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UDC 602.64 doi: 10.15407/biotech9.02.055
TARRAGON (Artemisia dracunculus L.) "HAIRY" ROOT CULTURE PRODUCTION
K. O. Drobot
A. M. Shakhovsky Institute of Cell Biology and Genetic Engineering
N. A. Matvieieva of the National Academy of Sciences of Ukraine, Kyiv
E-mail: katyadrobot@gmail.com
Received 20.02.2016
This paper is devoted the biotechnology development of Artemisia dracunculus L. genetic transformation. We obtained the transgenic A. dracunculus "hairy" root culture using A. rhizogenes A4-mediated transformation. The conditions of tarragon's genetic transformation were optimized. It was shown that leaves of in vitro cultivated plants were the optimal type of explants. The transgenic root formation frequency was up to 20% in case of leaves usage. The time of explants cocultivation with Agrobacterium suspension was found to be an important factor of biotechnology which affects the frequency of transgenic root growth. Transgenic root lines differed in morphological features and growth rate. Specific mass increase varied from 17 to 32 times after 3 weeks cultivation on 1/2 Murashige-Skoog medium.
Key words: Artemisia dracunculus L., genetic transformation, Agrobacterium rhizogenes A4, "hairy" root culture.
Biotechnological approaches are widely used to create plants with new properties and to obtain biologically active substances (BAS). At the moment such approaches are a topical trend of modern scientific researches in field of biology. Genetic engineering is one of the biotechnology approaches for construction of plants with new properties. Transformation method using soil bacteria Agrobacterium rhizogenes can be used to create transgenic roots which produce BAS. Regarding this the use of medicinal plants as an object for genetic transformation is of special interest. Artemisia spp plants may be used for achieving this goal.
Artemisia genus belongs to Asteraceae family and includes more than 500 plant species [1]. Tarragon — Artemisia dracunculus L. — is a well-known perennial plant, which is widespread throughout the world and also grows in Ukrainian steppe and forest steppe ecoregions [2]. The use of A. dracunculus was mentioned in ancient Greece, but some historicans considered that Asia is a real tarragon's origin. At present, there are two well-described cultivars (Russian and French [3]) of A. dracunculus, which differ in physiology, botanical features and phytochemical profile. French tarragon (2n = 36) is a sterile
chromosomal derivative of Russian tarragon (2n = 90). The last one can produce viable seeds, instead of French tarragon, which doesn't produce seeds [4].
Tarragon is widely used in folk medicine as well as in cosmetics and cuisine due to high essential oil content. It is also extremely popular in French cuisine as a spice and is also used for vinegar preparation. Light licoric or basil-, anise-like flavor could be referred to tarragon's culinary benefits [5].
Flavonoids, coumarins, phenilpropanoids, terpenes determine antimicrobial, antiviral, antifungal and antioxidant activities of A. dracunculus. Such a broad spectrum of biological activities could cause tarragons use in pharmaceutical industry for treatment of diseases such as inflammation [6], hepatitis [7] and different kind of infections (bacterial, viral) [8, 9]. Leaves of A. dracunculus accumulate artemisinin up to 0.27% [10]. Recent studies of A. dracunculus were devoted to plant micropropagation [11], medicine compounds accumulation [12] and artemisinin synthesis in particular [10].
"Hairy" root culture is a type of plant tissue culture, which is considered to be an alternative way for producing valuable plant derived compounds [13]. It could be obtained
via Agrobacterium rhizogenes-mediated transformation. A. rhizogenes is a soil bacteria which naturally cause hairy-root disease in plants owing to the presence of root-inducing plasmids. Transgenic hairy roots have been obtained in more than 89 different taxa, representing 79 species from 55 genera and 27 families using A. rhizogenes-mediated transformation [14]. This amount is rising from year to year.
"Hairy" root culture is characterized by unlimited and fast growth without use of exogenous hormones; it is unpretending to the growth conditions and doesn't require lighting, so the roots can be cultivated in bioreactors [15]. In contrast to cell culture, "hairy" root culture is characterized by high genetic stability. Establishment of transgenic roots in some cases takes only a few weeks [16]. This implies that the use of "hairy" root culture could attract particular attention and especially for study of tarragons transformation and BAS production possibility. A. rhizogenes-mediated genetic transformation was conducted for plants of some Artemisia representatives: A. annua [17 18], A. dubia and A. indica [19], A. aucheri [20], A. vulgaris [21], A. absinthium [22]. Transgenic root cultures obtained were used for producing BAS in bioreactors, especially for artemisinin content increasing. Nevertheless genetic transformation of tarragon has not been carried out yet. Establishment of biotechnology of genetic transformation of A. dracunculus using A. rhizogenes A4 wild strain was the aim of our work.
Materials and Methods
A. dracunculus were introducted in in vitro culture using seeds surface sterilization method. 14-days-old seedlings which were cultured on hormone-free half-strength Murashige-Skoog (V MS) [23] basal medium were used for genetic transformation experiments.
A. rhizogenes A4 wild strain was inoculated to the liquid LB medium (10 g/l casein hydrolyzed, 5 g/l yeast extract, 10 g/l NaCl, pH 7.0) and was left to grow overnight at 28 °C using rotation shaker SpeedVac Savant AES 2010 (Labconco, USA). Leaves, hypocotyls, stems and roots of 14-day tarragon seedlings were used as the explants for genetic transformation. Explants were sliced and cocultivated during 30 min with an overnight bacterial suspension. Then they were transferred to the agar-solidified V MS medium (20 g/l sucrose, pH 5.7) supplemented
with 600 mg/l cefotaxime for agrobacteria elimination. After cocultivation explants were placed onto V MS agar-solidified basal medium and were grown for 2-7 days. Well-grown roots formed on the explants after genetic transformation were excised from the explants and transferred to individual Petri dishes for further "hairy" root growth. The root formation frequency was calculated as a number of root producing explants in percents.
Extraction of DNA was carried out according to CTAB-method. The presence of rolB genes in established "hairy" roots was determined using PCR analysis on Mastercycle personal 5332 amplifier (Eppendorf). Amplification was carried out in following conditions: primary denaturation — 94 °C, 3 min, 30 cycles of amplification (94 °C, 30 s — 56 °C, 30 s -72 °C, 45 s), final polymerization —72 °C, 3 min. Products of reaction were separated in 1.5% agarose gel. O'GeneRuler 1 kb Plus DNA Ladder #1163 was used for sizing of rolB genes. Primers 5'-atggatcccaaattgctattccttccacga-3' and 5'-ttaggcttctttcttcaggtttactgcagc-3' (Size of amplified fragment is 780 b.p.) were used to confirm the presence of rol B gene in A. dracunculus "hairy" root lines.
Transgenic roots were subcultured every two weeks. Subcultivation was conducted at 24 °C and 16 h/d photoperiod. Growth rate of transgenic root lines was studied after 3 week of cultivation of excised root tips on V MS medium at the same conditions.
Results and Discussion
The results of our experiments approved that the time of explants cocultivating with Agrobacterium suspension is an important factor for transgenic root formation. The optimal time of explant cultivation on the medium without cefotaxim for agrobacterial genes transfer into plant cells appeared to be four days. Prolongation of this term has led to explants death, while reducing of this wasn't successful for root obtaining. Roots have formed on the 7-th day after cocultivation with bacterial suspension on leaf explants. "Hairy" roots weren't obtained using hypocotyls, stems and roots. Time of cocultivating of these explants with Agrobacterium suspension didn't effect transformation frequency. After two weeks of cultivation hypocotyls, stems and roots have lost capacity to grow and became dark. So the using of leaves as explants and transformational conditions mentioned above
b c
Fig. 1. A. dracunculus in vitro cultivated plant and "hairy" root culture: A. dracunculus 14-days seedling (a); initiation of "hairy" root growth (b); A. dracunculus "hairy" root culture (c)
were successful for A. dracunculus "hairy" roots induction.
So the using of leaves as explants and transformational conditions mentioned above were successful for A. dracunculus «hairy» roots induction.
The amplified products of 780 bp for rolB gene was determined using PCR analysis in all A. dracunculus "hairy" root lines obtained, as it shown in Fig 2.
The root formation frequency was found to be up to 20%. The root number per explant reached up to 5. It is known that frequency of transformation usually depends upon Agrobacterium strain, plant species and explant type [24]. For example Sujatha et al. [21] conducted transformation of A. vulgaris using four Agrobacterium strains: A4GUS, R1000, R1601and ATCC15834 and three explant
bp ■ 1000 760 500
250
2
4
M
Fig. 2. Electrophoregram which demonstrates the products of PCR analysis of "hairy" root clones transformed using A. rhizogenes A4 strain:
marker for sizing DNA fragments (M); DNA of A .dracunculus transgenic roots (1, 2); bacterial DNA (3); genomic DNAs of non-transformed roots (4) Results of typical experiment are presented
types: shoot tip, leaf and node. The authors proved that A. rhizogenes A4 GUS strain is more competent than other strains and the highest transformation rates were observed in leaf explant (92.6%). Giri et al. demonstrated that the ability of A. annua transformation by four different A. rhizogenes strains (LBA 9402, 9340, 9365, 15834 and A4) was also found to be different. The best root formation response (up to 100%) was observed for LBA 9402 strain, the worst one — 75% in case of using A4 strain [17]. Leaves often are the most susceptible to genetic transformation type of explant of another Artemisia species plants (A. dubia and A. indica (100%), A. aucheri (93%), A. absinthium(57,1%). High root formation frequency of A. vulgaris after A. rhizogenes transformation (up to 100%) we observed earlier [25]. The data cited demonstrate significant species-depended difference in transformational frequency among Artemisia representatives. Obviously the low transformation frequency of tarragon plants in our experiment was caused by species-specific susceptibility of this plant. It is necessary to take into account the growth rate as well as target compounds content in order to estimate metabolic activity and capacity to synthesize BAS in "hairy" roots. It is an important step for selecting the most productive "hairy" root lines. High growth rate allow to obtain more valuable substances in short time. Transgenic lines that are notable for high growth rate and target compounds content can be an alternative source of plant-derived valuable substances. In our investigation specific mass increase varied from 17 to 32 times afrer 30 days growth on V MS medium. So, biomass increase among different "hairy" root lines varied considerably in our investigation Such variability allowed to select the most productive "hairy" root
a
0,08
0,07
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8 0 05 и
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1 0,03 о
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0,02 0,01
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Samples
Fig. 3. A. dracunculus "hairy" root growth:
a, b — differences in A. dracunculus "hairy" root morphology (lenghth, branching); c — biomass increase of A. dracunculus control non-transformed roots (1) and "hairy" root lines (2-4) (P < 0.05)
lines, particularly lines №5 and line №7 (mass increase after 30 days of cultivation on hormone-free medium was up to 25 and 32 times respectively). It should be noticed that non-transformed roots had also ability to grow on hormone-free medium. However mass increase after 30 days of cultivation was only 1,08 times and they became dark in few weeks. "Hairy" roots obtained in our experiments didn't lose the capacity to grow even after 1.5 years of in vitro cultivation.
"Hairy" root lines obtained had a typical phenotype such as excessive and non geotropic roots, high branching growth on a hormone-free culture medium. We noted also morphological alterations among different transgenic lines. They differed in water cut range, fibril growth, root thickening and root branching (Fig. 3).
This phenomenon may be explained with variability in the expression level of rol genes or could be related to the peculiarities (site and number) of T-DNA integration into the plant genome [26].
Thus we firstly obtained biotechnology for genetic transformation of Artemisia dracunculus L. and have firstly obtainedА. dracunculus "hairy" root culture using A. rhizogenes A4 wild strain. Our overall results reveal that the induction of A. dracunculus transgenic roots by A. rhizogenes can be successfully established using leaves of 14-days-old seedlings. The results agree with the earlier findings in case of transformation of other Artemisia spp. plants and confirm that leaves are the most suitable explants for genetic transformation of plants which belong to Artemisia genus. The transgenic root formation frequency was up to 20%.
It was found that the time of explants cocultivating with Agrobacterium suspension is an important factor which affect the frequency of transgenic root growth. It was shown that the optimal period of cocultivation with Agrobacterium rhizogenes used to be 4 days. Thus, the use of the proposed biotechnology of A. dracunculus Arhizogenes-mediated genetic transformation enable to obtain "hairy" root lines which transgenic nature was confirmed by PCR analysis.
b
a
c
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ОТРИМАННЯ КУЛЬТУРИ «БОРОДАТИХ» КОРЕН1В ЕСТРАГОНУ
Artemisia dracunculus L.
К. О. Дробот А. М. Шаховський Н. А. Матвеева
1нститут ^i™HH0Ï бмлоги та генетично1 шженерп НАН Украши, Кшв
E-mail: katyadrobot@gmail.com
Метою роботи було розроблення б^техно-логи генетичноï трансформаци рослин естра-гону Artemisia dracunculus L. Як вектор було використано Agrobacterium rhizogenes дикого штаму А4. Уперше одержано культуру «боро-датих» корешв естрагону. Серед використаних експланмв (коренi, стебло, листя та гiпокотилi 14-добових культивованих in vitro проростшв естрагону) оптимальним типом виявилось листя. У разi його використання частота трансформаци сягала 20%. Встановлено, що важли-вим бмтехнолопчним чинником, який впливае на частоту трансформаци, е час кокультивуван-ня експланив з агробактерiею. Трансгеннi лши коренiв вiдрiзнялися за морфологiею та швид-кiстю росту. Прирiст маси «бородатих» коренiв через три тижш культивування на безгормональному середовишД Mурасiге-Скуга варт-вав вщ 17 до 32 разiв.
Ключовi слова: Artemisia dracunculus, гене-тична трансформащя, Agrobacterium rhizogenes А4, культура «бородатих» корешв.
ПОЛУЧЕНИЕ КУЛЬТУРЫ «БОРОДАТЫХ» КОРНЕЙ ЭСТРАГОНА
Artemisia dracunculus L.
Е. А. Дробот А. М. Шаховский Н. А. Матвеева
Институт клеточной биологии и генетической инженерии НАН Украины, Киев
E-mail: katyadrobot@gmail.com
Целью работы была разработка биотехнологии генетической трансформации растений эстрагона Artemisia dracunculus L. В качестве вектора был использован дикий штамм А-grobacterium rhizogenes А4. Впервые получена культура «бородатых» корней эстрагона. Среди использованных эксплантов (корни, стебли, листья и гипокотили 14-суточных in vitro культивируемых проростков эстрагона) оптимальным типом оказались листья. При их использовании частота трансформации достигала 20%. Установлено, что важным биотехнологическим фактором, влияющим на частоту трансформации, является время кокультивирования экс-плантов с агробактериями. Трансгенные линии корней отличались по морфологии и скорости роста. Прирост массы «бородатых» корней через три недели культивирования на безгормональной среде Мурасиге-Скуга варьировал от 17 до 32 раз.
Ключевые слова: Artemisia dracunculus, генетическая трансформация, Agrobacterium rhizo-genes А4, культура «бородатых» корней.
