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第四章 Micro-propagation(In Vitro Propagation )

(Ⅰ) Introduction--- Schwann (1839) expressed the view that each living cell of a multicellular

organism would be capable of developing independently if provided withthe proper external conditions.

--- Working independently of each other, White (1939), Gautheret (1939),and Novecourt (1939), almost simultaneously announced successfulmethods for the unlimited in vitro growth of plant tissues.

--- The most varied types of tissues and cells from an extraordinary numberof plant species have been grow in vitro using solid and /or liquid culturemedia.

--- The concept that the individual cells of an organism are totipotent isimplicit in the statement of the cell theory.

(II) Glossary of tissue culture terms.(Table 1)

(Ⅲ) Nutritional components of tissue culture media.1. Inorganic salts.(1). Macronutrient elements

Nitrogen:Added in the largest amount.Present in either a nitrate (NO3

-) or ammonium ion (NH4+), or a

combination of the two ions (NH4NO3).Magnesium sulfate (MgSO4.7H2O):

Satifies both the magnesium and sulfur requirements.Phosphorus:

Be represented by either NaH2PO4 or KH2PO4

Potassium:Is given as either KCl, KNO3 or KH2PO4.

Calcium: CaCl2.2H2O or Ca(NO3)2.4H2OChloride: KCl or CaCl2.2H2OSodium: is not generally required by higher plants.

May be an essential requirement of cultures of halophyte tissues.

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Plants with C4 photosynthetic pathways.Plants with Crassulacean acid metabolism (CAM).

(2). Micronultrients--- Plant cells require traces of certain micronutrients.--- Required by all higher plant cells are copper (Cu), zinc(Zn),

manganese(Mn), iron (Fe), boron (B), and molybdenum(Mo).--- Some media contain traces of cobalt (Co), iodine (I), nickel (Ni),

titanium (Ti),beryllium (Be), aluminum (Al).--- An iron stock solution is prepared separately because of the problem

of iron solubility. Usually the iron stock is prepared in a chelated formas ferric-sodium ethylenediamine tetra -acetate (NaFe-EDTA) or 2Na-EDTA with FeSO4.7H2O. Agar is also a source of numerous mineralelements.

--- One unresolved problem is that even the purest chemical reagentscontain traces of inorganic contaminats, and these elements constitutetraces of inorganic contaminats, and these elements constitute ahidden source of micronutrients .

2. Plant growth regulators:--- Two classes of plant growth regulators (auxin and cytokinin) are now

widely used in vitro propagation.--- Gibberellins and other synthetic growth regulators are occasionally used.

Auxins ：

Indole-3-acetic acid IAA (175) a nature auxin, unstableIndole-3-butyric acid IBA (203)α-naphaleneacetic acid NAA (186) stable

2,4 dichlorophenoxyacetic acid 2,4-D (221) strong, stableCytokinins ：

6-(4-hydroxy-3-methy1but-2-enyl)Zeatin: nature cytokininBenzyladenine BA (225)(6-benzylaminopurine)Kinetin (6-furfurylaminopurine)-(215)Isopentyladenine (dimethylally-aminopurine) DMAA (203), a naturalcytokininAdenine sulfate, Ad.SO4 (230), weak cytokinin activity

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Gibberellins ：

Gibberellic acid A3 GA3 (346)

(1). Auxin (some explants naturally produce enough auxin)a. The first role of auxins is the stimulation of division and cell enlargement

in existing buds, and the promotion of the creation of adventitious buds.b. The second role of auxins is the promotion of rooting .

－Higher concenration than for the first role applied.－Unfortunately, the concentrations that most strongly stimulate the

initiation of root primordia are usually too high for best root elongation.c. IAA, unstable－Degraded by light and enzymatic oxidation (because IAA oxidase may

be high in cultured tissues).Higher concentrations (1-30 mg/l) should be added.

d. NAA, stable－Lower concentrations (0.1-2.0 mg/l) may be added.

e. 2,4-D, strong, stable－Extent callus formation (0.1 mg/l-5 or 10 mg/l)－Cultures that require both an exogenous auxin and cytokinin for growth

frequently will respond with the addition of 2,4-D as the sole plantgrowth regulators.

f. Using higher concentrations of NAA or 2,4-D is likely to cause extensivecallus formation in explants and to inhibit early shoot bud regeneration.

(2). Cytokinin－They are powerful stimulants of cell division in explanted tissue.－They promote the proliferation of shoot buds.－Unfortunately, the concentrations of cytokinins that are most favorable

for shoot proliferation may inhibit root initiation and root growth .－Cytokinins interact with auxins in a way that makes the ratio of these two

compounds as important as their absolute concentrations.－Shoot formation : cytokinins/auxin : higher－Rooting : cytokinin/auxin : lower－Coconut milk: Diphenyl urea : a growth factor exhibits cytokinin -like

responses. 10-15% is convenient.

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Cytokinin (BA) concentration changes the kinds of shoot development in multiplication stage.

Although cytokinin promotes shoot formation, high concentrations also inhibit shoot elongation.

(3). Gibberellins:－May stimulate new growth and may influence shoot and root formation.－The addition of gibberellins is not usually required.

3. VitaminsVitamins gave catalytic functions in enzyme systems and are required only in

trace amounts.(1). Thiamine : thiamine - HCl.

--- Is the most important vitamin additive.--- To stimulate explant growth and may enhance root growth in later

stage.--- Added in amounts varying from approximately 0.1-30 mg/L.

(2). Nicotinic acid, pyridoxine, B complex vitamins--- Sometimes added--- However, little or no evidence of their importance

(3). Ascorbic acid and citric acid.

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--- Act as antioxidants--- Can be added to explant media in special cases where initial “browning

reaction” as a problem.4. Amino acid and complex organic supplements

With the exception of glycine (aminoacetic acid), which is a component ofseveral media, amino acids are not usually added to plant culture media.

If a mixture of organic nitrogen is considered necessary, the medium canbe enriched with either casein hydrolysate or casamino acids.

Peptone, yeast extract and malt extract are used infrequently today.5. Carbohydrates:

All media require the presence of a sugar as a carbon and energy source.(1). Sucrose

--- Is usually added in concentration of 20,000-45,000 mg/L.--- Nearly all cultures appear to give the optimum growth in the presence of

the disaccharide sucrose, whereas there can be considerable variabilityin growth when other disaccharides or monosaccharides are substitutedfor sucrose.

--- Many laboratories autoclave the sucrose with the remainder of thenutrient medium.

--- Sucrose is somewhat heat-labile, and these autoclaved media actuallyconsist of a combination of sucrose, D-glucose, and D-fructose.

(2). Myo -Inositol--- Is added to some culture media as a growth factor at a concentration of

10–100 mg/L.

6. Medium matrix--- Unless the culture is prepared in an aqueous medium as a suspension

culture, it is grown on a semisolid or solid matrix.--- Most stationary cultures are grown on an agar base in a concentration

range from abort 0.6-1.0% (w/v).--- Filter paper platforms were used by Heller (1965).

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(Ⅳ).Selection of culture media

--- The choice of a particular medium depends mainly on: the species of plant, the tissue or organ to be cultured, and the purpose of the experiment.

--- If the plant material has been cultured successfully in other laboratories,it is best to start with published methods.

--- A suitable start point for the initiation callus from a dicot. (dicotyledon)tissue explant would be the preparation of the MS basal medium.

--- For callus formation the addition of 2,4-D (0.2-2.0 mg/L) is effective formost tissue (The addition of a cytokinin (0.5-2.0mg/L) may be helpful).

--- Another combination for the production of callus is 2,4-D plus coconutmilk.

--- If these combinations fail to produce the desired results, a supplementof amino acids, or some natural plant extracts may be considered.

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Formulations of some plant tissue culture media (mg/L)

Component Murashige and Skoog,

(MS) 1962

Woody plant medium

(WPM)

Gamborg B5

NH4NO3 165.00 40.00 -KNO3 190.00 - 250.00MgSO4.7H2O 37.00 37.00 25.00KH2PO4 17.00 17.00 -CaCl2.2H2O 44.00 9.60 15.00Na2SO4 - - -K2SO4 99.00 -Ca(NO3) 2.4H2O - 55.60 -NH4SO4 - 13.40KCl - - -NaH2PO4.H2O a - 15.00 -MnSO4.H2O 1.69 2.23 1.00ZnSO4.7H2O 0.86 0.86 0.20H3BO3 0.62 0.62 0.30KI 0.083 - 0.075Na2MoO4.2H2O 0.025 0.025 0.025CuSO4.5H2O 0.0025 0.0025 0.0025CoCl2.6H2O 0.0025 - 0.0025FeSO4.7H2O 2.748 2.78 2.78Na2.DETA 3.724 3.73 3.725Nicotinic acid 0.05 0.05 0.10Thiamine . HCl 0.01 0.10 1.00Pyridoxine .HCl 0.05 0.05 0.10Myo-inositol 10.0 10.0 10.0Glycine 0.2 0.02 -

Use 10 mL of stock solution for preparing 1 L of the culture medium.

a. Commonly added as a supplement directly to the culture medium at 85 to 255 mg/L

(Ⅴ). Preparation of culture media

1. Water--- The water would be employed in all tissue culture media, including the

water used during the culture procedure.

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--- Should be double-distilled (DDH2O) or demineralized-distilled (deionizedwater).

--- It is mandatory that glass distillation is the final step.--- One should be cautioned against the prolonged storage of redistilled

water in polyethylene containers because these receptacles releasesubstance(s) that may be toxic to the cultures.

--- It is unwise to store double -distilled water in Pyrex vessels forprolonged periods because detectable amounts of bacteria mayaccumulate during storage under nonsterile conditions.

2. Stock solutions--- Weighing the individual constituents for each batch of culture medium is

only practical when the quantities required are large enough to beweighed accurately with available scales.

--- Those small amounts of agents, such as thiamine hydrochloride is oftenused at a concentration of 0.4 mg/L, that cannot be weighed with acommon centigram balance should be prepare the concentrated stocksolutions .

--- These stock solutions are then apportioned to the culture medium eitherby pipetting or by pouring, the volume of stock solution that contains therequired amount of the constitutent into the culture medium.

--- When the constituents are chemically compatible, stable, and requiredat constant rotios, as with the micronutrient minerals, severalcompounds can be dissolved in the same solution at the sameconcentration factor.

--- All the stock solution must be stored under refrigeration.

(1). Macronutrient salts--- Are required in relatively large quantities.--- The amounts needed to prepare as little as one liter can be weighed

with sufficient accuracy using a centigram balance.--- However, some workers prepare a 10-times concentrated stock solution

containing all of the macronutrient salts as a way of reducing theamount of weighing required for the preparation of small batches ofmedium.

--- One volume of such a stock solution added to nine volumes of culturemedium (i.e., 100 mg /L) yields the required final concentration of each

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salt.

(2). Micronutrient salts--- Are used in milligrams per liter, these quantities require a more

sophisticated analytical balance for accurate weighting.--- It is common to use a 100-times concentrated solution of the combined

micronutrient salts, excluding the iron source.--- One volume of this solution added to 99 volumes of culture medium (i.e.,

10 mg/L) will provide the desired final concentrations.--- Iron source--May be prepared separately as a 100-times concentrated stock solution.--It is more satisfactory in this case to dissolve the two components(FeSO4.7H2O, 2Na-EDTA) separately in half the volume of the stocksolution and then mix together.

-- One volume of this stock solution added to nine volumes of culturemedium (i.e., 100 mg/L) yields the required final concentration of eachsalt.

(3). Vitamins:--- It is common to use a 100-times concentrated solution of the combined

vitamins (e.g., glycine, nicotinic acid, pyridoxine-HCl, thiamine-HCl).--- Because of the possibility of heat degradation of the vitamins, the

vitamin supplement should be added after the remainder of the mediumhas been sterilized by autoclave.

--- The desired amount of vitamin stock can be sterlized by ultrafiltrationwith a syringe equipped with a sterile Swinney filter unit containing amillipore filter disc (0.22μm pore diameter).

--- The vitamin stock solution should be discarded after 30 days.

(4). Plant growth substances.--- Because of the low solubility of the commercially available forms of both

auxins and cytokinins, it is necessary to use some method is to preparethe water-soluble salt of the compound by adding a slight excess of anacid or base as required.

--- Ethyl alcohol or other organic solvents are sometimes used as co-solvents in preparation of stock solution of hormones. If this technique

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is used, one should be certain that the final concentration of the co-solvent in the culture medium is not high enough to cause toxicity.

--- The following precautions should be observed when using stocksolutions of hormones.

a. Solutions must be refrigerated or forzen during storage.b. Stocks should be carefully examined after storage for signs of

microbial growth, which will usually destory the compound.c. Concentrated solutions of some of the less soluble hormones may

precipitate from the solution during cold storage, and must be heatedto redissolve the compound before use.

d. IAA is unstable in solution, and to some extent even in the drycrystalline form. IAA solutions should therefore be stored no longerthan 1-2 weeks with refrigeration in darkness.

Auxins:--- A convenient stock solution for IAA, formula weight (F.W.)=175,

contains 1 mg / mL.--- To prepare 100 mL of this solution, place 100 mg of IAA crystalline

power in a container and add an equimolar amount of sodium hydroxide(F.W. = 40), e. q., 5 mL of 0.1 N solution.

--- Stirring and gentle heating will dissolve most of the crystals, and 95 mLof water can then be added with continued stirring to complete thepreparation.

--- Other auxins may be similarly prepared.

Cytokinins--- A convenient stock solution of the cytokinin kinetin (F.W.= 215) can be

prepared by placing 100 mg into a suitable container and adding anequimolar amount of hydrochloric acid (F.W.= 36.5),e.g., approximately5 mL of 0.1 N HCl.

--- Stirring and gentle heating will dissolve most of the crystals, and adding95 mL of water with continued stirring will complete the preparation, onemL will contain one mg of kinetin.

--- Other cytokinins may be similarly prepared.

3. Coconut milk.--- The collected milk is filted through several layers of cheesecloth.

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--- Boil the filtrate for approximately 10 min. in order to precipitate theproteins.

--- Cool to room tempreature, decant, and filter the supernatant through afairly rapid qualitative filter paper. (ex. Whatman No.4)

--- For the preparation of 1 liter of nutrient medium, add 100-150 cm3 ofcoconut milk.

--- Any unused coconut milk can be forzen for use at a later date.--- Melting and refreezing apparently does not diminish the cytokinin-like

properties of the substances present in the liquid endosperm.

4. pH adjustment of culture media.--- pH is very important, adjust it carfully.--- The adjustment of the pH of a culture medium is accomplished by

placing the calibrated meter probe in the solution and stirring the solutionwhile a dilute acidic or alkaline solution is added drop by drop until thedesired point is reached.

--- Generally, dilute hydrochloric acid (HCl) is used to lower pH, and dilutesodium hydroxide (NaOH) is used to raise pH.

--- Commercial concentrated HCl is actually approximately 36%-38% HClin water, for pH adjustment it is necessary to dilute the concentrate 10 to100-fold, so that when a single drop is added it will not cause large pHshifts.

--- Pure NaOH is a hydroscopic solid in the form of pellets or flakes,solutions of 0.4%-4.0% NaOH should be prepared for pH adjustment.

--- pH adjustment should be complete before adding agar.

5. The use of agar--- It is usually desirable to keep tissue explants in contact with, but not

submerged in, the culture medium.-- to provides better aeration.

--- Cultures in which explants are submerged in liquid media may requiresome kind of agitation to promote aeration.

--- It has long been a practice to add gelling substances to liquid culturemedia to obtain this condition.

--- Agar, gelatin, starch-derivative gels, and silica gel all have been usedsuccessfully, but agar remains the favored substance for plant tissue

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culture.--- When dissolved at concentrations ranging from 0.6% to 1.0% in water, it

forms a semi-solid gel suitable for in vitro media.--- To dissolve agar, heating it to 98℃ is sufficient, but brief, gentle boiling

generally will not damage the culture medium.--- After heating the medium to the proper temperature, then add the agar

and stir until the translucent pieces disappear completely.--- Solidilification occurs att approximately 40℃.

--- As long as the agar solution is kept hot, it can be poured, pipetted, orotherwise transferred to the culture containers.

(Ⅵ).Aseptic techniques

--- The most troublesome problem of in vitro work is that of producing andmaintaining aseptic condition.

--- Spores of bacteria and fungi are everywhere in our environment, and sosmall and lightweight that the slightest air movement lifts them.

--- Because of this ever-present problem, all in vitro procedures mustinclude precautions against microbial contamination.

--- It is essential to start with microbe-free plant pieces and to sterilize allglassware, media, and tools.

1. Sterilization methods(1).Dry heat

--- This method is used only for glassware, metal instruments, or othermaterials that are not charred by high temperature.

--- Objects containing cotton, paper, or plastic cannot be sterilized with dryheat.

--- Surgical blades and scalpels should not be sterilized by this methodbecause the high temperature will dull the cutting edges.

--- The objects to be sterilized are carefully wrapped in heavy-dutyaluminum foil before being placed in the oven.

--- A moderate temperature, approximately 160-180℃, for a period of 1.5

hours is sufficient for sterilization.(2) Wet heat

--- This proceduce employs an autoclave operated with water vapor underpressure.

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--- A steam pressure of 15 lb/in2 (1.2 kg/cm2) and a temperature of 121℃ in

applied for 20 min.--- Do not start timing the 20 mins period until the autoclave has reached

the proper temperature and all of the residual air in the chamber hasbeen displaced by steam.

--- At the end of the 20 mins period, the pressure must be allowed to returnslowly to the atmospheric level because rapid decompression will causethe liquid to boil out of the vessels.

--- Prolonged autoclaving must be avoided because it results in thedecomposition of the chimicals present in the medium.

--- Liquid volumes should not exceed 500 mL.--- Labware and paper goods may become wet from the steam that

condenses during the autoclaving process, the autoclaved meterials canbe dried in a warm oven or stored in the clean room area until dry.

(3) Ultrafiltration (microfiltration)--- Some media components are heat-labile and must be sterilized by

ultrafiltration at room tempreature.--- After ultrafiltration, the concentration should be stored aseptically in a

refrigerator or freezer until used.--- If agar media are to be used, the sterile concentrate should be added

after the rest of the medium has been autoclaved and cooled somewhat(45-50 ℃) but not yet solidified.

--- A filter membrane porosity of 0.22 microns (μm) is most frequentlyused, a pore size of 0.22μm is recommended for the retention of the

smallest bacteria.

(4) Chemical sterilization--- The working area is generally surface-sterilized with either ethanol (70%

v/v) or isopropanol (70% v/v).--- After immersion in the alcohol (80% v/v), the instrument is then passed

through the flame of a methanol lamp.--- Some commonly used disinfectants are as follow.

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Disinfectant Concentration Exposure time

Sodium hypochlorite* 0.5-5% 5-20 minutesEthyl alcohol 75-80% Several seconds-several

minuterBenzalkonium chloride 0.1-0.01% 5-20 minutesHydrogen peroxide 3% 15-30 minutesMercuric chloride 0.1-1% 20-30 minutes

*Most laboratories use a household bleaching agent such as clorox or purex.

These commercial products contain 5.25% NaOCl as the active agent.

--- The surface sterilization of plant material may be mostly accomplishedwith an aqueous solution of sodium hypochlorite (NaOCl).

--- Following the NaOCl treatment, the plant material must be rinsed withseveral change of sterile DDH2O in order to remove all traces of thedisinfectant.

--- Because of the corrosive effect on metallic instruments within thechamber, the NaOCl solution should be discarded immediately after use.

Note:--- Several chemicals employed in plant tissre culture media degrade on

exposure to the high temperature associated with steam sterilization.--- Gibberellins are readily degraded by elevated temperatures, and the

biological activity of a freshly prepared solution of GA3 was reduced bymore than 90% as a result of autoclaving.

--- The auxins NAA, IAA, and 2,4-D are thermostable.--- Aqueous solutions of kinetin, zeatin, and IPA have been

chromatographed before and after prolonged autoclaving with nobreakdown products detected.

--- Vitamins have varying degrees of stability, in general, the addition ofvitamins to culture media prior to heat sterilization is not advisable, filtersterilization at room temperature is preferable.

--- Thiamin-HCl can be heated to 110℃ in an aqueous solution without

decomposition, but if the pH of the solution is above 5.5, it is rapidlydestroyed.

--- Calcium pantothenate can not be autoclaved, whereas pyridoxine-HCl isthermostable.

--- Hypochlorite solutions should be used with care; inhalation can producesevere bronchial irritation and pulmonary edema, and skin contact can

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result in irritation. One should never use the mouth to pipette ahypochlorite solution.

--- Never use hypochlorite or other inorganic chloride preparations in thepresence of ultraviolet irradiation. The resulting release of free chlorinegas is a serious health hazard.

(Ⅶ). The culture room or chamber

--- Factors such as day and night temperature, light intensity, light quality(spectral balance) and photoperiod (length of daily light period) areimportant, and undoubtedly different stages and species has differentoptimal requirement.

1. Lighting--- Although the in vitro plantlets can obtain all the energy needed for

growth from the sugar is the culture medium, light is the important toproduce green plantlets with relatively normal leaves.

a. Light quality--- Most plants that are dependent upon artificial lighting as their sole

energy source grow somewhat better in a mixture of fluorescent andincandescent light.

--- A rule of thumb is to use cool or warm white fluorescent lampscombined with approximately 30% of the total installed lamp wattageas incandescent bulbs.

b. Light quantity--- Fluorescent illuminance levels reported in the literature range from

100 to 1000 ft-c (1000-10000 lux).c. Photoperiod

--- Most reports in the published literatures, use a “long day” photoperiod of 12-20 hours, with 16 hours being the most common.

--- It can be expected that continuous lighting or unusual photoperiodswill adversely affect some species.

2. Temperature--- Based on past experience with plants grown in greenhouses and

controlled environment chambers, one would expect that differentphotoperiod temperatures and somewhat lower dark periodtemperatures would be advantageous for many species.

--- The majority of published reports on in vitro culture, however, have

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used constant temperatures in the range of 20-28℃, with 25-27℃ the

most common.3. Relative humidity

--- Enclosed cultures maintain a nearly saturated humidity.--- Because of the desirability of some gas exchange between the

cultures and the room (chamber) air, most closures are deliberatelynot tight -fitting.

--- When the humidity of the room air is low, evaporation of the water inthe culture medium may become excessive. Under these conditionshumidification may be desirable.

(Ⅷ). Culture Methods:

1. Callus cultures -- Initiation and maintenance of callus(1). Sources of materials

--- Few plant tissues fail to respond to treatments designed to inducethe formation of a callus and it now seems clear that the isolationand successful establishment of a callus largely depends on theculture conditions employed and not on the source of plantmaterial.

--- Tissues from various parts of the plant can be grown in culture andproduce callus cultures: vascular combia, storage parenchyma,pericycle of roots, cotyledons, leaf mesophyll, provascular tissue.

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Callus culture. Left: Undifferentiated callus. Right: Callus of showing dark spots are

green meristemoid-producing areas that will initiate shoots.

(2). Sterilization of material--- There are a variety of chemical agents in common use for the

surface sterilization of plant material.--- The choice of agent and the time of treatment depend on the

sensitivity of the material to be sterilized.--- Frequently it is discovered that over-zealous sterilization leads not

only to the complete removal of all micro-organisms but is alsolethal to the plant tissue.

--- Sterilization procedures for different plant organs.

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TABLE: Sterilization procedures of different plant organs

PROCEDURETissue Pre-sterilization Sterilization Post-sterilization Remarks

SeedsSubmerge in absoluteethanol for 10 sec.and rinse in steriledistilled water

Seeds with intacttestas, submerge for20-30 min. in 10%W/V calciumhypochlorite or for 5min. in a 1% (W/V)solution of brominewater

Wash three times insterile water andgerminate in sterilewater. Wash fivetimes with steriledistilled water andgerminate on dampsterile filterpaper

Root and shoottissue for callusculture. Excellentfor tomato seeds

Fruits Rinse briefly withabsolute ethanol

Submerge for 10 min.in 2% (W/V) sodiumhypochlorite

Wash repeatedly withsterile water dissectout seeds or interiortissue

Good source ofsterile seedlings

Pieces of stem Scrub clean inrunning tap water andrinse with absoluteethanol

Immerse for 15-30min. in 2% (w/v)sodium hypochlorite,remove ends

Wash three times insterile water

Plant vertically inagar medium ordissect tissue outand culture inisolation

Storage organs Scrub clean inrunning tap water

Submerge for 20-30min. in 2% (w/v)hypochlorite

Wash three times insterile water. Dry withsterile tissue paper

Remove tissuefrom the inside ofthe structure

Leaves Rub surfaces brieflywith absolute ethanol

Immerse for about 1min. in 0.1% (w/v)mercuric chloride

Wash repeatedly withsterile water. Dry withsterile tissue paper

Difficult material tosterilize. Choosevery young leaves.Lamina laid on agar

(3). Isolation of plant tissues--- In certain instances the explant may appear to be a uniform piece

of tissue largely composed of one cell type. However, even in atissue in which the cells appear to be similar, they may be markeddifferences in the DNA content of the nuclei of the cells and thismay be the consequence of endopoly-ploidy.

--- The callus produced from a morphologically uniform tissue will, atleast in the initial stages, give a relatively uniform callus. On theother hand, explants which contain a variety of different cell typeswill tend to produce a mixed callus.

(4). Nutrient media--- The nutritional requirements for the initiation of callus vary

considerably for primary explants of different origin.--- The medium developed by Murashige and Skoog (MS) for tobacco

tissue culture is being used extensively for culturing callus on agaras well as cell suspension culture in liquid media.

--- The B5 medium for growung soybean tissue culture has also been

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used successfully to grow cells of a large variety of plant tissues.--- Both the MS and B5 media appear to contain the appropriate

amounts of inorganic nutrients to satisfy the needs of many plantcells in cultured.

--- The majority of excised tissues require the addition of one or moregrowth factors to the medium in order to stimulate callusdevelopment.

--- Explants can be subdivided, according to their growth factorrequirements, in the following manner: (a)auxins, (b)cytokinin,(c)auxin and cytokinin, (d)complex natural extracts.

(5).Subculture and preservation of cultures--- After callus has been grown for a period of time in association with

the original tissue, it becomes necessary to subculture the callusto a fresh medium.

--- The transferred fragment of callus must be of a sufficient mass toassure renewed growth on the fresh medium. If the transferredinoculum is too small, it may exhibit a very slow rate of growth ornone at all. Street has recommended that the inoculum be 5-10mm in diameter and weigh 20-100 mg.

--- Successive subcultures are usually performed every 4-6 weekswith culture tubes containing 30 cm3 medium.

--- Only healthy tissue (healthy -looking pieces) should be transferred,and brown or necrotic tissue must be discarded.

2. Suspension culture--- A suspension culture consists of cells and cell aggregates dispersed

and growing in a moving liquid medium.--- During incubation the amount of cell material increases; this increase

occurs only for a limited time and the culture reaches a point ofmaximum yield of cell material. If the culture is at this point, dilutedback (by subculture) to the same initial cellular content.

--- Thus the culture can be continuously propagated by successive batchcultures of appropriate duration and the number of stock culturesincreased at regular intervals.

--- Such suspension cultures are usually initiated by placing pieces offriable tissue (callus) culture in moving liquid medium.

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--- They have also been started from sterile seedlings or imbibed embryosby breaking up the soft tissues in a hand-operated glasshomogenizer and then transferring the homogenate, containing intactliving cells, dead cells and cell debris, to moving liquid medium.

--- Cell suspension cultures must be agitated or subjected to forcedaeration, and a platform (orbital) shaker is employed for this purposein the most laboratories. The best speed range for cultures in 250-cm3 Erlenmeyer flasks is 30-150 rpm; speeds above 150 rpm areunsuitable.

--- The volume of liquid medium in relation to the size of the flask isimportant for adequate aeration (i.e., the nutrition medium shouldoccupy about 20% of the total volume of the flask).

--- The dividing cells will gradually free themselves from the inoculumbecause of the swirling action of the liquid. It should be kept in mind,however, that no suspension culture has been shown to becomposed entirely of single cells.

--- The initiation of a cell suspension culture requires a relatively largeamount of callus to serve as the inoculum for example, approximately2-3 g for 100 cm3.

(1). Culture media--- There are multiplicity of media and it is often very difficult to find out

exactly what medium has been used in a particular investigationwithout working ones way back through previous published papers.

--- It may be necessary to use a different medium for suspensionculture growth than has been developed for satisfactory culture ofthe primary tissue culture.

--- Nevertheless the most widely used basic media developed forcallus cultures form the starting-suspension cultures.

--- Whenever it is desired to develop a satisfactory suspension culturefrom a callus culture, the first step is to study the influence ofmedium composition, and particularly the levels of auxin andcytokinin (increasing the auxin/cytokinin ratio will, in some cases,produce a more friable callus), on the texture of the callus culturegrowing on solid medium.

--- A medium which makes the callus culture friable-capable of beingeasily fragmented-not only gives a tissue mass which will readily

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break up when transferred to agitated liquid medium, but willprobably result in a suspension which remains well dispersed.

--- There is need for more critical studies on this aspect of plant tissueculture.

(2). Maintenance of stock suspension cultures--- When the plant material is first placed in the medium, there is an

initial lag period to any sign of cell division. This is followed by anexponential rise in cell number, and a linear increase in the cellpopulation. There is a gradual deceleration in the division rate.Finally, the cells enter a stationary or nondividing stage. In order tomaintain the viability of the culture, the cells should be subculturedearly during this stationary phase.

--- Because cells from different plant material vary in the length of time,it may be prudent to subculture during the period of progressivedeceleration.

--- Passage time can be learned only from experience, and a givensuspension culture should be subcultured at a time approximating

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the maximum cell density.--- Generally speaking, suspensions are maintained by using an

inoculum which establishes a relatively high initial cell density 5-2.5x 105 cells mL-1) and this density rises during incubation often towithin the range 1-4 x 106 cells mL-1. This means that the increasein cell number is such as would occur if all the cells underwent fourto six cell divisions. For many cell clones this increase is achievedin 18-25 days, although the passage time for some extremelyactive cultures may be as short as 6-9 days (particularly as suchcultures have no detectable lag phase or a very short lag phase).

3. Organogenesis--- Organogenesis refers to the process whereby tissue cultures or callus

may be induced to form shoots and complete plants.--- Cell and tissue cultures are able to differentiate and form a variety of

organs, e.g., roots, shoots, leaves and flowers. As a rule, theformation of these organs does not depend on the presence of pre-existing initials.

--- The formation of these new meristems involves two distinct growthphases:--- The first is the dedifferentiation of the original explant. This begins

shortly after the isolation of cell division and a consequentformation of a mass of undifferentiated cells.

--- In the second phase, different types of specialized cells, will againdifferentiate.

--- In a given callus mass only a few cells are involved in the initiationprocess, and the onset of the process is asynchronous andsomewhat unpredictable.

--- Torrey (1996) advanced the hypothesis that organogenesis in callus isinitiated with the formation of clusters of meristematic cells(meristemoids) capable of responding to factors within the tissue toproduce a primordium. Depending on the nature of the internalfactors, the stimuli can initiate, either root, shoot, or embryoid.

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台灣泡桐的器官發生：由葉片的切口長出許多不定芽

引用自：張淑華、何政坤（林試所簡訊）

--- Many observations on organ formation in cultured tissues support thehypothesis that localized meristematic activity precedes theorganized development of roots and shoots.

--- Regenerated plantlets from callus culture may show genetic variabilityand deviate from the normal diploids in being polyploids, which isreflected in various kinds of morphological abnormalities.

--- The studies reveal that callus masses are heterogeneous in cellcomposition and that the appearance and diversity of the constituentcells and their associations vary according to(1) the origin of the callus.(2) the extent to which it has been maintained in culture.(3) the composition of the culture medium.(4) endogenous compounds arising during culture.

Factors controlling organogenesis(1). Duration of culture

large vascuolated cells

meristematic zone

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--- Often the potential of a culture to form roots is lost or declines afterseveral subculture, however, there are exceptions to thisgeneralization.

--- This variability in tissues cultured in vitro, which for the present cannotbe satisfactorily explained, is matched by the instability of thegenome.

--- This instability had been clearly shown by investigations on diploidcallus from the meristem of pea roots (Torrey 1967). From this work itis evident that only a few weeks after the isolation of these tissuesthe number of diploid cells starts to decline and the callus comes toconsist mainly of tetraploid, and sometimes even of octoploid cells.

(2). Chemical factors--- The relative concentration of auxins and cytokinins is evidently

involved in initiating organogenesis.--- These two substances determine not only whether the tissue grows,

but also how it grows. The pith parenchyma of tobacco shoots, isgrown in vitro, (Skoog & Miller 1957) the agar medium containing 2mg/l of auxin and 1 mg/L of kinetin only an undifferentiated callusresults. The two substances in this case work additively. However ifthe kinetin concentrations is lowered to 0.02 mg/l without altering theauxin level, roots will arise in the cultures. Higher concentrations ofkinetin (0. 5 mg/L) lead, conversely, to the initiation of shoots, rootformation is then suppressed. Other compounds modify thisinteraction. Raising the phosphate concentration of the medium canreinforce the shoot formation and suppress or weaken the root -promoting effect of auxin. Adenine and amino acids, such as tyrosine,act similarly for in suitable concentrations they augment the kinetineffect and reduce that of auxin.

--- So far there are only a few observations with other tissues supportingthe principle that organogenesis is regulated by quantitative shifts inthe ration of hormones or other known substances.

--- The decisive factor for root or shoot formation in these culturesapparently was not the ratio between these substances, but ratherthe absolute concentration of the auxin in the medium.

--- The principle of regulation of organogenesis by quantitative changes ofthe ration between certain specific compounds cannot be generalized

Ⅱ-4-25

tissues.(3). Other factors

--- Organogenesis is generally dependent upon the size of the explantcultured.

--- The smaller the explant, the less has been the regenerative ability.--- The source of the explant cultured is important in determining the

regenerative potential.--- The physiological age of the explant is another factor which exercises

an influence on organ formation.--- Seasonal variations exercised a profound influence on regeneration.--- Oxygen gradient in a tissue culture may play an effective role in the

promotion of organogenesis as confirmed by Kessel and Carr (1972)in carrot. With reduction in the available oxygen, shoot formation wasfavored while rooting required an increased oxygen gradient.

--- The quality and intensity of light often played a key role inorganogenesis phenomenon. The blue region of the spectrumpromoted shoot formation and red light favored rooting.

--- Temperature, photoperiod, light intensity, pH, sugar concentration andeven quality of agar are other factors that may have a determiningrole in organogenesis and embryogenesis but on which the data aremeager.

4. Embryogenesis--- The capacity of flowering plant to produce embryos is not restricted to

the development of the fertilized egg; embryo (embryoids) can beinduced to form in culture plant tissues.

--- This phenomenon was first observed in suspension cultures of carrot bySteward and co-workers (1958) and in carrot culture grown on an agarmedium by Keinert (1959).

--- Although individual carrot cells are totipotent and carry all the genetictemplates necessary for the development of the whole plant, isolatedsingle cell do not generally become transformed into embryos byrepeated division.

--- By direct microscopic observations it could be proved that isolatedparenchymatous single cells from carrot cultures must first produce amulticellular aggregate before embryos can be initiated.

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--- The embryogenic cells in callus possessed protoplasmic strands linkingthem with neighboring cells during the initiation of embryos.

--- It appears that ”synergistic” cells are necessary for the embryogenesis process.

--- These results quite naturally raise the question of the criteria for definingadventive embryos, in particular how they are to be clearlydistinguished from developing buds.

--- The best definition states that the decisive feature for categorizing aplant structure as an embryo, besides other morphological properties, it isbiopolarity and the fact that at the earliest developmental stage it has atopposite ends a shoot and a radicular pole.

台灣油杉體胚癒合組織發育成許多早期胚（左）球形胚發育成具四枚子葉之成熟體胚（右）

引用自：何政坤、楊政川（林試所簡訊）

Somatic embryos at various stages. Precotyledonary stage to mature cotyledonary stage.

Ⅱ-4-27

--- Each proembryoid cell is capable of passing through the sequentialstages of embryo formation (i.e., globular, heart shape, and torpedoshape).

Factors controling embryogenesis--- Two distinctly different types of media may be required, one media for

the initiation of the embryogenic cells and another for the subsequentdevelopment of these cells into embryoids.

--- The most important chemical factors involved in the induction of somaticembryogenesis are the exogenous auxin content of the medium andthe composition of the nitrogen compounds added as nutrients.

--- The induction requires the presence of auxin, and an increased ration ofnitrogen to auxin may be important. Subsequent development of theembryogenic cells is severely restricted by the presence of auxin, andthe cultures are transferred to a medium very low or lacking inexogenous auxin after the induction stage.

--- The role of cytokinins in embryogenesis is somewhat obscure becauseof conflicting results.

--- In general, embryogenesis occur must readily in short-term culture.There are exceptions, however, and embryogenesis has been reported

Ⅱ-4-28

in some cultures maintained over a period of years.

5. Culture of the shoot apex.--- White (1933) made one of the first attempts in the culture of the shoot tip

using Stellaria media. Later, Loo (1945) succeeded in culturing stem tips,some 5 mm in length, excised from Asparagus officinalis.

--- The apical meristem refers only to the region of the shoot apex lying distalto the youngest leaf primordium, whereas the shoot apex refers to theapical meristem plus a few subjacent leaf primordia.

--- The in vitro culture of the shoot apex of woody plants requires severalconsecutive treatments.

--- Isolated buds generally require a prolonged low-temperaturetreatments prior to culture.

--- The excised bud, with the formation of a rosette of leaves, requiresexogenous gibberellin and cytokinin for growth.

--- The explant must then be transferred to another medium lackingexogenous growth regulators in order to promote stem elongation.

--- Finally, the culture must be transferred to a third medium containingexogenous auxin for the initiation of roots.

--- Although agar media have been successfully used for the culture of apicalmeristems, difficulty has been encountered with the culture of someisolated meristems. Goodwin (1966) developed a technique involving a

Ⅱ-4-29

filter-paper bridge. The explants are cultured individually in a rimlessPyrex test tube (16 x 150mm) containing a strip of Whatman No.1 filterpaper (9 x 90mm) folded in the shape of the letter M. The arms of thepaper strip are immersed in the liquid medium in the bottom of the tube.The explant, placed upright in the V of the bridge, is about 10-15 mm fromthe surface of the medium.

--- The required condition for each plant species vary and must be ascertainedexperimentally.

--- The methods described below provide guidelines on the choice of themedium, growth hormone ratio and culture conditions for meristem culture.

A. Materials and Reagents(1). Plant materials

--- The plant material should be from seedlings, new buds or youngshoots. Shoots with floral organs are unsuitable.

(2).Growth hormones(a) Cytokinins

Use either kinetin or BA in the medium at the following levels: 10-5,5 x 10-6, 5 x 10-7 and 10-7 M.

(b) AuxinsSimilar levels of NAA, IAA or IBA as indicated for cytokinins may beemployed in the medium. NAA is preferred, as IAA is unstable.Note: Combinations of cytokinins and auxin should include a wide

range of concentrations e.g. 25 combinations at the 5 levelsindicated.

(c) Gibberellic acid (GA3)At levels ranging from 10-6 M to 10-8 M, may be needed in addition toabove.Note: A level exceeding 10-6 M may suppress shoot initiation in

meristerms but it can enhance shoot development after initiation.(3). Nutrient agar medium

--- The MS medium is generally the most satisfactory, also B5 medium canbe employed.

--- Add the required amount of growth hormones.Note: IAA and IBA should be sterilized by filtering.

--- Adjust the pH to 5.7 with KOH or HCl.

Ⅱ-4-30

--- Add 0.6% Difco Bacto agar to the medium and heat until the agar isdissolved.

--- Dispense 25 mL into 25 x 100 mm tubes, plug the tubes with cottonand autoclave at 20 psi for 20 min.

Note: If any of the growth hormones need to be filter sterilized they areadded after autoclaving. The medium dispensed into the tubes.

--- After autoclaving, leave the tubes to cool at room temperature. Themedium can be stored for 6-8 weeks at 4℃.

(4). Growth conditions--- A growth cabinet programmed to provide a light intensity of 3000-4000

lux, a light and dark cycle of 16/8 hr, a temperature varying from 22-26℃, and a relative humidity of 70%.

B. Procedures(1). Preparation of sterile tissue.

--- Remove the shoot spices of young sprouts 3-5 cm long from the parentplant with a sharp razor blade.

--- Remove the leaves and rinse apices thoroughly in 70% ethanol.--- Soak in a 7% solution of sodium hypochloride for 5-10 min.Tween 20 or

80 (0.01%) may be added to the disinfectant to increase its wettingpower. Wash 5-6 times in sterile distilled water.

(2). Dissection of meristematic domes.--- Sterilize the instruments (knives, needles and forceps) by immersing in

70% ethanol, rinse in distilled water and dry with sterilized filter/blottingpaper.

Note : This should be done before each operation and as frequently aspossible.

--- Wipe the microscope and the dissection stage with 70% ethanol.--- Hold the disinflected shoot apex with a pair of forceps with one hand

under a workable magnification (10-50x) of the microscope.--- Remove the outer whorls of leaves with sharp sterile knives until themeristematic dome is reached. It will appear “glossy” under the microscope.

--- By manipulating the knives, make four cuts at the base of themeristematic domes at right angles to each other, gently remove it andplant immediately on the agar medium.

Note: The meristematic domes should contain portions of procambial

Ⅱ-4-31

tissue.--- It is not essential to remove the leaf primordia unless the culturing is

aimed at elimination of systemic viruses.--- Incubate the tubes containing meristems in the growth cabinet.

Note : It is desirable to test other growth conditions as well, e.g., 4000lux (some incandescent light ), 16/8 photoperiod at (alternative)temperatures ranging from 15-24℃.

--- When plantlets are formed, generally after 4-6 weeks, remove themfrom the tubes, wash the agar from the root system in running tapwater and plant them into plants should be kept covered with eitherglass beakers or plastic bags until they are established. Water withHoagland nutrient solution weekly.

Note: The size of the meristematic dome determines their ability tosurvive on nutrient medium even at optimal conditions. When themeristematic domes are isolated along with the adjoining leafprimodria and portions of the procambial tissue the survival rate isgreater.

牛樟未成熟種子誘導不定胚

紅檜徑頂培養

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Glossary of tissue culture terms

Adventitious : Roots, shoots, embryos, or other organs or tissues developingin an abnormal position.

Allotetraploid : Plant with four sets of chromosomes, usually with two sets fromeach parent.

Anther : Microsporangium bearing microspores which develop into pollengrains (microgametophytes).

Anther culture : The aseptic culture of anthers for the production of haploidplants from microspores.

Aseptic culture : Surface sterilization of parental explants, free from pathogens,but not necessarily free of internal symbionts.

Asexual propagation : Vegetative, somatic nonsexual propagation of plantparts without fertilization.

Auxins : Plant growth hormones of several types which cause cellenlargement , apical dominance and root initiation. One natural auxin isIAA(indoleacetic acid).

Axenic culture : Free of symbionts: not possible with surface sterilization ,and incorrectly used for aseptic culture.

Callus culture : Proliferation from a parental explant of many cells inprotoplasmic continuity, but having no equivalence with any normal tissue .Same as tissure culture.

Cell differentiation : Internal chemical or ultrastructural changes preceding oraccompanying specialization of function .

Cell suspension : Culture of single cells in moving liquid medium, often usedincorrectly to describe suspension cultures of cells and cell aggregates.

Clonal propagation : Asexual propagation from one individual (ortet ) of manynew plants (ramets)all with the same genotype.

Cytokinin : A class of plant growth hormones associated with cell division andcell differentiation.

Diploid : Having twice the number of chromosome sets found in normal sexcells, and reconstituted during fertilization or the somatic hybridization ofhaploid cells as the normal somatic (diploid ) number of chromosome setsper cell.

Embryo : The young plant developing in the megagametophyte from thefertilization of an egg cell , or without fertilization. In aseptic culture,

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adventitious embryos show polarization followed by the growth of a shootfrom one end a root from the other end.

Excise : Cut or isolate callus tissue from its parental explant, or to removeadventitious shoot from callus tissue for rooting.

Explant : A plant part excised and prepared for aseptic culture by surfacesterilization followed by the exposure of live cells to a nutrient medium.

Fertilization : The normal union of two gametes during sexual reproduction.Gamete : A sex cell, or nucleus, derived from a gametophyte and containing a

haploid wet of chromosomes, or one -half the number in somatic cells.Genotype : The genetic makeup of an individual carried in the chromosomes.Haploid : One -half the number of chromosome sets found in somatic cells ,

forming the typical chromosome number in sex cells, or gametes.Hybridization : The production of offspring of genetically different parents,

normally from sexual reproduction but also asexually by the fusion ofprotoplasts or by transformation.

Inoculum : A small piece of tissue cut from callus, or a small amount ofcell material from a suspension culture placed in contact with freshmedium for continued growth of the culture. Inocula (plural).

Karyotype : The characteristic number, size and shape of the chromosomes ina haploid set.

Megagametophyte : Produces the female egg, or sex cell. In gymnosperms, italso produces a nutritive tissue composed of haploid cells.

Meristem : A localized group of cells, actively dividing and undifferentiated butultimately giving rise to permanent tissue.

Meristimoid : A localized group of cells in callus tissue, characterized by anaccumulation of starch , RNA and protein, and giving rise to advantitiousshoots or roots.

Microspore : Haploid cell ultimately developing into a pollen grain(microgametophyte) that produces male gametes.

Nutrient medium : A solid or liquid combination of major and minor salts, anenergy source (sucrose), vitamins, hormones, and occasionally otherdefined or undefined supplements . Usuallyu made up from previouslyprepared stock solution, then sterilized by autoclaving or filtering through amicropore filter. Media (plural).

Organ culture : The aseptic culture of intact plant parts or organs that do notproliferate but maintain their original form .

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Organized tissue : Composed of regularly differentiated cells.Organogenesis : The initiation of adventitious shoots or roots from callus

tissue, or embryos from single cells or cell aggregates.Parasexual hybridization : Asexual fusion of protoplasts from genetically

different parents. Also called somatic, or vegetative, hybridization .Parenchymatous : Generally spherical and undifferentiated cells with primary

cell walls, capable of division and differentiation.Passahe : The duration of growth of callus or cell material from one subculture

to another.Plantlet : A small rooted shoot . Also, a piece of callus having both roots and

shoots that are not connected together inside the callus.Ploidy : The level of polyploidy, or the number of sets of chromosomes per cell

over two sets of chromosomes per cell over two sets.Polyploid : Containing three or more sets of chromosomes per cell, and also

referred to specifically as triploid, tetraploid, etc.Protoplast : A spherical cell without a wall, surrounded by a membrane.Somatic : Referring to vegetative, or nonreproductive, tissue.Suspension culture : Cells or cell aggregates dispersed and growing in

moving liquid medium .Tissue culture : Now used interchangeably with callus culture, but originally

used to describe what is now called organ culture.Transformation : Specifically, the transfer of genetic information to a plant cell

by DNA isolated from another cell, but now a general term for genetictransfer by different means, also called transgenosis.

Vacuole : A storage area in a cell surrounded by a membrane.Vegetative propagation : Same as asexual, or nonsexual, propagation.