Informe Trimestral
3. Predicción del Cuadro Macroeconómico
Phenotypic Expression & Heritability (Materials & Methods)} 5 1 3.2.2.4 Methods for estimating the heritability (h2) of characters
For the time-included characters, F-test, variance components of the effects, heritabillties and standard errors of heritabi!ities were estimated using the AOVFASBW statistical programme (l.L., Gordon unpublished). The method for estimating the heritabi!ities and the standard errors of heritabl!ities were based on the statistical models of Gordon et al. (1 972).
Phenotypic variance and heritabilities were estimated as follows:
where:
£I P::::phenotypic variance, u' ":::variance of block,
a2
fl::::variance of line (genotypic variance of lines), d(afll=variance of interaction between block and llne, £16 variance or error (a), cl8=within plot variance (genotypic variance of individual plant), dr=variance of time,a2
(tlrJ::::;variance of interaction between 1\ne and time, cl�::::variance of error (b).There were two genetic variances in the experiment, the variance arising from line (clp). and the variance arising from individual plants within the line (cl9(e)· Since a proportion of variance of individual plants within the line (cl
e)
is an environmental variance, the environment-free variance of line (cl9(eJ} can be estimated as follows:£1
g(e)
= K£1e
where the K ratio can be estimated using Smith (1936) equations as follows:
a2e(eJ
= cl jt' (as plants were each observed over t pooled times) clg
(6') = £1(1>) + cl.-(e)K ::::
dgce/dceJ
The environment-free variance of individual plants within the Jlne was estimated by multiplying the variance of individual plants within the line by the K
P/lenotypic Expression & Heritability (Materials & Methods)) 52 ratio (cl g(B) = Kd 8). Subsequently the three types of heritability, population (line), plant (within) and broadsense were estimated as follows:
Population (line) Plant (within) Broadsense h' = a' ,Ja', h2 :::: dg(o/dP h' = (a', + a'9l,,)ia',
As the standard error of each of these heritabilities were available, their significances were tested with the t-test.
The phenotypic variance of the drymass character was estimated as follows:
where:
dP::phenotypic variance, d.,.::variance of block, dp=variance of 1\ne (genotypic variance of population), d\.,.p)=variance of interaction between block and tine, dp=variance of herbicide, do=:=variance of individual plants (genotypic variance of population), d lPPl=variance of interaction between line and herbicide, d �=variance of residual (error). The population, plant and broadsense heritabilities for the drymass character (time-excluded) were estimated as described above for the time-included characters.
3.2.3 Inheritance o f dalapon resistance
For the 6 transgenic lines 49-1 , 51-1, 51-3, 51-5, 51-14 and 54-18, the number of independent functional deha!ogenase gene integration events was determined by a segregation analysis of dalapon resistance in progeny plants (R1) originating from seeds of selfed, tissue culture-derived, transgenic parents (for developed plants grown under greenhouse conditions). The number of resistant and susceptible phenotypes for each line at each levels of da!apon ( 1 . 5, 3.0, 6.0, 12.0, 24 and 48.0 kg ha"1) was recorded separately and used for a
.I
test. Since three blocks consisting of five plants per experimental unit were used for each line, a total of 1 5 plants was tested for each line at each level of herbicide.I n the
.I
test, since the degree of freedom was 1 , the approximation of the.I
distribution was improved and a more exact probability value was obtained fromPhenotypic Expression & Heritability (Materials & Methods)) 53
the
X
table by imposing a correction for continuity. This correction is intended to make the actual distribution, as calculated from discrete data, more nearly like theX
distribution based on normal deviation {Steel and Torrie, 1 980). Theapproximation calls for the absolute value of each deviation to be decreased by 0.5 as follows:
Homogeneity of the 3:1 segregation ratios over all levels of herbicide for each line tested using the pooled ;i formula (Steel and Torrie, 1 980) as follows:
L (n',(n,)
-n'.Jn
Where: }'=·1 or 2, P=1/4 or 3/4
In pooled segregation analysis, since three blocks consisting of five plants per experimental unit were used for each Hne and the results pooled over each level of herbicide, a total of 90 plants was tested for each line. The susceptible (negative) plants within each of the lines were recognized by two different procedures: a) plants which were phenotypically similar to those of the non transgenic line at each level of herbicide were recorded as susceptible plants to give a phenotypic based identification; b) the level of necrosis of each plant at each level of herbicide was determined based on the level of necrosis of the non transgenic control line to give a quantitative identification. Individual segregant plants for each of the lines were recognized on the basis of the level of leaf necrosis for each level of herbicide. Thus for 1 .5 kg herbicide ha·\ plants with more than 6% of necrosis were recorded as susceptible plants. For 3.0, 6.0, 12.0, 24.0 and 48.0 kg of herbicide ha·1 of herbicide, plants with more than 10, 25, 40, 50, and 65% of necrosis respectively were recorded as susceptible plants
Phenotypic Expression & Heritability (Materials & Methods))
54
(quantitative based identification). In the � test, since the degree of freedom was
1 , a more exact probability value was obtained from the � table by imposing a
correction for continuity as indicated above for the adjusted �.
3.3 Results
3.3.1 Herbicide resistance of transgenic white clover
3.3.1.1 The herbicide resistance of in vitro-grown transgenic white clover calli
The analysis of variance of white clover callus growth-gain data is presented in Table 3 . 1 . The significance {P<0.001) of the line, as a main effect, indicates that there are significant differences in average callus growth-gain of the lines in the presence of herbicide irrespective of other effects such as the herbicide and block effects. Furthermore, the significance (P<0_00.1) of the herbicide effect (another main effect) indicates that the levels of herbicide have significant effects on callus growth-gain irrespective of other effects.
The significant (P<D.001) effect of the interaction between line and herbicide levels indicates that as we!! as the main effects (line and herbicide levels), there are also variation amongst particular line x herbicide combinations which are departure from the main effects. Hence, each line shows a different phenotypic expressivity at the different levels of herbicide.
The mean total callus growth-gain of the white clover callus lines are presented in last column of Table 3.2. The transgenic line number 1 had the highest growth rate followed by line number 2, control and line number 3 .
All herbicide levels also have a significant effect on callus growth-gain such that by increasing the concentration of the herbicide, the rate of callus growth decreased significantly ( last row of Table 3.2). Lower levels of the herbicide have less effect on callus growth-gain, while higher levels of the herbicide are more effective in decreasing callus growth-gain. Increasing the concentration of herbicide from 0 to 100 mg L-1 or from 100 to 200 mg L-1 led to a decrease in the callus growth-gain of between 30-40%, while increasing the concentration from 200 mg L-1 to 300 mg L-1 led to a decrease of about 50%, and from 300 mg L-1 to 400 mg L-1 to a decrease of 72%.
Since the interactions between line and herbicide level are significant, comparing the callus fresh weight gain of the white clover lines at various dalapon levels is appropriate. The callus growth-gain of the white clover lines at various dalapon levels are indicated in Table 3.2. At a zero level of herbicide, the non transgenic control line showed the highest callus growth-gain, whi!e increasing
Phenotypic Expression & Heritability (Results) 56