Tissue culture: Tissue culture is the growth of tissues and/or cells separate from the organism.

This is typically facilitated via use of a liquid, semi-solid, or solid growth media, such as broth or agar. Plant tissue culture is a practice used to propagate plants under sterile conditions, often to produce clones of a plant. Different techniques in plant tissue culture may offer certain advantages over traditional methods of propagation, including:

The production of exact copies of plants that produce particularly good flowers, fruits, or have other desirable traits. To quic ly produce mature plants. The production of multiples of plants in the absence of seeds or necessary pollinators to produce seeds. The regeneration of whole plants from plant cells that have been genetically modified. The production of plants in sterile containers that allows them to be moved with greatly reduced chances of transmitting diseases, pests, and pathogens. The production of plants from seeds that otherwise have very low chances of germinating and growing, i.e.: orchids. To clean particular plant of viral and other infections and to quic ly multiply these plants as !cleaned stoc ! for horticulture and agriculture.

Totipotency is the ability of a single cell to divide and produce all the differentiated cells in an organism, including extraembryonic tissues. Totipotent cells formed during sexual and asexual reproduction include spores and "ygotes. #ygotes are the products of the fusion of two gametes $fertili"ation%. &n some organisms, cells can dedifferentiate and regain totipotency. 'or example, a plant cutting or callus can be used to grow an entire plant. The molecular mechanisms controlling totipotency are not well understood and are a sub(ect for current research. )ecently $*++,%, some reports in -cience suggest that in the model organism Caenorhabditis elegans, multiple mechanisms including )./ regulation maintain totipotency at different stages of development.

Explant: 0iving tissue, to transfer from an organism to an artificial medium for culture.

Uses of plant cell and tissue culture -cientific purpose and basic research 1icropropagation 2roduction of pathogen free plants 3rop improvement, plant breeding 4enetic engineering -ynthetic seed production 4ermplasm preservation -econdary product expression in vitro Plant propagators use plant tissue culture techniques in order to achieve one or more of the following objectives 5limination of viruses from infected plants )apid multiplication of clones 6egetative propagation of difficult to propagate species /ll the year round propagation of clones )apid multiplication of seedlings $in cases where seed is hard to get% Applications of tissue culture for crop improvement: &mportant applications of tissue culture in plant breeding are the production of new mutants, genomic recombination and selection of new recombinant by:

 /nther culture for haploid production 5mbryo/ovule culture after interspecific hybridi"ation 4enetic engineering In vitro selection In vitro germplasm conservation and exchange 1icropropagation 3ell and organ culture -omaclonal variation -omatic cell hybridi"ation $protoplast fusion% -omatic embryogenesis

aboratory facilities: Preparation of media 4as, water and electricity supplies and possibly compressed air and vacuum lines 7ater heater Different types of 4lassware 8otplate with magnetic stirrer -teamer for heating media 3oarse and sensitive balance

 -patula 1ixer 1icrowave oven 1illipore filter system /utomatic dispenser p8 meter Distillation apparatus De-ioni"er 7ide range of chemicals 9itchen timer 1etal rac s 1etal petridish holders Test tubes, flas s, plastic containers /utoclave or pressure coo er -torage space for chemicals, glasswares etc. -terile storage space for nutrient media, sterile water etc. -torage tan for distilled and/or de-ioni"ed water Drying and draining rac s 2ipette washer !solation of cultures 0aminar air flow cabinet

 :unsenbarner Dry sterili"er for nives, scalpels, forceps 'ilter paper 2etridishes 3entrifuge 8ypochlorite for sterili"ation ;,< alcohol 'ire-proof storage space for alcohol "eneral equipment Drying oven for drying glassware etc. after sterili"ation /utomatic dishwasher 3leaning materials $brushes etc.% Detergent Trolley )efrigerator for storing chemicals and nutrient media -mall deepfree"e #ulture room Temperature control $=>-*>o3% with cooling as well as heating 5lectricity supply essential for lighting, cooling and heating -helving for culture rac s 'luorescent tubes for lighting Timer for regulating day-length

 /larm system in case of malfunction Test tube rac s Table for ma ing observations -ha er or rotator $or%ing space for an efficient tissue culture laboratory 4reen house for cultivation and further growth of plant material 0aboratory where the nutrient media can be prepared, preferably with a lot of cupboard space -torage space for all apparatus $glassware, chemicals, nutrient media etc.% 7ashing-up area for autoclave and dishwasher preferably a separate room &noculation room with laminar air-flow cabinets, preferably with sterile $filtered% air under positive pressure )oom of balances, refrigerator, deep free"e, etc. 3ulture room Technicians room

Tissue culture media 3ommonly used culture media are,

=. 1urashige and - oog media $=;,*% *. -chen and 8ildebrandt media $=;>*% ?. 4authert medium $=;@*% @. 4amborgAs or :B media

B. 7hiteAs media $=;,?% ,. 8ellerAs media $=;B?% >. 0insmaier and - oog media $=;,B% C.

3hu or ., media $=;>C%

;. .itsch and .itsch media $=;,;% =+. 9ao medium $=;>@%

Tissue #ulture &edia'#omposition &E(!A #)&P)*E*T+ Dne of the most important factors governing the growth and morphogenesis of plant tissues in culture is the composition of the culture medium. The basic nutrient requirements of cultured plant cells are very similar to those of whole plants. 2lant tissue and cell culture media are generally made up of some or all of the following components: macronutrients, micronutrients, vitamins, amino acids or other nitrogen supplements, sugar$s%, other undefined organic supplements, solidifying agents or support systems, and growth regulators. -everal media formulations are commonly used for the ma(ority of all cell and tissue culture wor . These media formulations include those described by 7hite, 1urashige and - oog, 4amborg et. al., -chen and 8ilderbrandt, .itsch and .itsch, and 0loyd and 1c3own. 1urashige and - oogAs 1- medium, -chen and 8ildebrandAs -8 medium, and 4amborgAs :-B medium are all high in macronutrients, while the other media formulations contain considerably less of the macronutrients.

&acronutrients

The macronutrients provide the six ma(or elements-nitrogen $.%, phosphorus $2%, potassium $9%, calcium $3a%, magnesium $1g%, and sulfur $-%-required for plant cell or tissue growth. The optimum concentration of each nutrient for achieving maximum growth rates varies considerably among species. 3ulture media should contain at least *B-,+ m1 of inorganic nitrogen for adequate plant cell growth. 2lant cells may grow on nitrates alone, but considerably better results are obtained when the medium contains both a nitrate and ammonium nitrogen source. 3ertain species require ammonium or another source of reduced nitrogen for cell growth to occur. .itrates are usually supplied in the range of *B*+ m1E typical ammonium concentrations range between * and *+ m1. 8owever, ammonium concentrations in excess of C m1 may be deleterious to cell growth of certain species. 3ells can grow on a culture medium containing ammonium as the sole nitrogen source if one or more of the T3/ cycle acids $e.g., citrate, succinate, or malate% are also included in the culture medium at concentrations of approximately =+ m1. 7hen nitrate and ammonium sources of nitrogen are utili"ed together in the culture medium, the ammonium ions will be utili"ed together in the culture medium, the ammonium ions will be utili"ed more rapidly and before the nitrate ions. 2otassium is required for cell growth of most plant species. 1ost media contain 9, in the nitrate or chloride form, at concentrations of *+-?+ m1. The optimum concentrations of 2, 1g, -, and 3a range from =-? m1 when all other requirements for cell growth are satisfied. 8igher concentrations of these nutrients may be required if deficiencies in other nutrients exist.

&icronutrients The essential micronutrients for plant cell and tissue growth include iron $'e%, manganese $1n%, "inc $#n%, boron $:%, copper $3u%, and molybdenum $1o%. 3helated forms of iron and "inc are commonly used in preparing culture media. &ron may be the most critical of all the micronutrients. &ron citrate and tartrate may be used in culture media, but these compounds are difficult to dissolve and frequently precipitate after media are prepared. 1urashige and - oog used an ethylene diaminetetraacetic acid $5DT/%-iron chelate to bypass this problem.

3obalt $3o% and iodine $&% may also be added to certain media, but strict cell growth requirements for these elements have not been established. -odium $.a% and chlorine $3l% are also used in some media but are not essential for cell growth. 3opper and 3obalt are normally added to culture media at concentrations of +.= F1, 'e and 1o at = F1, & at B F1, #n at B-?+ F1, 1n at *+-;+ F1, and : at *B=++ F1. #arbon and Energy +ource The preferred carbohydrate in plant cell culture media is sucrose. 4lucose and fructose may be substituted in some cases, glucose being as effective as sucrose and fructose being somewhat less effective. Dther carbohydrates that have been tested include lactose, galactose, rafinose, maltose, and starch. -ucrose concentrations of culture media normally range between * and ? percent. Gse of autoclaved fructose can be detrimental to cell growth. 3arbohydrates must be supplied to the culture medium because few plant cell lines have been isolated that are fully autotropic, e.g., capable of supplying their own carbohydrate needs by 3D* assimilation during photosynthesis. ,itamins .ormal plants synthesi"e the vitamins required for their growth and development. 6itamins are required by plants as catalysts in various metabolic processes. 7hen plant cells and tissues are grown in vitro, some vitamins may become limiting factors for cell growth. The vitamins most frequently used in cell and tissue culture media include thiamin $:=%, nicotinic acid, pyridoxine $:,%, and myo-inositol. Thiamin is the one vitamin that is basically required by all cells for growth. Thiamin is normally used at concentrations ranging from +.= to =+.+ mg/liter. .icotinic acid and pyridoxine are often added to culture media but are not essential for cell growth in many species. .icotinic acid is normally used at concentrations of +.=-B.+ mg/literE pyridoxine is used at +.=-=+.+ mg/liter. 1yo-inositol is commonly included in many vitamin stoc solutions. /lthough it is a carbohydrate not a vitamin, it has been shown to stimulate growth in certain cell cultures. &ts presence in the culture medium is not essential, but in small quantities

myo-inositol stimulates cell growth in most species. 1yo-inositol is generally used in plant cell and tissue culture media at concentrations of B+-B+++ mg/liter. Dther vitamins such as biotin, folic acid, ascorbic acid, pantothenic acid, vitamin 5 $tocopherol%, riboflavin, and p-aminoben"oic acid have been included in some cell culture media. The requirement for these vitamins by plant cell cultures is generally negligible, and they are not considered growth-limiting factors. These vitamins are generally added to the culture medium only when the concentration of thiamin is below the desired level or when it is desirable to grow cells at very low population densities. Amino Acids or )ther *itrogen +upplements /lthough cultured cells are normally capable of synthesi"ing all of the required amino acids, the addition of certain amino acids or amino acid mixtures may be used to further stimulate cell growth. The use of amino acids is particularly important for establishing cell cultures and protoplast cultures. /mino acids provide plant cells with an immediately available source of nitrogen, which generally can be ta en up by the cells more rapidly than inorganic nitrogen. The most common sources of organic nitrogen used in culture media are amino acid mixtures $e.g., casein hydrolysate%, 0-glutamine, 0-asparagine, and adenine. 3asein hydrolysate is generally used at concentrations between +.+B and +.= percent. 7hen amino acids are added alone, care must be ta en, as they can be inhibitory to cell growth. 5xamples of amino acids included in culture media to enhance cell growth are glycine at * mg/liter, glutamine up to C m1, asparagine at =++ mg/liter, 0-arginine and cysteine at =+ mg/liter, and 0-tyrosine at =++ mg/liter. Tyrosine has been used to stimulate morphogenesis in cell cultures but should only be used in an agar medium. -upplementation of the culture medium with adenine sulfate can stimulate cell growth and greatly enhance shoot formation.

Undefined )rganic +upplements /ddition of a wide variety of organic extracts to culture media often results in favorable tissue responses. -upplements that have been tested include protein

hydrolysates, coconut mil , yeast extracts, malt extracts, ground banana, orange (uice, and tomato (uice. 8owever, undefined organic supplements should only be used as a last resort, and only coconut mil and protein hydrolysates are used to any extent today. 2rotein $casein% hydrolysates are generally added to culture media at a concentration of +.+B-+.=<, while coconut mil is commonly used at B*+< $v/v%. The addition of activated charcoal $/3% to culture media may have a beneficial effect. The effect of /3 is generally attributed to one of three factors: absorption of inhibitory compounds, absorption of growth regulators from the culture medium, or dar ening of the medium. The inhibition of growth in the presence of /3 is generally attributed to the absorption of phytohormones to /3. =-.apthaleneacetic acid $.//%, inetin, ,-ben"ylaminopurine $:/%, indole-?-acetic acid $&//%, and ,-H-H-dimethylallylaminopurine $*i2% all bind to /3, with the latter two growth regulators binding quite rapidly. The stimulation of cell growth by /3 is generally attributed to its ability to bind to toxic phenolic compounds produced during culture. /ctivated charcoal is generally acid-washed prior to addition to the culture medium at a concentration of +.B-?.+ percent.

+olidifying Agents or +upport +ystems /gar is the most commonly used gelling agent for preparing semisolid and solid plant tissue culture media. /gar has several advantages over other gelling agents. 'irst, when agar is mixed with water, it forms a gel that melts at approximately ,+I-=++I 3 and solidifies at approximately @BI3E thus, agar gels are stable at all feasible incubation temperatures. /dditionally, agar gels do not react with media constituents and are not digested by plant en"ymes. The firmness of an agar gel is controlled by the concentration and brand of agar used in the culture medium and the p8 of the medium. The agar concentrations commonly used in plant cell culture media range between +.B and =.+<E these concentrations give a firm gel at the p8As typical of plant cell culture media. /nother gelling agent commonly used for commercial as well as research purposes is 4elrite. This product is synthetic and should be used at =.*B-*.B g/liter, resulting in a clear gel which aids in detecting contamination.

/lternative methods of support have included use of perforated cellophane, filter paper bridges, filter paper wic s, polyurethane foam, and polyester fleece. 7hether explants grow best on agar or on other supporting agents varies from one species of plant to the next.

"rowth -egulators 'our broad classes of growth regulators are important in plant tissue cultureE the auxins, cyto inins, gibberellins, and abscisic acid. - oog and 1iller were the first to report that the ration of auxin to cyto inin determined the type and extent of organogenesis in plant cell cultures. :oth an auxin and cyto inin are usually added to culture media in order to obtain morphogenesis, although the ratio of hormones required for root and shoot induction is not universally the same. 3onsiderable variability exists among genera, species, and even cultivars in the type and amount of auxin and cyto inin required for induction of morphogenesis. The auxins commonly used in plant tissue culture media are =8-indole-?-acetic acid $&//%, =8-indole-?-butyric acid $&:/%, $*,@-dichlorophenoxy% acetic acid $*,@-D%, and =-napthaleneacetic acid $.//%. The only naturally occurring auxin found in plant tissues is &//. Dther synthetic auxins that have been used in plant cell culture include @-chlorophenoxyacetic acid or p-chlorophenoxyacetic acid $@32/, 232/%, $*,@,B-trichlorophenoxy%acetic acid $*,@,B-T%, ?,,-dichloro-*methoxyben"oic acid $Dicamba%, and @-amino-?,B,,-trichloropicolinic acid $2icloram%. The various auxins differ in their physiological activity and in the extent to which they move through tissue, are bound to the cells, or metaboli"ed. .aturally occurring &// has been shown to have less physiological activity than synthetic auxins. :ased on stem curvature assays, *,@-D has eight to twelve times the activity, *,@,B-T has four times the activity, 232/ and 2icloram have two to four times the activity, and .// has two times the activity of &//. /lthough *,@-D, *,@,B-T, 232/, and 2icloram are often used to induce rapid cell proliferation, exposure to high levels or prolonged exposure to these auxins, particularly *,@-D, results in suppressed morphogeneic activity. /uxins are generally included in a culture medium to stimulate callus production and cell growth, to initiate shoots,

particularly roots, and to induce somatic embryogenesis and stimulate growth from shoot apices and shoot tip cultures. The cyto inins commonly used in the culture media include ,-ben"ylaminopurine or ,-ben"yladenine $:/2, :/%, ,-H-H-dimethylaminopurine $*i2%, .-$*furanylmethyl%-=8-puring-,-amine $ inetin%, and ,-$@-hydroxy-?-mehty-trans-*butenylamino%purine $"eatin%. #eatin and *i2 are considered to be naturally occurring cyto inins, while :/ and inetin are synthetically derived cyto inins. /denine, another naturally occurring compound, has a base structure similar to that of the cyto inins and has shown cyto inin-li e activity in some cases. 1any plant tissues have an absolute requirement for a specific cyto inin for morphogenesis to occur, whereas some tissue are considered to be cyto inin independent, i.e., no cyto inin or a specific cyto inin may be required for organogenesis. The cyto inins are generally added to a culture medium to stimulate cell division, to induce shoot formation and axillary shoot proliferation, and to inhibit root formation. The type of morphogenesis that occurs in a plant tissue culture largely depends upon the ratio and concentrations of auxins and cyto inins present in the medium. )oot initiation of plantlets, embryogenesis, and callus initiation all generally occur when the ration of auxin to cyto inin is high, whereas adventitious and axillary shoot proliferation occur when the ration is low. The concentrations of auxins and cyto inins are equally as important as their ratio. 4ibberellins $4/?% and abscisic acid $/:/% are two other growth regulators occasionally used in culture media. 2lant tissue cultures can usually be induced to grow without either 4/? or /:/, although, certain species may require these hormones for enhanced growth. 4enerally, 4/? is added to culture media to promote the growth of low-density cell cultures, to enhance callus growth, and to elongate dwarfed or stunted plantlets. /bscisic acid is generally added to culture media to either inhibit or stimulate callus growth $depending upon the species%, to enhance, inhibit, or stimulate callus growth $depending upon the species%, to enhance shoot or bud proliferation, and to inhibit latter stages of embryo development.

#ompounds used for culture media

a. &acronutrients 9.D? .8@.D? 3a3l*.*8*D 1g-D@.>8*D 98*2D@ 3a$.D?%* $.8@%*-D@ .8@8*2D@ .a.D? .a8*2D@.@8*D 93l

b. &icronutrients 1n-D@. 8*D 9& 8?:D? #n-D@. >8*D 3u-D@. B8*D .a*1oD@. *8*D 3o3l*. ,8*D /l3l? .i3l*. ,8*D

c. !ron source 'e3l?.,8*D 'e-D@. >8*D 'e* $-D@%? .a*5DT/ d. vitamins 1yo-inositol Thiamine. 83l .icotinic acid 2yrodoxine. 83l 5ast extract 4lycine

e. Phytohormones /uxin: 2-chloro phenoxy acetic acid $232/% *,@-dichloro phenoxy acetic acid $*,@-D% &ndole acetic acid $&//% .apthelene acetic acid $.//% &ndole :utaric acid $&:/%

3yto inine:

,-:en"yl amino purine #eatin #ip-isopentyl adenine

4ibberellins: 4ibberellic acid

Preparation of &urashige and +%oog medium &urashige and +%oog medium or $MSO or MS0 (MS-zero)% is a plant growth medium used in the laboratories for cultivation of plant cell culture. 1-D was invented by plant scientists Toshio 1urashige and 'ol e 9. - oog during 1urashige!s search for a new plant growth regulator. &t is the most commonly used medium in plant tissue culture experiments.

+toc% +olutions The use of stoc solutions reduces the number of repetitive operations involved in media preparation and, hence, the chance of human or experimental error. 1oreover direct weighing of media components $e.g., micronutrients and hormones% that are required only in milligram or microgram quantities in the final formulation cannot be performed with sufficient accuracy for tissue culture wor . 'or these components, preparation of concentrated stoc solutions and subsequent dilution into the final media is standard procedure. &n addition, concentrated solutions of some materials are more stable and can be stored for longer periods than more dilute solutions. To prepare a stoc solution, weigh out the required amount of the compound and place it in a clean flas . &t is common practice to ma e a stoc solution =+x or =++x, depending upon the solubility of the compound. Dnce the chemical is in the flas , dissolved it in a small amount of water, ethyl alcohol, = . .aD8, or = .

830. .ext, slowly add double-distilled water to the flas , while agitating. 3ontinue this until the proper volume is reached. 0abel the flas with the name of the solution, preparation and expiration dates, and the name of the person who prepared the solution. 3ertain items, e.g., &//, must be prepared and stored in amber bottles to prevent photodecomposition. The volumes of stoc solutions prepared at various concentrations that must be used to achieve various final concentrations are presented in tabular form in the 2lant 4rowth )egulator -ection. &acronutrients -toc solutions of macronutrients can be prepared at =+ times the concentration of the final medium. / separate stoc solution for calcium salts may be required to prevent precipitation. -toc solution of macronutrients can be stored safely for several wee s in a refrigerator at *I-@I3. &icronutrients 1icronutrient stoc solutions are generally made up at =++ times their final strength. &t is recommended that micronutrient stoc s be stored in either a refrigerator or free"er until needed. 1icronutrient stoc solutions could be stored in a refrigerator for up to = year without appreciable deterioration. &ron stoc solutions should be prepared and stored separately from other micronutrients in an amber storage bottle. ,itamins 6itamins are prepared as =++J or =+++J stoc solutions and stored in a free"er $*+I3% until used. 6itamin stoc solutions should be made up each time media is prepared if a refrigerator or free"er is not available. 6itamin stoc solutions can be stored safely in a refrigerator for *-? months but should be discarded after that time. "rowth -egulators The auxins .// and *,@-D are considered to be stable and can be stored at @I3 for several monthsE &// should be stored at -*+I3. /uxin stoc solutions are generally prepared at =++-=+++ times the final desired concentrations. -olution of .// and *,@-D can be stored for several months in a refrigerator or indefinitely at -*+I3. 4enerally &// and *,@-D are dissolved in a small volume of ;B< ethyl alcohol or 9D8 and then brought to volume with double-distilled waterE .// can be dissolved in a small amount of = . .aD8 or 9D8, which also can be used to dissolve *,@-D and &//.

The cyto inins are considered to be stable and can be stored at -*+I3. 3yto inin stoc solutions are generally prepared at =++J to =+++J concentrations. 1any of the cyto inins are difficult to dissolve, and a few drops of either = . 830, = . .aD8, in 9D8 or D1-D, are required to bring them into solution. +torage of +toc% +olutions -torage conditions for most stoc solutions have already been pointed outE however, some additional points can be made. 'or convenience, many labs prepare stoc solutions and then divide them into aliquots sufficient to prepare from = to =+ liter of mediumE these aliquots are stored in small vials or plastic bags in a free"er. This procedure removes the inconvenience of having to un-thaw a large volume of fro"en stoc each time medium is prepared. -ome have found that heating in a microwave oven is a satisfactory and quic method of thawing concentrated medium.

+teps in the preparation of the &+ medium /0 litre volume1 a. Depending on the type of medium, the required amount of sucrose was ta en in =0 bea er, @++ ml filled with sterili"ed distilled water and agitated with a magnetic stirrer. b. .ecessary amount of stoc solutions were added to the bea er one by one. c. 1yo-inositol was measured and added to the medium. d. =+ mg &// was weighed and dissolved it in a few drops of =. .ao8 and then it was transferred to the medium mixture. e. -terili"ed distilled water was added until the total volume of liquid is approximately C++ ml f. The medium was transferred to a =0 volumetric flas and sterili"e distilled water was added to the final volume g. The p8 of the medium was ad(usted to B.C to , by delivering droplets of =. .D8 or =. 83l.

h. The medium was then transferred to =0 5rlenmeyer flas and required amount of agar was added to the medium. The flas was capped with aluminum foil. i. The medium was then sterili"e by autoclaving at a pressure of =B lbs/inch * and =*=o3 for =B minutes. 3D12D-&T&D. /.D 2)52/)/T&D. D' 1- 15D&G1 &.30GD&.4 -TD39 -D0GT&D.&ngradient -toc 1acronutrients 9.D? .8@.D? 1g-D@. >8*D 3a3l*. *8*D 98*2D@ 1icronutrients 1n-D@.@8*D #n-D@. >8*D 8?:D? 9& .a*1oD@. *8*D 3u-D@. B8*D 3o3l*. ,8*D g/0 $=+J% =;.++ =,.B+ ?.>+ @.@+ =.>+ mg/0 $=++J% **?+ C,+ ,*+ C? *B *.B *.B 3oncentration 1- medium mg/0$=+ ml gives% =;++ =,B+ ?>+ @@+ =>+ mg/0 $=+ ml gives% **.? C., ,.* +.C? +.*B +.+*B +.+*B

&ron source .a*5DT/ 'e-D@. >8*D 6itamins 4lycine .icotinic acid 2yrodoxine. 83l Thiamine. 83l 3yto inine 9inetine

mg/=++ ml $*+J% >@, BB, mg/=++ml $=++J% *+ +B +B += mg/=++ ml $=++J% =+

mg/0 $B ml gives% ?>.? *>.C mg/0 $=ml stoc gives% +.* +.+B +.+B +.+= mg/0 $= ml stoc gives% +.= mgl0

1yo-inositol &// -ucrose /gar $+.C< w/v%

=++ =+ ?+,+++ ;+++

+terillant An agent that destroys all viable forms of microbial life to achieve sterili2ation. +terili2ation is the destruction of all living matter. -in s are cleaned or disinfected, explants are disinfected, but media and transfer tools are sterili"ed.

(isinfection implies the use of chemical agents to ill pathogenic microbes, without necessarily sterili"ing the material to which the chemical is applied. +anitation is the processes of substantially reducing, and then maintaining, the microbial population in the air and on ob(ects in the laboratory to acceptable levelsE in short being clean by using water, soap, or detergent. +terili2ation of plant tissue 2lant material is surface sterili"ed using chemicals agents. 'or ovule, embryo, and endosperm culture, only the seeds are surface sterili"ed and the ovule or embryo or endosperm is dissected under aseptic conditions $usually in a laminar air flaw cabinet%. -imilarly for shoot apices, the buds are surface sterili"ed and the apices are dissected under aseptic conditions. /fter surface sterili"ation, plant material is rinsed ?-@ times in sterile distilled water to remove any remaining chemical sterili"ing agent. /ll operations including transfer of plant material are carried out under aseptic conditions, preferably under the hood of a laminar air flaw cabinet, which are commercially available for tissue culture laboratories. These cabinets have now replaced G6- sterili"ation wooden cabinets earlier utili"ed for this purpose. #hemicals: ' +odium 3ypochlorite /*a)#l1: &t is the most common chemical agent used to sterili"e plant tissue $+.+*B<-+.*B< .aD3l%. Diluted household bleach can also be used for this purpose, which normally contains B.*B< .aD3l. &t is equally effective and considerably less expensive. ' #alcium hypochlorite /#a)#l1: &t can be used as a substitute for .aD3l. 3aD3l causes slightly less damage to plant tissues but tends to precipitate out of solution. To avoid the accumulation of 3aD3l on the plant tissue surfaces, sterili"ation solutions should be filtered or decanted before use. ' 3ydrogent per oxide /34)41: 2lant tissues can also be surface sterili"ed using an 8*D* $?<-=+<%. &t is much easier to remove from tissues than .aD3l and 3aD3l. ' )ther substances: 2lant tissue can also be surface-sterili"ed by bromine water $=<-*<%, silver nitrate $/g.D? =<% and mercuric chloride $1g3l* +.=<-=<%%.

+urfactants ' Tween 45 or Triton 6'055: These are scientific reagent grade surfactants and are often added in low concentrations $+.+B<% to chemical sterili"ation solutions. Their use ensures that the sterili"ing agent come in contact with the entire plant tissue surface. ' +tirring the tissue: stirring the tissues during sterili"ation also facilitates good surface contact. ' Ultrasonic 7ath: &t is an effective method to ensure good surface contact during sterili"ation treatment in an ultrasonic bath li e the one those used to clean dentures. +terili2ation of media Tissue culture media are generally sterili"ed by autoclaving at =*=o3 and =.+B g/cm* $=B-*+ psi%. The time required for sterili"ation depends upon the volume of medium in the vessel.

&inimum Autoclaving Time 6olume of medium per vessel $ml% *B B+ =++ *B+ 1inimum autoclaving $min% *+ *B *C ?= 6olume of medium per vessel $ml% B++ =+++ *+++ @+++ 1inimum autoclaving $min% ?B @+ @C ,?

1inimum autoclaving time includes the time required for the liquid volume to reach the sterili"ing temperature $=*=o3% and =, min at =*=o3.

&f autoclave is not available, a pressure coo er may be used. 4rowth factors, such as 4/?, "eatin, /:/, urea, etc. $which are thermolabile% should not be autoclaved, but sterili"ed separately by membrane filtration. +terili2ation by filtration 'iltration is an excellent way to reduce the microbial population in solutions of heat-sensitive material, such as toxins, serum, and antibodies. )ather than directly destroying contaminating microorganisms, the filter simply removes them. :acteria can be stopped by using a filter of pore si"e less than +.>B Km. 1any small filters are used for viruses. 7hen using bacterial filters, the solution is not sterile because viruses and mycoplasma can pass through. The filter is tested by the fact that it should retain Serratia marcescens from broth culture. The following types of filters are nown. ' /sbestos and asbestos-paper dis s $-eit"% ' -intered glass filters ' 1icro filters ' Depth filters ' 3ellulose membrane filters =. 4radocol membrane $cellulose nitrate% *. 3ellulose acetate Air sterili2aton 0aminar flow biological safety cabinets- employs high-effeciency particulate air $852/% filters, which remove ;;.;>< of +.?Km particles. -terili"ation by )adiation $ultraviolet radiation usually *,+ nm%. &t can not penetrate glass, dirt, films, water, and other substances very effectively. G6 is harmful for man ind, so s illness is required. +terili2ation of glassware .ew glassware is always washed using detergents especially designed for the purpose to remove all traces of acids, etc. 'inally the glassware is rinsed in tap

water and then in distilled water. -ometimesE the glassware may need to be autoclaved before washing to remove agar medium and to destroy microbial contaminants. &icropropagation is the practice of rapidly multiplying stoc% plant material to produce a large number of progeny plants, using modern plant tissue culture methods. &t is the art and science of plant multiplication in vitro. The process includes many steps--stoc plant care, explant selection and sterili"ation, media manipulation to obtain proliferation, rooting, acclimation, and growing on of liners 1icropropagation is used to multiply novel plants, such as those that have been genetically modified or bred through conventional plant breeding methods. &t is also used to provide a sufficient number of plantlets for planting from a stoc plant which does not produce seeds, or does not respond well to vegetative reproduction. &ethod Establishment 1icropropagation begins with the selection of plant material to be propagated. 3lean stoc materials that are free of viruses and fungi are important in the production of the healthiest plants. Dften plants are first virus indexed to determine if they are clean and free of viruses. Dnce the plant material is chosen for culture, the collection of explant$s% begins and is dependent on the type of tissue to be usedE including stem tips, anthers, petals, pollen and others plant tissues. The explant material is then surface sterili"ed, usually in multiple courses of bleach and alcohol washes and finally rinsed in sterili"ed water. This small portion of plant tissue, sometimes only a single cell, is placed on a growth medium, typically containing sucrose as an energy source and one or more plant growth regulators $plant hormones%. Gsually the medium is thic ened with agar to create a gel which supports the explant during growth. -ome plants are easily grown on simple media but others require more complicated media for successful growE some media include vitamins, minerals and amino acids. The medium is sterili"ed during preparation to prevent fungal and bacterial contamination, which can outgrow and smother the growing explant. /utoclaves and filter sterili"ation are used to remove potential contaminates, under smaller scales of production a pressure coo er is often used. The plant tissue grows and differentiates into new tissues depending on the medium. 'or example, media containing cyto inins are used to create branched shoots from plant buds.

&ultiplication 1ultiplication is the ta ing of tissue samples produced during the first stage and increasing their number. 'ollowing the successful introduction and growth of plant tissue, the establishment stage is followed by multiplication. Through repeated cycles of this process, a single explant sample may be increased from one to hundreds or thousands of plants. Depending on the type of tissue grown, multiplication can involve different methods and media. &f the plant material grown is callus tissue, it can be placed in a blender and cut into smaller pieces and recultured on the same type of culture medium to grow more callus tissue. &f the tissue is grown as small plants called plantlets, hormones are often added that cause the plantlets to produce many small offshoots that can be removed and recultured. Pretransplant This stage involves treating the plantlets/shoots produced to encourage root growth and Lhardening.L &t is performed in vitro, or in a sterile Ltest tubeL environment. )oot growth does not always occur in the earlier stages in plant cell culture, and is of course a requirement for successful plant growth after the micropropagation procedure. &t is often performed in vitro by transferring the plantlets to a growth medium containing auxin$s% which stimulate root initiation. The pretransplant stage is not always performedE -ome plants are micropropagated and grown in culture and normal cuttings are made that are then rooted ex vitro. L8ardeningL refers to the preparation of the plants for a natural growth environment. Gntil this stage, the plantlets have been grown in LidealL conditions, designed to encourage rapid growth. Due to lac of necessity, the plants are li ely to be highly susceptible to disease and often do not have fully functional dermal coverings and will be inefficient in their use of water and energy. &n vitro conditions are high in humidity and plants grown under these condition do not form a wor ing cuticle and stomata that eep the plant from drying out, when ta en out of culture the plantlets need time to ad(ust to more natural environmental conditions. 8ardening typically involves slowly weaning the plantlets from a highhumidity, low light, warm environment to what would be considered a normal growth environment for the species in question. This is done by moving the plants to a location high in humidity, such as a green house with regular mist watering. Transfer from culture

&n the final stage of plant micropropagation, the plantlets are removed from the plant media and transferred to soil or $more commonly% potting compost for continued growth by conventional methods. This stage is often combined with the LpretransplantL stage. Advantages 1icropropagation has a number of advantages over traditional plant propagation techniques:

The main advantage of micropropagation is the production of many plants that are clones of each other. 1icropropagation can be used to produce disease-free plants. 1icropropagation produces rooted plantlets ready for growth, saving time for the grower when seeds or cuttings are slow to establish or grow. &t can have an extraordinarily high fecundity rate, producing thousands of propagules while conventional techniques might only produce a fraction of this a number. &t is the only viable method of regenerating genetically modified cells or cells after protoplast fusion. &t is useful in multiplying plants which produce seeds in uneconomical amounts, or when plants are sterile and do not produce viable seeds or when seed can!t be stored $vgr. recalcitrant seeds%. 1icropropagation often produces more robust plants, leading to accelerated growth compared to similar plants produced by conventional methods - li e seeds or cuttings. -ome plants with very small seeds, including most orchids, are most reliably grown from seed in sterile culture. / greater number of plants can be produced per square meter and the propagules can be stored longer and in a smaller area.

(isadvantages 1icropropagation is not always the perfect means of multiplying plants, conditions that limit its use include:

&t is very expensive, and can have a labor cost of more than >+< /s monoculture is produced after micropropagation, in case of infection whole crop can get damaged /n infected plant sample can produce infected progeny. This is uncommon if the stoc plants are carefully screened and vetted to prevent culturing plants infected with virus or fungus. -pecies specificity. .ot all plants can be successfully tissue cultured, often because the proper medium for growth is not nown or the plants produce secondary metabolic chemicals that stunt or ill the explant. 6ariability. -ometimes plants or cultivars do not come true to type after being tissue cultured, this is often dependent on the type of explant material utili"ed during the initiation phase or the result of the age of the cell or propagule line. 3ontamination. -ome plants are very difficult to disinfest of fungal organisms. 6ariability /cclimati"ation

The ma(or limitation in the use of 1icropropagation for many plants is the cost of productionE for many plants the use of seeds, which are normally disease free and produced in good numbers, readily produce plants in good numbers at a lower cost. 'or this reason, many plant breeders do not utili"e micropropagation because the cost is prohibitive, other breeders use it to produce stoc plants that are then used for seed multiplication. 1echanisation of the process could reduce labour costs, but has proven difficult to achieve, despite active attempts to develop technological solutions. Application of micropropagation 2lant regeneration 4enetic engineering )eforestation -econdary products

 3ultivars with high mar et value 2ropagation of difficult root plants or plants that are typically divided Techniques used to microprpagate plants +tructure 2lantlet -egeneration /uxilary shoot /dventitious shoot -eedling -eed culture 5mbryo culture 3allus 3allus cultures 2rotoplast cultures -omatic embryo -egenerating plants /uxilary shoot formation - meristem culture, auxiliary shoot cultures /dventitious shoot formation - Direct - &ndirect (evelopmental stages in micropropagation -tage &- establishment -tage &&- 1ultiplication Direct or indirect Explant source 1eristem or shoot tip 0eaf pieces, internode -eeds 1ature embryos or immature stem,

6egetative tissue -ingle cells 5mbryo, seedling or leaf

-tage &&&- )oot formation -tage &6- /cclimati"ation

+tage !' )bjectives To successfully place an explants into asceptic culture and an in vitro environment that promotes stable shoot production. 5xplant source selection 5xplants disinfestations 3ulture medium -tabili"ation

aboratory facilities ' &edia preparation &norganic salts Drganic compounds -ucrose 6itamins 8ormones -upports /gar

 1embrane boats 3ellulose plugs #ontainers Testubes :aby food (ars :ottles 3ulture vessels 3ontrol of the tissue culture environment Temperature 0ight intensity 2hotoperiod 0ight quality /ir exchange +tage !!' )bjectives To maintain the culture in a stabili"ed state and multiply microshoots to the number required for rooting. - 4rowth regulators - -ubculturing - 2ropagation ratio +ubculturing: transferring the explants to a fresh medium Explant8propgules: the piece of the plant used to initiate the micropropagation process. +tage !!!' )bjectives

To root microcuttings and prepare them for transfer to ex vitro conditions. - &n vitro rooting - 5x vitro rooting +tage !,' )bjectives To shift from a heterotrophic $sugar requiring% to autotrophic condition. - /cclimati"ation - &n vitro vs. ex vitro anatomy and physiology )rganogenesis The formation of organs such as leaves, shoots, or roots from cells or tissues The process of developing adventitious shoots and/or roots.

!ndirect organogenesis -omaclonal variation: genetic variation induced in plants produced from populations of cells in culture. 2rimary explants 3allus organ primordium

(edifferentiation' process of reverting to a non- speciali"ed or undifferentiated state (ifferentiation' process of initiating the growth of new and varied tissues or organs for speciali"ed functions. 5xplant Dedifferentiation induction Differentiation organ

Adventitious shoot formation /diploid plant regeneration1 =. 0eaf pieces *. Thin layer epidermal strips

?. 'ragmented soot apices @. 3otyledons and hypocotyls B. &mmature inflorescences on flower stems ,. :ulb scales #allus #ell division of nondifferentiated parenchyma cells. - 2roduced on explants in vitro as a response to wounding and medium supplementation with growth hormones - -eeds, stems, roots, leaves, storage organs, or fruits can be excised, disinfected and induced to form callus !solation9 inoculation and subculturing 7hy subculturing is necessary in tissue culture: =. The nutrient media is exhausted $deficiency phenomena% *. The nutrient media is dries out. ?. 4rowth has filled the tube or glass. @. The material is needed for further propagation. B. :rown and/or blac colour appears in the agar: plant species sometimes give off toxic substances during the first few wee s, which diffuses into the agar or liquid media. ,. &t is needed to give the isolated material a different growth and development pattern on a nown nutrient media. >. The medium has developed liquid due to a lowering of the p8 by the plant. 8ow to subculturing: 3allus culture is generally lower on solid media than liquid media for the following reasons,

=. The callus clump has limited contact with the solid medium and therefore ta es up less compounds from the media. *. Dxygen supply is better in liquid media. ?. 3allus clumps are able to fragment in liquid media producing a greater surface area with a resulting improvement in upta e of material from the medium. @. 4radients are present on a solid medium which can cause earlier differentiation, inhibiting the callus growth. / few general rules of liquid media of callus culture: =. )otary sha ers are generally used to prevent the liquid growth media from being lost from the flas s at high speed. *. The flas should not be more than *+-?+ percent full. ?. The amount of medium in the flas can normally be reduced if less cells or clumps of cells are inoculated. @. The optimal revolution of the machine should be about C+-=++ rpm. /lthough in some cases low $@+ rpm% and higher $=*+ rpm% may be used. B. The growth of cells, callus etc generally ta es place as follows: &nitially $the log phase% there is practically no growth and these increase exponentially, then becomes linear before showing down and finally stopping. ,. The rapid establishment of cells in suspension culture is proportional to the cell density at the beginning of the culture Anther culture -eproductive organs of flower &n the highly disciplined and programmed life cycle of angiosperms, they bear flowers $*n%, which are designated as reproductive organs. 'lowers possesses stamens $*n% $microsporophylls% and carpels $*n% $megasporophylls%. -tamens have pollen sacs or anthers $*n% i.e., microsporangia $*n%. 1icrosporangia contain pollen mother cells $213% or microspore mother cells $*n%. Through meiosis,

213s produce a large number of microspores or pollen grains, the first cells of the male gametophyte $n%. Dn the other hand, megasporangium usually contains a single diploid megaspore mother cell $*n%. :y meiosis, the megaspore mother cell generally produces a linear tetrad of $n%, a haploid megaspore i.e., female gametophyte of which the lowermost one usually functions and gives rise to the embryo sac while the other three degenerate. 3aploid plant The cells of haploid plants contain a single complete set of chromosomes, and these plants are useful in plant-breeding programs for the selection of desirable characteristics. The phenotype is the expression of single-copy genetic information, there being no mas ing of a trait through gene dominance. In vivo techniques of haploid production 4ynogenesis Dvule androgenesis 4enome elimination by distant hybridi"ation -emigamy 3hemical treatment Temperature shoc &rradiation effects In vivo methods yield a low frequency of haploid production. Dn the contrary, the success achieved using in vitro methods has been spectacular and there are reports on haploids being induced by anther culture or pollen culture from about *@> species.

Anther and pollen culture /nther and pollen culture is performed by the induction of embryogenesis from repeated divisions of haploid spores, either microspores or immature pollen grains. The purpose of anther and pollen culture is to produce haploid plants.

In vitro haploid cell culture with mature pollen grains Ginkgo biloba was initiated by Tulec e $=;B?%. 4uha and 1aheswary $=;,*E M,,E A,>% made the remar able discovery of angiospermic haploid cell culture with Datura inno ia. Their experiments produced numerous haploid plantlets. :ourgin and .itsch, $=;,>% obtained androgenic plants in !icotiana tabacum. -ince then, significant progress has been achieved in this field throughout the world, particularly in 3hina. "eneral anther culture procedure Gnopened flower buds of appropriate si"e are surface sterili"ed with effective sterilants. The time of sterili"ation may vary from species to species. The sterili"ed materials are washed @-B times with sterile distilled water and may be dipped in dehydrated alcohol. The buds are dissected by removing the sepals and petals or by cutting it into two halves. The anthers are carefully dissected from the flower buds and ept in petridish. )emoval of filaments from the anthers is done very carefully so as not to in(ure the anthers. &f the flowers are very small as in :rassica and /sparagus, the use of a stereo microscope may be preferred for dissecting the anthers. -ometimes, the whole inflorescence is used. Direct contact between anthers and sterilants is not desirable. &solated healthy anthers are cultured on an agar medium or in a liquid medium. Dn agar media, anthers are placed in such a way that they come into direct contact with the medium. -imilarly, pollen grains are removed from the anthers either by crushing the anthers mechanically or by natural dehiscence through float culture. The pollens are cultured on semi-solid media or liquid media. The optimum concentration of media components is the primary environment which induces androgenesis. Though there are no universal prescribed media for haploid cell culture for any species, 3hinese scientists are using the . , $3hu et al., =;>C%, for a wide range of species including rice, wheat, rubber, etc. :esides . ,, 1- $=;,*% and .itsch $=;,;% medium are also used. / schematic representation for the production of haploid plants through anther and pollen culture is shown in 'ig. =. :actors influencing anther cultures Physiological status of donor plant

2lants should be grown under controlled environmental conditions $photoperiod, light intensity, temperature and mineral nutrition% for better results. /nthers ta en from young plants and at the beginning of the flowering time may provide superiority. (evelopmental stages of anther This factor is related to the nowledge of the flowering time of a particular plant. The appropriate si"e of the flower bud reflects the stage of pollen development. This is a ey factor affecting the induction frequency of pollen plantlets. -underland $=;>C->;% mentioned five ideal stages for anther culture $'ig. *%. -tages &-&&& refer to anthers in which the pollen is predominantly in the early-, mid-, and late-uninucleate state. &n stage

&

&&

&&&

&6

6&

&6, the pollen undergoes the first mitotic division and may thus have dividing one-celled and two-celled pollens. -tage6 and 6& show two-celled pollens having a tube cell and a generative cell. /t the beginning of stage6, the generative cell is attached to the intine, at the middle stage the generative cell is detached and cytoplasmic synthesis ta es place in the vegetative cell i.e., in the tube cell. /t stage 6&, the generative cell is fully detached and the vegetative cell, i.e., the tube cell, is invested by the gametophyte cytoplasm. 'inally, it may be emphasi"ed that though the tetrad stage, late uninucleate to binucleate stages are important for anther culture, microspores (ust before or at the time of first mitosis is the most preferred stage. Pretreatment of flower buds and inflorescence

The following types of pretreatment are generally prescribed for enhanced response in anther/pollen culture. i% ii% iii% 0ow temperature pretreatment in the range of @-=+ o3 for *-B days before excision and culture of the anthers. 8igh temperature shoc in the range of ?+-BBo3 for =-@ days. 6ery low doses of radiation.

Effect of anther wall The anther wall plays an important role in anther culture. &t may act as a barrier by which direct effects of an exogenous media component on pollen is reduced to various degrees. /nther walls being diploid, act rapidly on the media component and the degeneration of these anther walls releases some inhibitory compound that adversely affects the development and growth of these microspores. 8owever, it is also reported that the anther walls release another substance that helps microspore division. These are nown as wall factors. 7all factors may be reduced by using a high concentration of sugars in the semi-solid media as well as the floating culture of anthers.

"enotype of the plant Though the theory of totipotency is universally acceptable for plant cells, androgenesis is genotype dependent. There are obvious differences in anther culture ability among the species, subspecies and varieties. Dut of @? cultivars of "yco#ersicon esculentum and =C lines of $rabid#osis thaliana, only three cultivars of each responded to androgenesis. The same has been noted in other important crops li e rice, wheat, rye and solanum. -ome scientists considered these differences as an impassable barrier for anther culture.

#ulture media The composition of the culture media plays an important role in androgenesis. 1any basal media formulations have been made for haploid cell culture over the decades. The .itsch and .itsch medium is nown as a

specific anther culture medium. :ut 1urashige and - oog media $=;,*% as well as .,medium of 3hu et al., $=;>C% and their modifications received wide acceptability for culturing anthers for a large number of plant species. The addition of undefined substances li e potato extract, coconut mil and casein hydrolysate in the culture medium have been shown to enhance anther response. Gse of liquid media for anther culture has proved useful in many crop species. The influence of various growth regulators on androgenesis has revealed that auxins are essential and cyto inin is beneficial in many cases. &nclusion of activated charcoal in the medium has proved stimulatory in many cases. -ole of +ucrose &n anther or pollen culture, sucrose plays an important role not only as a nutrient but also as an osmoticum. &n general tissue culture, it is used in the range of *-@<. :ut its requirement in haploid cell culture varies from species to species. /n elevated sucrose level of ,< is an essential requirement for most cereals. 2otato and wheat require ,< sucrose. &n rice, a low concentration of sucrose $?<% favours androgenic initiation and callus induction while ,< sucrose helps differentiation frequency of green plants. %rassica sp requires =*-=?< sucrose for androgenesis. )ther culture conditions i% ii% &ncubation temperature: Desirable temperature varies from *Bo3-*Co3. 2hotoperiod: -ome plants require continuous light immediately after inoculation, some prefer absolute dar until a response is noted, while others require =,/C hours light/dar . 'loat culture: -underland et al., $=;>>% described the float culture technique whereby, anthers are floated on the surface of the liquid media. 7ithin a few days, the release of embryogenic microspores ta es place through natural dehiscence in the liquid medium. This is noted in !icotiana& Datura and 'ryza.

iii%

!n vitro pathways of androgenesis Pathways of microspore divisions

'our pathways based on the few initial divisions in the microspores have been identified as leading to in vitro androgenesis $'ig. ?% i% 2athway & The microspores divide by an equal division and two identical daughter cells contribute to the sporophyte development. 6egetative and generative cells are not distinctly formed in this pathway $ Datura inno ia%. ii% 2athway && The division of uninucleate microspores is unequal, resulting in the formation of a vegetative and a generative cell. The sporophyte arises through further divisions in the vegetative cell while the generative cell either does not divide or does so once or twice before degenerating $!icotiana tabacum& (ordium vulgare& )riticum aestivum& and Ca#sicum annuum% iii% 2athway &&& The uninucleate microspore undergoes a normal unequal division but the pollen embryos are predominantly formed from the generative cell alone $(yoscyamus niger%. The generative cell either does not divide at all or does so only to a limited extent. iv% The division of microspore is asymmetrical as in 2athway &&. :oth vegetative and generative cells divide further and contribute to the development of the sporophyte $Datura inno ia, occasionallyE Datura metel and $tro#a belladonna%.

Application of anther and pollen culture Nuic est method of haploid production Nuic est method of advancing hetero"ygous breeding line to homo"ygosity &ncreases the selection efficiency /llows the early expression of recessive genes

 4ametoclonal variation 3reating recombinants, as well as variants and new forms

+omaclonal variation 7ac% ground and definition 0ar in and -cowcroft $=;C=% introduced the term somclonal variation. 2lant variant obtained from tissue culture is called somaclones. 5vans et al., $=;C@% coined the term gametoclonal variation for variant clones specifically raised from gametic or gametophytic cells. +omaclonal variation &t is the term used to describe the variation seen in plants that have been produced by plant tissue culture. 3hromosomal rearrangements are an important source of this variation. /lthough, generally a callus phase is involved before the cells can undergo redifferentiation leading to regeneration of a whole plant, but rarely the dedifferentiated cells can give rise to whole plants directly without an intermediate callus phase. These aspects of tissue culture and its application in clonal propagation and in the general on of hereditary variation called somaclonal variation. 1eristem tip culture and micropropagation produce plants that are, with few exceptions, true-to- type, but regeneration of plants through callus or cell suspension culture can induce unexpectedly rich and novel changes among cells, tissues and organs, which in turn, provide a new and exciting variability in regenerated plants. The recovery of genetic changes in plants regenerated from tissue culture offers an opportunity to unravel natural variability and to use this variation for development of new varieties. 3alliclones $plants regenerated from callus% and protoclones $plants regenerated from protoplast% may not be identical to their parents. The origins of somaclonal variation have been studied extensively, but remain largely theoretical or un nown. The amount of variation that can be expected in vitro will vary with the clone, age of the clone, use of mutagenic agents, and use of selection pressure applied to single cells for stress conditions such as salt level, herbicides, microorganisms or their byproducts, and specific metabolites.

-omaclonal variation has been reported by so many different researchers wor ing with so many different crops, that is reasonable to expect somaclonal variation in all tissue culture experiments. &f no visual, morphogenic changes are apparent, other plant screening procedures must be applied. There are both benefits and disadvantages to somaclonal variation. The phenomenon of high variability in individuals from plant cell cultures or adventitious shoots has been named somaclonal variation. 7enefits 7hile recombinant D./ techniques offer promise for modification of crops, the relative paucity of nowledge of plant genetics and biochemistry has delayed development of recombinant D./Obased products using higher plants. 8owever, somaclonal variation offers an opportunity to uncover the natural variability in plants and to use this genetic variability for new product development. The ma(or li ely benefit of somaclonal variation is in plant improvement. -omaclonal variation leads to the creation of additional genetic variability. 3haracteristics for which somaclonal mutants can be enriched during in vitro culture include resistance to disease pathotoxins, herbicides and tolerance to environmental or chemical stress, as well as for increased production of secondary metabolites. The natural variability associated with tissue culture represents a pool upon which selection pressure can be imposed to isolate unique forms of a clone. (isadvantages -omaclonal variation has proved to be a serious problem for investigators and propagators who require extreme uniformity, as in the horticulture and forestry industries where tissue culture is employed for rapid propagation of elite genotypes. $ays of reducing somaclonal variation Different steps can be used. &t is well nown that increasing numbers of subculture increases the li elihood of somaclonal variation, so the number of subcultures in micropropagation protocols should be ept to a minimum. )egular reinitiation of clones from new explants might reduce variability over time. /nother way of reducing somaclonal variation is to avoid *,@-D in the culture medium, as this hormone is nown to introduce variation.6itrificationPhyperhydracityQ may be a

problem in some species. &n case of forest trees, mature elite trees can be identified and rapidly cloned by this technique. 8igh production cost has limited the application of this technique to more valuable ornamental crops and some fruit trees. ;inds of variation -omaclonal variation is not restricted to, but is particularly common in plants regenerated from callus. The variations can be genotypic or phenotypic, which in the later case can be either cytogenetic, genetic or epigenetic in origin. Typical cytogenetic alterations are: changes in chromosome numbers $polyploidy and aneuploidy%, chromosome structure $translocations, deletions, insertions and duplications%. 4enetic alteration include changes in D./ sequence $base mutations%. Typical epigenetic related events are: gene amplification, gene methylation, 8istone acetylation, ubiquitilation, sumoylation etc. *ature of variation The variation is indisposed 3hanges can occur at high frequencies 3hanges can occur in qualitative and quantitative traits 3hanges can occur in homo"ygous form -omaclonal variations involve the whole range of genetic variation

Possible mechanisms of variation =. The pre-existing genetic variation in the explants tissue is a possible mechanism of variation. 2olysomaty, i.e., a variable chromosome number in different cells of a particular tissue in a mother plant may cause somaclonal variation. /s plant cells mature $age%, their ploidy level tends to change. &n tobacco that diploid and polyploidy cells exist in equal percentage B cm below the apical meristem, while more than CB< cells are polyploid, *+ cm below the apical meristem. -uch differentiated and mature cells may undergo endoreduplication or endomitosis or spindle fusion and are bound to

behave differently in culture. 2olytene cells and high polyploid cells are common in cotyledonary tissues. *. The spontaneous mutation that can accumulate during the many division cycles that cells of the explant go through before differentiating into an in vitro plant. The recessive mutations will naturally require a method by which they can express even diploid cells. -omatic crossing over followed by segregation is a li ely mechanism, for the homo"ygosity and thus phenotypic expression of the recessive. ?. .umerical and structural changes in chromosomal during in vitro growth. @. &ntracellular mutagenic agents produced during in vitro growth. B. /ctivation of transposable elements or (umping genes, are genetic entities which have the locus at which they get integrated is matured. ,. 6ariability generated due to the effects of the culture process namely: duration of culture, role of plant growth regulators, nutritional stress. Duration and age of culture and frequency of subculturing may disturb normal plant regeneration. 0ong-term callus cultures, except in a few cases, exhibit hetero"ygosity. Dne of the triggers of change in ploidy level of a culture is a plant growth regulator. :oth auxin and cyto inin particularly *,@-D and inetin are instrumental in bringing aryotypic alteration in cultured cells. These growth regulators can trigger a high rate of cell division in cultured explants and sometimes may directly induce mutation by altering the state of D./ methylation. *,@-D is considered to be responsible for various genetic abnormalities such as aneuploidy, polyploidy, endoreduplication and micronucleus formation. :esides growth, other aspects of the culture medium i.e., levels of 9.D?, chelating agents and some microsalts and metals have the property to destabili"e the normal genetic ma eup. Gndefined substances li e coconut mil and yeast extract may induce somaclonal variation. (etection and isolation of somaclonal variants =. 1orphologically distinct cells such as non-photosynthetic $non-green% cells or cells that accumulate anthocyanins and other plant pigments are detected visually. *. To isolate herbicide and antibiotic resistant variants, plant cells are simply grown on media containing of the wild-type cells in a culture.

?. The surviving cells are then subcultured and retested for growth on herbicide or antibiotic supplemented medium. @. Through this method, one can eliminate any remaining wild-type cells that may have inadvertently survived the first round of selection. Application in plant breeding -omaclonal variation is the important sources of introducing genetic variations that could be of value to plant breeders. -ingle gene mutation in the nuclear or organelle genome usually provides the best available variety in vitro which has a specific improved character. -omaclonal variations are used to uncover new variants retaining all the favourable characters along with an additional useful trait, e.g., resistance to diseases or herbicide. These variants can then be field-tested to ascertain their genetic stability. 6arious cell lines selected in vitro and plant regenerated through it prove potentially applicable to agriculture and industry specially resistance to herbicide, pathotoxin, salt or aluminium, useful in the synthesis of secondary metabolites on a commercial scale, etc. +omaclonal variation is not species specific Tomato $variations are noted in fruit colour, plant architecture% 2otato $generation of new breeding lines including resistance to late blight of potato% -ugarcane $recovered disease resistance plants% Tobacco )ice $change of phenotypic traits such as number of tillers per plant, number of fertile tillers per plant, frequency of fertile tillers etc.% Dats $altered phenotype with plant height, awn morphology% :arley and mai"e $abphyl syndrome i.e., decaussate leaf and ear arrangement giving twice the number of leaves on a normal node% :rassica $exponentially slow growth, precocious flowering from the apex, altered leaf wax, reduced lamina etc.%

 4arlic $improved bulb shape and si"e% 3arrot $disease resistant line against the leaf spot pathogen $lternaria dauci.% -omaclones for various characters have been raised from different fruit crops li e iwi, strawberry, citrus, sweet cherry, apple and pear.

Embryo rescue /bortion of embryos at one or the other stage of development is a characteristic feature of distant hybridi"ation. 5.g the crossing of two species or varieties often fails when using distant parents who do not share many chromosomes. 5mbryo rescue is the process when plant breeders rescue inherently wea , immature or hybrid embryos to prevent degeneration. 3ommon in lily hybridi"ing to create new interspecific hybrids between the various lily groups $such as /siatic, Driental, Trumpet, etc%. 7hen cross-pollination occurs between genetically widely different plants, the resulting embryo may be aborted because of parental mutual incompatibility. -uch embryos may be excised and grown on a congenial medium such as nutrient agar. Embryo'rescue technique Techniques were developed in the early =;++!s enabling unripe seed or embryos from adult plants to be rescued to form small plants. This was done mainly with seed which had a very long dormancy period or when the seeds were particularly heterogeneous. 7ith the continuing developments in tissue culture this technique was also used to save embryos from ovules, which had been fertili"ed but had never developed into viable seed on the mother plant.

&nitially, complete ovaries were put in tissue culture whereby seedlings were obtained from embryos which would have died in a later stage of development. -aving embryos that died in an early stage of development came at a later stage, resulting in high-tech ovule- and embryo-culture techniques. Dften a combination of these techniques is used: parts of the ovaries are put in tissue culture following which ovule and/or embryo culture is applied. :actors affecting the success of embryo culture Development of a viable plant from an embryo depend on many factors. "enotype- &n some plant species the embryos are easy to grow and some are difficult. There are even differences between cultivars of a given species. (evelopmental stage of the embryo at isolation' 4enerally very small undifferentiated embryos are virtually impossible to grow in vitro. The more developed the embryo in vivo the easier it is to grow in vitro. -ometimes it is the easier to culture it in vitro. -ometimes it is possible with the use of speciali"ed techniques to culture very small embryosE a piece of endosperm from a mature seed or a piece of hypocotyls tissue is incubated in close contact with the embryo. -ometimes young embryo can also be transplanted in the endosperm of a normal seed from the same plant species. "rowth condition of the mother plant' Gsually mother plants are grown in green house. &mproving the growth of the mother plant under controlled conditions generally results in better endosperm development, and therefore, better growth of the isolated embryos. The growth of the embryo is also promoted if the cotyledons are more developed, this also being dependent on the growth of the mother plant. #omposition of the nutrient media - &mmature embryos call for a more critical nutrient medium composition than required for mature embryos. 8owever, both mature and immature embryos require macro and micro elements and sugar. Gsually a solid medium with p8 B-, is used. -accharose is generally used as sugar source, although glucose and fructose are sometimes suitable. /gar concentration of +.,-+.C< is usually chosen, higher concentration resulting in the inhibition of growth. /uxins and 3yto inines are not generally used with embryo cultures since they often induce callus formation. -ince the nutrient requirements changed during growth and development of the embryos, subculturing is sometimes necessary.

)xygen' This is an important factor, and the oxygen requirement of embryo culture appears sometimes to be higher than the oxygen concentration normally present in air. ight- -ometimes isolated embryos need to be grown in dar ness for >-=@ days, after which can be transferred to the light to allow chlorophyll formation. Temperature' The optimum temperature is dependent on the plant species used. .ormally a relatively high temperature is used for growth $**-*C o3%, although some species require a lower temperature $=>o3%. / cold treatment $@o3% might be necessary to brea dormancy.

+terillants 3aD3l, .aD3l, 8*D*, :romine water, -ilver nitrate, 1ercuric chlorite, different types of antibiotics, 5thanol, propanol, isopropyl alcohol, 3etavlon or 3etramide. Primary sterillants 5thanol, iso-propyl alcolho and 3etavlon. #oncentration and duration of treatment Types of seed, age of seed .ame of the sterillant 3aD3l .aD3l 8*D* :romin water /g.D? 3onc. &n < =+ * =+-=* =-* = Time $min.% B-?+ B-?+ B-=B *-=+ B-?+

8g3l* /ntibiotics

+.=-= @-B+ mg/l

*-=+ ?+-,+

The effectiveness of the sterilants /mong these, 3aD3l, .aD3l and :romine water is more effective. -oft seed coat require less time for treatment whereas, bean seed coat will not be harmed if for long time. Dverdose is simply toxic to all plant material. -o, density and time should be maintained carefully. Excision or dissection /ta%e out the embryos from seed1 'or the invitro culture of embryos, generally it is necessary to free them from their surrounding tissues. The mature embryos are isolated by splitting open the seed. -eed of the hard seed coat are dissected after soa ing them in water. -maller embryos require very careful dissection with aid of a stereoscope and quic transfer to the culture vial or vessels. Embryo nurse endosperm transplant /s a rule, very young embryos are difficult to culture on artificial culture medium. This problem is overcome by embedding or transplanting or inserting the proembryo in the full endosperm or dissected out endosperm tissue. The endosperm tissue wor s as nurse cell. Phases of embryo development - /utotrophic - 8eterotrophic #ulture requirement The most important aspect of the embryo culture is the selection of the right culture media that would support progressive and orderly development of the excised embryos at different stages of development. - .atural plant extracts

/ccording to 0arue experiment, +.B m1 or slight less than that plant extract is required in the medium 6an Dver :ee - 1edium contains =< dextrose, =< agar, some mineral salt, 4lycin, Thymine, nicotinic acid, 6it :, succinic acid and pentothinic acid, coconut mil give good result. 9ant and :rin - Dates, :ananas, wheat glutin hydrolysate, mai"e and tomato (uice $TR% substitutes of chemicals. Tomato (uice was most effective on the base of medium containing ??-,,< tomato (uice, >-; days old embryos under normal embryogenic development. 7ithout TR these embryos grew irregularities. TR is the source of ascorbic acid. 4rowth regulators may not require for embryo culture. p8 varies from medium to medium. #ulture storage' low temperature and low light is required until embryos are germinated.

)vule or ovary culture Dvule culture is important with reference to in vitro pollination. :esides these it serves as an experimental system to study the in vitro response to "ygote and very young embryo. The first attempt to isolated ovules and culture them under aseptic conditions was made by 7hite $=;?*% in $ntirrhinum ma*us . 8owever, the technique of ovule culture was developed and perfected at the department of :otany, Gniversity of Delhi, &ndia. )aising matured seeds by culturing ovules containing globular or older embryos is comparatively easy. 'or in vitro pollination it is absolutely essential to be able to grow very young ovules excised soon after pollination. The first report of a successful culture of ovules containing "ygote was published by 1aheswary $=;BC%. -imilar to that of embryo culture the medium and its compounds are determined by the age of the ovule and types of plant materials are in use. Dvule culture holds a great potential for raising hybrids which normally failed to grow due to the abortion of the embryo at the early stage of development when its excision and / or culture is either very difficult or impossible. The loss of the hybrid embryo due to pre mature abscission of fruits, as happens in many

interspecific crosses of cotton can be prevented by ovule culture $-tewart 8su, =;>C%. 'or example the cross Gossy#ium arboreum J Gossy#ium hirsutum the hybrid embryo develops only up to C-=+ days after pollination $D/2% subsequently, numerous abnormalities occur leading to the failure of further embryo development. :easley $=;@+% and 7eaver $=;BC% cultured the immature embryos to raise full plants, but excised embryos failed to grow. Dvary culture- 0a )ue $=;@*% was probably the first to raise asceptic cultures of angiosperm flowers $ovary% later on =;B= .itsch develop the technique of ovary culture who grow detached ovaries of Cucumis anguria, "yco#ersicon esculentum, !icotiana tabacum, and +asiolus vulgaris on synthetic medium. Dvary culture is essential to raise interspecific hybrids between sexually incompatible parents %rassica cam#estris J %. oleracea. Application of embryo9 ovary and ovule culture - Dbtaining rare hybrids - 8aploid production - -hortening of the breeding cycle - )apid seed viability test
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2ropagation of rare plants