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Research Report A leading global R&D planning and evaluation institute for technological innovation in food, agriculture and forestry

LAST UPDATE : 2016-01-01

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Subject Clarifying the Causes of Flower Colour Degradation in Chrysanthemum and Developi
Date 2016-12-29 14:50:42 Hit 546
Contents Title : Clarifying the Causes of Flower Colour Degradation in Chrysanthemum and Developing
New Cultivar with Thermotolerance

Discoloration of chrysanthemum flowers was found to be serious in summer
season, resulting in declination of ornamental value in the market. Hence,
enhancement of flower color by genetic engineering to overcome extreme
temperature during summer season is necessary. In this study, we produced
transgenic chrysanthemums (cvs. Peach Red, Peach ND, and Vivid Scarlet)
using Agrobacterium-mediated transformation system, which contains
anthocyanin regulatory RsMYB1 gene isolated from radish (Raphanus sativus L.)
by placing under the control of either cauliflower mosaic virus 35S or petal
specific promoter InMYB. In order to consider the copy number of target gene
(RsMYB1), transgenic plants confirmed by PCR were further analyzed by
southern hybridization, In addition, expression level of anthocyanin biosynthetic
genes was also confirmed by reverse-transcription(RT) PCR. Furthermore,
expression level of anthocyanin in the transgenic plants were examined in the
Background: Several MYB genes belonging to R2R3 MYB transcription factors
have been used in several plant species to enhance anthocyanin production,
and have shown various expression or regulation patterns. This study focused
on the effect of ectopic expression of an RsMYB1 isolated from radish
(Raphanus sativa) on chrysanthemum cv. ‘Shinma’. Results: The RT-PCR results
confirmed that RsMYB1 regulated the expression of three key biosynthetic
genes(CmF3H, CmDFR, and CmANS) that are responsible for anthocyanin
production in transgenic chrysanthemum, but were not detected in the
non-transgenic line. In all transgenic plants, higher expression levels of key
biosynthetic genes were observed in flowers than in leaves. However, the
presence of RsMYB1 in chrysanthemum did not affect any morphological
characteristics, such as plant height, leaf shape or size, and number of flowers.
Furthermore, no anthocyanin accumulation was visually observed in the leaves
and floral tissue of any of the transgenic lines, which was further confirmed by
anthocyanin content estimation. Conclusion: To our knowledge, this is the first
time the role of an MYB transcription factor in anthocyanin production has
been investigated in chrysanthemum.
This research was conducted to develop genetic transformation of the
recalcitrant chrysanthemum cv. Shinma by application of appropriate antibiotics
and selective agents. Clavamox had the least inhibitory effect on shoot
regeneration compared to timentin, carbenicillin, and cefotaxime. Clavamox, at a
concentration of 125 mg L−1, was found to be the most suitable for shoot
regeneration and production of quality shoots, suppressing the growth of
Agrobacterium in explants infected with strains GV3101 or C58C1 for 3 and 4
weeks, respectively. The concentration of phosphinothricin (PPT) was found to
be 1.0 mg L−1 for screening of putative transgenic shoots. Moreover,
transgenic chrysanthemums were obtained by culturing explants co-cultivated
with A. tumefaciens strain GV3101 harboring an anthocyanin regulatory gene
RsMYB1 isolated from radish (Raphanus sativus), which was placed under the
control of cauliflower mosaic virus promoter (CaMV) 35S and petalspecific
promoter InMYB1 isolated from the morning glory (Ipomoea nil), on shoot
regeneration medium supplemented
with recommended concentration of antibiotic and selective agent. Flow
cytometry analysis revealed that there was no
variation in ploidy level between transgenic plants and donor plants
(non-transformants). To our knowledge, this is the first
report of the use of Clavamox and MYB transcription factor for genetic
transformation of this chrysanthemum.
When encounter harsh environmental conditions such as drought, salinity, low
temperature and high temperature, plants constantly monitor the environmental
signals. Therefore, severe abiotic stresses cause adverse effects on the
growth, development and productivity followed by generating morphological,
physiological, biochemical and molecular changes in plants. Of abiotic stresses,
high temperature especially emerged in vigorously growing season can have
dramatic impacts on the productivity and quality of crops resulting in farmers’
low income. Many of researches using the Arabidopsis as a model plant have
clarified responses to heat stress, though these findings still remain to be
obscure to crops.
Chrysanthemum is one of the most important commercial cut flowers in the
world. In summer, heat stress by high temperature has effects on
chrysanthemum like low productivity, flowering delay and discoloration.
Monothiol glutaredoxins play important roles in maintaining redox homeostasis
in living cells and share some conserved function across species. Arabidopsis
thaliana monothiol glutaredoxin AtGRX3 is critical for protection from oxidative
stress in cytosol and/or nucleus. Collectively, we here report that
over-expression of AtGRX3 in chrysanthemum plants confers tolerance to heat
stress. To improve the tolerance to heat stress in chrysanthemum, independent
fourteen lines constitutively over-expressing AtGRX3 that isolated from A.
thaliana were generated via Agrobacterium-mediated genetic transformation
technology. All lines were characterized by polymerase chain reaction, Southern
hybridization, semi-quantitative reverse transcription-PCR, and bioassays.
Over-expression of AtGRX3 in Chrysanthemum plants enhanced photosynthetic
performance and insistence of cell membrane permeability, and decreased
oxidative damage in addition to promotion of plant growth under heat stresses.
These findings suggest a specific protective role of the redox protein against
high temperature stress, and provide a genetic engineering strategy to improve
crop thermotolerance.



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