Fruit yield and quality of strawberry plants transformed with a fruit specific strawberry pectate lyase gene
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Fruit yield and quality of strawberry plants
transformed with a fruit specific strawberry
pectate lyase gene
Youssef M.S., Silvia J.M., Bellido M.L., Carmen M.P., Marta B., Samia A.A., Jose L.C., Jose M.L.,
Fernando P.A., Juan M., Miguel A.Q. and Jose A.M. Scientia Horticulturae. 2009.119: 120–125.
Presented by Nittaya Pitiwittayakul
ID 50890029
Strawberry
Strawberry (Fragaria x ananassa Duch.)
is one of the most valuable and worldwide cultivated small
fruits
is considered as non-climacteric and its ripening is
characterized by a fast softening, acquiring a melting texture
in few days
The rapid loss of firm texture constrains the postharvest
shelf life of strawberry and also the harvest practices, and
frequently
fruits are harvested before being fully mature
to accommodate shipping practices, with the
consequent reduction in quality
Strawberry
Strawberry (Fragaria x ananassa Duch.)
is one of the most valuable and worldwide cultivated small
fruits
is considered as non-climacteric and its ripening is
characterized by a fast softening, acquiring a melting texture
in few days
The rapid loss of firm texture constrains the postharvest
shelf life of strawberry and also the harvest practices, and
frequently
So, Improvement of textural properties is
therefore one of the main objectives of
Strawberry breeders
Mechanism of strawberry softening
Cell wall degradation seems to be the main factor
responsible for strawberry softening
the degradation of the middle lamella of cortical
parenchyma cells as well as a dramatic increase in pectin
solubilization, although the total quantity and length of
pectins is little affected
Polygalacturonase (PG) and pectate lyase are among the
different enzymes that could be involved in pectin
degradation during fruit ripening.
Pectate lyase Pectate lyases (PL, EC 4.2.2.2), otherwise known as pectate transeliminases, catalyse the eliminative cleavage of de- esterified pectin, which is a major component of the primary cell walls of many higher plants The role of Pectate lyase on strawberry softening, studies performed with transgenic strawberry plants indicate a crucial role of pectate lyase on pectins metabolism
Antisense-RNA
From: commons.wikimedia.org/wiki/File:Antisense_DNA...
Co-suppression
Fig 3. A variegated petunia. Upon injection of the gene
responsible for purple coloring in petunias, the
flowers became variegated or white rather than
deeper purple as was expected
From:www.rsc.org/ej/OB/2004/b404932m/b404932m-f2.gif
In 2002,The researchers have demonstrated that the inhibition of this
enzyme by insertion of an antisense sequence of a fruit specific pectate
lyase gene (FaplC) reduces strawberry softening, extends postharvest
shelf life and improves quality of processed fruits
However, most of transgenic lines containing the antisense sequence
showed a significant reduction in fruit yield, due to a reduction of fruit set
and fruit weightObjective • To evaluate the effect of transformation with a sense sequence of a fruit specific strawberry pectate lyase gene on strawberry fruit yield and softening
Materials and Methods
2.1 Plant material and Agrobacterium-mediated transformation
FaplC gene
in the sense
orientationMaterials and Methods
2.2 Molecular analysis of transgenic plants
Confirm test FaplC gene expression QRT-PCR
The transgenic nature of plants was RNA was isolated from a pool of 5-6 red fruits
confirmed by PCR amplification The reverse transcription reaction contained
220-bp fragment nptII gene 1 RT buffer, 10 mM DTT, 1 mM each dNTP, 0.2 mM
specific FaplC primers, 1 mg of DNase-treated
540-bp fragment chimaeric gene total RNA and 40 U of SuperscriptTM RT II.
35S-FaplC.
The reaction was heated at 70°C for 5 min and
For Southern blotting, 5–10 g of DNA cooled to room temperature.Afterwards, the
reaction was incubated at 42°C for 5 min, followed
were digested overnight with EcoRI,
by 50 min at 50°C and 15 min at 70°C.
fractionated in a 0.8% agarose gel and
transferred to a Hybond N+ membrane. Transcribed cDNA was subjected to PCR
The filter was hybridized at 64ºC with a amplification in a reaction mixture containing 30
digoxigenin labelled probe obtained by ml of: 1 PCR buffer, 1.5 mM MgCl2, 0.2 mM dNTPs,
PCR amplification of the 35S-FaplC gene 0.2 mM of each specific primers, 3 ml of SYBR
Green I (1:5000 diluted), 3 ml of transcribed cDNA,
from pJPJ4 plasmid. The EcoRI and 2 U of Taq polymerase
digestion of our plasmid yields a DNA
fragment of about 2 kb which hybridizes The PCR program was: 94°C for 2 min, followed by
35 cycles of 94°C 1 min, 55°C for 30 s and 72°C for
with the probe. 30 s, and a final step of 72°C for 5 min.Materials and Methods
2.3 Phenotypic analysis of transgenic plants
yield estimated as total number of fruits per plant and g of fruits per plant
Fruit quality evaluated using only normal shape fruits of uniform size and
coloration, and weight larger than 5 g.
Color estimated using the CTIFL (Centre Technique Interprofessionel des
Fruits et Legumes, France) code. This code comprise eight categories,
increasing the red color from 1 (light orange-red) to 8 (dark wine-red)
Soluble solids measured by using a refractometer Atago N1
firmness measured by using a hand-penetrometer (Effegi) with 9.62 mm2
surface needle
For histological study, blocks of tissue were fixed in Bouin reagent for 24 h,
dehydrated in an ethanol series to 95% ethanol and polymerized with the Historesin
Embedding kit. Sections (4 mm thick) were cut from the polymerized samples and
stained with 0.2 g/l ruthenium red for 2 h.Results and discussions
Molecular analysis of transgenic plants
This result indicates the
stable integration of the
transgene in Pel lines
Fig. 1. (A) DNA amplification by PCR of a 220 bp fragment from the nptII gene and a
540 bp fragment from the chimaeric 35S-FaplC gene. P: plasmid; C: control
nontransformed DNA; 1: DNA from Pel 1 line; 3: DNA from Pel 3 line. (B)
Southern blot analysis of DNA from control and transgenic Pel lines. Arrowhead
on the right indicates the inserted copy of FaplC gene.Results and discussions
Molecular analysis of transgenic plants
Both Pel lines showed a significant
reduction in FaplC mRNA levels when
compared with control untransformed
fruits, with a gene silencing of 84% in
Pel 1 and 71% in Pel 3
71%
84%
Fig. 1. (C) Relative FaplC gene expression estimated by QRT-PCR in ripen fruits from
control and transgenic Pel linesResults and discussions
Molecular analysis of transgenic plants
Table 1. Fruit yield of control and transgenic plants (Pel) transformed with a sense
sequence of FaplC gene, during a 2 years evaluation
Thirty plants per genotype and year were employed. Mean separation was performed by Tukey test at P = 0.05Results and discussions
Molecular analysis of transgenic plants
more than 40% of fruits had a fresh weight lower than 5 g
and only a small percentage of fruits showed a weight
higher than 15 g
more than 60% of fruits were included in the lowest
class, fruit weight lower than 5 g
Fruits from Pel 3 line were similar in weight than those of
control non-transformed plants, although the production
of fruits with medium weight, 5-15 g, was increased in
this transgenic line.
Fig. 2. Fruit weight distribution of control and
transgenic pectate lyase plants (Pel),
obtained during the first year assay. Bars
with different letters within each fruit
weight category indicate statistically
significant differences by Tamhane T2
test at P = 0.05.Results and discussions
Molecular analysis of transgenic plants
Table 2. Fresh weight, color, soluble solids and firmness of full ripen selected fruits
from control and transgenic pectate lyase plants
Data correspond to the first year of analysis. Mean separation was performed by Tamhane T2 test at P = 0.05.
Fruits from the three transgenic pectate lyase lines were significantly
firmer than those of control non-transformed plants, ranging the
increments between 33% for Apel 39 and 18% for Pel 3.
A positive correlation between pectate lyase gene inhibition and
fruit firmness can be drawn from these results.Results and discussions
Molecular analysis of transgenic plants
Pel fruits showed cells with an extensive area of
cell to cell contact and a dense cell wall staining,
indicating that these walls were enriched with
pectin when compared with control fruits.
Fig. 3. Cross-sections of cortical cells from full
ripen fruits. (A) Control nontransformed
fruits; (B) Pel 1 fruits; (C) Pel 3 fruits. Bars
correspond to 50 m.Results and discussions
Molecular analysis of transgenic plants
both Pel lines also showed
higher firmness values
than control
Fig. 4. Firmness of transgenic pectate lyase fruits at different
developmental stages. Firmness is expressed as percentage of
control, non-transformed fruits. Data correspond to mean S.D.
of a minimum of 25-50 fruits per line and stage.Conclusion A significant inhibition of FaplC gene expression has been achieved by co-suppression. In spite of the fact that the inhibition is lower than that obtained with antisense transformation, ripe fruits also showed an increment in fruit firmness that was maintained after 3 days of postharvest. The use of this approach to manipulate pectate lyase gene expression does not have negative effects on yield, and lines with higher fruit set, similar fruit size, and increased fruit firmness can be obtained.
Thank you for your attention
Nomenclature commonly used in real time quantitative RT-PCR: Baseline is defined as PCR cycles in which a reporter fluorescent signal is accumulating but is beneath the limits of detection of the instrument. ΔRn is an increment of fluorescent signal at each time point. The ΔRn values are plotted versus the cycle number. Threshold is an arbitrary level of fluorescence chosen on the basis of the baseline variability. A signal that is detected above the threshold is considered a real signal that can be used to define the threshold cycle (Ct) for a sample. Threshold can be adjusted for each experiment so that it is in the region of exponential amplification across all plots. Ct is defined as the fractional PCR cycle number at which the reporter fluorescence is greater than the threshold. The Ct is a basic principle of real time PCR and is an essential component in producing accurate and reproducible data.
• แปลตรงตัวได้ความหมายที่สุดคือ PCR ในขณะที่กาลังเกิดขึ้นจริงๆ ทา
ให้สามารถติดตาม PCR ในช่วง exponential phase ได้อย่าง
แม่นยา ซึ่งนาไปสู่การวัดปริมาณได้ โดยมีสมการของ PCR ดังนี้
• Xn = X0(1 + E)n
• โดย Xn คือ ปริมาณ PCR product ที่ cycle ที่ n
X0 คือ ปริมาณเริ่มต้นของ template
E คือ Amplification efficiency
n คือ cycle numberYou can also read
