Characterization of Carnivorous Plants Sarracenia Purpurea L. Transformed with Agrobacterium Rhizogenes

Carnivorous plant of Sarracenia genus are used by human in folk medicine for centuries. The reason for this phenomenon is biochemical composition of Sarracenia plants, which possess many bioactive compounds with anti-inammatory, antioxidant, antiviral and antibacterial properties. The subject of this research was genetic transformation of Sarracenia purpurea L. with Agrobacterium rhizogenes strain 15834, 9402 and A4 by using two alternatively methods of bacterial injection or co-culture the bacteria with plants explants. These studies conrmed the possibility of hairy roots induction in S. purpurea using the strain of A. rhizogenes 15834 and the injection method. Seven lines of transformed plants, exhibiting the integration of rolB gene, were obtained. The formed hairy roots showed morphological differences in comparison to the roots of unmodied plants. A mathematical model was used to optimize the conditions for the extraction of bioactive compounds. Extracts isolated under optimal conditions from transformed plants showed biochemical changes i.e., the increase in the accumulation of total polyphenols and triterpenes in comparison to untransformed plants, especially when induced roots were analyzed. HPLC analysis showed increase in the level of betulinic acid in some transformed Sarracenia lines. Betulinic acid remains pentacyclic triterpenoid compound with high pharmacological signicance. The further work connected with isolation and identication of the other bioactive compounds will be done in the nearest future.


Introduction
Sarracenia purpurea L. belongs to the carnivorous plants, which remain interesting as the ornamental plants, but what is more important they possess many valuable compounds. S. purpurea L. is well known as Cree medicinal plant (Cieniak et al., 2015). The plants from Sarracenia genus were found to possess antioxidant, antidiabetic, antiviral and antibacterial constituents (Kannan et (Table 1). Because hairy roots cultures are promising method for the production of interesting secondary metabolites in plants (Tian, 2015) and these cultures can be applied for enhanced synthesis of bioactive compounds, the aim of this study was to establish the tissue culture of Sarracenia purpurea L. plants and transformation of them via agroinfection method with Agrobacterium rhizogenes strain.
It should be pointed out that hairy roots cultures are characterized by high genetic stability and high growth rate without addition of any plant growth regulators (Shakeran et al., 2017, Rana et al., 2017. Thus these cultures can be considered as the tool for plant physiology and interaction analysis i.e. allelopathy (Stanišić et al., 2019) or for the production of medical compounds i.e. heterologous proteins.
In this case human tissue-plasminogen activator was produced in hairy roots of oriental melon (Cucumis melo L.) (Kim et al., 2012) or human gastric lipase in Arabidopsis hairy roots (Guerineau et al., 2020). The level of produced metabolites in hairy root cultures is also often higher than in mother plant or root, for example it was exhibited that for hairy root cultures of Isatis tinctoria L. total avonoids content in 24day-old cultures was about 30% higher than in 2-year-old roots, derived from eld cultivation (Gai et al., 2015).
Additionally, the elicitation of the hairy root cultures can enhance the production of interesting compounds (Shakeran et al., 2017), the second strategy may be genetic transformation of A. rhizogenes with desired genes. However, as it was shown genetic transformation with application of this bacterium strain can cause reduction in the effectiveness of hairy root formation in plants (Rana et al., 2017). It should be also pointed out that many plant species, which remain recalcitrant to A. To our best knowledge this is the rst report concerning transformation of plants from Sarracenia genus with Agrobacterium rhizogenes strain.

Plant material
The plant material used in this study was a carnivorous plant Sarracenia purpurea L. Plants were obtained thanks to the Botanical Garden of the University of Wrocław.

Plant transformation
The explants of Sarracenia purpurea L. were infected with Agrobacterium rhizogenes 15834, A4, LBA 9402 (OD 600 = 0,6) to obtain the hairy roots cultures. In this purpose two methods of transformation were used: by injection and co-culture. In the rst method cultures of A. rhizogenes were injected with the use of the sterile needle to the plant explants and then the explants were incubated at 26°C for 24h on MS medium without antibiotic. Then explants were rinsed in 45 ml of sterilize water with 200µl carbenicillin (100 mg/L) and were placed on solid medium with claforan (400 mg/L) and carbenicillin (100 mg/L). After one week, the concentration of claforan was reduced to 200 mg/1. After 14 days of incubating these explants in the dark, they were cultivated in medium with 3% of sucrose and PPM (750µl/ 1L). After next two weeks explants were transferred to medium without antibiotics. Explants were transferred to fresh medium every week. After about 6 weeks the rst hairy roots were formed at the wound sites. There were only in explants transformed with strain Agrobacterium rhizogenes 15834.
In the second method of co-culture of leaf explants with Agrobacterium strain, 50µL of bacterial suspension (OD600 = 0,6) and plant explants were placed in 25 ml of sterile distilled water and shaken for 48 hours at 26°C. Then the transformed explants were placed on medium described above.
The con rmation of T-DNA integration The genomic DNA isolated from the untransformed Sarracenia roots serve as a negative control and was used as template in PCR method, performed in the same conditions as described above.

Selection of optimal extraction method of bioactive compounds
Ultrasound extraction has been found to be the best method to isolate compounds from Sarracenia purpurea plants (

Total polyphenol content
Total polyphenol content of prepared extracts was determined using the Folin-Ciocalteu method. Extracts were prepared by ultrasound extraction of 6 mg of plant tissue with 6 ml of 80% EtOH. Extraction was performed for 10 minutes at 64°C. 80% EtOH was used to dilute the samples. 250 µL of diluted samples and serial standard solutions, which was gallic acid were placed in glass test tubes. 1.25 ml of 10 times diluted Folin-Ciocalteu reagent was placed into the tubes. After 5 min of incubation in the darkness at room temperature, 1 ml of Na 2 CO 3 (75 g/L) was added and incubated for 5 min (50°C, in the darkness).
After incubation, the tubes were cooled to 4°C and absorbance was measured at 760 nm. The results were expressed as mg Gallic Acid Equivalents (GAE) per gram of dry weight (mg GAE g -1 DW) (Debetić at all., 2020). All analyses were done in triplicate.

Total triterpenes content
Total triterpenes content of prepared extracts was determined using modi ed spectrophotometric method using sulphuric acid (Le at all, 2018). 50µL of plant extracts, reagent blank and standards were incubating at 60°C for 15 min in a shaker bath, with 50 µL of 8% (w/v) vanillin in ethanol and 0,5 mL of 72% (v/v) sulphuric acid. After incubation, the samples were cooled for 5 min and then the absorbance (560 nm) was measured.

HPLC analyses of betulinic acid
HPLC analyses were performed on the system Agilent 1200 series HPLC (Agilent, San Jose, CA, USA) with Phenomenex C18 column (Kinetex, 2.6µm, 100A, 150 x 4.6 nm) and a diode array detector (DAD). The mobile phases: water/ formic acid (99.9:0.1 v/v) (solvent A) and acetonitrile/ water (99.9:0.1 v/v) (solvent B) were used for betulinic acid analysis. Flow rate was 0.9 ml/ min. Before analysis, samples were ltered through polytetra uoroethylene lters (0.22 µm). The autosampler temperature was kept at 4°C and the column 30°C. Standard of betulinic acid (Sigma) was used for identi cation. Betulinic acid in biomass was quanti ed with an external standard of betulinic acid (1 mg L -1 ) at 210 nm.
Samples for HPLC analysis were prepared in triplicate. Content of polyphenols were expressed as mg of compound per g of dry weight (DW).

Analysis of rolB gene integration
The integration of rolB gene from Ri plasmid into plant genome was performed via PCR method as described in Materials and methods section. Among 70 analyzed roots the positive results were obtained for 7. Applied in PCR reaction primers ampli ed fragment of rolB gene of 423 bp (Fig. 2). The presence of this gene was con rmed for 7 transgenic lines of transformed roots and it was not detected in the genome of Sarracenia root, derived from control, untransformed plants. It should be pointed out that the integration of rolB gene is the most important in the transformation process (Sevón and Oksman-Caldentey, 2002).

Optimization of the extraction of bioactive compounds
The most e cient extraction of bioactive compounds (polyphenols) from obtained plants Sarracenia purpurea are: 10 minutes, 64% (v/v) EtOH and 60°C. The highest values of the phenylpropanoids and triterpenes were obtained in predicted conditions. Thus, the content of determined compounds under the established conditions were consistent with the assumptions resulting from Box-Behnken design ( Table 3). All the extracts for determining the content of individual compounds were prepared under these optimal conditions.

Analysis of total polyphenols content
The increase in the content of total polyphenols was observed for all induced hairy roots when compared to untransformed roots (Fig. 3). The highest increase was noticed for two lines 7#1 and 7#2, for which 9fold and 7-fold, higher polyphenols content was measured.
The stems of plants did not exhibit this tendency and the total polyphenols amount was not changed when compared to wild type plants.

Analysis of total triterpenes content
The highest level of total triterpenes content was observed for three lines of transformed roots i.e. lines 4#5, 7#2 and 7#1, and the increase was respectively 4, 7 and 9 fold in comparison to wild-type Sarracenia roots (Fig. 4). In contrast to the level of polyphenols, the amount of measured triterpenes high increase was determined for most stems and this increase was higher than observed for induced transformed roots. It should be pointed out that reason for this phenomenon is unknown, probably it may be the effect of infection of plants by A. rhizogenes. It is known that triterpenes remain compounds intermediating plant-pathogen interactions.It was also exhibited that triterpenes play a crucial role in plant defense system (Cárdenas et al., 2019) and in the allelopathy (Wang et al., 2014). Thus, it can be speculated that transformation of S. purpurea with A. rhizogenes caused the plant response to the pathogen attack.

Betulinic acid content
All analyzed composites plants were used for isolation and measurements of betulinic acid, the compound of antibacterial, antiviral and anticancer properties. The activity of betulinic acid against Escherichia coli and Staphylococcus aureus (Taralkar and Chattopadhyay, 2012), as well as anti-HIV and even antimalarial properties were described (Cichewicz and Kouzi, 2004). It should be pointed out that this compound exhibits also cytotoxic effect against tumor cells (melanoma) (Cichewicz and Kouzi, 2004; Weber et al., 2020) Five tested transgenic lines exhibited higher betulinic acid amount in leaves than in control Sarracenia plants (Fig. 5). The best results were obtained for lines 7#1, 7#2 and 4#4, for which the highest amount of betulinic acid was measured. These lines showed 70%, 62% and 66%, respectively, higher level of betulinic acid than wild-type, control plants. Transformed roots did not exactly show the same tendency and for two lines: 4#2 and 4#4 elevated amount of betulinic acid was noticed, for line 4#4 the highest amount was determined and it was 34% higher than in untransformed roots.
Obtained results indicated that transformation of A. rhizogenes caused increase in the betulinic acid amount, which remains the compound with medicinal signi cance.

Conclusions
Herein, we described the transformation of Sarracenia purpurea L., insectivorous plants to generate composite plants with hairy roots. The obtained composite plants and wild-type differed in the aspect of biochemical composition i.e. the increased accumulation of phenolic compounds and triterpenes in the obtained hairy roots was noted. Transformation of plants with Agrobacterium rhizogenes 15834 may have a positive effect on the content of compounds with pharmacological potential in the composite plants. Further experiments will focus on studying the effect of plant extracts, derived from transgenic Sarracenia hairy roots on the proliferation of cancer cells and pathogenic microorganisms.

Declarations
Author Contribution Statement KMP and MWK designed research and wrote ms. KP performed plant transformation, selection of hairy roots, performing compounds determination.MP and KMP analysis of polyphenol and triterpenes content and HPLC results. All the authors approved the manuscript.   Total triterpenes content determined in the investigated hairy roots and composite plants of S. purpurea.
The analyses were performed as described in "Materials and methods" section. The data (±SD) resulted from three samples per line.