NORMA INTERNACIONAL DE CONTABILIDAD 16 PROPIEDADES, PLANTA Y EQUIPO.
2 Esta Norma debe ser aplicada en la contabilización de los elementos de propiedades, planta y equipo, salvo cuando otra
cii/urer::, ll«
Protein estimation© were started late in 1972, to see if variations from test to test could be correlated with variation© in the protein content of the sti w
extracts. The method used was that of Lowry et al (1951).
The Lowry reagent© wero prepared as follows i-
Reagent A - 2f eodiuo carbonate in C.l K sodium hydroxide.
Reagent B - 1$ copper sulphate. ©agent C-f/ potassium tartarate.
Reagent D - One pert B plus one port C plus ICO parts A, in that order.
Fe&gents A, 3 and 0 were stored, reagent I) was made up
fresh before each test. All chenioale used were supplied
by Ji. *T. Ltd. The method woo to take 0.5 ml© of sample
to be tested one add 2.5 ale of reagent 3. The mixture
wee shaken and left for 10 ninutes. Folia and Cloo<e&u’s
phenol reagent supplied by B.l • • Ltd. was diluted in the proportion of five parts reagent to seven prrts die tilled
water. 0.25 ale of the diluted rea ent was added to the
e&mple and the mixture thoroughly shaken immediately after
nixing. It was then allowed to stand for thirty rcinutcis.
Hie optical density of the sample m e read at 750 nun on a Bsckmai 02500 Spectrophotometer.
A calibration curve wae plotted usin Bovin©
serum Albumin supplied by Jalbloohem Ltd. Dilutions
were prepared containing between 0 and 0.2 ng/nl. The
optical density of each protein concentration wne measured - 147 -
aad the curve plotted urn in ,-:raph 16* the protein
concentration of an extract could then be determined froa it# optical density by reading froa this crsph*
•he standard stigma extract of 25 atlases in v *1 ole of buffer woe found to have a high protein content* dilutions of extract of between 1/30 and 1/2Q0 gave m 4 l e g » in the accurate range of the gxmph betveen protein contents
0*1 anc 0,3 ag/ed* A freehly«*prapared extract of s fixture
of kwle one *alboglabra sti&jsns hue s protein content
of 5*25 ag/al* After the extract hau been stored in the
deep frees© for 10 days, thawed and centrifuged, it had
s protein content of 3»?5 ng/al* 36 of th© protein in
the original sti& m extract had been lost after frcesizv';*
J2BStiddHjkfiS&jL
A considerable tusount of protein was lost when the stigssa. extracts were stored in the deep free*© ano
subsequently thawed out for use in eel tests* This protein
appeared as a precipitate in the tube and was &ntl, enlc protein whose loss could be detected in the gel plates
(photo* raph 10)* the precipitated protein did not appeal
to Include the — protein which was still detectable in gel
plates us in* stlgaa extract whicii had been frosen* 3Df
of the
total
protein content was lost after frees In* andthawing*
Tlis unreliability of the detection of the — protein, particularly In antiserira 8 did not appear to be due to variations in the protein content of th©
stlgaa extract froa test to test* Variation in
detection occurred froa gel to gel in tests set up at the
sane time with the same batch of stigma extract and gels
prepared together# Ber this ramson, and also because
of the time involved, the protein content of ell extracts wee not measured#
JBJg£gfr.
One of the aims of this study was to produce a purified or partly-purified 3-protein extract to use as
vaccine# This was to enable easier Identification of the
3- protein antibody than was possible after absorption
of all the other antibodies present in tho serun# harin
1971 and 1972 purification of the stigma extract was not
ex tend eel beyond freezing of the extract as the only aorey
for the 13—protein was gel dif fusion of absorber eerua.
As already stated, thie was not an efficient method ac titre was low and repeatability suspect#
An alternative assey for the presence of
-protein would be to allow it to carry out in vitro its role in the plant, that is, inhibition of germination
of pollen containing the same S-allele# Brassies
pollen germinates very poorly in vitro. Investigations
were commenced in 1971 to find a germinating medium which would increase the in vitro germination of Brasrlca
pollen so that inhibition of germination could be used
as an aeaey for the B- protein.
This is assuming a number of unproven feeto.
Although Moral lah showed the association be'veen the
r— protein and the B-allele of a plan* by comparing the segregation of the two in the progeny, there is no evidence that the 3-protein is the factor exerting the
effect of the S-allele. Thus, inhibition of like
pollen rasy not be achieved by the 3~pr©tein itself. Another link In the chain between allele and effect
nay be responsible for inhibition. It le also possible
that* assuming inhibitory action* the B»preteln cannot function in purified extract* but m y need the aid of sense
other undetected factor* Another assumption is that
germination in vitro is a condition directly comparable
with germination in vivo* This cannot be so as the
nature of the oonplex interrelationships between pollen
tuiK and stl m are unknown and so canno t be reproduce-
in vitro.
iollen tubes were first observed by Ardcl in 1824 while examining the stigma of foptulaea oleracea. In vitro pollen gemination was studied by Schleiden
(1849)* Van Tieghea (1869) and others* They observed
the pollen germination of several plants and concluded that gemination varied from species to species and even
from variety to variety. Stone pollen* si ch as lyruc will
geminate in distilled water (Knight 1917)* but most pollen requires an aqueous solution of sucrose before i will
gerairate* In 1955* Sdhnuekor discovered that boron as
borate was a stimulant to pollen gemination and tube growth in many species*
Br&naloa pollen is trlnuoleate and* in gsaoral* trlnucleatc pollen is more difficult to geminate in
vitro than btnuoleat© pollen (iirevbaker and :■*jusacer 1961).
tiisa (19501 1935) found that cruciferous pollen oust be
cultivated in a medium containing sucrose and agar* In 1950 he found that a pU of 5 or 6 was unsuitable for
germination of x m b: cu pollen* but that a cacrcs e-^elatin
medium adjusted to a pi! of 7*5 with ©odium aostat* wr s
a suitable besal medium. Schw&nlts (1942) found that
boron Improved the genainaion of Braseloa pollen no had
been found for nany other genera. Loo and Hwang (1944)
ft ermine ted pollen of J,WKM» on a banal medium
of 0*6 H sucrose and 5£ gelatin* They obtained a
nomination percentage of between 23 and 29, with pollen
tube len :the of between 12 mod 14 ml ora* i langi tneee
sulphate, colchicine and indol*-3-ac©Uc a d d all lnere&eec pollen gemination and tube length although the latter two treatment© produced abnormal pollen tubes*
Zn 1967 Minaeva and (Jorboleva found that some flavanolde had a stimulatory effect on pollen gemination
and tube growth* Quercetin, lsorhamnetln, rutin and
ieoqucrcotin inoreaeed the number of gerdnatiiv: pollen grain© per 1000 grain© and increased the pollen tube
length in all except the last case. Tho basal medium
waa a 20f micros* solution and the pollen was from
aone itobelliferae* Tho effect of flavanolde on the genstna*
tion of . ■oleraooa pollen was to be measured and conperec with paper chromatograph!c test© on the flavanolde
present in the pollen and etlgaa* thio ie described in
the next section*
*he sodium used for the germination of the
pollon m e as follow®t
Qxoid Zonagar Ho* 2 1*5#
sucrose 15%
Boric A d d 0*01^
calcium nitrate 0*035
Magnesites sulphate C*C2>