Chemical Profile of Cell Cultures of Kalanchoë gastonis-bonnieri Transformed by Agrobacterium rhizogenes

Abstract

Kalanchoë gastonis-bonnieri Raym.-Hamet & Perrier is a plant used for medicinal purposes in the treatment of several ailments. The aim of this study was to analyze the chemical profile of extracts from K. gastonis-bonnieri embryogenic calli, generated from genetically transformed roots by Agrobacterium rhizogenes. Putative transformants were verified by PCR. Hydroalcoholic extracts were obtained and the chemical profile was analyzed by LC-ESI-MS/MS. Root formation was obtained from 80% of infected seedlings. Fifteen root lines were isolated, and two lines showed prominent longitudinal growth and profuse branching in the B5 semi-solid medium. In all lines, the formation of nodules and later embryogenic callus was observed. Putative transgenic root lines were cultivated in free-plant growth regulators B5 medium. In the two selected lines, the PCR amplification of rolA, rolB, rolC, rolD, and aux1 genes was detected. The extract of embryogenic calli showed 60 chemical compounds tentatively identified, such as ferulic acid, quinic acid, neobaisoflavone, and malic acid, among others, and the chemical profile was different in comparison to wild-type extracts. This is the first study reporting the analysis of the chemical profile of hairy root extracts derived from Kalanchoë gastonis-bonnieri. This work displays the great potential for obtaining chemical compounds of pharmacological importance from hairy roots and facilitates the identification of new useful drugs against human chronic-degenerative diseases.

1. Introduction

The main drawback of obtaining bioactive compounds from plants is the variation in the accumulation of secondary metabolites due to development, plant growth cycles, and diversity of environmental conditions [1]. The biosynthesis and accumulation of chemical compounds of interest in highly specialized tissues occurs at specific stages of development [2]. The hairy roots induction through Agrobacterium rhizogenes infection is a biotechnological alternative to obtain specific secondary metabolites in vitro cultures free of plant growth regulators [3]. A. rhizogenes is a Gram-negative bacterium of the Rhizobiaceae family that induces hairy roots disease by infecting higher plants and inserting root loci (rol genes) from the root-inducing plasmid (Ri) into the plant genome [4]. From hairy roots, induction of embryogenic calli and regeneration of whole plants has been observed in species such as Hypericum perforatum [5], Tylophora indica [6], Gentiana utriculosa [7], and Pentalinon andrieuxii [8].

It has been reported that hairy roots synthesize chemical compounds that are not detected in wild plants [9]. Furthermore, some authors have reported that both shoots and transgenic plants from hairy roots accumulate higher levels of metabolites in comparison to wild plants. Tusevski et al. (2014) [5], reported the accumulation of naphthodiatrons and specific phenolic compounds in transgenic shoots of Hypericum perforatum from hairy roots. Vinterhalter et al. (2019) [10], reported a higher accumulation of xanthones in transgenic plants of Gentiana utriculosa regenerated by somatic embryogenesis derived from hairy roots. Hiebert-Giesbrecht et al. (2021) [8], reported the accumulation of terpenoids in leaf of transgenic plants, obtained from hairy roots, compared to wild Pentalinon andrieuxii.

The species of the genus Kalanchoë (Crassulaceae) are succulent plants, used in traditional medicine to treat several health conditions such as gastric ulcers, asthma, infections, tumors, and blood glucose regulation [11]. Additionally, it is an important ornamental plant. Various studies have been developed around the Kalanchoe genus, which provide knowledge on human health [12]. The importance of these plants is found in the diversity of chemical compounds that accumulate which represent the interest in the medicine industry, and as an ornamental plant due to the diversity of the colors and leaf shapes, these characteristics contribute to the economic importance of the Kalanchoe genus [13]. In any case, agronomic management for plant production is extremely important because chemical compounds accumulate at different parts of the plant and in response to environmental factors. The production of plants in controlled environments represents an option for maintaining germplasm and keeping plant diversity with all its benefits [14,15]. The application of biotechnological tools to develop technologies that can provide interesting metabolites and generate new plant materials with specific characteristics represents the option to make the most of natural resources. Thirukkumaran et al. [16], reported the application of technologies allowing the production of transgenic plants without selectable marker genes and described that marker-free transgenic K. blossfeldiana could be produced using ipt-type MAT vector system carrying the chimeric ipt. The transformation of Kalanchoe pinnata by Agrobacterium tumefaciens with ZsGreen1 by Cho et al. [17] selected optimum succulent species for future genetic transformation efforts and the development of an efficient transformation method using a novel fluorescent gene, was accomplished. This method achieves new cultivars of succulents with eye-catching colors or patterns in the leaves and flowers. The potential to develop new cultivars with predictable traits in a reduced period is a great advantage of the genetic transformation approach.

Kalanchoë gastonis-bonnieri Raym.-Hamet & H. Perrier is used in traditional Latin American medicine as a contraceptive and in the treatment of genital and urinary infections, diabetes, kidney infections, gastric ulcers, leishmaniasis, and cancer [18,19,20,21]. Few studies have reported the chemical profile of K. gastonis-bonnieri [19,21,22]. The aim of this study was to analyze the chemical profile of extracts from the embryogenic calli of Kalanchoë gastonis-bonnieri generated from hairy roots.

2. Materials and Methods

2.1. Plant Material

Kalanchoë gastonis-bonnieri was collected in Centro de Desarrollo de Productos Bióticos-IPN, Yautepec, Morelos, México (18°49′53″ N, 99°05′37.40″ W at 1064 m.a.s.l.). The in vitro plants were obtained from vegetative shoots (size: 2–3 cm) that grew from meristematic tissue, at the tip of acuminated adult plant leaves from wild-growing plants that were gently washed with tap water, then with a Tween 20 (Sigma Company, St. Louis, MO, USA) solution (1%) for 1 min, ethanol (70%) for 2 min and NaOCl (0.5%) during 17 min, and rinsed 3 times with sterile distilled water between each solution [23]. Vegetative shoots were cultivated on semi-solid MS (Sigma Company, St. Louis, MO, USA) medium [24], to which a sterile medium was added with 30 g/L sucrose (Sigma Company, St. Louis, MO, USA) and 3 g/L Phytagel (Sigma Company, St. Louis, MO, USA) and the pH was adjusted to 5.8 before autoclaving at 125 °C for 15 min. In glass Gerber-type containers with 20 mL of medium, cultures were incubated in a growth chamber at 25 ± 2 °C with a photoperiod of 16 h light/8 h dark, at 30 μmol/m²s provided by cool white fluorescent tubes, for 35 d.

2.2. Induction of Transformed Roots

The A. rhizogenes strain A4 was used and cultured in YMB medium (2.8 g/L) with 3 g/L of phytagel (Sigma-Aldrich^®, St. Louis, MO, USA) and incubated at 29 °C [25] for 3 days. Subsequently, it was kept at 4 °C and reseeded every 30 d. In vitro seedlings of K. gastonis-bonnieri were inoculated in the internodal zone by a longitudinal scalpel wound with the A. rhizogenes strain A4 and incubated in semi-solid MS medium [24] free of growth regulators added with 30 g/L of sucrose (Sigma Company, St. Louis, MO, USA), and 2.6 g/L of Phytagel (Phytotech, St. Lenexa, KS, USA) [26]. The transformation frequency was determined [27]. The bacterium was eliminated from the plant culture with cefotaxime (Phytotech, St. Lenexa, KS, USA) according to Tavassoli and Safipour-Afshar [28], and the cultures were maintained in semisolid MS medium [24] with cefotaxime for 30 days with subcultures every 7 days to eliminate A. rhizogenes residues. The bacteria-free cultures were transferred to a B5 liquid medium for subsequent analyses. Root segments were individualized and transferred to semi-solid B5 medium [29] phytohormones free, supplemented with 30 g/L sucrose, 2 g/L polyvinylpyrrolidone, and 2.6 g/L phytagel (Sigma Company, St. Louis, MO, USA).

Finally, after 55 days, the selected lines were transferred to liquid B5 medium, and the cultures were maintained at the above-mentioned conditions at 100 rpm and sub-cultured every 30 days.

2.3. Morphological Description of In Vitro Cultures

The analysis of the culture development was carried out as previously described [30]. The specific growth rate (μ) and the doubling time (T2) were determined as follows:

μ = ln (X − X0/t − t0) × 100       T2 = ln2/(μ)

were, X: Final dry weight, X0: Initial dry weight, t: Final time, t0: Initial time. The plant material morphology was observed in a stereoscopic microscope (Nikon, SMZ-1500, Tokyo, Japan), coupled to a PC (Data image, DS33, Tokio, Japan) with a video camera, controller, and integrated interface. The plant material was disaggregated, and the samples were kept hydrated with B5 liquid culture medium. The samples were displayed in triplicate [31].

2.4. DNA Extraction and PCR Analysis

Genomic DNA from plant material was extracted using the method of Doyle and Doyle [32]. Plasmid DNA of A. rhizogenes was extracted with Wizard^® Plus SV Minipreps DNA Purification System kit (Promega Corporation, A1460 Madison, WI, USA), following the manufacturer’s protocol. DNA from the A. rhizogenes A4 strain was used as a positive control, DNA from wild-type K. gastonis-bonnieri plants was used as a negative control, and sterile distilled H₂O was used as a negative control, to develop the polymerase chain reaction (PCR). The amplification was carried out in a thermal cycler (Applied Biosystems, Gene Amp PCR Systems 9700, Waltham, MA, USA), using specific primers according to reports in each case. rolA: 5′-CGTTGTCGGAATGGCCCAGACC-3′ and 3′-CGTAGGTCTGAATATTCCGGTCC-5′ to amplify a 248 bp fragment, rolB: 5′-ACTATAGCAAACCCCTCCTGC-3′ and 3′-TTCAGGTTTACTGCAGCAGGC-5′, to amplify a 652 bp fragment [33], rolC: 5′-TGTGACAAGCAGCGATGAGC-3′ and 3′-GATTGC AAACTTGCACTCGC-5′ to amplify a 487 bp fragment, rolD: 5′-CCTTACGAATTCTCTTAGCGGCACC-3′ and 3′-GAGGTACACTGGACTGAATCTGCAC-5′ to amplify a 477 bp fragment [34] and aux1: 5′-CCAAGCTTGTCAGAAAACTTCAGGG-3′ and 3′-CCGGATCCAATACCCAGCGCTTT-5′ to amplify a 815 bp fragment [35]. DNA electrophoresis was performed in a 1.5% agarose gel at 95V for 60 min. The visualization of the amplified fragments was mixed with a SYBR ^®Green (Lonza, Hayward, CA, USA) solution. Electrophoresis was analyzed in a photo-documenter (ChemiDoc™ MP Imaging System BIO-RAD, 170-01402, Hercules, CA, USA).

2.5. Ultrasound Assisted Extraction (UAE)

Metabolites were extracted in an ultrasound bath Branson (Branson Ultrasonics™, 2510R-MTH, Brookfield, CT, USA) with automatic control of time and temperature and ultrasound frequency of 40 kHz. A total of 50 mg of dry and ground biomass were mixed in 2 mL of ethanol (80%, v/v), and were sonicated at 40 ± 5 °C for 30 min. Samples were centrifuged at 3500 rpm for 5 min. The supernatants were recovered and filtered through cellulose membranes (0.22 μm) (MILLEX^® GS). Samples were dryness at 25 ± 2 °C and the dried extracts were stored at 4 °C until analyzed [36].

2.6. Chemical Characterization of Kalanchoë Gastonis-Bonnieri Extracts

The chemical profile of extracts was obtained by LC-ESI-MS/MS. Samples were solubilized in 500 µL of MeOH, HPLC grade (Sigma-Aldrich^®) and filtered through nylon membrane, (0.45 µm, Agilent Technologies, Santa Clara, CA, USA). The mobile phase for gradient elution consists of two solvents: solvent A (0.1% formic acid (FA) Sigma-Aldrich^® in H₂O) and solvent B (0.1% FA in CH₃CN/MeOH (1:1; v/v) Sigma-Aldrich^®. The linear gradient profile was as follows: 95% A (5 min), 95–90% A (10 min), 90–50% A (55 min), 50–95% A (65 min), and 95% A (70 min). The injection volume was 10 µL. The flow rate (0.6 mL/min) was split 1:1 before the MS interface. Electrospray ionization analysis (ESI) was performed using a micrOTOF-Q II mass spectrometer (Bruker Daltonics, Bremen, Germany). The mass spectrometer was operated in negative ion mode with a capillary potential of 2.5 kV, gas temperature of 180 °C, drying gas flow of 6 L/min, and nebulizer gas pressure of 1.0 Bar. Detection was performed at 50–3000 m/z.

The tentative identification of compounds was based on the comparison of the MS fragmentation profile obtained by the analytical equipment, with the mass spectra of MassBank of North America (https://mona.fiehnlab.ucdavis.edu accessed on 15 July 2021) and Competitive Fragmentation Modeling for Metabolite Identification (http://cfmid3.wishartlab.com accessed on 30 July 2021).

2.7. Statistical Analysis

Relative abundance data of tentatively identified metabolites in K. gastonis-bonnieri extracts were analyzed by clustered color mapping, using a Pearson distance measurement mean bond clustering method, using the Heatmapper software (http://www.heatmapper.ca/, accessed on 15 September 2021).

3. Results

3.1. Morphology of the Kgb1 and Kgb2 Cultures and Molecular Analysis

K. gastonis-bonnieri seedlings were obtained from in vitro cultures. The infection with A. rhizogenes A4 strain was accomplished and the root formation at the infection site was observed after 15 d with a transformation efficiency of 80%. Fifteen root lines were individualized in a semi-solid B5 medium, most of the lines were characterized by slow growth and poor branching, and the formation of cell aggregates was observed 30 d after isolation from the initial explant. In vitro cultures of K. gastonis-bonnieri were successfully initiated from aseptically vegetative shoots isolated from wild-growing plants. The in vitro shoots were subjected to Agrobacterium-mediated transformation (Figure 1). Figure 1a shows an uninfected explant (negative control), demonstrating that mechanical injury did not result in root formation. In Figure 1b–d, the response of different infected seedlings and the development of hairy roots that emerge from the infection site with plagiotropic growth is shown; Figure 1b shows abundant proliferation of hairy roots, while in Figure 1c, d explants with few roots were obtained; Figure 1d shows callus formation and few roots scarcely hairy developed from the infection site.

A total of 15 lines were individualized and 2 lines, Kgb1 and Kgb2, were selected due to accelerated growth and abundant secondary roots. Figure 2 shows the follow-up of the development of Kgb1 and Kgb2 cell cultures at 9 d (Figure 2a), 18 d (Figure 2b), and 25 days (Figure 2c) of the subculture. The images were captured 90 days after remaining in liquid B5 medium. The asynchronous growth in different stages of somatic embryogenesis was observed. The globular (GB), torpedo (T), heart-shaped (H), and embryo (SE), structures were identified. Embryogenic calli cultures were morphologically heterogeneous, with asynchronous development, meaning that the growth of dedifferentiated cells, the cell differentiation, and the development of embryos occur at uncoordinated times, hence the cellular aggregates in Kgb-1 as Kgb-2 show different stages of embryogenesis through the 25-days of subculture. Figure 2d shows the initial embryogenic aggregates, which were observed since the roots were isolated from the infected explant and remained in a liquid medium. These structures were observed in all stages of culture. The lines currently remain stable with the same characteristics.

Figure 3 shows the amplified fragments of the rolA, rolB, rolC, rolD, and aux1 genes from the DNA of Kgb1 and Kgb2 lines; none of the analyzed genes was amplified from K. gastonis-bonnieri wild-type plants. These results suggest that the Kgb1 and Kgb2 lines were induced in the infection mediated by A. rhizogenes A4 strain. In this work, it is suggested that both TL-DNA and TR-DNA of A. rhizogenes A4 strain were inserted into Kgb1 and Kgb2 genomes. It has been reported that the response of a plant species to genetic transformation by A. rhizogenes is in the function of the integration and combined expression of rolA, rolB, rolC, and rolD genes [31,32].

3.2. Analysis of the Chemical Profile of Kgb1 and Kgb2 Extracts

Figure 4 shows changes during the cell growth of Kgb1 and Kgb2 lines. The stages were defined as follows: stage (I) 1–10 days of culture, as an adaptation period, and changes in cell growth were observed, stage (II) 11–21 d, increase accelerated biomass with doubling time for Kgb1 (T2) = 3.31 d and cell growth speed (μ) = 0.20 d⁻¹; while for Kgb2, T2 = 4.15 d and μ = 0.16 d⁻¹, stage (III) 21–25 d, a decrease in biomass growth was observed, for Kgb1 the T2 = 20.3 d and μ = 0.03 d⁻¹; finally, for Kgb2 T2 = 48.5 d, and μ = 0.01 d⁻¹; stage (IV) 25–35 d, considerable decrease in cell growth was observed in both lines. The chemical profile of Kgb1 and Kgb2 was analyzed at 9, 18 and 25 days of culture, which were selected taking quantity biomass as a selection criteria.

Table 1. Tentatively identified compounds in Kgb1 and Kgb2 cell lines extracts of Kalanchoe gastonis-bonnieri (- means no detected, while +, ++ and +++ refer to different peak intensity).

RT (min)	Tentative Identification	Match Factor	Monoisotopic Mass	Main Fragments (m/z)	Parent Ion [M-H]-	Kgb1			Kgb2			Wild Type Plants
						Days of Culture						Leaves	Roots
						9	18	25	9	18	25
	Flavonoids
5.9	3,7-Dihydroxy-3’,4’-dimethoxyflavone	79	314.0863	637.1386	313.0684	-	-	-	-	-	-	+	-
				638.1423
				653.1331
6.1	Syringetin-3-O-glucoside	78	508.1272	477.1005	507.1111	-	-	-	-	-	-	+	-
				508.1177
				535.2139
6.2	Quercetin-3-O-pentosyl (1-2) acetilpentosida	80	608.1295	327.0844	607.1259	-	-	-	-	-	-	+	-
				623.1247
				623.2683
6.2	3,4-dimethoxy-myricetin-3-O-dideoxyhexosyl(1-2)-dideoxyhexoside	80	638.1445	521.2012	637.1383	-	-	-	-	-	-	+	-
				623.1227
				638.1445
6.3	Guaijaverin	79	434.0762	434.0762	433.0762	-	-	-	-	-	-	+	-
				491.0744
				519.2203
6.5	Apigenin-6-C-glucoside-7-O-glucoside	90	594.1500	461.1064	593.1466	-	-	-	-	-	-	+	-
				506.0978
				594.1500
6.6	6,7,3’,4’-Tetrahydroxyflavanone	86	288.0149	146.9666	287.1465	-	-	+	-	-	-	-	-
				288.1511
				309.1295
6.7	Kaempferol-3-O-arabinoside	95	418.0852	418.0852	417.0791	-	-	-	-	-	-	+	+
				491.1174
				607.2728
6.9	Epiafzelechin (2R,3R)(-)	80	274.1671	187.0955	273.1671	-	-	+	-	-	-	-	-
				289.1635
				607.2757
7.0	Naringenin	80	272.1627	112.9843	271.1528	-	-	+	-	-	-	-	-
				289.1627
				607.2727
7.1	Kaempferide 3-glucuronide	95	476.0914	597.2469	475.0873	-	-	-	-	-	-	-	+
				607.2738
				608.2776
7.2	Diosmine	89	608.1664	475.0857	607.1641	-	-	-	-	-	-	+	-
				608.1683
				643.1410
7.7	Quercetin	90	302.0368	146.9683	301.0317	-	-	-	-	-	-	+	+
				157.0095
				302.0331
8.2	2’,7-Dihydroxy-4’-methoxy-8-prenylflavan 2’,7-diglucoside	83	664.2705	653.2342	663.2626	-	-	-	-	-	-	+	-
				680.2472
				707.2874
8.6	Fisetin	80	286.0392	112.9833	285.0360	-	-	-	-	-	-	+	+
				286.0392
				315.0477
10.5	Kaempferol-7-neohesperidoside	93	594.2728	197.9606	593.2716	+	-	-	+	-	-	+	-
				201.0350
				594.2728
11.1	Quercetin-3-O-vicianoside	94	596.2922	197.9612	595.2873	+	+	-	+	-	-	-	-
				596.2922
				723.3794
11.9	Neobavaisoflavone	90	322.1733	322.1762	321.1724	+	+	-	-	+++	-	-	+
				406.1516
				421.0987
	Fatty acids
6.3	Deacetoxy (7)-7-Oxokhivorinic acid	78	520.2216	509.1912	519.2210	-	-	-	-	-	-	+	-
				520.2216
				536.2101
7.6	3-Hydroxytetradecanedioic acid	83	274.171	146.9682	273.1676	-	-	+	-	-	-	-	-
				173.9999
				274.1710
8.4	(9S,10E,12S,13S)-9,12,13-Trihydroxy-10-octadecenoic acid	81	330.2366	174.0007	329.2320	-	+	-	-	-	-	-	-
				201.0377
				330.2366
8.7	Corchorifatty acid F	83	328.2277	201.0351	327.2141	+	+	+	+	+	+	+	-
				263.1309
				328.2158
9.2	(Z)-9,12,13-trihydroxyoctadec-15-enoic acid	80	330.9998	157.0100	329.2300	+	++	+	+	+	+	+	-
				330.2365
				397.2198
11.3	13-HOTrE	81	294.2131	275.2000	293.2095	+	+	+	+	-	+	-	-
				294.2131
				613.9943
11.7	Vernolic acid	83	296.2289	116.9269	295.2255	+	+	+	+	+	+	+	-
				296.2289
				363.2136
12.4	Altamisic acid	80		112.9851	279.1628	-	-	-	-	-	+	-	-
				135.9701
				309.1725
12.8	α-Linolenic acid	80	278.7812	135.9699	277.2160	+	-	-	-	-	+	-	-
				278.2192
				400.2108
13.3	Linoleic acid	80	280.2351	278.7270	279.2322	+	-	-	-	-	-	-	-
				280.2351
				325.1854
14.9	Heneicosanoic acid	90	326.1831	326.1873	325.1829	+++	-	-	-	+++	++	-	-
				339.2004
				340.2043
	Coumarins
1.4	4-Hydroxycoumarin	83	162.0495	117.0193	161.0451	+	-	-	+	-	-	-	-
				128.0353
				292.1378
4.9	3,4,5-Trihydroxy-6-{[2-oxo-6-(3-oxobutyl)-2H-chromen-7-yl]oxy}oxane-2-carboxylate	85	408.0981	341.0851	407.0945	-	-	-	-	-	-	+	-
				408.0981
				443.0710
6.3	Hallactone B	80	440.1090	112.9848	439.1031	-	-	-	+	-	-	-	-
				174.0002
				440.1090
11.4	Rugosal A	87	266.1482	134.8628	265.1446	-	-	+	+	-	+	++	+++
				135.9684
				817.1497
11.8	Corylifol A	85	390.2340	321.1705	389.2295	-	-	-	-	-	-	+	-
				390.2340
				411.2125
	Phenolic acids
5.4	Caffeic acid	93	180.0365	135.0425	179.0325	-	-	-	-	-	-	+	+
				180.0365
				755.1999
5.7	1-Caffeoyl-4-deoxyquinic acid	90	338.0910	191.0536	337.0887	-	-	-	-	-	-	+	-
				338.0910
				359.0706
5.9	4-Coumaric acid	81	164.0467	164.0467	163.0378	+	-	+	+	-	+	-	-
				279.0519
				475.1830
6.0	Ferulic acid	86	194.0537	194.0537	193.0486	+	-	++	+	-	++	-	-
				309.0612
				311.1114
	Phenolic compounds
5.2	Syringate	81	198.0453	198.0453	197.0428	-	-	-	-	-	-	+	-
				313.0536
				431.1863
5.5	Verbasoside	83	462.1709	415.1603	461.1640	+	-	+	+	-	+	-	-
				451.1389
				462.1709
15.2	p-Coumaraldehyde	81	147.8759	178.8796	146.9643	+	-	-	-	+	+	-	-
				220.9484
				231.9439
	Terpenes
6.4	Kanokoside D	89	624.2753	577.2622	623.2694	-	-	-	+	-	-	-	+
				613.2428
				624.2753
10.3	Heliannuol A	94	250.1536	248.9578	249.1497	+	++	++	+	+++	+++	++	++
				250.1536
				251.1476
12.3	Ipecoside	80	565.3214	116.9282	564.3191	+	-	-	-	-	-	-	-
				554.2898
				581.3077
	Carboxylic acids
1.0	Malic acid	80	134.0215	128.0336	133.0119	+++	+++	+++	-	-	+++	++	+
				341.1061
				377.0831
1.0	DL-Pyroglutamic acid	80	129.0345	290.0858	128.0336	-	+	-	+	-	-	-	-
				310.0687
				403.1352
1.2	Citrate	90	192.0301	111.0072	191.0175	-	-	-	-	-	-	+	-
				128.0334
				173.0081
1.3	D-(-)-Quinic acid	91	192.0579	157.0341	191.0520	-	-	-	-	++	-	+++	-
				377.0813
				379.0805
	Alkaloids
6.1	Voacristine	80	384.0108	384.0108	383.0066	-	-	-	-	-	-	-	+
				481.1331
				563.2139
	Amino acids
0.8	D-Glutamine	87	146.0324	179.0556	145.0598	+	-	-	+	-	-	-	-
				215.0324
				307.1131
4.3	Tryptophan	82	204.0841	204.0841	203.0800	-	-	-	-	-	-	+	-
				261.0370
				271.0673
	Carbohydrate
1.1	Sarmentosin epoxide	90	291.0908	133.0134	290.0859	+++	++	++	+++	++	++	-	-
				200.0561
				632.2048
	Others
0.7	Ribose-1-arsenate	89	273.9598	158.9785	272.9565	++	-	-	+	-	-	-	-
				273.9598
				274.9575
0.9	3-(4-hydroxy-3,5-dimethoxyphenyl)-1-(2,4,6-trihydroxy-3-methoxyphenyl)propane-1,2-dione	90	378.0889	341.1077	377.0854	-	+++	-	-	-	-	-	-
				379.0830
				404.1046
1.0	2-(1,3-dihydroxy-4-oxocyclohex-2-en-1-yl)-5-hydroxy-3,6,7-trimethoxy-4H-chromen-4-one	79	378.0822	341.1083	377.0846	+	-	-	-	-	-	-	-
				404.1043
				470.1521
4.4	Cusparine	82	307.1179	112.9835	306.1163	-	-	-	-	-	-	+	-
				296.0879
				350.1422
5.1	1,6-Dihydroxy-3,7-dimethoxy-2-(3-methyl-2-butenyl)-8-(3-hydroxy-3-methyl-1E-butenyl)-xanthone	86	440.1819	393.1746	439.1785	-	-	+	+	-	-	-	-
				429.1495
				440.1819
5.8	1-(2H-1,3-benzodioxol-5-yl)-2-[2,6-dimethoxy-4-(prop-2-en-1-yl)phenoxy]propyl benzoate	83	476.1831	429.1758	475.1800	-	-	-	-	-	-	+	-
				476.1831
				521.1996
8.5	3-(1,2-dihydroxypropyl)-1,6,8-trihydroxyanthracene-9,10-dione	83	330.2334	285.0364	329.2298	+	-	+	+	-	+	+	-
				286.0402
				330.2334
8.7	Isocyclocalamin	85	502.2151	491.1836	501.2109	-	-	-	-	-	-	-	+
				493.1828
				518.2009

shows 60 tentatively identified metabolites in the extracts of Kgb1, Kgb2 lines at 9, 18, 25 daysof culture, and the wild plant of K. gastonis-bonnieri; among them, 18 flavonoids, 11 fatty acids, 5 coumarins, 4 phenolic acids, 3 phenolic compounds, 3 terpenes, 4 carboxylic acids, 1 alkaloid, 2 amino acids, 1 carbohydrate and 8 compounds grouped as others, based on chemical structure. The highest number of flavonoids was identified in the wild type, while fatty acids and carbohydrate were mainly identified in Kgb1 and Kgb2 lines.

The metabolites were identified according to LC-ESI-MS/MS parameters: retention time, match factor values database, molecular formula, and monoisotopic mass. The tentative identification of compounds was based on the comparison of the MS fragmentation profile obtained by the analytical equipment, with the mass spectra of MassBank of North America and Competitive Fragmentation Modeling for Metabolite Identification.

The Kgb1 and Kgb2 lines showed a differential chemical profile compared to the wild plant. Particularly, in Kgb1 and Kgb2 extracts the following chemical compounds were detected: ribose-1-arsenate (0.7), 3-(4-hydroxy-3,5-dimethoxyphenyl)-1-(2,4,6-trihydroxy-3-methoxyphenyl)propane-1,2-dione (RT = 0.9), 2-(1,3-dihydroxy4-oxocyclohex-2-en-1-yl)-5-hydroxy-3,6,7-trimethoxy-4H-chromen-4-one (RT = 1.0), sarmentosin epoxide (RT = 1.1), 4-hydroxycoumarin (RT = 1.4), sarmentosin epoxide (RT = 1.1) D-glutamine (RT = 1.8), 1, 6-dihydroxy-3,7-dimethoxy-2-(3-methyl-2-butenyl)-8-(3-hydroxy-3-methyl-1E-butenyl)-xanthone (RT = 5.1), verbasoside (RT = 5.4), 4-coumaric acid (RT = 5.9), ferulic acid (6.0), hallactone B (RT = 6.3), 6,7,3’,4’-tetrahydroxyflavanone (RT = 6.6), epiafzelechin (2R,3R)(-) (RT = 6.9), naringenin (RT = 7.0), 3-hydroxytetradecanedioic acid (RT = 7.6), (9S,10E,12S,13S)-9,12,13-trihydroxy-10-octadecenoic acid (RT = 8.4), 13-HOTrE (RT = 11.3), ipecoside (RT = 12.3), altamisic acid (12.4), a-linolenic acid (RT = 12.8), linoleic acid (RT = 13.3), and heneicosanoic acid (RT = 14.9), p-coumaraldehyde (RT = 15.2). Although, neobavaisoflavone, malic acid, heneicosanoic acid, heliannuol A, 3-(4-hydroxy-3,5-dimethoxyphenyl)-1-(2,4,6-trihydroxy-3-methoxyphenyl)propane-1,2-dione,sarmentosin epoxide, were major compounds in the Kgb1 and Kgb2 cell lines, in comparison to wild plant.

Figure 5 shows the comparison of the relative abundance of the identified metabolites; the lowest concentration in red light, while the highest in green light. According to the row dendrogram, it is observed that the relative abundance of the compounds is different between extracts of the transformed lines Kgb1, Kgb2, leaf, and wild type root extracts. Column dendrogram allows for visualizing the formation of two main groups: on the left the extracts obtained from Kgb1 of 9, 18, and 25 days of culture and Kgb2 of 9 and 25 days, and on the right side the leaf and wild root extracts. Subgroups are formed between the Kgb1 and Kgb2 lines, showing the chemical compound diversity between the different culture days analyzed. Regarding the relative abundance of 4-coumaric acid, ferulic acid, verbasoside, and 3-(1,2-dihydroxypropyl)-1,6,8-trihydroxyanthracene-9,10-dione. They were similar at 9 and 25 days between Kgb1 and Kgb2, while the wild type of leaf and root extracts were similar in relation to the abundance of rugosal A, fisetin, quercetin, kaempferol-3-O-arabonoside, and y caffeic acid. Dendrogram also shows the identification of specific compounds in Kgb1 and Kgb2 extracts in the different culture periods. In the Kgb1-d9 extract, particular compounds were identified as ipecoside, linoleic acid, 2-(1,3-dihydroxy-4-oxocyclohex-2-en-1-yl)-5-hydroxy-3,6,7-trimethoxy-4H-chromen-4-one, and linolenic acid. In the Kgb1-d18 extract, specific compounds were identified as 3-(4-hydroxy-3,5-dimethoxyphenyl)-1-(2,4,6-trihydroxy-3-methoxyphenyl) propane-1,2-dione, 9,12,13-trihydroxy-10-octadecenoic acid, pyroglutamic acid, quercetin-3-O-vicianoside, and y 13-HOTrE, while in the Kgb1-d25 extract, the specific identified compounds were epiafzelechin (2R,3R), 6,7,3’,4’-tetrahydroxyflavanone, and naringenin y 3-hydroxytetradecanedioic acid.

Likewise, specific compounds were observed in the leaf and/or root extracts: 3-(1,2-dihydroxypropyl)-1,6,8-trihydroxyanthracene-9,10-dione, kaempferol-7-neohesperidoside, rugosal A, fisetin kaempferide 3-glucuronide, quercetin, kaempferol-3-O-arabinoside, and caffeic acid. Similar relative compound abundance was observed for Kgb1 and Kgb2 extracts at 9 days of culture: ribose-1-arsenate, 4-hydroxycoumarin, D-glutamine, sarmentosin epoxide, 4-coumaric acid, and ferulic acid; at 25 days of culture: 4-coumaric acid, ferulic acid, and verbasoside.

4. Discussion

Some data on transformation efficiency have been reported: 72% in leaf explants of Solanum erianthum D. Don. [36]; 65% in shoot explants and 60 in leaf explants of Althaea officinalis [28]; 56% in leaf explants and 29% in seedlings’ inter nodal explants of Salvia bulleyana [37]. According to this information, the transformation efficiency of A. rhizogenes strain A4 is diverse; in this work, A4 strain was capable of infecting K. gastonis-bonnieri tissue which led to genetically modified embryogenic calli. Also, Chaudhuri et al. [38] and Tavassoli and Safipour-Afshar [28] reported that transformation efficiency was obtained as a function of culture conditions, Agrobacterium-host interaction, age, and explant type. It has also been associated with actively dividing cells showing higher transformation rates.

In this work, 15 lines were individualized, and 2 lines, Kgb1 and Kgb2, were selected, due to accelerated growth and abundant secondary roots. Kgb1 and Kgb2 showed spontaneous callus formation. Vinterhalter et al. [10] reported calli and somatic embryo formation at 35 d of culture, from roots induced with the A. rhizogenes A4M70GUS strain, in Gentiana utriculosa. Hiebert-Giesbrecht et al. (2021) [8] reported in Pentalinon andrieuxii the formation of embryogenic callus 6 months after obtaining hairy roots with the A. rhizogenes ATCC15834 strain in leaves and hypocotyls. Results obtained in this work represent the possibility of whole plant regeneration; moreover, is an interesting tool to carry out advanced studies on the secondary metabolism of K. gastonis-bonnieri transgenic culture. The response of K. gastonis-bonnieri agrees with previous reports on the spontaneous formation of embryogenic calli from hairy roots obtained by Agrobacterium infection, on different plant species such as Gentiana utriculosa with the A. rhizogenes A4M70GUS strain [1,10], Pentalinon andrieuxii with the A. rhizogenes ATCC15834 strain [8]; all these strains carrying the same agropine-like Ri plasmid [39]. It has been suggested that abnormal morphological features of hairy roots can be a result of the combined participation of rol genes in plant cells since each gene might be associated with specific phenotypic alterations in Kalanchoë species; in addition, the response of each plant species against infection by A. rhizogenes is diverse [38]. In this work, it is suggested that both TL-DNA and TR-DNA of A. rhizogenes A4 strain were inserted into Kgb1 and Kgb2 genomes. It has been reported that the response of a plant species to genetic transformation by A. rhizogenes is in function of the integration and combined expression of rolA, rolB, rolC, and rolD genes [40,41].

The observed difference in the chemical profile of the transformed embryogenic calli extracts is possibly due to asynchronous growth in the different culture periods [42]. There is wide variability in the compounds reported in K. gastonis-bonnieri, which could be attributed to the analyzed plant material that includes the plant development stage, the collection season, and extraction conditions, even among the transformed cell lines analyzed in this work.

Kgb1 and Kgb2, at 9 days of culture, showed a greater number of detected compounds, in contrast to culture analyzed at 18 and 25 days, which could be related to the adaptation of subculture towards the beginning of a new cycle [43]; a lower number of compounds were detected at 18 days of culture which could be attributed to accelerated cell growth [43,44], therefore the cell metabolism is redirected toward multiplication and cell growth. Finally, at 25 days of culture, the increase of some compounds was observed, which could be attributed to the accumulation of compounds related to the embryogenic process. The accumulation and changes in metabolites detected are related to different stages of development and growth in the in vitro culture, which is also observed at different developmental stages of wild plants [45,46,47] as a part of plant development or in response to epigenetic factors.

The extracts of Kgb1 and Kgb2 lines and leaves showed corchorifatty F acid, a compound identified in rice and other cereals, with antibacterial activity [48]. In Kgb1 and Kgb2 extracts, sarmentosine epoxide was detected, which has shown antihepatotoxic activity [49]. In this work, malic acid, caffeic acid, rugosal A, campferol 3-O-arabinoside, quercetin, fisetin, and heliannuol were detected in leaves and roots of K. gastonis bonnieri which have not been previously reported in wild plants.

Interestingly, hairy root cultures synthesize compounds that have not been detected in wild plants [9]. Furthermore, some authors have reported that shoots and transgenic plants obtained from hairy roots accumulate specific metabolites compared to the wild plant. Tusevski et al. [5], reported an accumulation of naphthodiatrons and specific phenolic compounds in transgenic shoots obtained of hairy roots from Hypericum perforatum. Vinterhalter et al. [7] reported xanthones in transgenic Gentiana utriculosa plants regenerated by somatic embryogenesis from hairy roots. Hiebert-Giesbrecht et al. [8] reported terpenoids accumulation in leaf extracts of transgenic plants, which were obtained from hairy roots of Pentalinon andrieuxii.

The phenotypic changes showed in transformed cultures, could be due to (a) position and (b) number of copies of the T-DNA inserted in the genome of the host cell, (c) the regulation of gene expression, and (d) protein biosynthesis encoded by rol genes in the plant cell, among others [7,8].

The biological activity of some of compounds identified in this work has been reported, quinic and malic acid inhibit the growth of S. aureus and P. aeruginosa [50]; the neobavaisoflavone is a compound that has antioxidant, anti-inflammatory, and anticancer properties [51]; ferulic acid has a wide variety of effects, especially on oxidative stress, inflammation, vascular endothelial injury, fibrosis, apoptosis, and platelet aggregation [52]. The compounds detected in Kgb1 and Kgb2 suggest the effect of genetic transformation through the differential biosynthesis of chemical compounds. It has been reported that highly specialized plant organs such as roots and leaves are needed for the biosynthesis of phytochemicals [26]; therefore, Kgb1 and Kgb2 do not develop highly specialized organs, which could explain the limited accumulation of flavonoids.

5. Conclusions

This is the first report of the chemical profile of transformed cell cultures of K. gastonis-bonnieri. Specific compounds that have not been reported in K. gastonis-bonnieri wild-type plants were identified. In vitro, culture of genetically transformed embryogenic calli could be an alternative to produce metabolites of commercial interest in the pharmacological industry as treatment against various diseases, such as cancer. In addition, obtaining transgenic K. gastonis-bonnieri plants from embryogenic callus could be an opportunity in the ornamental industry since genetic modification could produce plants with different phenotype.