion at 60--180 rpm.
(C) Spinning culture: The volume of the culture bottle is10 Lit, which rotates in a culture spinner at 120 rpm at 45° .
(D) Stirred culture:
(i) It is used large-scale batch culture.
(ii) The cell suspension of the vessel is kept dispersed continuously by bubbling sterile air through culture medium.
Demerits of Batch Culture:
(i) In batch culture cells grow upto a certain point and then remain stationary and cell size remain constant.
(ii) In this process growth factors of the medium exhausted or toxic metabolites accumulated.
(II) Continuous Culture:
The type of cell suspension culture with a steady state supply of fresh medium to maintain the physiological state of growing cells is termed as Continuous Culture.
Advantage:
Due to the continuous addition of the culture medium, nutrient is not depleted.
Types:
(i) Closed Continuous culture:- Cells are separated from the drained medium and added back to suspension culture. Thus, addition of fresh medium is balanced by outflow of old medium and biomass of the cells are constantly maintained.It is also called Turbidostat method.
(ii)Open Continuous Culture:- In this case addition of the fresh medium is associated with hervest of an equal volume of suspension containing both cell and culture medium. It is also called chemostat method.
8.Explain different phases of growth occured during Cell Suspension Culture.
Different Phases of Growth:-
(i) Lag phase: It is the first phase with a little growth.During this phase the cells adjust to the replenished supplies of nutrients and perform all important synthesis prior to cell division.
(ii) Log Phase: It is the exponential growth phase where cells divide very fast. Under optimum condition in every 20--50 hr. cell number doubled.
(iii) Liniar phase: It is a period of very rapid division of cells , hence, number of cells increase and some nutrients become limited.
('iv) Stationary Phase :- At the last phase rate of cell division decreases, and number of cells stabilized . Hence, finally growth halts.
9.What are the different uses of Cell Suspension Culture.
Use of cell suspension culture:
(i) induction of somatic embryo and shoots,
(ii) in vitro mutagenesis and selection of mutants,
(iii) genetic transformation studies,
(iv) production of secondary metabolites.
10. Mention the advantages of cell suspension culture.
Advantages of cell suspension culture:
Cell suspension culture have advantages over callus culture, like:
(i) The suspension can be pipetted,
(ii) They are less heterogeneous and cell differentiation is less pronounced,
(iii) They can be cultured in volumes upto 1,500 liters,
(iv They can be subjected to more stringent environmental controls,and
(v) They can be manipulated for production of natural products by feeding precursors ( Kurz and Constable,1979)
11. What are the different methods of measuring growth in the Cell Suspension Culture.
2/2020 BotH,TU
Cell growth during cell suspension culture is measured by the use of Hemocytometer,Hartley funnel,etc in a lab.
During this (i) Number of cells are counted, (ii) Packed cell volume is obtained, (iii) Fresh weight of cells are measured, (iv) Dry weight of cells are measured,and (v) Measurement of cell conductivity or osmolarity.
By performing the above said activities (i) change in medium composition ,and (ii) rate of agitation is decided.
12. Write a note on measuring growth in Cell Suspension Culture.
(i) Fresh Cell Weight and Dry Cell Weight:- FCW and DCW both require the manipulation of samples in nonsterile conditions.
(ii) Settle Cell Volume and Packed Cell Volume : SCV is the percentage of the total volume of suspension occupied by the cell mass. PCV is the total volume of suspension occupied by the cell mass after compacting the suspension by centrifugation.
(iii) Cell number/ mL of media: Cell counting is done by pipetting 1 ml of the suspension culture and counting the total number of cells under under the microscope/ cell counting chamber.
(iv) Culture Cell Density: It is performed after the complete disaggregation of cell , by incubating with an 8% chromium trioxide solution or with hydrolytic enzymes like cellulase and pectinase.
(v) Protein and / or DNA content: Ethidium bromide or propidium cell staining is used for this .
(vi) Medium Conductivity: It represents the ionic concentration os the culture medium and decrease inversely to biomass gain in an electric field of an electroporator.
(vii) Oxygen and Mass transfers : It is effected by the factors like viscocity, density, cell buoyancy, and aggregation.
(viii) Cell Viability: Fluorescein diacetate is used to check cell viability.
(ix) Osmolarity: OP and Ionic Strength of culture medium influence the membrane transport pH, extracellular enzymes and secondary metabolic production(Saurabh Bhatia,2015).
Callus Culture
13.Define callus . 2/2020 BotH,TU
The term "callus" originates from Latin word Callum (Callum= hard). A callus is an amorphous mass of loosely arranged thin walled parenchyma cells of the parent tissue ( Dodds and Roberts,1985) or a callus is collective disorganised cell masses (Ikeuchi,M. et al.2013).
14. How callus is formed? 2/2020 BotH,Tu
Naturally, callus develops by infection caused by microorganisms from wounds due to stimulation by auxin and cytokinin , and artificially by tissue culture:
(i) At first 2 mm sterile segment of an Explant is incubated at 25-28°C in an alternate light and dark regime of 12 hr keeping in nutrient medium.
(ii) Auxin of the medium induces cell division and upper surface of explant is covered by callus.
(iii) The three developmental stages are : induction, cell division and differentiation.
Callus can be produced from a single differentiated cell, and many callus cells are totipotent ,being able to regenerate the whole plant body( Steward et al ,1958; Nagata and Takebe,1971).
15. What is Callus Culture?
Callus Culture is the culture of dedifferentiated plant cells induced on media usually containing relatively high auxin concentration or a combination of auxin and cytokinin under in vitro conditions ( P.Srivastava and R. Chaturvedi,2014).
16. How growth is measured in Callus Culture?
The growth index (GI) of the Callus Culture is measured by fresh and dry weight measurement.
(i) Hervested tissue is carefully weighted on the balance and tissue dedication is avoided.
(ii) In case of dry weight measurement tissue is either lyophilized or dried ( microwave oven drying at 60°C) until the constant weight is not achieved (S.Bhatia ,2015).
17. Define transgenic plant.
A plant which contains transgenes ( genes of an unrelated organism) obtained through the process of genetic engineering is termed as a transgenic plant.
The first transgenic plant was produced in 1983 .
18. What is a vector ?
A circular DNA molecule of independent existence and replication capability is used as a in genetic engineering.
e.g. Ti plasmid ,Ri plasmid, Gemini virus and caulimoviruses ,etc.
19. Why do DNA of viruses are not preferred in genetic engineering?
Virus genomes are neither integrated into plant genomes nor transmitted through seed. So, these vectors are not preferred in stable gene transfer.
20 . What is agroinfection?
When an intact or modified plant virus is introduced into plant cells via Ti plasmid or Ri plasmid , the viral genome being placed within the T-DNA, this phenomenon is then called as agroinfection.
21. Write a note on Agrobacterium mediated gene transfer:
There are two methods of Agrobacterium mediated gene transfer:
(1) Coculture with tissue explants and
(2) in planta transformation.
The methods are described below:
Coculture with tissue explants:
Procedure of leaf fragment method:
1. At first gene of interest is inserted within the T-region of disarmed Ti-plasmid ,thus recombinant Ti-plasmid is made.
2. The recombinant Ti-plasmid is placed in Agrobacterium.
3. Leaf discs of a few mm dia. are excised from surface-sterilized leaves and cocultured in petri dish by submerging the discs in bacterial M.S.medium.
4. The petri dished are sealed with paraffin and incubate at room temperature for 2-3 days for the transfer of T-DNA from recombinant Agrobacterium to leaf cells.
5. The leaf discs are then washed in carbenicillin or cefotaxime to kill bacteria from leaf tissue surface.
Meanwhile plant cells express the transgene and turned to Kanammycin resistant.
6. Next leave discs are transferred to plant regeneration medium containing Kanammycin (100 mg/lit.) and Cefotaxime (200 mg/lit).
7.Petri dishes are again incubated with 20° -- 25°C temperature.
8.After 4-6 weeks, callus developed and finally shoot appear from cut edges of leaves.
9. When shoots turn 0.5 to 1.0 cm tall, transferred to half strength M.S. medium having Kanammycin and Cefotaxime/carbenicillin.
10. Then shoots transferred on solid medium rich in high auxin to cytokinin for rooting.
11.After 3 weeks plantlets are transferred to soil for accllimatisation or hardening.
References
Bhatia, N., (2021),Cell Suspension Culture: an overview,Lab associates.
Dodds,J.H. and Roberts,L.W.(1985).Experiments in Plant Tissue Culture,2nd Ed.Cambridge University Prass, Cambridge.
Ikeuchi,M., Sugimoto, K. and Iwase ,A.(2013) Plant Callus: Mechanisms of Induction and Repression . Plant Cell.2013Sept;25(9):3159-3173.
Kurz,W.G.W.and Constable,F.(1979).In Microbial Technology,Vol.I,2ndEd.,(eds.Peppler,H.J.and Perlman,D.),pp.389-416,Academic Press:London.
Nagata T.,Takebe I.(1971).Plating of isolated tobacco mesophyll protoplasts on agar medium. Plants 99: 12-20[PubMed] [Google Scholar]
Srivastava P., Chaturvedi R.Herbal Medicine and Biotechnology for the Benifit of Human Health, Animal Biotechnology,2014[ Science Direct] .
Saurabh Bhatia, Plant Tissue Culture. Modern Applications of Plant Biotechnology in Pharmaceutical Sciences,2015 [ScienceDirect] .
Steward F.C., Mapes M.O., Mears K.(1958). Growth and organized development of cultured cells,II. Organization in cultures grown from freely suspended cells .Am.J.Bot.45: 705-707[Google Scholar]
Vasil, I.K., and Nitsch,C.(1975).Z.Pflanzenphysiol. 76:191-212.
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