Page 151 - Zbornik_Konf_2019

https://doi.org/10.15414/2019.9788055220703

4 International Scientific Conference Abstracts Book
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'FLORAL‐DIP' GENETIC TRANSFORMATION OF AMARANTHUS CAUDATUS L. WITH
HETEROLOGOUS GENES
Olha Yaroshko, Bogdan Morgun, Liudmyla Velykozhon, Mykola Kuchuk

Institute of Cell Biology and Genetic Engineering, National Academy of Sciences of Ukraine, Kyiv,
Ukraine; E-mail.: 90tigeryaroshko90@gmail.com
Nowadays, amaranth becomes popular due to its rich biochemical composition and
unique medicinal properties. In connection with this, it is promising to improve its properties
with the help of biotechnological methods.
The aim of this work was to evaluate the presence of the transferred bar gene and nos
terminator in amaranth tissues, and to obtain transgenic plants of amaranth after the 'floral-
dip' genetic transformation.
The objects of the investigation were local cultivars of species Amaranthus caudatus L.:
Helios, Karmin. The above-mentioned cultivars were transformed by method 'floral-dip'. After
'floral-dip' transformation with Agrobacterium tumefacience strain GV3101 with vector pCB
125 and A. rhizogenes strain A4, with vector transgenic seeds were obtained. All mentioned
vectors contained bar as a selectable screenable marker.
The incorporation of the T-DNA into amaranth cells was confirmed with herbicide
selection (herbicide Basta®, Bayer CropScience AG, Germany). For this purpose, the lowest
lethal dose of Basta herbicide on amaranth seedlings was detected in our previous
experiments (160 mg/l of herbicide).
Seeds obtained from plants not subjected to agroinfiltration were grown in plastic pots
with soil in greenhouse conditions (22–26°C, 14-hour light period, illumination 3000–4500
lx). At the age of two weeks, the seedlings were sprayed with concentration 160 mg/l of
herbicide. The amaranth plants were used as negative control grown without herbicide
spraying. Spraying of seedlings was conducted in order to fulfil the selection of assumedly
transformed plants with the presence of bar gene.
After spraying with the herbicide, tolerant plants were obtained for cultivars: Helios and
Karmin.
Percentage of tolerant plants A. caudatus cv. Helios (with vector pCB125) to the effect of
the herbicide (160 mg/l) was 4.05 %, cv. Karmin (with vector pCB125) to the effect of the
herbicide (160 mg/l) was 2.4 %. Percentage of tolerant plants A. caudatus cv. Karmin (with
vector pCB131) was 1.2 %.
After conducting of PCR analysis, positive results were obtained for the presence of bar
and nos terminator. The percentage of bar-positive plants was 0.6 % ('Helios', vector
pCB125); 0.1 % ('Karmin', vector pCB131) from total initial quantity of seeds. The percentage
of nos terminator-positive plants was 0.15 % ('Helios', vector pCB125) from total initial
quantity seeds.
After conducting a series of experiments, transgenic plants of amaranth were obtained
after the 'floral-dip' genetic transformation. The transgenic plants were obtained for cultivars
Helios and Karmin, species Amaranthus caudatus. The presence and functioning of the
transferred bar gene and nos terminator in amaranth tissues were confirmed by PCR analysis.

Keywords: transformation, Agrobacterium, Amaranthus.
Acknowledgments
The study was supported by a project (State Registration No. 0118U003663) granted by the National
Academy of Sciences of Ukraine.

4 International Scientific Conference Agrobiodiversity Nutrition, Health and Quality of Human and Bees Life |151
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September 11–13, 2019