Agrobacterium Rhizogenes Strain A4 Research Articles

Tapping the potential of Calotropis procera hairy roots for cardiac glycosides production and their identification using UHPLC/QTOF-MS.

The present work deals with the establishment of hairy root cultures from different explants of C. procera using Agrobacterium rhizogenes strain A4. A high transformation frequency (95%) was obtained from leaves followed by cotyledons (81.6%) and hypocotyls (38.3%). Genetic transformation of hairy roots was confirmed through PCR by amplifying a 400 bp fragment of the rolB gene. Hairy roots were highly branched, possessed plagiotropic and rapid growth on hormone-free ½ B5 medium. Ten cardiac glycosides, including calotropagenin, calotoxin, frugoside, coroglaucigenin, calotropin, calactin, uzarigenin, asclepin, uscharidin, and uscharin, based on their specific masses and fragmentation properties were identified in ethanolic extracts of hairy roots by ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry UHPLC/QTOF-MS. This protocol could be used as a powerful tool for large-scale in vitro production of highly valued cardiac glycosides and for further transcriptomics or metabolomics studies.

PECULIARITIES OF THE GROWTH OF Artemisia tilesii Ledeb. “HAIRY” ROOTS WITH DIFFERENT FOREIGN GENES

Aim. To compare Artemisia tilesii “hairy” root lines with different transferred genes in terms of the relationship between the total content of flavonoids, the levels of antioxidant activity (AOA) and reducing power (RP), as well as the activity of phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), rolB and rolC genes. Methods. We compared the root lines Nos. 10 and 16, obtained by transformation with the wild Agrobacterium rhizogenes strain A4, lines Nos. 2 and 4, obtained using A. rhizogenes carrying pSV124 vector with ifn-α2b and nptII genes, as well as the roots of non-transformed plants that were cultivated in vitro. The presence and activity of rolB, rolC, PAL, and CHS genes were determined by PCR and real-time PCR, respectively. The content of flavonoids, AOA and RP were determined by standard tests with AlCl3, DPPH (2,2-diphenyl-1-picrylhydrazyl) and K3[Fe(CN)6] accordingly. Results. The content of flavonoids in most of the lines was higher than in the control, and correlated with AOA and RP. Roots No. 10 were characterized by the fastest growth, which coincided with higher activity of rolB and rolC genes. The activities of PAL and CHS in “hairy” roots were lower than those in non-transformed ones. Conclusions. Root lines carrying only rolB and rolC and lines with additional ifn-α2b and nptII genes had similar ranges of flavonoids concentration, AOA and RP levels that exceeded those in the control. The dependence of the root growth rate, and lack of the dependence of the flavonoids content with the activity of the rol genes were demonstrated. PAL activity inversely correlated with flavonoids content in all experimental lines, which may be the result of overproduction of compounds in transgenic roots.

Genetic engineering of tropane alkaloid biosynthesis of hyoscyamus reticulatus L. hairy roots by pmt gene overexpression and feeding with putrescine

The use of biotechnology strategies to increase the production of natural products in medicinal plants, can be very useful and commercially profitable. In this study, control and transgenic hairy roots were obtained from Agrobacterium rhizogenes strain A4 and Agrobacterium tumefaciens strain C58C1(pRiA4-pBMI) carrying the pmt gene C58C1, respectively. The effect of pmt gene overexpression on growth, biochemical traits, and phytochemical ingredients were determined in Hyoscyamus reticulatus hairy roots, which were also fed with different concentrations of the precursor compound putrescine (0, 1, 2, 3, and 4 mM) for two exposure times (24 and 48 h). The growth rate was higher in transgenic hairy roots. The maximum total content of phenol, flavonoid, and alkaloid were obtained in transgenic hairy roots fed with putrescine (4, 3 and 1 mM, respectively) after 48 h. According to the half maximal inhibitory concentration (IC50) values, the antioxidant activity was lower in the control than transgenic hairy roots. High performance liquid chromatography indicated that the hyoscyamine content (125.09 μg g−1 DW with 3 mM putrescine after 24 h; 203.83 % compared with the control) was higher in control hairy roots, whereas scopolamine levels (161.88 μg g−1 DW with 3 mM putrescine after 48 h; 207.34 %) was higher in transgenic hairy roots. The highest increase in the expression level of the pmt (2.6-fold with 3 mM putrescine) and h6h (2.83-fold with 2 mM putrescine) genes occurred after 48 h in both transgenic and control hairy roots, respectively. The combination of genetic engineering and precursor feeding strategies improved the accumulation of secondary metabolites in vitro.

Hairy root cultures of Salvia viridis L. for production of polyphenolic compounds

Salvia viridis is a containing bioactive phenols plant, which has been used in Turkish medicine. The aim of the study was to establish hairy root culture of the species in order to increase the production of the compounds with pharmacological activity. Transformed roots are promising biotechnological systems that produce great amounts of secondary metabolites; this is attributed to their fast growth and their genetic and biosynthetic stability. Stable hairy root lines of S. viridis were established from shoot explants using Agrobacterium rhizogenes strain A4. The highest frequency of transformation (45%) was achieved when the shoot was inoculated at the node or internode by bacteria cultivated on Yeast/Mannitol/Broth (YMB) medium supplemented with acetosyringone. The transformation of the root clones was confirmed by polymerase chain reaction using aux1, aux2, rolB and rolC primers. Of the five obtained transformed root clones, the highest biomass was achieved for clone K3 (13.63 ± 0.5 g/L after 5 weeks) grown in Woody Plant (WP) medium in darkness. The UPLC-PDA-ESI–MS analysis of hydromethanolic extracts of transformed roots of S. viridis revealed the presence of 10 compounds identified as caffeic acid derivatives. Quantitative analysis showed that rosmarinic acid (RA) was the predominant compound in all clones. The highest RA (35.8 mg/g dry weight) and total polyphenol (41.24 mg/g dry weight) content were evaluated in clone K3 cultured in WP medium. These values were 8-fold higher than those of the non-transformed roots, indicating that hairy root cultivation could be a promising technique for the production of valuable compounds from S. viridis.

Growth kinetics and withanolide production in novel transformed roots of Withania somnifera and measurement of their antioxidant potential using chemiluminescence

Markedly increased withanolide content was found in transformed roots (TR) of Withania somnifera germplasm grown in low mineral minimal media and withanolides showed high antioxidant potential when analysed using acidic potassium permanganate chemiluminescence. Transformation frequency of explants infected with Agrobacterium rhizogenes strain A4 varied between the three germplasms tested with the highest observed as 75 ± 0.9. Transformed root production was explant specific with leaves being the most productive among the different explants used. Withanolides, namely withaferin A, withanolide A, withanolide B and 12-deoxywithastramonolide were detected in TR cultures and differences in their content were found between germplasms. The highest concentrations of secondary metabolites were found in 4-week-old cultures and concentrations declined by the 8th and 12th week of culture. In 4-week-old cultures, the biomass of TR cultures was 4.5 fold higher than their respective non-transformed roots (NTR). Withaferin A was found in TR at levels that were 28–34 times higher than that found in NTR. A rapid method for the determination of the antioxidant potential of W. somnifera TR extracts was developed using post-column acidic potassium permanganate chemiluminescence (APPC) detection. The APPC chromatographic peaks for extract constituents showed strong alignment with those found for ultraviolet absorbance detection. The methods developed in this study for TR culture establishment and the use of a fast and sensitive way for the qualitative and quantitative determination of the antioxidant activity of their metabolites provides a new platform that will have use for similar studies in other species.

Pyrethrin accumulation in elicited hairy root cultures of Chrysanthemum cinerariaefolium

The flowers of Pyrethrum (Chrysanthemum cinerariaefolium) are known to contain Pyrethrins that are naturally occurring potential insecticide. Hairy roots were induced from leaves of C. cinerariaefolium using Agrobacterium rhizogenes strain A4. The root clones were characterized in to four groups i.e. thick, unbranched (D2 and D5), thin, highly branched (D3), thick, branched (B2) and thick, highly branched (D1, D6). Six established hairy root clones showed the presence of pyrethrin and were selected for elicitation studies. Growth kinetics studies revealed highest growth index in hairy root clone D1 (592.0) followed by D6 and D3 on dry weight basis after 40 days of culture. The maximum pyrethrin content was found in the clone D3 (7.2 mg/g dw) which is comparable to the flowers obtained from the variety “Avadh”. Hairy root clone D2 (5.2 mg/g dw) and D6 (1.3 mg/g dw) contained pyrethrin but in less amount as compared to clone D3. The PCR analysis showed the presence of rol B and rol C genes in all the six hairy root clones while rol A was detected only in D2 clone. The methanolic extract of D3 clone showed antifungal activities against phytopathogenic fungal strains which were found maximum against Curvuleria andropogonis followed by Colletotrichum acutatum and Rhizoctonia solani. Hairy root clones D2, D3 and D6 were elicited with culture filtrate of endophytic fungus (Fusarium oxysporum) and bacteria (Bacillus subtilis). The culture filtrate (4.0 %v/v) of both the fungal and bacterial origin was found to be effective in enhancing the pyrethrin content in all the tested hairy root clones. Clone D3 showed maximum pyrethrin content on elicitation with F. oxysporum (9.7 mg/g dw) and B. subtilis (9.7 mg/g dw) culture filtrate, which is 32 % higher than the non elicited D3 hairy roots (7.2 mg/g dw). F. oxysporum also enhanced the hairy root growth resulting into the higher biomass yield of D3 (50 %) and D2 (76 %) in comparison to control non elicited hairy root clones of D3 and D2, respectively leading to higher pyrethrin yield.

The identification and quantitative determination of rosmarinic acid and salvianolic acid B in hairy root cultures of Dracocephalum forrestii W.W. Smith

Dracocephalum forrestii, an important plant in Tibetan medicine, was transformed with Agrobacterium rhizogenes strain A4 to obtain hairy root culture. A maximum transformation frequency of 82% after seven weeks was obtained by inoculation of internodal stem segments. Six lines of hairy roots were obtained and cultured in darkness in Woody Plant (WPM) liquid medium. The transgenic nature of the hairy roots was investigated by PCR and RT-PCR. Three main compounds: rosmarinic acid (RA), salvianolic acid B (SAB) and caffeic acid (CA) were identified in extracts of D. forrestii hairy roots by UPLC-PDA-ESI-MS analysis. Quantitative HPLC analysis showed that RA levels were greatly increased in hairy roots (max. 19.97mg/g DW) compared with the roots of one-year-old field grown plants (4.7mg/g DW), but SAB levels were significantly lower in transformed roots (max. 11.7mg/g DW) than in untransformed roots (16.4mg/g DW). Caffeic acid was detected only in transformed roots. A time course experiment with the most productive hairy root line showed that a culture period of 30days and a medium supplemented with 3% of sucrose was optimal for biomass and production of analysed phenolic compounds. Production remained stable over at least 3.5 years of continuous subculturing of the hairy roots.

The influence of methyl jasmonate, cholesterol and l‐arginine on solasodine production in hairy root culture of Solanum mammosum

The increasing demand of diosgenin for high‐revenue synthesis of steroid hormones by the pharmaceutical industries has driven researchers to look for other alternatives. Solasodine which was reported to be present in Solanum mammosum is known to be a potential source. The present study highlighted that added methyl jasmonate, cholesterol and l‐arginine into the modified liquid full‐strength Murashige and Skoog (MS) medium (with ammonium to nitrate ratio 10.3 mM: 39.4 mM, and 4% (w/v) sucrose) could influence the solasodine production in the hairy roots of S. mammosum. The findings showed that both hairy root line‐ATCC31798 and line‐A4 (which were separately induced by Agrobacterium rhizogenes strain ATCC31798 and A4) acquired solasodine productivity of 4.5 mg/g dry weight roots with average dry biomass of 190 mg after 32 days culture, when using 50 mg fresh weight roots as initial inoculum size, with 100 mM cholesterol, 1000 μM l‐arginine and 300 μM methyl jasmonate added simultaneously into the culture medium on day 20 of culture. The amount of solasodine obtained was five times higher than those without both the elicitor and precursor treatment. The improved solasodine production with a high‐biomass growth could reduce the production cost of steroid synthesis in the long run.

Enhanced trans-resveratrol production in genetically transformed root cultures of Peanut (Arachis hypogaea L.)

In the present study, 30 Ri-transformed root lines of peanut (Arachis hypogaea) cv. JL-24, a popular Indian cultivar, obtained following infection with Agrobacterium rhizogenes strains LBA9402, A4 and R1000 were selected on the basis of growth index and maintained in vitro for 3 years. The root lines showed high degree of branching and rapid, plagiotropic growth on phytohormone free solid N/5 medium but were devoid of root hairs. Trans-resveratrol was isolated by preparative HPLC from Ri-transformed roots and identified by ESI–MS/MS. Strain independent variability was observed among 30 Ri-transformed root lines on the basis of lateral root density per cm (7.60 ± 0.30 to 4.5 ± 0.5), relative thickness (0.54 ± 0.07 to 1.54 ± 0.1 mm), growth index (9.16 ± 1.1 to 17.79 ± 1.35 FW basis and 10.77 ± 0.95 to 19.46 ± 1.78 DW basis) and trans-resveratrol content 0.27 ± 0.03 (root line R1000-1) to 0.969 ± 0.141 mg g DW−1 (root line RIX-19) in solid N/5 medium, which was 4.1–14 fold greater than in excised non-transformed root cultures (0.06 ± 0.01 mg g DW−1). Optimum growth and productivity in liquid culture was achieved in N/5 medium supplemented with 0.01 % activated charcoal. Root line RIX-19 showed maximum trans-resveratrol accumulation (1.21 ± 0.09 mg g DW−1) and productivity (0.37 ± 0.08 mg per flask), which was 19 fold higher than non-transformed root cultures. This optimized protocol can be utilized for large scale cultivation of transformed root cultures in industrial bioreactors for mass synthesis of trans-resveratrol.

Iridoid and phenylethanoid glycoside production and phenotypical changes in plants regenerated from hairy roots of Rehmannia glutinosa Libosch.

Spontaneous shoot regeneration was obtained from the hairy roots of Rehmannia glutinosa cultured in liquid woody plant medium for 4 weeks in darkness after induction by Agrobacterium rhizogenes strain A4. These shoots were proliferated on solid Murashige and Skoog (MS) medium in 300 mL glass jars and in liquid MS medium in a 5 L nutrient sprinkle bioreactor. These media were supplemented with 0.1 mg L−1 of indole-3-acetic acid (IAA) and 1.0 mg L−1 of 6-benzylaminopurine. All shoots became rooted on MS agar medium containing IAA or indole-3-butyric acid within 4 weeks. The plantlets were then transferred into soil and grew in a greenhouse. The transgenic nature of the plants was confirmed by polymerase chain reaction (PCR) and reverse-transcriptase PCR analyses. The pRi-regenerated transformants demonstrated a few phenotypical alterations. Production of six iridoid (catalpol, aucubin, loganin, catalposide, harpagide, harpagoside) and two phenylethanoid (verbascoside and isoverbascoside) glycosides was determined in shoots cultured in vitro and leaves and roots of 6- and 12-month-old plants by ultra high performance liquid chromatography. The shoot culture was able to synthesize low amounts of catalpol, verbascoside and isoverbascoside. Leaves and roots of pRi-regenerants accumulated similar (harpagoside, isoverbascoside), higher (catalposide, aucubin, harpagide, verbascoside) or lower (catalpol, loganin) levels of the metabolites in comparison with untransformed R. glutinosa plants and their content varied with the age of the plants and the organ analyzed. However, the increase in shoot and root biomass of transformed plants resulted in a significantly greater yield of all analyzed compounds.

Evaluation of Total Phenolic Compounds and Insecticidal and Antioxidant Activities of Tomato Hairy Root Extract

Tomatoes are one of the most consumed crops in the whole world because of their versatile importance in dietary food as well as many industrial applications. They are also a rich source of secondary metabolites, such as phenolics and flavonoids. In the present study, we described a method to produce these compounds from hairy roots of tomato (THRs). Agrobacterium rhizogenes strain A4 was used to induce hairy roots in the tomato explants. The Ri T-DNA was confirmed by polymerase chain reaction amplification of the rolC gene. Biomass accumulation of hairy root lines was 1.7-3.7-fold higher compared to in vitro grown roots. Moreover, THRs efficiently produced several phenolic compounds, such as rutin, quercetin, kaempferol, gallic acid, protocatechuic acid, ferulic acid, colorogenic acid, and caffeic acid. Gallic acid [34.02 μg/g of dry weight (DW)] and rutin (20.26 μg/g of DW) were the major phenolic acid and flavonoid produced by THRs, respectively. The activities of reactive oxygen species enzymes (catalase, ascorbate peroxidase, and superoxide dismutase) were quantified. The activity of catalase in THRs was 0.97 ± 0.03 mM H2O2 min(-1) g(-1), which was 1.22-fold (0.79 ± 0.09 mM H2O2 min(-1) g(-1)) and 1.59-fold (0.61 ± 0.06 mM H2O2 min(-1) g(-1)) higher than field grown and in vitro grown roots, respectively. At 100 μL/g concentration, the phenolic compound extract caused 53.34 and 40.00% mortality against Helicoverpa armigera and Spodoptera litura, respectively, after 6 days. Surviving larvae of H. armigera and S. litura on the phenolic compound extract after 6 days showed 85.43 and 86.90% growth retardation, respectively.

Hairy roots of Dracocephalum moldavica: rosmarinic acid content and antioxidant potential

Hairy roots of Dracocephalum moldavica L. were induced using Agrobacterium rhizogenes strain A4. Transformed roots were obtained from shoot explants with low transformation frequency of up to 3 %. The effects of different liquid media: Murashige and Skoog (MS), Gamborg et al. (B5) and Woody Plant (WP) with full- and half-strength (½MS, ½B5, ½WP), on biomass accumulation and rosmarinic acid (RA) content were investigated. The hairy roots were cultured in photoperiod (16 h light/8 h dark) and darkness. Biomass of D. moldavica hairy roots was the highest (7.23 g flask−1 of fresh weight and 0.89 g flask−1 of dry weight) in the cultures grown in WP medium under periodic light. Ultra performance liquid chromatography analysis revealed the highest RA content (78 mg g−1 dry wt) in roots cultured in ½B5 medium under photoperiod conditions. It was about tenfold higher compared to roots of field-grown mother plants. Antioxidant activities and total phenolic contents of methanolic extracts of D. moldavica hairy roots cultured in ½B5 and WP media under photoperiod and darkness and roots of field grown plants were compared. All extracts were investigated using 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging and phosphomolybdenum reduction assays. Total phenolic contents were estimated by the Folin–Ciocalteu method. The methanolic extract of D. moldavica hairy roots grown in ½B5 medium under photoperiod possessed the strongest effects on reducing Mo and DPPH radical scavenging. The activities were significantly higher (p ≤ 0.05) than those of methanolic extract of roots of intact plants grown in the field. The most active methanolic extract of hairy roots was characterized by the highest level of rosmarinic acid and total content of phenolic compounds.

Effects of Phytohormones and Jasmonic Acid on Glucosinolate Content in Hairy Root Cultures of Sinapis alba and Brassica rapa

Although some study have established hairy root cultures from brassicaceous plants with glucosinolates (GS) as characteristic secondary metabolite, studies are missing which compare hairy roots with the corresponding mother plants. Therefore, two different plant species-Sinapis alba and Brassica rapa subsp. rapa pygmeae teltoviensis-were transformed with the Agrobacterium rhizogenes strain A4. Aliphatic and indolyl GS were present in B. rapa, exhibiting larger quantities in leaves than in roots. Aromatic p-hydroxybenzyl GS were found particularly in the leaves of S. alba. However, the proportion of indolyl GS increased suddenly in transformed hairy roots of S. alba and B. rapa. Cultivation with the phytohormone kinetin (0.5 mg L(-1)) enhanced GS accumulation in B. rapa hairy roots, however not in S. alba, but 2,4-D (0.4 mg L(-1)) induced de-differentiation of roots in both species and reduced GS levels. GS levels especially of 1-methoxyindol-3ylmethyl GS increased in hairy roots in response to JA, but root growth was inhibited. While 2 weeks of cultivation in 100 to 200 μM JA were determined at optimum for maximum GS yield in S. alba hairy root cultures, 4 weeks of cultivation in 50 to 100 μM JA was the optimum for B. rapa.

Strategies to Improve the Production of Forskolin from Hairy Root Cultures of Coleus forskohlii Briq.

The aim of this study was to elucidate the effect of elicitors and precursors on the production of forskolin from the hairy root cultures of Coleus forskohlii Briq. Hairy root cultures were established from leaf explants by infecting with Agrobacterium rhizogenes strain A4 on MS basal medium. Suspension cultures of hairy root cultures were initiated in MS medium containing IBA (1.0 mg/L), casein hydrolysate (600 mg/L). We investigated the growth of biomass and forskolin production in suspension cultures of hairy roots. The production of forskolin was parallel to the growth of biomass. The maximum production of forskolin was observed after 5 weeks. With the objective to increase the yield of forskolin, abiotic elicitors such as salicylic acid (100 μM and 500 μM), copper sulphate (100 μM and 500 μM), methyl jasmonate (100 μM and 500 μM) and precursors such as α-ketoglutaric acid (0.2 mM and 1.0 mM), L-phenylalanine (0.2 mM and 1.0 mM) were added to hairy root cultures on different days of incubation period and evaluated their effects on production of forskolin. Elicitor, methyl jasmonate (500 μM) and the precursor, L-phenylalanine (1 mM) on day-14 addition significantly enhanced the production of forskolin over the control hairy root cultures C. forskohlii. Given forskolin’s limited commercial supply, this study provides avenues for improving the production of forskolin in the hairy root culture of C. forskohlii.

Analysis of 6-methoxy podophyllotoxin and podophyllotoxin in hairy root cultures of Linum album Kotschy ex Boiss

Linum album is a herbaceous medicinal plant that contains some lignans with antiviral and anticancer properties such as podophyllotoxin (PTOX) and 6-methoxy podophyllotoxin (MPTOX). In this research, hairy root cultures of L. album were established by transformation with Agrobacterium rhizogenes strains LBA9402, A4, AR15834 and Agrobacterium tumefaciens strain C58C1 (pRiA4). The presence of PTOX and MPTOX in the hairy roots was verified by ESI/MS in positive ion mode. MPTOX was confirmed and its enantiomer determined by nuclear magnetic resonance spectroscopy and circular dichroism spectroscopy, respectively. PTOX and MPTOX production was determined by HPLC, in different lines of hairy roots. The results showed that all obtained hairy root lines produced higher yield of lignans than mother plant roots. In addition, the lignan content in the roots derived from A. rhizogenes strain LBA9402 was higher than in those obtained from A. tumefaciens strain C58C1.

Agrobacterium rhizogenes-mediated hairy root induction in Taraxacum officinale and analysis of sesquiterpene lactones

Hairy roots were efficiently induced from leaf and petiole explants of Taraxacum officinale after infection with the Agrobacterium rhizogenes strains A4 and ATCC 15834. The highest frequency of hairy root initiation was observed after transformation of leaf explants with the A4 strain. Hairy roots developed from leaf tissue produced more biomass than non-transformed roots. A quantitative study of sesquiterpene lactones showed that A. rhizogenes induced root cultures accumulated higher levels of these compounds than non-transformed and wild plant roots. The present results demonstrate that T. officinale hairy root culture is a valuable alternative approach for the production of sesquiterpene lactones.

Diterpenoids in transformed root culture of Salvia austriaca Jacq.

Salvia austriaca Jacquin (Austrian sage), is medicinal herbaceous plant native of high altitudes across Russia and eastern Europe [1]. It has been described that the roots of this species produce abietane diterpenoids [2]. In this work we established of S. austriaca hairy root culture by infection of aseptic shoots with Agrobacterium rhizogenes strain A4. Transformation of the roots was confirmed by PCR method by detection of rolB and rolC genes of Agrobacterium in the root cells [3]. The roots were maintained in half-strength hormone-free B5 liquid medium [4]. The dried and powdered hairy roots were extracted 3 times with n-hexane using an ultrasonic bath. The filtrated and evaporated extract was purified by column chromatography on Sephadex LH-20 and eluting with acetone to obtain a fraction rich in abietane diterpenoids. Further separation of the constituents of this fraction by preparative thin layer chromatography (TLC) led to isolation of taxodione, 6-deoxy-taxodione, 15-deoxy-fuerstione and a few other diterpenoids.

Influence of Rhizobacterial and Agrobacterial Inoculation on Selected Physiological and Biochemical Changes of Banana Cultivar, Berangan (AAA) Plantlets

A series of experiments were carried out to observe the effects of rhizobacterial and agrobacterial inoculation, singly orcombined on the total content, concentration and distribution of the biochemical components (total soluble protein,soluble nitrogen, proline, peroxidase activity, total soluble phenolic, nitrate reductase activity, nitrate, chlorophyll),physiological characteristics (percentages of growth, number of roots, fresh and dry weight of roots, maximum and totallength of roots) and mineral contents (N, P, K, Ca and Mg) of in vitro banana plantlets, Berangan cultivar (AAA) usingMS basal medium. The aims of this study are to determine the influence of various rhizobacteria sp. and Agrobacteriasp. inoculation, singly and combined on biochemical and physiological changes of the important banana plantlets inMalaysia, Berangan cultivar (AAA).Results from the inoculation study using MS basal medium were indicated that inoculation with rhizobacteria (Azospirillum brasilense Sp7, Bacillus sphaericus UPMB10 and Microbacteriumoxydens UPMB11) or agrobacteria (Agrobacterium rhizogenes strains, AR9402 and A4) showed positive response ongrowth of in vitro banana plantlets compared to uninoculation after one month of experiment. The inoculationtreatments also increased the number of root, fresh and dry weight of roots and total length of root. In addition withinoculation, the total content or concentration of the respective biochemical activity as total soluble protein, peroxidase,nitrate reductase, proline, nitrate, soluble nitrogen, phenolic and chlorophyll of the host plants increased and variedaccording to the type of bacteria used. Inoculation with these bacterial also enhanced the accumulation of N and P in thebanana plantlets. Co-inoculation with rhizobacteria (Azospirillum brasilense Sp7, Bacillus sphaericus UPMB10 andMicrobacterium oxydens UPMB11) and agrobacteria (Agrobacterium rhizogenes strains AR9402 and A4) also showedsimilar response as in single inoculation; UPMB10+AR9402 treatment was the most effective treatment. The abovefinding provided evidence that Azospirillum brasilense Sp7, Bacillus sphaericus UPMB10, Microbacterium oxydensUPMB11, Agrobacterium rhizogenes strains AR9402 and A4, singly or combined are potentially effective in promotinggrowth of in vitro banana plantlets. Inoculation of rhizobacteria was showed beneficial to the banana plantlet in salineconditions through increment of growth and improvement in rooting system. Thus, these bacterial strains could be usedas a bioenhancer for growth of in vitro banana plantlets

The effect of yeast extract and methyl jasmonate on rosmarinic acid accumulation in Coleus blumei hairy roots

The leaves of axenically grown Coleus blumei were inoculated with Agrobacterium rhizogenes strain A4 and hairy root were established. PCR and Southern hybridization analysis confirmed transgenic nature of hairy root clones. Cultures of normal roots, induced by α-naphthaleneacetic acid on leaf explants, and hairy roots were evaluated for growth and rosmarinic acid content. Significantly better growth and up to 2.8 higher amount of rosmarinic acid was detected by HPLC analysis in hairy root clones. Methyl jasmonate stimulated rosmarinic acid accumulation in 6 out of 11 tested clones, while yeast extract induced RA accumulation in two and diminished it in 5 out of 11 tested hairy root clones.

Terpenoid indole alkaloid production by Catharanthus roseus hairy roots induced by Agrobacterium tumefaciens harboring rol ABC genes

We have established Catharanthus roseus hairy root cultures transgenic for the rol ABC genes from T(L)-DNA of the agropine-type Agrobacterium rhizogenes strain A4. The rol ABC hairy root lines exhibit a wild-type hairy root syndrome in terms of growth and morphology on solid medium. However, they differ from wild-type hairy root lines in that they more frequently have excellent adaptability to liquid medium and do not appear to form calli during cultivation. Moreover, they do not produce detectable levels of mannopine and agropine which, in contrast, are often synthesized abundantly in wild-type hairy root lines. The absence of these opines does not appear to cause the rol ABC lines to have higher levels of terpenoid indole alkaloids than wild-type hairy root lines. Unlike wild-type lines, rol ABC lines produce very similar levels of total alkaloids despite wide variations in individual alkaloid contents. This work demonstrates that the three genes rol ABC are sufficient to induce high-quality hairy roots in Catharanthus roseus.