plant practicals bs1003 2010-2011

7/26/2019 Plant Practicals BS1003 2010-2011

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MODULEBS-1003

CELLANDDEVELOPMENTALBIOLOGY

DEPARTMENTOFBIOLOGY

LECTURERS

Lectures 1 & 8 15, and practicals 1,4 & 5 will be given by Prof Pat He!o"-Harr#o$.

Email: phh4le.ac.u!

Pra%t#%a! &ea&!#$e

"and in y#ur practical answer sheets t# the $i#l#gy %epartment ''ice ()drian *+a- by 1. a.m.

#n the /uesday a'ter each practical. 0#u can submit y#ur w#r! anytime up t# the deadline. Note:

this is theBIOLOGY office on the third floor, NOT the College office on the first floor.

"and in ractical 1 with the ractical 4 write2up.

0#u will receive a receipt '#r y#ur submitted w#r! which y#u sh#uld stic! int# the bac! page #' this

handb##!. 0#u will als# have t# sign in y#ur w#r!.

LECTURE1

PROFPATHESLOP-HARRISON

Le%t're 1( I$tro&'%t#o$ to BS-1003) Ce!! a$& De*e!o"+e$ta! B#o!o,

3n this lecture we will discuss basic cell and devel#pmental bi#l#gy with emphasis #n cell structure,ways t# study cells, what we !n#w ab#ut cells and h#w they w#r!, and appr#aches t# cell bi#l#gy.

e will als# discuss h#w and where t# learn ab#ut cell and devel#pmental bi#l#gy.

PRACTICAL1

PLANTTISSUECULTUREANDGENETICTRANSFORMATION

Part 1( P!a$t t#'e %'!t're. %a!!' "ro&'%t#o$ a$& or,a$ re,e$erat#o$ o$

%'!t're& t#'e e/"!a$t of toa%%o

3ntr#ducti#n/he #bective t#day is t# set up plant tissue cultures 'r#m t#bacc# lea' tissue, and y#u will study the

results in detail later, in ractical 4.

/issue culture e6periments dem#nstrate a phen#men#n that is '#und in plants and 'ungi: that is, that

'ully di''erentiated cells can be induced, under certain h#rm#nal c#nditi#ns, t# underg# a new

devel#pmental pr#gramme, and regenerate an entire new #rganism. /his devel#pmental 'le6ibility

#' plants is termedplasticity, and the capability #' regenerating a new #rganism 'r#m previ#usly

di''erentiated cells is termed totipotency. /he pattern #' #rgan regenerati#n seen in t#bacc# tissue

cultures is in'luenced t# a large e6tent by the type and c#ncentrati#ns #' h#rm#nes (auxins andcytokinins- that are supplied t# the explant tissues in the agar culture medium. /ypically,

#rgan#genesis in t#bacc# tissue cultures is preceded by the '#rmati#n #' an undi''erentiated (n#n2


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$721* ell and %evel#pmental $i#l#gy

specialised- tissue, termed callus.

E6perimental3n this e6periment y#u will study the e''ect #' the relative levels #' tw# plant h#rm#nes (au6ins and

cyt#!inins- #n callus '#rmati#n and #rgan regenerati#n 'r#m cultured t#bacc# lea' e6plants.

0#u will carry #ut the w#r! in pairs, in sterile air 'l#w cabinets. Each gr#up #' students will beta!en by their dem#nstrat#r t# the 'l#w cabinets, where the tissue manipulati#ns will be

dem#nstrated. hen w#r!ing in the cabinets, ta!e care t# !eep y#ur e6perimental material 'ree

'r#m micr#bial c#ntaminati#n, by 'laming all dissecting e9uipment (scalpels, '#rceps- and all#wing

it t# c##l immediatelybe'#re use. #r! at all times #n a sterile sur'ace (ie #n the lid #' a etri dish-

situated well inside the sterile cabinet. %# n#t lean #ver, breathe #n, #r t#uch with y#ur 'ingers the

plant material, #r y#u will c#ntaminate it. %# n#t put anything behind the material y#u are handling

in the 'l#w h##d. $e care'ul n#t t# set the alc#h#l #n 'ire by putting very h#t dissecti#n instruments

in it. 3' y#u d#,LEAVE IT and ask for assistance(

)'ter the intr#duct#ry tal!, c#llect andlabel with your names and demonstrator group number the

etri dishes c#ntaining the media. eep the dishes uprightat all times andopen only in a sterile aircabinet.

0#u are pr#vided with sterilised lea' material 'r#m ; wee!2#ld t#bacc# seedlings, and * types #'

tissue culture medium, tw# #' which each c#ntain di''erent levels #' the plant h#rm#nesyl amin#purine (a cytokinin- and ?)) = napthalene acetic acid (a synthetic auxin-. /he

media are designated as '#ll#ws:

@7 2 @7 medium with#ut h#rm#nes A?/BLC

@73 2 @7 medium plus .5 mgDl $) & 4 mgDl ?))

@7%46+ 2 @7 medium plus 1 mgDl $) & .1 mgDl ?))

A@7 = @urashige and 7!##g medium 2 a balanced mi6ture #' in#rganic mineral salts and trace

elementsC

r#t#c#l

1. 7terilise y#ur '#rceps and scalpel by dipping in alc#h#l and 'laming.

+. ut e6plants 'r#m the lea' tissue, each appr#6imately .5 t# 1 cm s9uare, and #n each plate

arrangesixe6plants, evenly spaced apart. /ry n#t t# damage the e6plants t## much by handling:

remember they are alive. Ensure that each e6plant is 'irmly but gently pressed int# the agarmedium, t# ensure g##d c#ntact.

*. 7eal the plates with sealing 'ilm, t# prevent the medium drying #ut.

4. %raw ar#und the #utline #' each e6plant #n the b#tt#m #' the petri dish with a mar!er pen , s#

that y#u have a rec#rd #' #riginal e6plant si>e.

5.Label your plates with your initials and demonstrator groupusing a permanent mar!er and leave

#n the white tray pr#vided. lates will be incubated at +5 in the light (1; h day length- '#r the

ne6t * wee!s.

rite2up3t is imp#rtant that y#u ma!e ?37E n#tes c#ncerning y#ur #bservati#ns #' the original

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explants and at weekly interals#' the numbers and types #' tissuesD#rgans that devel#p during the

tissue culture pr#cess s# that y#u can use this data in answering the 9uesti#ns p#sed during y#ur

detailed analyses in practical 4.!ake sure you examine and make notes about your samples during

both "racticals # and $%

Part ( Ce!! "ro!#ferat#o$ a$& or,a$o,e$e# +eate& Agrobacterium

3ntr#ducti#nlant and animal devel#pment can be perturbed by disease. $#th plants and animals can su''er

oncogenic diseases, in which the n#rmal c#ntr#l #ver cell divisi#n is l#st and tum#urs '#rm. 3n

plants, species #' the s#il bacterium &grobacteriumcause disease sympt#ms in in'ected plants that

are characterised by tum#urs, the s#2called r#wn Fall disease (&grobacterium tumefaciens- #r by

the aberrant pr#ducti#n #' Ghairy r##tsG (&grobacterium rhi'ogenes-. /he disease sympt#ms are

caused by the trans'er 'r#m the bacteria, and e6pressi#n in the plant nucleus, #' genes carried #n a

circular %?) m#lecule (a plasmid, the Gtum#ur2inducingG Ti(plasmid< #r the Hr##t2inducingI )i(

plasmid-, '#ll#wing e6cisi#n and trans'er #' part #' that plasmid. /his trans'er #' genes 'r#m

bacterium t# plant represents a natural '#rm #' genetic engineering, and has been e6pl#ited

e6perimentally as a means #' m#di'ying plant gr#wth, devel#pment and metab#lism, '#r b#th basicand applied research purp#ses.

lant tum#ur tissues are characterised by an ability t# gr#w #n media lac!ing h#rm#nes,

since the genes trans'erred 'r#m the bacteria t# the plant cells enc#de genes that pr#m#te the

synthesis #' au6ins and cyt#!inins.

E6perimental3n this e6periment y#u will in#culate plant tissues (carr#t tap r##ts- with three di''erent strains #'

&grobacterium, and study the e''ects #n the in'ected tissue. /he e6plants will be maintained #n a

minimal culture medium that c#ntains n# added h#rm#nes. /here'#re, any callus #r #ther #utgr#wth

#' the e6plants will be determined by the trans'#rming e''ect #' the bacteria.

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0#u are pr#vided with sterilised carr#t tap r##ts, 4 plates #' B% culture medium, and cultures #'

three strains #'&grobacterium:

L$)444 Ac#ntr#lC = a GdisarmedG&. tumefaciensstrain (lac!ing h#rm#ne bi#synthesis genes-.

/*J = a wild type&. tumefaciens strain.

L$)4+ = a wild type&. rhi'ogenesstrain.

#r! ne6t t# a $unsen 'lame #n the bench, and be care'ul n#t t# c#ntaminate y#ur plant material #r

agar plates:keep the lids one6cept when in#culating, and use sterilised instruments. Label all etri

dishes clearly with the strain #'&grobacterium, y#ur gr#up and dem#nstrat#r name.

r#t#c#l

1. Ksing 'lamed and c##led instruments, and w#r!ing #n a large etri dish in 'r#nt #' a $unsen

burner 'lame, care'ully cut the r##t int# 5 mm thic! discs where the r##t is + t# 4 cm diameter.

+. lace the tissue slices #nt# 4 plates, 5 dis!s per plate.

*. Ksing a sterile wire l##p ('lamed and c##led between &grobacteriumstrains-, in#culate each

tissue slice within a single plate with one#' the bacterial strains: i.e. use #ne etri dish #' each plant

species per strain.Leae one plate of explants free from inoculation, as a second control.

4. 7eal the dishes with nesc#'ilm, label with y#ur name and place #n a white tray. /hese will be

c#llected and incubated at +5 in the light.

?/E: Beturn&grobacteriumst#c! culture tubes t# the b#6 y#u c#llected them 'r#m at the end #'

the dem#nstrati#n bench. )ll tubes must be acc#unted '#r be'#re the practical can end.

3n ractical 4 y#u will assess the results #' this e6periment.

rite2up3t is imp#rtant that y#u ma!e ?37E n#tes c#ncerning y#ur #bservati#ns #' the #riginal e6plants

and at 2ee! #$ter*a!#' the numbers and types #' tissuesD#rgans that devel#p s# that y#u can use

these data in answering the 9uesti#ns p#sed during y#ur detailed analyses in ractical 4.

Yo'r e 2#!! e +a&e a*a#!a!e &'r#$, Pra%t#%a! a$& 3 o o' %a$ %4e% o$ t4e "ro,re

of o'r e/"er#+e$t(

PRACTICAL5(

PLANTTISSUECULTUREANDGENETICTRANSFORMATION

I$tro&'%t#o$

0#u are re9uired t# ma!e #bservati#ns #n the material y#u set up in ractical 1.

3t is p#ssible that micr#2#rganisms will have c#ntaminated s#me #' y#ur cultures. %# n#t #pen the

dishes i' c#ntaminati#n is very heavy. 3' y#ur tissues have n#t resp#nded, l##! at material 'r#m#ther gr#ups (c#nsult y#ur dem#nstrat#r i' in d#ubt-

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Part 1( P!a$t t#'e %'!t're. %a!!' "ro&'%t#o$ a$& or,a$ re,e$erat#o$ o$

%'!t're& t#'e e/"!a$t of toa%%o

E6perimental

Part 1a General obser!ations of e"#lants and regenerating organ s$stems/a!e lids #'' dishes and #bserve the appearance #' the e6plants using the dissecting micr#sc#pes

n#ting especially di''erences between the resp#nses #n the di''erent culture media (@7, @73 &

@7%4M+-. Kse the 'ull range #' magni'icati#ns. $ased up#n y#ur #bservati#ns answer the

9uesti#ns presented #n the answer sheets.

Part 1b %icrosco#ic anal$sis of callus and regenerating organ s$stems

#mparis#n #' callus cells with cells in tissues #' intact plants.

r#t#c#l

1. ut #'' s#me t#bacc# callus (#r use established callus pr#vided- and tease apart with'#rcepsDm#unted needles int# li9uid culture medium #n a micr#sc#pe slide.

+. lace #n a c#verslip and e6amine at l#w p#wer under a dissecti#n micr#sc#pe and at high p#wer

under a c#mp#und micr#sc#pe.

*. ut thin secti#ns #' t#bacc# lea' tissue with a ra>#r blade and e6amine under the c#mp#und

micr#sc#pe.

resent labeled diagrams #' the #rgani>ati#n #' b#th callus and lea' tissue and their c#nstituent cells

#n the answer sheets pr#vided.

Part ( Ce!! "ro!#ferat#o$ a$& or,a$o,e$e# +eate& Agrobacterium

E6perimental

/a!e lids #'' dishes and #bserve the appearance #' the carr#t e6plants y#u set up in ractical 1,

using the dissecting micr#sc#pes and 'ull range #' magni'icati#ns.

n the answer sheets pr#vided answer the '#ll#wing 9uesti#ns:

a6%escribe the appearance #' the e6plants ineach of the four treatments. 0#u sh#uld ma!e

?37E n#tes and drawings, describing any micr#bial c#ntaminati#n, the appearance #' any

tum#urs, callus #r di''erentiated structures.

/hen, answer the '#ll#wing 9uesti#ns:

6 hy d# y#u s#metimes #bserve limited callus pr#ducti#n in the un2in#culated c#ntr#l e6plantsN

%6 hy d# e6plants in#culated with L$)444 ust '#rm rare callusN

&6E6plain h#w in#culati#n with /*J causes tum#ur '#rmati#nN

e6E6plain why callus tissue, resulting 'r#m h#rm#ne treatment, w#uld n#t c#ntinue t# gr#w a'ter

trans'er t# h#rm#ne 'ree medium, whilst /*J tum#ur tissue w#uld d# s#.

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f6an y#u suggest tw# mechanisms by which the Bi (B##t2inducing- /2%?) which is trans'erred

t# plant cells by L$)4+ c#uld cause r##t inducti#n. A#nsider the trans'er #' genes in'luencing

h#rm#ne levels #r sensitivity t# particular h#rm#nesC

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PRACTICAL7

LIGHTINDEVELOPMENT. PHOTOMORPHOGENESIS

I$tro&'%t#o$

@any aspects #' the devel#pment #' plants are c#ntr#lled by light (ph#t#2m#rph#genesis-. /hese

e''ects #' light are mediated by signal2transducing ph#t#recept#rs (as distinct 'r#m energy2

transducing ph#t#recept#rs such as chl#r#phyll- that detect light signals and then alter devel#pment.

/he best characterised signal2transducing ph#t#recept#r isphytochrome. /his ph#t#recept#r has the

uni9ue pr#perty #'photochromicity, i.e. it can e6ist in either #' tw# '#rms that are interc#nvertible

by light #' di''erent wavelengths. /he tw# '#rms #' phyt#chr#me are called "r(red light2abs#rbing

'#rm- and"fr('ar2red light2abs#rbing '#rm- and the inter2c#nversi#ns can be summarised as:red light

far-red light

Pr Pfr

3n dar!2gr#wn plants #nly the inactive '#rm r is present, and irradiati#n #' the plants with visible

light (c#ntaining red wavelengths- leads t# the c#nversi#n #' s#me #' the r t# the active '#rm, 'r.

r#ducti#n #' active 'r initiates a range #' devel#pmental changes. "#wever, the ph#t#chr#micity

#' phyt#chr#me all#ws '#r m#st #' the 'r t# be c#nverted bac! t# inactive r i' the plants are

subse9uently irradiated with 'ar2red light. /his ph#t#reversibility can be used t# establish the

inv#lvement #' phyt#chr#me in the resp#nses #' dar!2gr#wn plants t# light. /he e6periments

described bel#w will all#w y#u t# investigate s#me aspects #' phyt#chr#me acti#n.

Part 1( P4oto%o$tro! of a$t4o%a$#$ a%%'+'!at#o$ a$& &e*e!o"+e$t #$ +'tar&

ee&!#$,)nth#cyanins are redDpurple pigments #'ten '#und in 'l#wers and 'ruits. /hey als# accumulate in

devel#ping seedlings, in a light2dependent manner, alth#ugh their r#le in seedlings is n#t clear. 3n

this part #' the practical y#u will study the e''ect #' di''erent light regimes #n anth#cyanin

accumulati#n.

E6perimental0#u are pr#vided with seedlings #' white mustard (*inapis alba- which are * days #ld. Each batch

#' seedlings was germinated in the dar! '#r + days and then given the '#ll#wing treatments:

%: c#ntinu#us dar!ness '#r 1 dayyme that is

easy t# assay (a s#2calledreporter gene-. /# av#id inter'erence with the assay it is imp#rtant that the

rep#rter en>yme is n#t naturally '#und in plants. 7uch hybrid fusion genescan then be trans'erred

bac! int# plants by genetic transformation, and the rep#rter gene activity can be m#nit#red by asimple en>yme assay.

ne c#mm#nly2used en>yme is Q2glucur#nidase (FK7-, which is a bacterial hydr#lase ('r#m

+. coli- that brea!s Q2glucur#nic b#nds. 3n the presence #' the substrate 42methylumbelli'eryl

glucur#nide (!--, FK7 releases a KP2'lu#rescent pr#duct 42methylumbelli'er#ne (!- that can

be measured in a 'lu#rimeter.

E6perimentalA#+: t# investigate h#w the pr#m#ter #' an imp#rtant light regulated gene resp#nds t# di''erent

light treatments using transgenic plantsharb#uring areporter gene fusion.

) !ey light2regulated gene which is e6pressed in all plants enc#des the:light(haresting chlorophyll

ab binding protein (#rC&/ protein-. /he )$ pr#tein is synthesi>ed in all green tissues and is

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l#cated within the chl#r#plasts, where it binds chl#r#phyll t# assist in presenting chl#r#phyll '#r

e''icient light abs#rpti#n.

/he regulat#ry regi#n #' the cab gene has been 'used t# the c#ding regi#n #' the +. coli FK7 gene

(Oig. 1-, and then trans'erred int# t#bacc# using&grobacterium2mediated gene trans'er.

/rans'#rmed t#bacc# plants have been gr#wn under the same light regimes as the mustard seedlingsin the Ganth#cyaninG e6periment (%, , B, BDOB, OB-.

0#u are re9uired t# m#nit#r the levels #' FK7 gene e6pressi#n (which rep#rt the activity #' the cab

gene pr#m#ter- by per'#rming assays '#r FK7 activity in the seedlings given di''erent light

treatments.

Plant promoter(light-regulated)

BacterialGUS promoter

cab

GUS

Plant promoter(light-regulated)

GUS

HYBRID GENE FUSION

Oigure 1. %iagram #' gene 'usi#n and trans'er.

r#t#c#l1(/a!e 15 seedlings gr#wn under each light c#nditi#n and crush well in 15 Rl #' FK7 e6tracti#n

bu''er (FE$- in a micr#centri'uge tube using a plastic r#d. (FE$ c#ntains the reacti#n substrate

@KF-. @a!e sure that the crushing is very th#r#ugh and the tissue is c#mpletely br#!en up and

'ully e6tracted in the bu''er.

(repare micr#centri'uge tubes c#ntaining Rl .+ @ ?a+*. /hese s#luti#ns will be used

t# st#p the reacti#n (7/ /K$E7-.

3( O#r only the white light(grown sample, add a 'urther 5 Rl #' FK7 e6tracti#n bu''er, mi6, andthen immediately rem#ve a 1 Rl ali9u#t and add t# Rl #' .+ @ ?a+*t# st#p the reacti#n

(/ = sample-. 3ncubate all samples at *J.

5( )gain, '#r only the white light(grown sample, rem#ve and trans'er 1 Rl ali9u#ts int# separate

st#p tubes at / = 5 min, 1 min, 15 min, and * min. O#r the #ther samples, ust all#w the reacti#n

t# pr#ceed at *J '#r * min and then st#p a 1 Rl ali9u#t in .+ @ ?a+*.

7( Estimate the 9uantitative di''erences in en>yme activity by e6amining (with y#ur dem#nstrat#r-

the am#unt #' 'lu#rescence under KP illuminati#n in c#mparis#n t# thestandard solutions proided

#' the reacti#n pr#duct 42methylumbelli'er#ne (@K-.

9( /rans'er 1 Rl samples #' each reacti#n mi6ture t# the wells #' a micr#titre plate with tw#

replicates '#r each sample. @easure 'lu#rescence with the 'lu#rimeter as dem#nstrated using 1 Rl

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.+ @ ?a+* in microtitre well &0as y#ur $L)? t# G>er#G the machine.

)nalysis and 9uesti#nsa6 l#t a graph #' y#ur 'lu#rescence data m#nit#red #ver the time c#urse 'r#m the white light

treatment A/he reacti#n sh#uld be appr#6imately linearC.

6 hy d# y#u #bserve an increase in 'lu#rescence in the reacti#n with timeN

%6 Or#m the gradient #' this line calculate the change in 'lu#rescence per minute '#r the white light

treatment.

&6 Ksing the data pr#vided '#r the 'lu#rescence (arbitrary units- #' standard s#luti#ns #' @K t# pl#t

a graph #' 'lu#rescence (y2a6is- versus @K c#ncentrati#n (62a6is-.

e6 Kse this graph t# determine the am#unt #' @K pr#duced in * min during incubati#n #' each #'

y#ur e6tracts. /hen use these data t# calculate the speci'ic activity #' FK7 in y#ur e6tracts using the

units nm#l @KDminDseedling. ABemember y#u started with J seedlingsC. resent a value '#r each #'the treatments (%, , B, BDOB, OB- as a table.

f6 $ased up#n y#ur 9uantitative data what can y#u deduce ab#ut the regulati#n #' cab gene

pr#m#terN

,6 hat advantages and disadvantages can y#u see #' using rep#rter genes t# study the transcripti#n

#' a particular gene, rather than measuring the level #' the native transcript (mB?)- #r pr#tein in

wild2type plantsN

age 1 +1 D +11

plant practicals bs1003 2010-2011

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