2.3.1 Synthesis of ABA-BSA conjugate for use in indirect immunoassay
The method of Weiler ( 1 980) was used with minor modifications. To 45mg (±)ci s-trans abscisic acid (Sigma A 1 0 1 2) dissolved in 1 5ml methanol was added 1 45mg tyrosine hydrazide (TH) (Sigma T3 1 35). Oxygen free nitrogen gas was bubbled through to purge oxygen from the solution and headspace. A balloon filled with O2 free N2 was placed over the vial, the vial wrapped in aluminium foil and incubated at 50GC for 5 days. Purging as above was repeated daily. Methanol was evaporated and the residue re dissolved in O.5ml H20. A small sample was loaded onto a semi-prep HPLC column (Econosphere C 1 8, internal diameter l Omm, length 250mm) and the absorbance (280 nm) trace compared with standard elution times for TH and ABA (Table 2.6) using a linear gradient of increasing acetonitrile (Table 2.5).
Table 2.5 Sol vent gradient for the separation of ABA -TH from un-reacted tyrosine hydrazide on a preparative C l 8 HPLC column. Flow rate was 5ml/min. Curve 6 is a constant rate of increase over the time period. TEA is 40mM acetic acid in water adjusted to pH 3.38 with triethylamine.
Time (minutes) %Acetonitrile %TEA Curve
o 10 90 *
1 0 30 70 6
3 5 5 0 5 0 6
No attempt was made to identify the unknown products. Unknown peaks 1 and 2 were assumed to be the syn- and anti- i somers of ABA-TH referred to by Weiler ( 1 980). As the conjugate was not required for production of anti-sera, no further attempt was made to separate these i somers. The remainder of the preparation was injected using the same
2-26 Chapter 2: General Materials and Methods. ABA Analysis protocol and eluent was collected from 1 7 to 20 minutes. The collected fraction was taken to dryness and re-dissolved in 500j.l1 methanol.
Table 2.6 Retention times of compounds present in a conj ugate preparation of ABA and tyrosine hydrazide on a semj-preparative C I 8 HPLC column.
Chemical Start Stop
TH 8 : 28 1 4:00 Unknown 1 1 7:20 1 8:34 Unknown 2 1 8: 34 1 9:50 ABA 20: 1 5 22:33 Unknown 3 23.30 26.00 Unknown 4 27.50 3 1 .30
P-aminohippuric acid (pAH) (Sigma A3759) (200 mg) was dissolved in 1 20rnl water, 200mg BSA (Sigma A3350) was added and the pH adjusted to 8.0 with O. l M NaOH. The mixture was warmed slightly until the BSA completely dissolved. 1 -ethyl-3-(3- dimethyl aminopropyl) carbodiimide-HCL (EDC) (200 mg, Sigma E 1 769) was added, the pH adjusted to 6.4 with 2M HCL and the solution stirred for 6 hours at room temperature on a well insulated magnetic stirrer. A further 1 00 mg EDC was added and stirring continued for a further 1 4 hours. The BSA-pAH conjugate formed was dialysed against H20 (20 1, 1 2 changes) at 4°C and lyoprulised.
To 25mg lyophilised BSA-pAH was added 5ml H20, the mixture cooled on ice and 0.5m] NaN02 in water ( 1 20 mg/rnl) was added dropwise. The mixture was allowed to incubate for five minutes and 0.5rnl ammonium sulphamate (Sigma A4630) in water (60 mg/ml) was added dropwise. The ABA-TH in methanol was added to O. l M borate buffer pH 9.0 ( l Ornl). The protein solution was added dropwise and the solution turned a deep gold orange colour. The solution was gently stirred for 30 minutes and immediately dialysed against H20 (20 litre, 1 2 changes) at 4°C. The ABA-BSA conj ugate was
2: General Materials and Methods. ABA Analysis 2-27 lyophilised and then re-dissolved in 3 .6ml of 0.05M NaHC03 (pH 9.6). Aliquots of 30 III were stored at -20°e.
2.3.2 Indirect enzyme linked immunosorbent assay (ELISA) for ABA
Indirect ELISA was used to assay samples for ABA usmg the method of Walker Simmons ( 1 987) .
A tris buffered saline buffer (TBS) was prepared by dissolving 6. 1 g Tris, 0.2g MgCb, 8.8g NaCl in one litre of water and adjusting the pH to 7.8. On the day of use, a washing buffer (TBST -BSA) was prepared by adding O.Smlll Tween-20 and 1 mg/ml BSA (fraction V, Sigma A33S0) to TBS buffer.
Nunc 'maxisorp' 96 well immuno plates were used for ELISA. An Eppendorf 4780 multi-pette was used for delivery of all bulk reagents to plates due to the speed and high precision obtained between aliquots. Monoclonal antibody to +ABA (Mab) was purchased from Idetec Inc., Calif. Mab was mixed with TBS containing 2 mg/ml BSA to give a concentration of 33.3 Ilg/ml MAb and aliquots of I ml were stored at -20°e. Immediately prior to use, one aliquot was thawed and diluted with l Oml TBS containing 2 mg/ml BSA. Standards of (±)cis-trans-abscisic acid were used, however as the antibody only recognises +ABA, the results had to be corrected by a factor of SO%.
Conjugate (30Ill) was diluted to 20ml with NaHC03 buffer and 200j.lI aliquots were delivered to each well of a new ELISA plate, The plate was wrapped in aluminium foil and incubated at 4°C overnight. Diluted Mab (3S0 j.ll) was delivered to polypropylene micro-centrifuge tubes containing 3S01l1 of standard or sample in TBS . Standards used were in the range O.S to SOO pgll 00111 +ABA, although the assay was most sensitive between 2.S and 1 00 pgll OOj.lI. At least two Bo (nil ABA) and one NSB (nil Mab) tubes were included per plate. Tubes were vortexed briefly to mix, covered in aluminium foil and incubated at 4°C overnight.
On the following morning, the conjugate was emptied from plate wells by inverting. Each well was rinsed with 200 III TBST-BSA three times, with the second and third rinses left
2-28 2: General Materials and Methods. ABA
in wells for five minutes to block non-specific binding sites. Aliquots (200111) of Mab+sample/standard was added to triplicate wells and the plate incubated in the dark at 20°C for 2.S hours. Plates were drained and rinsed as above. Rabbit anti-mouse alkaline phosphatase conjugate (Sigma A33S0, 200 Ill) diluted 1 : 1 000 with TBS was added to each well and the plate incubated as above for two hours. Plates were drained and rinsed as above. P-nitrophenyl phosphate (Sigma 1 04- lOS) was dissolved in O.OSM NaHC03 pH 9.6 at 1 mg/ml and 200111 aliquots were added to each well .
Plates were read i n a microplate reader (Dynatech M RSOOO). Readings of absorbance at 4 1 0nm were taken at regular intervals, until absorbance of the Bo wells was approximately 1 .0 (usually between 1 to 1 .S hours). Results were converted to ABA concentrations by using absorbance values converted to 10git[BIBo] and plotted against 10gl O [ABA].
B I B / Bo Logit[
= ( 1 -B /
Results were validated using recovery plots (Jones, 1 987; Walker-Simmons, 1 987) which involves determination of ABA concentration determined in samples at several dilutions spiked with different concentrations of standard ABA (Figure 2.9). The monoclonal antibody has very high specificity for +ABA (Mertens et aI., 1 983) and results obtained in a similar indirect ELISA for ABA have been validated by GC-MS (Norman et aI., 1 986) cited in Walker-Simmons ( 1 987). This strongly suggests that the ELISA procedure described here allows specific quantification of +ABA.
2: General Materials and Methods. ABA 2-29
Table 2.7 A validation of a sample of kiwifruit outer peJicarp in the indirect ELISA for abscisic acid showing the intercept (Sample ABA pgll 00 Ill) and the slope due to addition of standard ABA. In the absence of interference, the slope should be close to one.
Dilution Intercept Slope
1 1 1 00 252.9 1 .04 1 /500 52 0.96 1 1 1 000 33.5 0.97 Nil Sample 1 .2 0.95 400 11100 dilution � - - rJ.l � 3 00 ... bJ) c. � � rJ.l
6
2 00 til c:!�
11500 dilution 111000 dilution < 1 00 Nil Sample � < o o 2 0 4 0 6 0 8 0 1 00 1 20A B A added (pg)
Figure 2.9 Example of the validation of a kiwifruit outer peJicarp sample in an indirect ELISA for abscisic acid (ABA). The bottom line is standard ABA with no sample added. Assay validity is indicated by the parallel slopes of each dilution of the sample to the standard (nil sample) and the proportional decrease in ABA concentration with increasing dilution (Table 2.7). Values are means and standard error bars of ABA
2-30
2.4
Mineral Analysis
2: General Materials and Methods. Mineral Analysis
Fruit for mineral analysis were dried and ground to a fine powder ( 1 mm screen) in a bench grinder. Dried and ground fruit was held at 60°C for 24 hours and cooled in a dessicater prior to weighing and analysis.
Analysis of potassium, magnesium and calcium was by wet nitric acid digestion followed by atomic absorbtion spectroscopy . Concentrated nitric acid (4ml) was added to 1 00 mg of dried and ground fruit matter in glass digestion tubes and the mixture was refluxed at 1 50°C for at least four hours until it had cleared. At this stage the temperature was increased to 250°C until all acid was boiled off. Hydrochloric acid (0.2 M, sOrnl) containing 250 ppm Sr(N03)z and 250 ppm CsCI was added to the residue. After appropriate dilution, samples were analysed on a GEC 904 atomic absorbtion spectrophotometer.
Analysis of nitrogen and phosphorus was by kjeldahl digestion followed by colorimetric autoanalysis (Twine and Williams, 1 97 1 ; Technicon, 1 973). Kjeldahl digestion solution was prepared by dissolving 1 00 g K2S04 and 1 g Selenium powder in one litre of concentrated sulphuric acid and heating until the solution cleared. Kjeldahl digestion solution (4rnl) was added to 1 00mg fruit matter in a glass digestion tube and the mixture was heated to 350°C for at least four hours until it had cleared. The residue was diluted to sOml with distilled water. After appropriate dilution, samples were analysed for atomic emission on a Technicon autoanalyser.
2 : General Materials and Methods. Seed Reduction and Estimation 2-3 1
2.5 Manipulation and Estimation of Seed Numbers in Kiwifruit
2.5.1 Reduction of fruit seed number by style excission
Style number restriction has been u sed in the past to obtain fruit with experimentally induced low seed numbers (Hopping, 1 976a; Lai et al., 1 990). By removing the majority of styles prior to flower opening and thus pollination by honeybees, pollination of most ovules is prevented. However in several experiments, de-styling was found to be relatively inconsistent, with de-styling not resulting in low seed numbers in many fruit. An experiment was set up to ascertain if the procedure could be optimised to provide fruit with low seed numbers more consistently.
Six treatments of style reduction to two, three, four, five and eight styles or control were applied randomly to unopened kiwifruit flowers over a period of three days. Styles were removed close to the ovary with dissecting scissors on 1 8 flowers per treatment. An effort was made to ensure that the styles remaining were evenly distributed around the ovary so that the fruit would not be misshapen. Following treatment, flowers were immediately hand pollinated by rubbing a dehiscing male flower against the styles. Fruit were harvested at commercial maturity, weighed and all seeds extracted for counti ng and measurement of total seed weight.
Style manipulation followed by hand pollination successfully reduced seed numbers in kiwifruit to as low as 1 06 seeds when two styles were left intact (Table 2.8). Each style manipulation treatment had a significantly lower (contrast P=O.OOO 1 ) mean seed number than the control treatment and as the number of styles left intact was increased from two through to eight, average seed numbers in the fruit increased from 252 up to 629. Variation of seed numbers was lower at the lower style numbers and appeared to increase at higher style numbers as a result of an increase in maximum seed number. Although data transformation was able to stabilise the variance, non-transformed data have been presented to illustrate the potential for style manipulation treatments to successfully and consistently reduce seed numbers. When between four and eight styles were left intact, fruit were present in the dataset which would have been considered to be
2-32 2: General Materials and Methods. Seed Reduction and Estimation well pollinated (Table 2.8). It appears that reduction in the number of style to two styles per fruit can be successful in providing a guaranteed low seed number, below that present in a well pollinated fruit.
Table 2.8 Average seed numbers (arithmetic means) in kiwifruit that had styles excised prior to hand pollination (n= 1 8).
Styles intact after de-styling Mean Standard error Minimum Maximum procedure 2 252 1 0 1 1 06 425 3 372 1 80 1 0 1 693 4 363 225 1 1 2 822 5 588 1 87 306 906 8 629 340 96 1 067
Control (not de-styled) 1 265 1 68 992 1 545
2.5.2 Estimation of seed numbers in kiwifruit
A correlation has previously been obtained between seeds on 1 4 exposed surfaces of a kiwifruit cut into defined pieces and the total number of seeds in the fruit (D.1. Woolley, Personal communication, 1 992). However the counting of seeds on 1 4 surfaces is relatively time consuming. In order to improve the procedure, a correlation was made between total seed number and seed number on six cut surfaces.
The fruit used were from the style manipulation experiment described in section 2.5 . 1 . Frui t were sliced through the centre transversely and each half again cut through it's centre transversely (Figure 2. 1 0) . This gave six cut surfaces which could be easily and consistently obtained with different kiwifruits. The total number of seeds exposed on the six exposed surfaces were counted (a cut seed was counted as half a seed, so both halves together would contribute one count). Seeds were extracted, dried, the total number of
2 : General Materials and Methods. Reduction and Estimation 2-33 seeds was counted and a regression of total seed number against surface seed number made (Figure 2. 1 1 ).
the counting of surface seed numbers was found to be simple, reproducible and fast which made this technique experimentally viable for estimating seed numbers of reasonably large numbers of fruit. By dividing fruit into three equal sections, longitudinal variation in distribution of seeds throughout a fruit was accounted for. This may be important especially in differentiating well pollinated fruit where seeds are distributed evenly throughout the entire fruit length and poorly pollinated fruits where there may be fewer seeds apparent at the end of the fruit distant from the styles. Seed weight was not obtained directly from surface counts, but was able to be estimated quickly by removal and drying of seeds present on the cut surfaces which appeared to be representative of the average seed size.
Figure 2. 1 0 Diagram of transverse sectioning of kiwifiuits into four equal sections giving six surfaces for seed counting.
2-34 2: General Materials and Methods. Seed Reduction and Estimation / /