Plant tissue culture
Plant tissue culture
Introduction Introduction
Most methods of plant transformation applied to GM crops require that Most methods of plant transformation applied to GM crops require that a whole plant is regenerated from isolated plant cells or tissue which a whole plant is regenerated from isolated plant cells or tissue which have been genetically transformed. This regeneration is conducted have been genetically transformed. This regeneration is conducted inin vitro
vitro so that the environment and growth medium can be manipulatedso that the environment and growth medium can be manipulated
to
to enensursure e a a hihigh gh frfreqequeuencncy y of of rregegenenereratatioion. n. In In adaddiditition on to to a a hihighgh frequency of regeneration, the regenerable cells must be accessible to frequency of regeneration, the regenerable cells must be accessible to gene transfer by whatever technique is chosen. The primary aim is gene transfer by whatever technique is chosen. The primary aim is the
therereforfore e to to prproduoduce, as ce, as easeasily and ily and as as quiquicklckly y as as pospossibsible, le, a a larlargege number of regenerable cells that are accessible to gene transfer. The number of regenerable cells that are accessible to gene transfer. The subsequent regeneration step is often the most difficult step in plant subsequent regeneration step is often the most difficult step in plant transformation studies. However, it is important to remember that a transformation studies. However, it is important to remember that a high frequency of
high frequency of regeneratiregeneration does on does not necessarily correlate with highnot necessarily correlate with high transformation efficiency.
transformation efficiency.
Plant tissue culture Plant tissue culture
Practically any plant transformation experiment relies at some point on Practically any plant transformation experiment relies at some point on tissue culture. There are some exceptions to this generalization, but tissue culture. There are some exceptions to this generalization, but the ability to regenerate plants from isolated cells or tissues
the ability to regenerate plants from isolated cells or tissues in vitroin vitro
underpins most plant transformation systems. underpins most plant transformation systems.
Plasticity and totipotency Plasticity and totipotency
Two concepts, plasticity and totipotency, are central to understanding Two concepts, plasticity and totipotency, are central to understanding plant cell culture and regeneration. Plants, due to their long life span, plant cell culture and regeneration. Plants, due to their long life span, hav
have e devdeveloeloped ped a a grgreateater er abiabilitlity y to to endendurure e exextrtrememe e conconditditionions s andand predation than have animals. Many of the processes involved in plant predation than have animals. Many of the processes involved in plant gr
growtowth h and and devdeveloelopmepment nt adaadapt pt to to envenvirironmonmentental al condconditiitionsons. . ThiThiss pl
plasastiticicity ty alallolows ws plplanants ts to to alalteter r ththeieir r memetatabobolilismsm, , grgrowowth th anandd dev
develoelopmepment nt to to besbest t suisuit t thetheir ir envenvirironmonmentent. . PPartarticuicularlarly ly imimporportantantt as
aspepectcts s of of ththiis s adadapapttatatioion, n, as as fafar r as as plplanant t ttisissusue e cuculltuturre e anandd regeneration are concerned, are the abilities to initiate cell division regeneration are concerned, are the abilities to initiate cell division from almost any tissue of the plant and to regenerate lost organs or from almost any tissue of the plant and to regenerate lost organs or under
undergo go differdifferent ent develdevelopmenopmental tal pathwpathways ays in in reresponse sponse to to partiparticularcular st
stimimululi. i. WhWhen en plplanant t cecelllls s anand d titissssueues s arare e cucultlturureded in in vivitrtroo theythey
generally exhibit a very high degree of plasticity, which allows one generally exhibit a very high degree of plasticity, which allows one type of tissue or organ to be initiated from another type. In this way, type of tissue or organ to be initiated from another type. In this way, whole plants can be subsequently regenerated. This regeneration of whole plants can be subsequently regenerated. This regeneration of whole organisms depends upon the concept that all plant cells can, whole organisms depends upon the concept that all plant cells can, giv
given en the the corcorrrect ect stistimulmuli, i, exexprpress ess the the tottotal al gengenetietic c potpotentential ial of of thethe pa
parreent nt plplanantt. . ThThis is mmaiaintnteenanancnce e of of gegenenettic ic popotetenntitial al is is cacallleledd ‘totipotency’. Plant cell culture and regeneration do, in fact, p
‘totipotency’. Plant cell culture and regeneration do, in fact, provide therovide the most compelling evidence for totipotency.
The culture environment The culture environment When cultured
When cultured in vitroin vitro, all the needs, both chemical (see Table 1) and, all the needs, both chemical (see Table 1) and
phy
physisicacal, l, of of ththe e plplanant t cecelllls s hahave ve to to memet t by by ththe e cucultlturure e vevesssselel, , ththee gr
growtowth h memediudium m and and the the exexterternal nal envenvirironmonment ent (li(lightght, , temtemperperatuaturere,, etc.). The growth medium has to supply all the essential mineral ions etc.). The growth medium has to supply all the essential mineral ions rreqequiuirred ed fofor r grgrowowtth h anand d dedevvelelopopmmenentt. . In In mmanany y ccasasees s ((as as ththee biosynthetic capability of cells cultured
biosynthetic capability of cells cultured in vitroin vitro may not replicate thatmay not replicate that
of the parent plant), it
of the parent plant), it must also supply additional must also supply additional organic supplementsorganic supplements such as amino acids and vitamins. Many plant cell cultures, as they are such as amino acids and vitamins. Many plant cell cultures, as they are not photosynthetic, also
not photosynthetic, also requirrequire the addition e the addition of a fof a fixed carbon source inixed carbon source in the form of a sugar (most often sucrose). One other vital component the form of a sugar (most often sucrose). One other vital component that must also be supplied is water, the principal biological solvent. that must also be supplied is water, the principal biological solvent. Physical factors, such as temperature, pH, the gaseous environment, Physical factors, such as temperature, pH, the gaseous environment, lig
light ht (qu(qualiality ty and and durduratiation) on) and and osmosmotiotic c prpressessurure, e, alsalso o havhave e to to bebe maintained within acceptable limits.
maintained within acceptable limits. Plant cell culture media
Plant cell culture media Culture media used for the
Culture media used for the in vitroin vitro cultivation of plant cells arecultivation of plant cells are
composed of three basic components: composed of three basic components:
(1) essential elements, or mineral ions, supplied as
(1) essential elements, or mineral ions, supplied as a complex mixturea complex mixture of salts;
of salts;
(2) an organic supplement supplying vitamins and/or
(2) an organic supplement supplying vitamins and/or amino acids; andamino acids; and (3) a source of fixed carbon; usually supplied
(3) a source of fixed carbon; usually supplied as the sugar as the sugar sucrose.sucrose. For practical purposes, the essential elements are further divided into For practical purposes, the essential elements are further divided into the following categories:
the following categories: (1) macroelements (or
(1) macroelements (or macronutriemacronutrients);nts); (2) microelements (or micronutrients); and (2) microelements (or micronutrients); and (3) an iron
Com
Compleplete, te, plaplant nt celcell l culculturture e medmedium ium is is usuusuallally y madmade e by by comcombinbininging several different components, as outlined in Table 2.
several different components, as outlined in Table 2. Media components
Media components
It is useful to briefly consider some of the individual components of the It is useful to briefly consider some of the individual components of the stock solutions.
stock solutions. Macroelements Macroelements
As is implied by the name, the stock solution supplies those elements As is implied by the name, the stock solution supplies those elements required in large amounts for plant growth and development. Nitrogen, required in large amounts for plant growth and development. Nitrogen, phosphorus, potassium, magnesium, calcium and sulphur (and carbon, phosphorus, potassium, magnesium, calcium and sulphur (and carbon, which is added separately) are usually regarded as macroelements. which is added separately) are usually regarded as macroelements. These elements usually comprise at least 0.1% of the dry weight of These elements usually comprise at least 0.1% of the dry weight of
plants. plants.
Nitrogen is most commonly supplied as a mixture of nitrate ions (from Nitrogen is most commonly supplied as a mixture of nitrate ions (from the KNO
the KNO33) and ammonium ions (from the NH) and ammonium ions (from the NH44NONO33). Theoretically, there). Theoretically, there
is an advantage in supplying nitrogen in the form of ammonium ions, is an advantage in supplying nitrogen in the form of ammonium ions, as
as ninitrtrogeogen n mumust st be be in in ththe e rrededucuced ed forform m to to be be inincocorprpororatated ed inintoto mac
incorporation. However, at high concentrations, ammonium ions can incorporation. However, at high concentrations, ammonium ions can be toxic to plant cell cultures and uptake of ammonium ions from the be toxic to plant cell cultures and uptake of ammonium ions from the medium causes acidification of the medium. In
medium causes acidification of the medium. In order to use ammoniumorder to use ammonium ions as the sole nitrogen source, the medium needs to be buffered. ions as the sole nitrogen source, the medium needs to be buffered. Hi
High gh coconcncenentrtratatioions ns of of amammomoninium um ioions ns cacan n alalso so cacaususe e cucultltururee pr
probloblemems s by by incincrereasiasing ng the the frfrequequencency y of of vitvitrifrificaicatiotion n (th(the e culculturturee appears pale and ‘glassy’ and is usually unsuitable for further culture). appears pale and ‘glassy’ and is usually unsuitable for further culture). Using a mixture of nitrate and ammonium ions has the advantage of Using a mixture of nitrate and ammonium ions has the advantage of weakly buffering the medium as the uptake of nitrate ions causes OH¯ weakly buffering the medium as the uptake of nitrate ions causes OH¯ ions to
ions to be excretbe excreted.ed.
Phosphorus is usually supplied as the phosphate ion of ammonium, Phosphorus is usually supplied as the phosphate ion of ammonium, sodium or potassium salts. High concentrations of phosphate can lead sodium or potassium salts. High concentrations of phosphate can lead to the precipitation of medium elements as insoluble phosp
to the precipitation of medium elements as insoluble phosphates.hates. Microelements
Microelements
These elements are required in trace amounts for plant growth and These elements are required in trace amounts for plant growth and development, and have many and diverse roles. Manganese, iodine, development, and have many and diverse roles. Manganese, iodine, copper, cobalt, boron, molybdenum, iron and zinc usually comprise the copper, cobalt, boron, molybdenum, iron and zinc usually comprise the microelements, although other elements such as nickel and aluminium microelements, although other elements such as nickel and aluminium are frequently found in some formulations. Iron is
are frequently found in some formulations. Iron is usually added as usually added as ironiron sul
sulphaphate, te, altalthouhough gh iriron on citcitratrate e can can alsalso o be be useused. d. EthEthylylene ene DiaDiaminminee Tetra acetic Acid (EDTA) is usually used in conjunction with the iron Tetra acetic Acid (EDTA) is usually used in conjunction with the iron
sulphate. The EDTA comple
sulphate. The EDTA complexes with the iron so as to allow the xes with the iron so as to allow the slow andslow and con
contintinuouuous s rerelealease se of of iriron on intinto o the the memediudium. m. UncUncompomplelexxed ed iriron on cancan precipitate out of the
precipitate out of the medium as ferric oxide.medium as ferric oxide. Organic supplements
Organic supplements
Only two vitamins, thiamine (vitamin B
Only two vitamins, thiamine (vitamin B11) and myo-inositol (considered) and myo-inositol (considered
a B vitamin) are considered essential for the culture of plant cells a B vitamin) are considered essential for the culture of plant cells inin vitro
vitro. . HowHoweveever, r, othother er vitvitamiamins ns arare e alsalso o addadded ed to to plaplant nt celcell l culculturturee
media. media.
Amino acids are also commonly included in the organic supplement. Amino acids are also commonly included in the organic supplement. Th
The e mosmost t frfrequequentently ly useused d is is glyglycincine e (ar(arginginineine, , aspasparaaragingine, e, aspaspartarticic acid, alanine, glutamic acid, glutamine and proline are also used), but acid, alanine, glutamic acid, glutamine and proline are also used), but in many cases its inclusion is not essential. Amino acids provide a in many cases its inclusion is not essential. Amino acids provide a source of reduced nitrogen and, like ammonium ions, uptake causes source of reduced nitrogen and, like ammonium ions, uptake causes aci
acidifdificaicatiotion n of of the mediuthe medium. m. CasCasein ein hydhydrorolyslysate can ate can be be useused d as as aa relatively cheap sourc
relatively cheap source of a e of a mix of amino acids.mix of amino acids.
Carbon source Carbon source
Sucrose is cheap, easily available, readily assimilated and relatively Sucrose is cheap, easily available, readily assimilated and relatively stable and is therefore the most commonly used carbon source. Other stable and is therefore the most commonly used carbon source. Other carbohydrates (such as glucose, maltose, galactose and sorbitol) can carbohydrates (such as glucose, maltose, galactose and sorbitol) can
also be used, and in specialized circumstances may prove superior to also be used, and in specialized circumstances may prove superior to sucrose.
sucrose.
Gelling agents Gelling agents Media for plant cell
Media for plant cell cultureculture in vitroin vitro can be used in either liquid or ‘solid’can be used in either liquid or ‘solid’
forms, depending on the type of culture being grown. For any culture forms, depending on the type of culture being grown. For any culture types that require the plant cells or tissues to be grown on the surface types that require the plant cells or tissues to be grown on the surface
of the medium, it must be solidified (more correctly termed ‘gelled’). of the medium, it must be solidified (more correctly termed ‘gelled’). Agar, produced from seaweed, is the most common type of gelling Agar, produced from seaweed, is the most common type of gelling agent, and is ideal for routine applications. However, because it is a agent, and is ideal for routine applications. However, because it is a natural product, the agar quality can vary from supplier to supplier and natural product, the agar quality can vary from supplier to supplier and fr
from om batbatch ch to to batbatch. ch. FoFor r mormore e demdemandanding ing appappliclicatiationsons, , a a ranrange ge of of purer (and in some
purer (and in some cases, considerably more expensive) gelling agentscases, considerably more expensive) gelling agents are available.
are available. Summary Summary
These components, then, are the basic ‘chemical’ necessities for plant These components, then, are the basic ‘chemical’ necessities for plant
cel
cell l culculturture e medmedia. ia. HowHoweveever, r, othother er addiadditiotions ns arare e madmade e in in ororder der toto manipulate the pattern of growth and development of the plant cell manipulate the pattern of growth and development of the plant cell culture.
culture.
Plant growth regulators Plant growth regulators W
We e havhave e alralreadeady y bribrieflefly y conconsidsiderered ed the the conconcepcepts ts of of plaplastisticitcity y andand totipotency
totipotency. The . The essential point as essential point as far as far as plant cell culture is plant cell culture is concernedconcerned is
is ththatat, , dudue e to to tthihis s plplasastiticicity ty anand d totottipipototeencncyy, , spspeecicifific c mmedediiaa manipulations can be used to direct the development of plant cells in manipulations can be used to direct the development of plant cells in culture. Plant growth regulators are the critical media components in culture. Plant growth regulators are the critical media components in determining the developmental pathway of the plant cells. The plant determining the developmental pathway of the plant cells. The plant growth regulators used most commonly are plant hormones or their growth regulators used most commonly are plant hormones or their synthetic analogues.
synthetic analogues.
Classes of plant growth regulators Classes of plant growth regulators
There are five main classes of plant growth regulator used in
There are five main classes of plant growth regulator used in plant cellplant cell culture, namely: culture, namely: (1) auxins; (1) auxins; (2) cytokinins; (2) cytokinins; (3) gibberellins; (3) gibberellins; (4) abscisic acid; (4) abscisic acid; (5) ethylene. (5) ethylene. Each class of
Each class of plant growth regulator will be briefly looked at.plant growth regulator will be briefly looked at. Auxins
Auxins
Auxins promote both cell division and cell growth The most important Auxins promote both cell division and cell growth The most important naturally occurring auxin is IAA (indole-3-acetic acid), but its use in naturally occurring auxin is IAA (indole-3-acetic acid), but its use in plant cell culture media is limited because it is unstable to both heat plant cell culture media is limited because it is unstable to both heat and light. Occasionally, amino acid conjugates of IAA (such as and light. Occasionally, amino acid conjugates of IAA (such as indole-acety
acetyl-L-l-L-alanialanine ne and and indoleindole-acet-acetyl-Lyl-L-gly-glycine)cine), , whicwhich h are are mormore e stablstable,e, are used to partially alleviate the problems associated with the use of are used to partially alleviate the problems associated with the use of IAA. It is more common, though, to use stable chemical analogues of IAA. It is more common, though, to use stable chemical analogues of IIAA AA aas s a a ssoouurrcce e oof f aauuxxiin n iin n ppllaannt t cceelll l ccuullttuurre e mmeeddiiaa. . 22,,4 4--Dichlorophenoxyacetic acid (2,4-D) is the most commonly used auxin Dichlorophenoxyacetic acid (2,4-D) is the most commonly used auxin and
available (see Table 3), and some may be more effective or ‘potent’ available (see Table 3), and some may be more effective or ‘potent’ than 2,4-D in some instances.
than 2,4-D in some instances.
Cytokinins Cytokinins
Cytokinins promote cell division. Naturally occurring cytokinins are a Cytokinins promote cell division. Naturally occurring cytokinins are a la
largrge e grgrououp p of of ststruructctururalally ly rrelelatated ed (t(thehey y arare e pupuririne ne dederirivavatitiveves)s) compounds. Of the naturally occurring cytokinins, two have some use compounds. Of the naturally occurring cytokinins, two have some use in plant
in plant tissue culture media (see Ttissue culture media (see Table 4). These able 4). These are zeatin and are zeatin and 2iP (2-2iP (2-isopentyl adenine). Their use is not widespread as they are expensive isopentyl adenine). Their use is not widespread as they are expensive (particularly zeatin) and relatively unstable. The synthetic analogues, (particularly zeatin) and relatively unstable. The synthetic analogues, ki
kinenetitin n anand d BABAP P (b(benenzyzylalamiminopnopururinine)e), , arare e ththererefeforore e usused ed momorree fr
freeququenentltlyy. . Non-Non-pupuririnene-b-basaseed d chechemmicicalals, s, susucch h as as susubsbsttititututeded phenylureas, are also used as cytokinins in plant cell culture media. phenylureas, are also used as cytokinins in plant cell culture media. These substit
These substituted phenyluruted phenylureas can eas can also substitutalso substitute e for auxin for auxin in in somesome culture systems.
Gibberellins Gibberellins T
Theherre e arare e nunumemerrouous, s, nanatuturaralllly y ococcucurrriringng, , ststruructctururalally ly rrelelatateded compou
compounds nds tertermed med ‘gibbe‘gibberelrellins’lins’. . They They are involved in are involved in reregulatigulating ng cellcell elo
elongatngationion, , and and arare e agragronomonomicaically lly imimporportantant t in in detdetererminmining ing plaplantnt height and fruit-set. Only a few of the gibberellins are used in plant height and fruit-set. Only a few of the gibberellins are used in plant tissue culture media, Gibberelic Acid 3 (GA3) being
tissue culture media, Gibberelic Acid 3 (GA3) being the most common.the most common. Abscisic acid
Abscisic acid
Abscisic acid (ABA) inhibits cell division. It is most commonly used in Abscisic acid (ABA) inhibits cell division. It is most commonly used in plant tissue culture to promote distinct developmental pathways such plant tissue culture to promote distinct developmental pathways such as somatic embryogenesis.
as somatic embryogenesis. Ethylene
Ethylene
Ethylene is a gaseous, naturally occurring, plant growth regulator most Ethylene is a gaseous, naturally occurring, plant growth regulator most commonly associated with controlling fruit ripening, and its use
commonly associated with controlling fruit ripening, and its use in plantin plant tissue culture is not widespread. It does, though, present a particular tissue culture is not widespread. It does, though, present a particular pr
probloblem em for for plaplant nt tistissue sue culculturture. e. SomSome e plaplant nt celcell l culculturtures es prproduoducece ethylene, which, if it builds up sufficiently, can inhibit the growth and ethylene, which, if it builds up sufficiently, can inhibit the growth and development of the culture. The type of culture vessel used and its development of the culture. The type of culture vessel used and its means of closure affect the gaseous exchange between the culture means of closure affect the gaseous exchange between the culture vessel and the outside atmosphere and thus the levels of ethylene vessel and the outside atmosphere and thus the levels of ethylene present in the culture.
present in the culture.
Plant growth regulators and tissue culture Plant growth regulators and tissue culture
Generalisations about plant growth regulators and their use in plant Generalisations about plant growth regulators and their use in plant cell culture media have been developed from initial observations made cell culture media have been developed from initial observations made in
in ththe e 19195050s. s. ThTherere e isis, , hohowewevever, r, sosome me coconsnsididererabable le didifffficicululty ty inin predicting the effects of plant growth regulators: this is because of the predicting the effects of plant growth regulators: this is because of the great differences in culture response between species, cultivars and great differences in culture response between species, cultivars and eve
even n plaplants nts of of the the samsame e culcultivtivar ar grgrown own undunder er difdifferferent ent conconditditionions.s. However, some principles do hold true and have become the paradigm However, some principles do hold true and have become the paradigm on
on whiwhich ch mosmost t plaplant nt tistissue sue culculturture e reregimgimes es arare e basbased. ed. AuxAuxins ins andand cytokinins are the most widely used plant growth regulators in plant cytokinins are the most widely used plant growth regulators in plant tissue culture and are usually used together, the ratio of the auxin to tissue culture and are usually used together, the ratio of the auxin to th
the e ccytytokokininiin n dedetterermiminining ng ththe e tytype pe of of cuculltuturre e eeststabablilishsheed d oror regenerated (see Figure 1). A high auxin to cytokinin ratio generally regenerated (see Figure 1). A high auxin to cytokinin ratio generally favours root formation, whereas a high cytokinin to auxin ratio favours favours root formation, whereas a high cytokinin to auxin ratio favours shoot formation. An intermediate ratio favours callus production.
Culture types Culture types
Cultures are generally initiated from sterile pieces of a whole plant. Cultures are generally initiated from sterile pieces of a whole plant. T
Thehese se pipiececes es arare e tetermrmed ed ‘e‘expxplalantnts’s’, , anand d mamay y coconsnsisist t of of pipiececes es of of organs, such as leaves or roots, or may be specific cell types, such as organs, such as leaves or roots, or may be specific cell types, such as pollen or endosperm. Many features of the explant are known to affect pollen or endosperm. Many features of the explant are known to affect the
the efficefficiency of iency of cultuculture re initiinitiation. Generalation. Generally, ly, youngyounger, er, mormore e rapidlrapidlyy growing tissue (or tissue at an early stage of development) is most growing tissue (or tissue at an early stage of development) is most effective. Several different culture types most commonly used in plant effective. Several different culture types most commonly used in plant transformation studies will now be examined in bit detail.
transformation studies will now be examined in bit detail. Callus
Callus
Explants, when cultured on the appropriate medium, usually with both Explants, when cultured on the appropriate medium, usually with both an auxin and a cytokinin, can give rise to an unorganised, growing and an auxin and a cytokinin, can give rise to an unorganised, growing and dividing mass of cells. It is thought that any p
dividing mass of cells. It is thought that any plant tissue can be used aslant tissue can be used as an
an eexpxplalantnt, , if if ththe e cocorrrrecect t cocondndititioions ns arare e fofounund. d. In In cucultlturure, e, ththisis proliferation can be maintained more or less indefinitely, provided that proliferation can be maintained more or less indefinitely, provided that the callus is subcultured on to fresh medium periodically. During callus the callus is subcultured on to fresh medium periodically. During callus formation there is some degree of dedifferentiation (i.e. the changes formation there is some degree of dedifferentiation (i.e. the changes that occur during development and specialization are, to some extent, that occur during development and specialization are, to some extent, rreveverersesed)d), , boboth th in in momorprphoholology gy ((cacallllus us is is ususuaualllly y cocompmpososed ed of of un
unspspececiaialilisesed d paparrenenchchymyma a cecelllls) s) anand d memetatabobolilismsm. . OnOne e mmajajoror consequence of this dedifferentiation is that most plant cultures lose consequence of this dedifferentiation is that most plant cultures lose the ability to photosynthesise. This has important consequences for the ability to photosynthesise. This has important consequences for the culture of callus tissue, as the metabolic profile will probably not the culture of callus tissue, as the metabolic profile will probably not match that of the donor plant. This necessitates the addition of other match that of the donor plant. This necessitates the addition of other components—such as vitamins and, most importantly, a carbon source components—such as vitamins and, most importantly, a carbon source —to the culture medium, in addition to the usual mineral nutrients. —to the culture medium, in addition to the usual mineral nutrients. Callus culture is often performe
Callus culture is often performed in the dark d in the dark (the lack of photosynthetic(the lack of photosynthetic capability being no drawback) as light can encourage differentiation of capability being no drawback) as light can encourage differentiation of th
the e cacallllusus. . DuDuriring ng lolongng-t-tererm m cucultlturure, e, ththe e cucultlturure e mmay ay lolose se ththee rrequequirirememenent t for for auauxixin n anand/od/or r cycytotokikininnin. . ThThis is prprococesess, s, knknowown n asas ‘habituation’, is common in callus cultures from some plant species ‘habituation’, is common in callus cultures from some plant species (such as sugar beet). Callus cultures are extremely important in plant (such as sugar beet). Callus cultures are extremely important in plant biot
biotechechnolnologyogy. . ManManipuipulatlation ion of of the the auxauxin in to to cytcytokiokinin nin ratratio io in in thethe me
emb
embryoryos s frfrom om whiwhich ch whowhole le plaplants nts can can subsubseqsequenuently tly be be prproduoducedced.. Callus cultures can also be used to initiate cell suspensions, which are Callus cultures can also be used to initiate cell suspensions, which are used in a variety of ways in plant transformation studies.
used in a variety of ways in plant transformation studies. Cell-suspension cultures
Cell-suspension cultures Ca
Callllus us cucultlturureses, , brbroaoadldly y spspeaeakikingng, , fafall ll ininto to onone e of of twtwo o cacatetegogoririeses:: compact or friable. In compact callus the cells are densely aggregated, compact or friable. In compact callus the cells are densely aggregated, whereas in friable callus the cells are only loosely associated with each whereas in friable callus the cells are only loosely associated with each oth
other er and and the the calcallus lus becbecomeomes s sofsoft t and and brbreakeaks s apaapart rt easeasilyily. . FFriariableble callus provides the inoculum to form cell-suspension cultures. Explants callus provides the inoculum to form cell-suspension cultures. Explants from some plant species or particular cell types tend not
from some plant species or particular cell types tend not to form friableto form friable callus, making cell-suspension initiation a difficult task. The friability of callus, making cell-suspension initiation a difficult task. The friability of ca
callllus us cacan n sosomemetitimemes s be be imimprprovoved ed by by mamaninipulpulatatining g ththe e memedidiumum components or by repeated subculturing. The friability of the callus components or by repeated subculturing. The friability of the callus cancan also sometimes be improved by culturing it on ‘semi-solid’ medium also sometimes be improved by culturing it on ‘semi-solid’ medium (medium with a low concentration of gelling agent).
(medium with a low concentration of gelling agent). When friable callusWhen friable callus is placed into a liquid medium (usually the same composition as the is placed into a liquid medium (usually the same composition as the solid medium used for the callus culture) and then agitated, single cells solid medium used for the callus culture) and then agitated, single cells and/or small clumps of cells are released into the medium. Under the and/or small clumps of cells are released into the medium. Under the correct conditions, these released cells continue to grow and divide, correct conditions, these released cells continue to grow and divide, eve
eventuntuallally y prproduoducincing g a a cecell-ll-sussuspenpensiosion n culculturture. e. A A rrelaelativtively ely larlargege inoculum should be used when initiating cell suspensions so that the inoculum should be used when initiating cell suspensions so that the released cell numbers build up quickly. The inoculum should not be too released cell numbers build up quickly. The inoculum should not be too large though, as toxic products released from damaged or stressed large though, as toxic products released from damaged or stressed cells can build up to lethal levels. Large cell clumps can be removed cells can build up to lethal levels. Large cell clumps can be removed dur
during ing subsubculculturture e of of the the celcell l sussuspenpensionsion. . CelCell l sussuspenpensiosions ns can can bebe maintained relatively simply as batch cultures in conical flasks. They maintained relatively simply as batch cultures in conical flasks. They are continually cultured by repeated subculturing into fresh medium. are continually cultured by repeated subculturing into fresh medium. This results in dilution of the suspension and the initiation of another This results in dilution of the suspension and the initiation of another batch growth cycle. The degree of dilution during subculture should be batch growth cycle. The degree of dilution during subculture should be determined empirically for each culture. Too great a degree of dilution determined empirically for each culture. Too great a degree of dilution will result in a greatly extended lag period or, in extreme cases, death will result in a greatly extended lag period or, in extreme cases, death of the
of the transferrtransferred cells.ed cells. A
Aftfter er susubcbculultuturre, e, ththe e cecelllls s didivivide de anand d ththe e bibiomomasass s of of ththe e cucultltururee increases in a characteristic fashion, until nutrients in the medium are increases in a characteristic fashion, until nutrients in the medium are exhausted and/or toxic by-products build up to inhibitory levels—this is exhausted and/or toxic by-products build up to inhibitory levels—this is called the ‘stationary phase’. If cells are left in the stationary phase for called the ‘stationary phase’. If cells are left in the stationary phase for too long, they will die and the culture will be lost. Therefore, cells too long, they will die and the culture will be lost. Therefore, cells should be transferred as they enter
should be transferred as they enter the stationary phase. It the stationary phase. It is thereforeis therefore important that the batch growth-cycle parameters are determined for important that the batch growth-cycle parameters are determined for each cell-suspension
each cell-suspension culture.culture. Protoplasts
Protoplasts
Protoplasts are plant cells with the cell wall removed. Protoplasts are Protoplasts are plant cells with the cell wall removed. Protoplasts are mo
most st cocommmmononly ly isisolaolateted d frfrom om eieithther er leleaf af memesosophyphyll ll cecelllls s or or cecellll suspensions, although other sources can be used to advantage. Two suspensions, although other sources can be used to advantage. Two
general approaches to removing the cell wall (a difficult task without general approaches to removing the cell wall (a difficult task without dam
damagiaging ng the the prprotootoplaplast) st) can can be be taktaken—en—memechachanicnical al or or enzenzymymatiaticc isolation. Mechanical isolation, although possible, often results in low isolation. Mechanical isolation, although possible, often results in low yields, poor quality and poor performance in culture due to substances yields, poor quality and poor performance in culture due to substances released from damaged cells. Enzymatic isolation is usually carried out released from damaged cells. Enzymatic isolation is usually carried out in
in a a sisimpmple le sasalt lt sosolulutition on wiwith th a a hihigh gh ososmomotiticucum, m, plplus us ththe e cecell ll wawallll de
degrgradiading ng enenzyzymemes. s. It It is is ususuaual l to to ususe e a a mimix x of of boboth th cecellllululasase e andand pectinase enzymes, which must
pectinase enzymes, which must be of be of high quality and high quality and puritypurity.. Pr
Protootoplaplasts sts arare e frafragilgile e and and easeasily ily damdamageaged, d, and and thetherereforfore e musmust t bebe cultured carefully. Liquid medium is not agitated and a high osmotic cultured carefully. Liquid medium is not agitated and a high osmotic potential is maintained, at least in the initial stages. The liquid medium potential is maintained, at least in the initial stages. The liquid medium must be shallow enough to allow aeration in the absence of agitation. must be shallow enough to allow aeration in the absence of agitation. Protoplasts can be plated out on to solid medium and callus produced. Protoplasts can be plated out on to solid medium and callus produced. Wh
Wholole e plplanants ts cacan n be be rregegenenereratated ed by by ororgaganonogegenenesisis s or or sosomamatiticc em
embrbryoyogegenenesisis s frfrom om ththis is cacallllusus. . PProrototoplplasasts ts arare e idideaeal l tatargrgetets s forfor transformation by a variety of means.
transformation by a variety of means. Root cultures
Root cultures Root culture
Root cultures can s can be establishedbe established in vitroin vitro from explants of the root tip of from explants of the root tip of
either primary or lateral roots and can be cultured on fairly simple either primary or lateral roots and can be cultured on fairly simple media. The growth of roots
media. The growth of roots in vitroin vitro is potentially unlimited, as is potentially unlimited, as roots areroots are
indeterminate organs. Although the establishment of root cultures was indeterminate organs. Although the establishment of root cultures was one of the first achievements of modern plant tissue culture, they are one of the first achievements of modern plant tissue culture, they are not widely used in
not widely used in plant transformation studies.plant transformation studies. Shoot tip and meristem culture
Shoot tip and meristem culture
The tips of shoots (which contain the shoot apical meristem) can be The tips of shoots (which contain the shoot apical meristem) can be
cultured
cultured in in vitvitroro, producing clumps of shoots from either axillary or, producing clumps of shoots from either axillary or
adventitious buds. This method can be used for clonal propagation. adventitious buds. This method can be used for clonal propagation. Sho
Shoot ot memeririststem em cucultlturures es arare e potpotenentitial al alalteternrnatativives es to to ththe e momorree commonly used methods for cereal regeneration (see the Case study commonly used methods for cereal regeneration (see the Case study be
belolow) w) as as ththey ey arare e leless ss gegenonotytypepe-de-depependndenent t anand d momorre e efeffificicienentt (seedlings can be used as donor material).
(seedlings can be used as donor material). Embryo culture
Embryo culture Em
Embrbryoyos s cacan n be be usused ed as as exexplplanants ts to to gegenenerarate te cacallllus us cucultlturures es oror somatic embryos. Both immature and mature embryos can be used as somatic embryos. Both immature and mature embryos can be used as explants. Immature, embryo-derived embryogenic callus is the most explants. Immature, embryo-derived embryogenic callus is the most popular method of monocot
popular method of monocot plant regeneration.plant regeneration.
Microspore culture Microspore culture
Haploid tissue can be cultured
Haploid tissue can be cultured in vitroin vitro by using pollen or anthers as anby using pollen or anthers as an
explant. Pollen contains the male gametophyte, which is termed the explant. Pollen contains the male gametophyte, which is termed the ‘microspore’. Both callus and embryos can be produced from pollen. ‘microspore’. Both callus and embryos can be produced from pollen.
Two main approaches can be taken to produce
Two main approaches can be taken to produce in vitroin vitro cultures fromcultures from
haploid tissue. haploid tissue.
The first method depends on using the anther as the explant. Anthers The first method depends on using the anther as the explant. Anthers
(somatic tissue that surrounds and contains
(somatic tissue that surrounds and contains the pollen) can be the pollen) can be culturedcultured on solid medium (agar should not be used to solidify the medium as it on solid medium (agar should not be used to solidify the medium as it co
contntaiainsnsininhihibibitotorry y susubsbsttananceces)s). . PolPollelen-n-dederirivveed d eemmbrbryyos os araree sub
subseqsequenuently tly prproduoduced ced via via dehdehisciscencence e of of the the mamaturture e antantherhers. s. TheThe dehiscence of the anther depends both on its isolation at the correct dehiscence of the anther depends both on its isolation at the correct st
stagage e anand d on on ththe e cocorrrrecect t cucultlturure e cocondndititioionsns. . In In sosome me spspececieies, s, ththee reliance on natural dehiscence can be circumvented by cutting the wall reliance on natural dehiscence can be circumvented by cutting the wall of
of ththe e ananththerer, , alalththougough h ththis is dodoeses, , of of cocourursese, , tatakke e a a coconsnsididererabablele amount of time. Anthers can also be cultured in liquid medium, and amount of time. Anthers can also be cultured in liquid medium, and poll
pollen en rerelealeased sed frfrom om the anthethe anthers rs can can be be indinduceuced d to to forform m emembrybryos,os, al
alththouough gh ththe e efeffi-fi-cicienency cy of of plplanant t rregegenenereratatioion n is is ofofteten n vevery ry loloww.. Immature pollen can also be extracted from developing anthers and Immature pollen can also be extracted from developing anthers and cultured directly, although this is
cultured directly, although this is a very time-consuming process.a very time-consuming process.
Both methods have advantages and disadvantages. Some beneficial Both methods have advantages and disadvantages. Some beneficial ef
effefectcts s to to ththe e cucultlturure e arare e obobseservrved ed whwhen en ananththerers s arare e usused ed as as ththee ex
explaplant nt matmaterierial. al. TheThere is, re is, howhowevever, er, the dangethe danger r thathat t somsome e of of thethe embryos produced from anther culture will originate from the somatic embryos produced from anther culture will originate from the somatic anther tissue rather than the haploid
anther tissue rather than the haploid microspormicrospore cells. If isolated pollene cells. If isolated pollen is
is usused ed ththerere e is is no no dadangnger er of of mimixxed ed emembrbryo yo foforrmamatitionon, , bubut t ththee efficiency is low and the
efficiency is low and the process is time-consuming.process is time-consuming.
In microspore culture, the condition of the donor plant is of critical In microspore culture, the condition of the donor plant is of critical importance, as is the timing of isolation. Pretreatments, such as a cold importance, as is the timing of isolation. Pretreatments, such as a cold tr
treeatatmmenentt, , arare e ofofteten n fofounund d tto o iincncrreaease se ththe e efeffificcieiencncyy. . ThTheesese pretreatments can be applied before culture, or, in some species, after pretreatments can be applied before culture, or, in some species, after placing the anthers in culture.
placing the anthers in culture.
Plant species can be divided into two groups, depending on whether Plant species can be divided into two groups, depending on whether they require the addition of plant growth regulators to the medium for they require the addition of plant growth regulators to the medium for po
pollllenen/a/antntheher r cucultlturure; e; ththosose e ththat at do do alalso so ofofteten n rreqequiuirre e ororgaganinicc supplements, e.g. amino acids.
supplements, e.g. amino acids. Many of the cereals (rice, wheat, barleyMany of the cereals (rice, wheat, barley and maize) require medium supplemented with plant growth regulators and maize) require medium supplemented with plant growth regulators for pollen/anther
for pollen/anther culture.culture. R
Regeegenerneratiation on frfrom om micmicrorosporspore e exexplaplants nts can can be be obtobtainained ed by by dirdirectect embryogenesis, or via a callus stage
embryogenesis, or via a callus stage and subsequent embryogenesis.and subsequent embryogenesis. Ha
Haplploioid d titissssue ue ccululttururees s ccan an alalso so be be ininiititiatateed d frfrom om tthe he fefemmalalee gam
gametetopophyhyte te (t(the he ovovulule)e). . In In sosome me cacaseses, s, ththis is is is a a momorre e efeffificicienentt method than using pollen or anthers.
method than using pollen or anthers.
The ploidy of the plants obtained from haploid cultures may not be The ploidy of the plants obtained from haploid cultures may not be haploid. This can be a consequence of chromosome doubling during haploid. This can be a consequence of chromosome doubling during th
the e cucultlturure e pepeririodod. . ChChroromomososome me dodoububliling ng (w(whihich ch ofofteten n hahas s to to bebe induced by treatment with chemicals such as colchicine) may be an induced by treatment with chemicals such as colchicine) may be an adv
advanantatagege, , as as in in mamany ny cacaseses s hahaploploid id plplanants ts arare e not not ththe e dedesisirreded outcome of regeneration from haploid tissues. Such plants are often outcome of regeneration from haploid tissues. Such plants are often
referred to as ‘di-haploids’, because they contain two copies of the referred to as ‘di-haploids’, because they contain two copies of the same haploid genome.
same haploid genome.
Plant regeneration
Plant regeneration
Ha
Havvining g lolookokeed d at at ththe e mmaiain n ttypypees s of of plplanant t cculultturure e ththat at ccan an bebe established
established in in vivitrotro, we can now look at how whole plants can be, we can now look at how whole plants can be
regenerated from these
regenerated from these cultures.cultures.
In broad terms, two methods of plant regeneration are widely used in In broad terms, two methods of plant regeneration are widely used in pl
planant t trtranansfsforormamatition on ststududieies, s, i.i.e. e. sosomamatitic c emembrbryoyogegenenesisis s anandd organogenesis.
organogenesis.
Somatic embryogenesis Somatic embryogenesis
In somatic (asexual) embryogenesis, embryo-like structures, which can In somatic (asexual) embryogenesis, embryo-like structures, which can devel
develop op into whole plants in into whole plants in a a way analogous to way analogous to zygotzygotic embryosic embryos, , areare formed from somatic tissues (Figure 2). These somatic embryos can be formed from somatic tissues (Figure 2). These somatic embryos can be produced either directly or indirectly. In direct somatic embryogenesis, produced either directly or indirectly. In direct somatic embryogenesis, the embryo is formed directly from a cell or small
the embryo is formed directly from a cell or small group of cells withoutgroup of cells without the production of an intervening callus. Though common from some the production of an intervening callus. Though common from some tissue
tissues s (usua(usually reprolly reproductiductive tissues ve tissues such as such as the nucelluthe nucellus, s, stylestyles s oror pollen), direct somatic embryogenesis is generally rare in comparison pollen), direct somatic embryogenesis is generally rare in comparison with indirect somatic
with indirect somatic embryogenesis.embryogenesis.
In indirect somatic embryogenesis, callus is first produced from the In indirect somatic embryogenesis, callus is first produced from the explant. Embryos can then be produced from the callus tissue or
explant. Embryos can then be produced from the callus tissue or fromfrom a cell suspension p
a cell suspension produced from that callus.roduced from that callus.
Somatic embryogenesis usually proceeds in two distinct stages. In the Somatic embryogenesis usually proceeds in two distinct stages. In the initial stage (embryo initiation), a high concentration of 2,4-D is used. initial stage (embryo initiation), a high concentration of 2,4-D is used. In the second stage (embryo production) embryos are produced in a In the second stage (embryo production) embryos are produced in a medium with no or very low
In many systems it has been found that somatic embryogenesis is In many systems it has been found that somatic embryogenesis is improved by supplying a source of reduced nitrogen, such as specific improved by supplying a source of reduced nitrogen, such as specific amino acids or casein
amino acids or casein hydrolysate.hydrolysate. Organogenesis
Organogenesis
Somatic embryogenesis relies on plant regeneration through a process Somatic embryogenesis relies on plant regeneration through a process analogous to zygotic embryo germination. Organogenesis relies on the analogous to zygotic embryo germination. Organogenesis relies on the production of organs, either directly from an explant or from a callus production of organs, either directly from an explant or from a callus c
cuullttururee. . TThheerre e aarre e tthrhreee e mmeetthhodods s oof f ppllaannt t rreeggeennereraattiioon n vviiaa organogenesis.
organogenesis.
The first two methods depend on adventitious organs arising either The first two methods depend on adventitious organs arising either
fr
from om a a ccalalllus us cculultturure e or or didirreectctlly y frfrom om an an eexpxpllanant t (F(Fiigugurre e 33).). Alternatively, axillary bud formation and growth can also be used to Alternatively, axillary bud formation and growth can also be used to rregegeenenerratate e wwhoholle e plplanantts s frfrom om sosomme e ttypypees s of of titissssue ue cculultturure.e. Organogenesis relies on the inherent plasticity of plant tissues, and is Organogenesis relies on the inherent plasticity of plant tissues, and is regulated by altering the components of the medium. In particular, it is regulated by altering the components of the medium. In particular, it is the
the auxauxin in to to cytcytokiokinin nin ratratio io of of the the memediudium m thathat t detdetererminmines es whiwhichch developmental pathway the regenerating tissue will take. It is usual to developmental pathway the regenerating tissue will take. It is usual to induce shoot formation by increasing the cytokinin to auxin ratio of the induce shoot formation by increasing the cytokinin to auxin ratio of the culture medium. These shoots can then be rooted relatively simply.
Summary
Summary
Tissue culture and plant regeneration are an integral part
Tissue culture and plant regeneration are an integral part of most plantof most plant tr
tranansfsforormamatition on ststraratetegigieses, , anand d cacan n ofofteten n prprovove e to to be be ththe e momostst challenging aspect of a
challenging aspect of a plant transformation protocol. Kplant transformation protocol. Key to success iney to success in integrating plant tissue culture into plant transformation strategies is integrating plant tissue culture into plant transformation strategies is the realisation that a quick (to avoid too many deleterious effects from the realisation that a quick (to avoid too many deleterious effects from so
somamaclclononal al vavaririatationion) ) and and efeffificicienent t rregegenenereratation ion sysyststem em mumust st bebe developed. However, this system must also allow high transformation developed. However, this system must also allow high transformation efficiencies from whichever transformation technique is adopted.
efficiencies from whichever transformation technique is adopted.
Not all regeneration protocols are compatible with all transformation Not all regeneration protocols are compatible with all transformation techniques. Some crops may be amenable to a variety of regeneration techniques. Some crops may be amenable to a variety of regeneration and transformation strategies, others may currently only be amenable and transformation strategies, others may currently only be amenable
to one particular protocol. Advances are being made all the time, so it to one particular protocol. Advances are being made all the time, so it is impossible to say that a particular crop will never be regenerated by is impossible to say that a particular crop will never be regenerated by a particular protocol. However, some protocols, at least at the moment, a particular protocol. However, some protocols, at least at the moment, are
are cleaclearly rly mormore e efficefficient ient than than otherothers. s. RRegenegeneratioeration n frofrom m immimmaturaturee embryo-derived somatic embryos is, for example, the favoured method embryo-derived somatic embryos is, for example, the favoured method for regenerating monocot species.
