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5.3.3.1 A genotype with promising growth features with reduced gs

This genotype originated from self-pollination of the parent plant 59F2. The F2

plant had visually higher leaf angle from the stem and had the greyish leaf phenotype

similar to the sp5 plants having low stomatal conductance (Table 5-6). The 59F3 seeds Figure 5.10. Quantitative-PCR data obtained from the 102F2-101F3 family derived from

selfing of 102F2 plant number 101F3. There were three biological and two technical repeats

in each reaction. 101F3-1 and 101F3-2 were two separate DNA samples from the same

parent plant (101F3), used as heterozygous control and all F4 plants were its progeny. wild

type, 3F4 and 4F4 contained only two copies of the LeNCED1 and lacked Tr-Ds element,

confirmed by PCR. Plants 31F4 and 26F4 were homozygous for the LeNCED1. Error bars

128 germinated at the same frequency as the wild type without any chemical treatment. The

early seedling establishment and growth of this plant was similar (data shown in

Chapter-6) to wild type and no signs of interveinal flooding or chlorosis were observed.

Table 5-6: Leaf stomatal conductance (mmol m-2 s-1) of 59F2 in comparison to the

wild type and sp5 genotypes

GT No.of plants Mean

gs

*SEM 1p-value

**59F2 1 290.5 33.4 n/a

wild type 6 529.5 47.5 0.01

***sp5 6 205.9 31.7

* represent standard error of means, calculated from multiple gs values obtained from each plant.

**Genotype 59F2 germinated on water alone

***sp5 germinated on 3.28 M norflurazon

1

p-value indicates the data analysis through t-test assuming equal variance.

5.3.3.2 Genotypic analysis of 59F3

PCR results obtained by using the primers Ds1For2 and notRev5 (Appendix II) for detection of the Ds element in the 59F3 family (accession AT1679) showed that, out

of 50 plants tested, 14 had lost the Ds element while 36 still retained an insertion. Chi- square test was in agreement that this genotype was heterozygous for a single Ds locus (Table 5-7).

129

Table 5-7: Calculation of segregation ratio of Ds element in genotype 59F3 by using

Chi square test.

5.3.3.3 Southern Hybridization Analysis

Genomic DNA from randomly selected plants containing the Ds element, as determined by PCR, was analysed by Southern blot. A probe was prepared using the

complete ORF of LeNCED1 gene. Data showed that there were two transposition events in the 59F3 family (Figure 5-11); these were named as Tr-Ds-6 and Tr-Ds-7. In total, 29

plants from genotype 59F3 preselected for the Ds element through PCR were analysed

using Southern blotting. The results showed that all plants contained Tr-Ds-6, Tr-Ds-7 and the two T-DNA-Ds bands. This suggested that the F2 parent was homozygous for all

these bands, and so the family is uninformative with respect to genetic linkage.

GT Number of plants observed Number of plants expected x2 calc x 2 0.05,1 Ratio 3:1 59F3-Ds+ 36 37.5 59F3-Ds - 14 12.5 Total 50 50 0.24 3.841 yes

Where x2cal.was the calculated Chi Square value

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5.3.3.4 Stomatal conductance measurements in genotype 59F3

To find out the effect of the Tr-Ds elements on the stomatal conductance, these plants were grouped in blocks each containing six plants with one wild type and one sp5

plant. There were six blocks in each screen. Data for the gs was collected four times a

day for two days. The gs of 59F3 was significantly lower (40%) than the wild type

plants but higher (20%) than sp5 plants (Figure 5-12). Also, the gs of sp5 plants was

significantly lower than the wild type plants under controlled environmental conditions,

as expected (P<0.001). Endogenous NCED bands Tr-Ds-6 Tr-Ds-7 517-1 WT T-DNA-Ds bands

Figure 5.11. A Southern blot analysis of 59F3 plants preselected for the presence of Ds element. Blot hybridised with a probe containing complete ORF of LeNCED1

gene. Where T-DNA-Ds represent bands inherited from 517-1 line, Tr-Ds shows the transposed bands.517-1 line and wild type were used as control.

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5.4 Discussion

Complex physiological parameters control the movement of stomata in response

to environmental factors, and ultimately control conductance of the leaf and the rate of

transpiration. These factors are wide ranging and include light (Yu et al., 2004, Kositsup

et al., 2010), CO2 (Robredo et al., 2007), VPD (Elsharkawy and Cock, 1984, Day, 2000,

Pou et al., 2008), drought stress (Miyashita et al., 2005) and time of the day (Jarvis and

McNaughton, 1986, Correia et al., 1997). Under glasshouse conditions, controlling most

Figure 5.12. Means of stomatal conductance of genotype 59F3 under

controlled environmental conditions analysed using ANOVA. Ailsa Craig Tm2a(WT) and sp5 were used as control. Error bars represent the standard error of means. Different letters represent statistically significant differences at P < 0.01. n = 6

132 of these parameters was not possible therefore all of the screening for the gs was

performed under controlled environmental conditions to reduce experimental variation.

The genotypes tested for gs during the present study were randomly chosen with

the main objective to select a line which had reduced gs but no associated problems (e.g.

poor seed germination and slow growth and establishment) (Thompson et al., 2000,

Tung et al., 2008). Stomatal conductance can be a good indicator of plant water use

efficiency (Earl, 2002, Thompson et al., 2007, Pou et al., 2008), hence, it was decided to

select genotypes with reduced gs to achieve higher WUE.