1.2 Curado del concreto 1 Conceptos del curado
1.3.3 Aprovechamiento de la fibra
To c haracterise the expression pattern of TM1 0, total RNA from flower buds, earl y fruit (7 - 1 0 days post-anthesis (d.p.a)), young l eaves, shoot tips and roots were analysed by northern hybridisation. However, TAN 0 transcript could not be detected i n these t i ssues using this technique. even at reduced stringency . F urther experiments using total RNA fro m different fruit samples also did not detect any TM1 0 transcripts. These res ults suggested T/vfl 0 may be expressed at very low levels in these tissues or there may be a post-transcriptional regulatory pathway affecting m RN A accumul ation (Kuhn e t al . . 200 I ) . The more sensitive reverse transcriptase polymerase chain reaction ( RT-PCR) was there fore used to further examine T,'H} 0 expression i n tomato tissues. In RT-PCR, a reverse transcriptase synthesises DNA copies o f m RN A transcript, which is then amp l i fi ed by a DNA polymerase ( Had idi and Yang, 1 99 0 ) .
L 1 2 3 1 2.0 6.0 3 .0 1 .6 0.5
Figure 4.5. Southern hybridisation of tomato genomic DNA digested with EcoRI ( Lane 1 ), HindlfI ( Lane 2) and XbaI (Lane 3), using labelled TMJO
specific probe. Four to five fragments in the EcoRI digestion hybridised to the probe. The HindIII and XbaI digestions resulted i n single major hybridised bands . Lane L contained the l -kb DNA ladder and the numbers on the left-hand side represent the fragment sizes in ki lobasepairs.
Gene-speci fic pnmers ITM-O 1 and ITM-02 were used i n a one-step RT-PCR (as described i n Chapter 2) to amplify a 0.6-kb DNA fragment using 1 �g total RNA as template i n a 50 )11 reaction. The RT-PCR products were e lectrophoresed on agaros e g e l , transferred t o Hybond N+ membrane (Arnersham) and probed with a l abelled TJ\;fj a-specific probe (Hadidi and Yang, 1 990). The hybridi sation was analysed with Storm phosphorimager and Imagequant software (Molecular Dynamics) . Total RNA from young leaves (0.5- 1 . 0 c m long), large leaves (2-5 c m long), growing shoot tips, roots, flower buds, fruit of 7- 1 0 d.p.a and 1 4-2 1 d.p.a were analysed. The results showed that TMI a is expressed in both small and immature big leaves, shoot tips, fl ower buds, early and late fruit tissues but, not in roots (Figure 4 . 6A) . This pattern suggests that TMI a expression is specific to shoot tissues.
4 . 2 . 6 TMI O
is exp ressed in fru its
To further characterise TMI a expressIOn In deta i l , total RNA from sepals, peta l s , stamens, ovary of flower buds and ovary at anthesis were analysed. TMI O expression i n the d i fferent floral organs was barely detected but expression i s relatively h igher 1 11 anthesis ovary (Figure 4 . 6B), i ndicating a possible increase in TMI a expression at anthesis. To analyse TMI a expression d uring frui t deve lopment, total RNA from fru i ts at 2 , 4, 6, 9, 1 4 and 2 1 d.p.a were also analysed for levels o f transcript. Tlvfl a expressi o n was detected a t comparable levels i n a l l fruit samples examined ( F i gure 4 . 6C). The ex pression o f TMI O i n a l l the fru it ti ssues exam ined suggests i t may be i nvo l ved i n the m u l ti p l e stages of tomato fruit development.
Taken together, T/v/l () expression i s at very low l eve l s spec i fic to shoot ti ssues o f" tomato w i t h re lative l y high level of expression i n leaves. I n the fl ower, TJ'v/ l O expression was relatively hi gher in the ovary a t anthes is and t h i s conti nued through f"ru i t development to at least 2 1 days post-anthesis.
1 2 3 4 5 6 7
A
se pe st ov an-ovB
d.p.a 2 4 6 9 1 4 21c
Figure 4.6. RI-PCR expression analyses of TMIO in tomato tissues. RI PCR products (after 25 cycles) were subjected to electrophoresis in 1 % agarose gel, transferred to a membrane and hybridised with IM 1 O-specific probe. Total RNA used as templates were isolated from: A. S mall leaves 0 ), l arge leaves (2), shoot tips (3), roots (4), flower buds (5), early fruit (6) and late fruit. B. Pre-anthesis sepals ( se), petals (pe), stamens (st), ovary (ov) and anthesis ovary. C. Fruits of 2, 4, 6, 9, 14 and 21 days post anthesis (d.p.a).
4.2.7 In situ
hybridisation
In situ hybridisation technique was used to exami ne TMI O expression i n early floral tissues. D i goxigenin-labelled TMI O-speci fi c RNA probes were prepared from TMI O DNA fragment amplified usi ng ITM-O l and I TM-02 primers. The primers ITM-O l and ITM-02 c arried promoter sites for the T3 and T7 RNA polymerases, whic h were used for in vitro transcription. The anti sense RNA probe was used to detect TMI O transcript whil e the sense probe was u sed for background levels. Here also, t he l evel of TMI O transcript was very low. However, in vegetative tissues, TMI O transcript was detected at the tip of the sympodial bud, i n the l eaf axi l and i n leaf blade (Figures 4 . 7 A, 4 . 7C). Hybridisation with sense probe was used as i ndication of background level (Fi gure 4.7B). TMI O transcripts were also detected i n the floral meristem and in the stamen and ovary of mature flower bud (F igures 4.7C, 4 . 7D).
4.2.8
Tomato transformation
Agrobacterillm transformation vectors, pART2 70S and pART270AS, carryIng the
TM1 0 cDNA in sense and antisense orientations to the CaMV 3 5 S promoter, were constructed as described in C hapter 2. Transgenic tomato plants were generated with
Agrobacterillm t umefaciens harbouring pART270S, the TMf 0 sense vector. A total o f
4 9 6 tomato cotyledon explants were i nocu lated with this vector and sel ected o n kanamyc in containing medi um. Overal l , 1 1 7 e x plants (23 .5% o f explants inoculated) produced putative transgen i c shoots at an average of 1 .2 shoots per explant. A fter repeated transfers to selection medium, 32 putative transgenic plants were selected and rooted on kanamycin-contai ning medi um.
Figure 4.7. RNA in situ hybridisation analysis of TMIO expression ill tomato tissues. A. TMIO transcript was detected in leaf blades, sympodial buds and in floral meristems . B. Sim ilar section in A probed with sense RNA as control. C . TMlO expression was not expressed in emerged sepal primordia but was detected in the floral meristem at this stage. D. In mature floral bud, TM 1 0 transcripts were detected at low levels in the petals, stamen and ovary .
Bars= 1 50 )lm. ov :ovary; pe: petal; sb: sympodial bud; se: sepal; st: stamen.
A CaMV 355 B L 1
2
36.0
2.0
1.0
TM10 cDNA 1 . 1 -kb 4 56
�-02
7 8 9 OC53'Figure 4.8. PCR of TMI O transgenic tobacco plants. A. A schematic diagram showing the binding sites of the primers p35S- 1 and ITM-02, used to amplify a 1 . I -kb fragment. B . Gel photograph of PCR products from some tobacco transgenic l ines. Lanes 1 -8 contain PCR products of putative transformed plants,; lane 9, product of non-transgenic control plant. The lane marked L contains the l -kb DNA ladder (GIBCO-BRL). The size of the ladder fragments in kilobasepairs is indicated on the left-hand side.
These were transferred to soil together with 1 0 non-transgenic tomato p l ants regenerated through tissue culture. Transgenic plants were confirmed by peR using P 3 5 S - 1 primer, specific to the 3 5 S promoter and I TM-02 ( F igure 4.8).