tissue culture

1. Principal Applications of Plant Tissue Culture in Plant Science
The principal applicationsof planttissueculture inplantscienceare discussed under the following sub-
headings:
 Micro Propagation
 Clonal Propagation
 Production of Genetically Variable Plants
 Plant Pathology and Plant Tissue Culture
 Plant Breeding, Plant Improvement and Plant Tissue Culture
 Production of Useful Bio-chemicals
 Preservation of Plant Genetic Resources or Gene Conservation Banks
 Importance of Tissue Culture in Biotechnology
i. Micro Propagation:
The regenerationof whole plantthroughtissue culture ispopularlycalled“micro-propa­gation”.Thisisa
technique where a callus mass has been initiated from a single explant taken from any living part of a
donor plant and within very short time and space, a large number of plantlets can be produced from
such callustis-sue. Again, it is possible to make a large number of callus pieces from the original stock
culture during sub-culturing.
Then it is possible to pro-duce hundreds of plantlets that develop on each of these callus pieces.
Therefore, the most obvi-ous advantage of micro-propagation is the numer-ical one. Suspension
cul-tures can also be used to exploit this numerical advantage as they produce numerous cell
aggre-gates relatively rapidly, generally growing faster than callus tissues.
The numbersof plantletpro-ductiondependsuponthe numberof shoot primordia that can be induced
to formwithinthese cell aggregates.Alternatively,if the cell suspen-sionculture happens to be embryo
genic,thenthispropagationpotential dependsuponthe rate atwhichembryoidsare formedby the cell
aggre-gates and the rate at which new embryo genic ag-gregates are formed in culture.
Again, the shoot tip and nodes of the regenerated plantlets de-rived from callus culture and cell
suspensioncul-ture canbe multipliedfurtherfollowingorganculture method.Asaresult,large numbers
of a plant species or variety can be propagated all the year around. The plant breeder or grower is no
longer restricted by season in the production of large numbers of plants.
ii. Clonal Propagation:
In vitroclonal propagationisa type of mi-cro-propagation.The culturedplantsraisedfromtissue culture
are derivedasexuallyandalsomul-tiplywithinculture vessel by asexual means. A sexual reproduction,
on the other-hand, gives rise to plants which are genetically identical to the parent plant.

2. Multiplication of genetically iden-tical copies of a cultivar by asexual reproduction is called clonal
propagation and a plant popu-lation derived from a single donor plant in tis-sue culture constitutes a
clone.So,the vari-ability that can arise from sexual reproduction and seed formation in a crop plant is
omitted.
More specifically,asingle plantwithdesirable characters can be selected from a breeding pro-gramme
and propagatedsothat furthertrialsandselectionscanbe carriedout as quickly as possible. The plants
with long seed dormancy can be raised faster by in vitro clonal propaga-tion than in vivo seed
propagation.
The undesir-ablejuvenile phase associated with seed raised plants in some variety does not appear in
the vegetatively propagated plants from adult mate-rial. For the orchids, in vitro clonal propagation is
the only commercially viable method of micro- propagation. Clonal multiplication of cultivar is very
important in horticulture and silviculture.
iii. Production of Genetically Variable Plants:
In some callusculture,there isamajorten-dencyof the callustissue towardsthe numerical variation of
chromosomes in the cells that oc-curs after a number of serial subcultures. Such chromosomal
variations in culture may arise be-cause of two factors. First, the cells of various ploidy and genetic
constitution of the initial ex- plant may be induced to divide and secondly, culture condition may
contribute new irregulari-ties.
The chromosomal instabilityinthe culturedcells play an important role in polyploidization of cells and
genetically variable plants can be raised from such polyploidized cells by subse-quent micro-
propagation. Thus, tissue culture is proving to be rich and novel sources of vari-ability with a great
potential in crop improve-ment without resorting to mutation or hybridiza-tion.
Variantsselectedthroughtissue culture hasbeenvariously termedtoascalliclones(fromcallus culture)
or protoclones (from protoplast culture). Larkin and Scowcroft have proposed a general term “soma
clones” for plant variants achieved from tissue cultures, irrespective of their origin.
Such variant plants may show some useful characters such as resistance to a particu-lar disease,
herbicide resistance, stress tolerance etc. Such changes are valuable for crops which are normally
propagated by vegetative methods. Moreover, plant breeders can exploit such vari-ants for their
breeding programme.
iv. Plant Pathology and Plant Tissue Culture:
There have been many valuable contribu-tions of plant tissue culture to problems concern-ing plant
pathology.One outstandingsuccess is the virus eradication by apical meristem culture and the second
success of tissue culture in plant pathology is the result of its application to the problems of plant
tumors, especially crown gall.

3. 1. Virus Eradication:
In virus infected plants, the distribution of viruses in plant body is uneven. It is well known that the
apical meristems are generally either free or carry a very low concentration of viruses. The apical
meristem culture is the only way to obtain a clone of virus free plant which can be multiplied
vegetatively under control conditions that would protect them from the chance of reinfection. The
elim-inationprocedure of viruscanusuallybe im-proved by combining it with heat therapy of the host
plant or the culture.
Virus eradication by apical meristem culture has enormous horticul-tural and agricultural value e.g. in
the produc-tion of plants for the cut flower industry when stock plants of registered line must be
main-tainedinas near-perfect condition as possible. Any infection by virus that affects growth rate or
physical characteristics of size and shape is obviously very serious if it afflicts these nuclear stock, for
they are the basis of all propagation and breeding.
In the agricultural world, the production or yield of a crop can fall dramatically as a result of viral
infection and render that particular va-riety no longer saleable or commercially viable. Tissue culture
techniquescouldbe of value inrestoringthe original propertiesof the variety,byremovingthe infection
and so bringing it bank into the commercial market. These virus tested stocks could provide ideal
material forthe na-tional andinternational distribution of plants, either for further propagation or use
as breeding material. It is hoped that these selected virus free cultures would be acceptable to
quarantine authorities.
2. Study of Crown Gall by Plant Tissue Culture:
Smith and Townsend (1907) discovered that crown gall or plant tumor formation was induced by a
bacterium, Agrobacterium tumefaciens. Braun (1941) showed that in sunflower Agrobac-terium could
induce tumorsnotonlyat the in-oculatedpointbutata considerable distance,where secondarytumors
free of bacteria are formed.
Cells of these secondary tumors could be cultivated by tissue culture technique and mul-tiplied on a
mediumwithoutaddingauxinandcytokinin,whereasnormal tissuerequiredaux-insand,insome cases,
cytokinins.
Crowngall tissuesdeprivedof bacteriagive rise to tumors by grafting. Kulescha (1952) established that
theysynthesizedmore auxinthannormal tis-sues. Braun demonstrated that crown gall tis-sues free of
bacteria contain a tumor-inducing principle (TIP) which may be a macromolecule.
The biochemistryof the crowngall problemwasstudiedbyLidret(1957) whodiscoveredanami-no acid
called lysopin which might be specific for the tumor. Menage and Morel (1965), Gold- mann-Menage
(1970) and Morel (1971) isolated two substances of the same chemical family octopine and nopaline
and concluded that these opines were not characteristic of the plant but of the bacterium that had
induced the tumor.

4. Crowngallsinducedbysome strainsof A.tume-facienssynthesize octopin, while tumors produ-ced by
other strains elaborate nopaline. Follow-ing these observations, Morel (1971) suggested that the
synthesis of opines depends on the pres-ence of TIP in the tumor cells of genes coming from the
bacterium. In other words, the TIP con-sists of DNA. Zwnen et al. (1974) discovered the segment of
bacterial DNA which is responsible for the tumoral transformation and opine syn-thesis.
This segment belongs to a large plasmid. Only a small part (about 8%) of the plasmid is stably
incorporated and replicated in plant cells. Therefore, we can say that this transferred DNA (T-DNA)
contains the genetic information which promotes the tumoral transformation and gene coding for
octopine or nopaline.
Finally,geneti-cistsandmolecularbiologistswere able to estab-lish the map of the crown gall plasmid.
But the basic mechanismof tumoral transformation has not been clarified. It is hoped that plant tissue
culture technique can throw some light on the basic mechanism of tumoral transformation.
v. Plant Breeding, Plant Improvement and Plant Tissue Culture:
The conventional breeding methods are the most widely used for crop improvement. But in certain
situations, these methods have to be sup-plemented with plant tissue culture techniques either to
increase their efficiency or to be able to achieve the objective which is not possible through the
conventional methods.
Embryoculture isnow routinelyusedinrecoveryof hybridplants from distant crosses. Some examples
are recoveryof hybridsfromHordeumvulgare x Secale cereale,Triticumaes-tivumx Agropyron repens,
H. vulgare x Triti-cum species. In case of Triticale, a rare hy-brid between Triticum and Secale develop
viable seeds.
But most of the tetraploid and hexaploid wheat carry two dominant genes Kr1 and Kr2 which prevent
seed development in crosses with Secale. The hybrid seeds are minute, poorly de-veloped and show
verypoor germination.Byembryoculture,50-70% hybridseedlingshasbeenobtained.Hybridseedlings
from T. aestivum x H. vulgare are not obtained. But it has been achieved by embryo culture.
When H. vulgare or T. aesiivum (used as male) is crossed with H. bulbosum (used as fe-male) the
chromosome complement of H. bulbo-sum is eliminated from the developing embryo. Most of the
seedling obtained from such crosses are haploid, having only one set of chromosomes either from H.
vulgare or T. aestivum parent.
Embryoculture isalso useful forpropaga-tionof orchids;shorteningthe breedingcycle and overcoming
seeddormancy.Inmeristemculture,shoot apical meristem along some surrounding tissue is grown in
vitro. It is used for clonal propagation and recovery of virus free plants and is potentially useful in
germplasm exchange and long-term storage of germplasm through freeze preservation.

5. Anther and pollen culture has a potential application in plant breeding and plant improve-ment
programme for the production of haploid as well as homozygous diploid plant. All-year-round rapid
clonal propagation us-ing plant tissue culture techniques has highlight-ed possibilities for new plant
improvement tech-niques.
Protoplast culture and somatic hybridiza-tion is a promising line for plant breeding and pl ant
improvementtechniques.But,atpresent,techniquesforselectionandmultiplicationof so-matic hybrid
and regenerationof hybridplantsisverylimitedtoafew classical plantspecies.So it is expected that in
near future it would be possible to use this technique for a wide variety of plant species.
Anothermostimportantapproachisthe mutationof tissue culture cells to produce a mu-tant line from
whichplantscan be raised.Pro-ductionof mutantline ishighlydesirableforplantbreeding. Callus cells,
producedeitherfromvegetative cell or reproductive tissues, can be subjected to a range of mutagenic
chemicals e.g. N-nitroso-N-methyl urea or ionizing radia-tions e.g. gamma rays.
The hope is that per-manentchangesinthe DNA patternof some of the cellswouldbe achievedby such
treatment.Plantscouldbe raisedfromthe treatedculturesandanymutant whole plants selected from
the population either by physical differences or by metabolic/biochemical differences. Biochemical
mutantscouldbe selectedfordisease resistance,resistance tophytotoxin,improvement of nutri-tional
quality, adaptation of plants to stress con-ditions e.g. saline soils and to increase the bio-synthesis of
plant products used for medicinal or industrial purposes.
vi. Production of Useful Bio-chemicals:
Man dependsonplantsformanycom-poundsotherthan food such as medicines, pig-ments, vitamins,
hormones, flavoring agents, la-tex and tanins. If most plant somatic cells are totipotent, it should be
possible totake a cul-ture of cells from a plant that naturally produces a certain biochemical and cause
the culture to produce that chemical underinvitroconditions. The main difficulty is that we do not yet
un-derstandthe regulationmechanismsthatcontrol the productionof mostbiochemical substance and
so cannot manipulate them.
Evenso, a surprisingnumberof cell cultureshave beenfoundthatdoproduce specializedbio- chemicals
foundinthe intact parent,includingalkaloidsuchasnicotine,atropine,ephedrine,caffeine andcodeine
and their precursors and derivatives. Production of cardiac glycosides and other steroids,
benzoquinones, latex, phenolics, anthocyanin’s, organic acids, anti-tumor agents, antimicrobials and
various flavors and odours have also been reported.
The firstpatentsfor producingbiochemical’scommerciallybylarge-scale plant cell cul-ture was issued
in1956. The general approachsince thenhasbeentoselecthighproduct-yield-ingcell lines, preferably
as suspension cultures and to enhance their efficiency either by feed-ing them inexpensive product
precursors or by manipulating their biosynthetic control mecha-nisms.

6. Here the expertise of the microbial prod-uct industry is valuable. Large-volume auto-mated culture
vessels called fermenters have been used successfully to mass produce cultured plant cells. This
technology will soon be produc-ing selected pharmaceuticals and other high-cost bio-chemicals
commercially.
vii. Preservation of Plant Genetic Resources or Gene Conservation Banks:
The need for a programme for the conser-vation of plant genetic resources arises from the rapid
changesthat are occurringinmodernagri-culture practice. The primitive cultivars and wild relatives of
crop plantsconstitute a pool of genetic diversity which is invaluable for future breeding programmes.
But these have alreadyledtothe replacementbynew cultivars which encompass a much narrow range
of genetic di-versity.
As a result,there isaveryreal dangerof future breedingbeingimpededbythe shrink-ing genetic bases
of some crops. Therefore, stor-age of this sort of irreplaceable breeding material or germplasm (gene
combinations available for breeding) and establishment of a centralised gene bank are the practical
ways to solve these prob-lems.
Conventionally, germplasms are stored in the form of seeds because they occupy a rela-tively small
space and can be storedformany years. But there are a number of important species, particularly root
and tuber crops, which are normally propagated vegetatively.
These in-clude potato, sweet potato, yam and cassava. So conventional preservation methods is not
appli-cable to vegetatively propagated plants. On the other hand, the cost of maintaining a target
pro-portionof the available genotypeof these plantin nurseriesorfieldisveryhighandthere isa risk of
the plants being lost as a result of disease or environmental hazards. It is now possible with modern
tissue cul-ture techniques to provide a germplasm storage procedure which uniquely combines the
possibil-ities of disease elimination and rapid clonal mul-tiplication.
In addition,the possibilityof usingliquidnitrogenfreeze-storagetechniquesforthe preser-vationof cell,
tissue andapical meristemisbeingstudied.The advantages of this technique sire that cell division and
normal cellular reactions are totally arrested at the very low temperature of liquid nitrogen (-196°C),
which means that there should be a high level of genetic stabil-ity and that the chemical reaction
responsible for cellular damage will not occur.
The plant materials can be stored in liquid nitrogen for desirable period. This technique could be
par-ticularlyvaluable forstorage of anygermplasmwhichneedsto be maintained in a clonal form. This
technique is known as cryopreservation or freeze preservation of tissue or cell.
It has al-ready been shown to be successful with a range of cell cultures e.g., carrot cell and sycamore
sus-pension cells and meristem tips from a number of crop plants including asparagus, tomato and
potato.Planttissue ormeristemsof suchplantshave beensuccessfullyrecovered from liquid ni-trogen
and grown into normal, mature fertile plants.

7. viii. Importance of Tissue Culture in Biotechnology:
In 1981, the European Federation of Bio­technology defined “biotechnology as the integrated use of
biochemistry,microbiologyandchemical engineeringinorderto achieve the tech-nological application
of the capacitiesof micro-besand cultured tissueand cells”.Some peo­ple equate it with the new field
of geneticen-gineering,while others take a broader viewpoint defining it as the evaluation and use of
biological agents and materials in the production of goods and service for industry, trade and
commerce.
To reduce confusion we will limit our interpre-tation to the two areas most often equated with
biotechnology.One of these,the genetic engi-neering of organisms, is the endeavor that in-spired the
coinage of the term “biotechnology” in the 1970’s. The other area consists of recent developments in
the fields of tissue and cell cul-ture, most notably those that have enabled us to fuse two different
eukaryotic cells into a sin-gle cell that possesses the combined properties of both.
Cell suspension culture in liquid medium is a relatively young field of biotechnology. This technology
involvesthe large-scaleculture of iso-latedplantcell underconditionwhichinduces them to synthesize
the natural secondary meta-bolites characteristic of parent plants from which they were obtained.
In recent years, the tech-nique of callus culture and cell suspension cul-ture has also been viewed,
particularly from the view-point for the study of biosynthesis and metabolism of steroids and cardiac
glycosides. Biotechnologists are also trying to increase the synthesis of natural compounds or new
com-poundsbyhigherplantcellsculture asaresultof mixingorfeedingtransformable precursorsinthe
culture medium.
The metabolic process of transformation or conversion of such added pre-cursors into the natural or
new compound within the cell is known as biotransformation. Biotech-nologists are also trying to
augment the synthesis of medicinally important alkaloids in culture by means of fungal elicitor
Thismeansthat cells are culturedina liquidmediumbyaddingre-quired quantity of the bacterial filter
sterilizedextractof certain fungi. It has been observed that the fungal extract in certain cases helps to
increase the synthesis of a desirable compound by the higher plant cell.
Biotechnologists are also trying to modify the genetics of the cultured cells by three ways such as:
 Mutagenesis and selection of cell lines in cell suspension culture,
 Transplanta-tionof foreigngeneticmaterial inprotoplastsbymeansof genetic engineering and
 Somatichybridizationbythe fusionof distantlyrelated plant proplast just to widen the genetic
diversity of hybrids.

8. In the field of mutagenesis and selection of cell lines in vitro and the exploitation of totipotency,
biotechnologists are trying to improve plants, for example by producing crop spe-cies that are more
resistant to draught, disease, poor soil condition, chemical pesticides and her-bicides.
Anothergoal of biotechnologyusingplanttis-sue culture technique is to produce self-fertilizing plants
that wouldprovide theirownusable ni-trogen.Plantsrelyonafew typesof bacte-ria and cyanobacteria
to fix atmosphericnitro-genintoabiologicallyusuableform.Geneti-callyengineeredplantsthat contain
the bacterial genes for nitrogen fixation could grow well even in nitrogen-poor soil.
Geneticengineersare alsodevelopingplantsthatproduce theirownpesticides(one biteand pest dies).
In 1986, the first of such genetically engineered plants successfully passed a field test.
The value of plantproteinto humandietis being improved by creating corn or beans that manufacture
a complete protein, one with all the amino acids essential to the human diet.
In anothereffort (W. David et al., Science, Vol. 234, pp. 856, 1986) the luciferase gene from the firefly,
Photinuspyrahswasusedasa re-porterof gene expressionbylightproductionintransfectedplantcells
and transgenic plant.
A complementary DNA (cDNA) clone of the firefly luciferase gene under the control of a plant virus
promoter (cauliflower mosaic virus 35 S RNA promoter) was introduced into plant protoplast cells
(Dancus carota) and into plants (Nicotiana tobacum) by the use of the Agrobactenum (time- facience
tumor-inducing plasmid.
In thisexper-iment, stableexpressionof the fireflyluciferase geneinplantcell andtransgenicplants has
been achieved. The transgenic plant incubated in luciferin also emits light like firefly. The successful
introductionof animal DNA intoplantgenome hasopenedanew avenue inthe field of biotech-nology.
Withthe helpof thistechnologyproduc-tionof antibodyhasalsobeenpossible inplant(Nature 342 No.
6245, 1989).

9. Summary:
The importance and application of plant cell and tissue culture in plant science are vast and varied.
There have been many valuable con-tribution of plant tissue culture in the field of fundamental and
applied botany. The regeneration of whole plants through tissue culture is popularly called “micro-
propaga­tion”. By this method, a large number of plant species can be propagated all the year round.
The plant breeder is no longer restricted by sea-son in the production of large numbers of plants.
In vitroclonal propagationisa type of mi-cro-propagation.Bythismethod the variabil-ity that can arise
fromsexual reproductionandseedformationinacrop plant,can be omit-ted. The plant with long seed
dormancycan be raisedfasterbyinvitro clonal propagation. For the orchid, in vitro clonal propagation
isthe onlycommerciallyviablemethodof micro-propa-gation.Clonal multiplication of a cultivar is very
important in horticulture and silviculture.
The plant tissue culture isalsoprovingtobe richand novel sourcesof variabilitywithagreatpotential in
crop improvementwithoutre-sortingtomutationorhybridization.LarkinandScowcroft have proposed
a general term “Soma­clones” for plant variants obtained from tissue cultures, irrespective of their
origin.Suchvari-antplantsmayshow some useful characters such as resistance to a particular disease,
herbicide resistance, stress tolerance etc. Plant breeders can exploit such variants for their breeding
pro-gramme.
There have been many valuable uses of plant tissue culture to solve the problems con-cerning plant
pathology.One outstandingsuc-cessisthe viruseradication by apical meristem culture and the second
success of tissue culture in plant pathology is the result of its applica-tion to the problems of plant
tumors, especially crown gall.
The convention, breeding methods are most widely used for crop improvement. But in cer-tain
situations, these methods have to be sup-plemented with plant tissue culture technique either to
increase their efficiency or to be able to achieve the objective which is not possible through
conventional methods.
Embryoculture isnow routinelyusedtorecoveryof hybridplantfromdistantcrosses.Meristemculture,
anther and pollen culture, plant protoplast culture and somatic hybridization contribute a lot of
poten-tial applications in plant breeding and plant im-provement programmes. Plant tissue culture,
particularlycell sus-pensionculture,hasbeenexploited for the pro-duction of useful alkaloids, cardiac
glycosides and other steroids.
Preservation of plant genetic resources can be achieved by plant tissue culture. Cryopreservation of
planttissue,cell ormeristeminliquidnitrogenfordesirable period and the recovery of whole plant via
organogenesis is also a valuable tool for the preservation of very rare germplasm and to maintain a
clone for crop improvement.

10. Planttissue culture isrelativelyayoungfieldof biotechnology.Biotechnologistsare try-ingtomodifythe
geneticsof the culturedcellsbythree ways—mutagenesisandselection of cell lines; transplantation of
foreigngeneticmaterial inprotoplastbygeneticengineering,andsomatichybridizationby the fusion of
distantly related plant protoplasts just to widen the genetic diver-sity among the existing plants.