Effects associated with insertion of rol genes on morphogenic potential in explants derived from transgenic Bacopa monnieri (L.) Wettst

The present study deals with the establishment of rolA-transgenic and rolB-transgenic plants for the first time through Agrobacterium tumefaciens mediated transformation, exploiting the inherent morphogenic potential of an important medicinal plant, Bacopa monnieri (L.) Wettst. The rolA-transgenic and rolB-transgenic plants showed integration and expression of rolA and rolB genes respectively, whereas Ri-transformed plants showed integration and expression of rolA, rolB, rolC and rolD genes. Morphogenic potential of different types of explants derived from rolA-transgenic, rolB-transgenic and Ri-transformed plants on basal medium was evaluated. Shoot organogenesis was enhanced significantly in leaf (1.6-fold) and internode (1.4-fold) explants derived from rolA-transgenic plants, rolB-transgenic leaf (2.4-fold) and internode (1.6-fold) explants as well as leaf (5.2-fold) and internode (3.3-fold) explants derived from Ri-transformed plants compared to explants from non-transformed plants. Substantial increase in root organogenesis was also noticed in rolA-transgenic leaf (1.7-fold) explants, rolB-transgenic leaf (3.6-fold) and internode (1.4-fold) explants as well as leaf (4.1-fold) and internode (1.9-fold) explants derived from Ri-transformed plants compared to non-transformed ones. In addition to this, growth of root tip and shoot regeneration was also noticed from Ri-transformed root explants, but not in rolA-transgenic, rolB-transgenic and non-transformed roots. Clones of each transgenic plant line harboring rol genes depicted notable phenotypic changes including reduced shoot and internode length, increased number of nodes/plant, leaves/plant and roots/plant. The leaf morphology was altered in rolB-transgenic and Ri-transformed plants but not in rolA-transgenic plants. Insertion of rol genes, individual or in combination, of Agrobacterium rhizogenes resulted in enhanced morphogenic potential of excised explants and alteration in phenotype of transgenic Bacopa monnieri plants maintained in vitro.


Introduction
Plant cells are highly totipotent to undergo morphogenesis in vitro through shoot organogenesis and/or somatic embryogenesis (Bhojwani and Razdan 1983). Although both the developmental programmes are regulated by several factors, Skoog and Miller's (1957) classical findings on quantitative auxin/cytokinin ratio plays a pivotal role in morphogenesis. Since then, several pioneer works have concluded that the fate of regenerating organs during morphogenesis can be altered either exogenously by hormone application in the culture media (Tran Than Van 1980) or endogenously within the plant body by introducing Agrobacterium T-DNA genes synthesizing auxins and cytokinins (Owens et al. 1988;Wabiko and Minemura 1996).
For centuries, the complex plant-A. rhizogenes pathogenic interaction and functions of Ri T-DNA oncogenes have been deciphered (Mauro et al. 2017;Bahramnejad et al. 1 3 2019). This bacterium induces neoplastic root growth in host plants at infection sites by transferring its T-DNA to produce opines for food and energy (Tepfer and Tempé 1981;Chilton et al. 1982). Molecular analysis has revealed four rooting loci (rolA, rolB, rolC, rolD) of T-DNA involved in rhizogenesis by manipulating endogenous hormone metabolism/ sensitivity in transformed plant cells although exact mechanism of action is unknown (White et al. 1985;Mauro et al. 2017). Due to hormonal disequilibrium, developmental programmes are altered, as a side-effect, in transformed plants affecting morphogenesis. The Ri-transformed rhizoclones regenerate spontaneously or induced in hormonesupplemented media into Ri-transformed plants with altered phenotypes through direct or indirect morphogenesis (Roychowdhury et al. 2013). Although Ri-transformed plant regeneration was amply demonstrated, reports on transgenic plant production harbouring individual rol genes are scarce which explained the contribution of these genes in morphogenesis (Sarkar et al. 2018). The inter-relationship between rol genes and morphogenesis is further confirmed when explants of Ri-transformed or rol-transgenic plants were investigated for organogenic response although the number of such reports is very limiting. Among them, rolB gene has been extensively studied and is considered as a meristeminducing gene (Altamura 2004). Casanova et al. (2003) demonstrated dual activities of rolC gene due to improved shoot and root regeneration in transgenic Dianthus caryophyllus explants in presence of cytokinin and auxin respectively. However, the effects of rolA and rolD genes on organogenesis has been very less explored. van Altvorst et al. (1992) reported root induction in rolA or rolB or rolABC-expressing tomato leaf explants in presence of auxin but not in controls. But rolD gene enhanced flower induction in flowering and rooting media in transgenic tobacco thin cell layer (TCL) explants indicating its commitment towards floral organogenesis (Mauro et al. 1996).
Bacopa monnieri (L.) Wettst. (Plantaginaceae), due to its diverse pharmacological potential, has been widely studied for micropropagation and de novo organogenesis (Saha et al. 2020). We have reported earlier high morphogenic potency of excised leaf and internode explants on basal medium and suggested this species to be an ideal system to study morphogenesis in vitro, circumventing the effects of exogenous phytohormones (Sarkar and Jha 2017).
Induction of Ri-transformed roots followed by Ri-transformed plant regeneration through A. rhizogenes-mediated transformation was reported in B. monnieri (Majumdar et al. 2011;Paul et al. 2015). Ri-transformed roots, unlike nontransformed roots, regenerated spontaneously either directly or from Ri-transformed calli on basal medium (Majumdar et al. 2011;Paul et al. 2015;Largia et al. 2016), suggesting their higher shoot organogenic competence due to T-DNA oncogenes. As excised B. monnieri explants inherently undergo morphogenesis on basal medium, the interaction of rol genes with endogenous hormones during in vitro organogenesis can easily be investigated in this species. Reports are available on A. tumefaciens-mediated transformation to develop transgenic B. monnieri plants (Majumdar et al. 2012;Yadav et al. 2014;Paul et al. 2015). However, attempt to produce transgenic plants expressing individual rol genes and to study the role of rol genes on morphogenesis in B. monnieri has not been made. Therefore, in the present research work, B. monnieri was transformed by individual rolA and rolB genes and also by Ri T-DNA through Agrobacterium-mediated transformation. The objectives of the study were to evaluate the effects associated with insertion of rol genes, either individual or in combination, on plant phenotype and morphogenic potential of explants from transgenic plants.

Tissue culture
Axenic cultures of B. monnieri were established from shoot tip explants (Sarkar and Jha 2017). The cultures were maintained with regular sub-culture on MS (Murashige and Skoog 1962) basal medium containing 3 % (w/v) sucrose and 0.75 % (w/v) agar (Sigma, India).

Sensitivity test of antibiotics on shoot organogenesis from explants
Prior to transformation, the amount of kanamycin and hygromycin that inhibit shoot organogenesis in leaf and internode explants of non-transformed plants was tested to select transgenic shoots. The explants were cultured on MS medium containing different concentrations of kanamycin (25, 50, 75, 100 mg l − 1 ), hygromycin (5, 10, 15, 20, 25, 30 mg l − 1 ) and cefotaxime (250, 500 mg l − 1 ). After four weeks, frequency of explants showing shoot organogenesis was recorded. For each experiment, 30 explants of each type were used per treatment and the experiment was repeated thrice.

Transformation procedure
Leaves (1-1.5 cm) and internodes (~ 0.9 cm) excised from 15-days-old micropropagated plants were used as explants. Transformation was done following the protocol of Paul et al. (2015). For each experiment, 50 explants of each type from different plants were infected and the experiment was repeated thrice.

Transformation with A. rhizogenes strain
After surface-sterilization, control and infected explants were cultured on MS medium containing cefotaxime (500 mg l − 1 ). Roots induced at wound sites of infected explants were excised and cultured on the same medium under 16/8 h photoperiod or under complete darkness. Each excised root was propagated as a separate root line. Fast growing axenic root lines were maintained on cefotaximefree MS medium after six months of culture initiation with regular subculture at 4-weeks interval. Roots induced from uninfected explants (control) were similarly excised and cultured on MS medium containing cefotaxime (500 mg l − 1 ).

Shoot organogenesis and establishment of rolA-transgenic and rolB-transgenic plants
After two months of shoot bud induction from wound sites of infected explants on respective selection media, infected explants with developing micro shoots (< 1.5 cm) were transferred to selection media supplemented with 1.0 mg l − 1 6-benzylaminopurine (BA). Micro shoots regenerated from shoot buds induced from wound sites of different explants derived from different plants were propagated as separate clones. After four weeks, excised elongated shoots were cultured on BA-free selection media with sub-culturing at 4-weeks interval (cefotaxime concentration was gradually reduced to zero). Micro shoots were rooted spontaneously to develop complete rooted plantlets. After six months of culture initiation, putatively transgenic plants were cultured on respective liquid bacterial media on a gyratory shaker at 28ºC in dark at 180 rpm for 24-48 h to determine their axenic nature. Finally, clones of rolA-transgenic and nptIItransgenic plants were established on MS medium containing kanamycin (100 mg l − 1 ) and rolB-transgenic plants on MS medium containing hygromycin (15 mg l − 1 ).
Shoots regenerated from leaf and internode explants of non-transformed plants via organogenesis on MS medium and MS containing cefotaxime (500 mg l − 1 ) were maintained separately as control.

Spontaneous regeneration from Ri-transformed roots and establishment of Ri-transformed plants
Shoot buds (~ 1 cm) spontaneously regenerated from Ritransformed roots were excised and cultured on MS medium containing cefotaxime (500 mg l − 1 ). Micro shoots regenerated from shoot buds induced from different Ri-transformed root lines were multiplied as separate clones. Micro shoots were rooted spontaneously on this medium to develop complete rooted plantlets. Cefotaxime was withdrawn after six months of regular sub-culture at 4-weeks interval and the axenic cultures were finally established on MS basal medium. Excised non-transformed roots were cultured on MS containing BA (0.01 mg l − 1 ) for shoot induction (control).

Confirmation of transformation by PCR and RT-PCR
Genomic DNA was extracted from different transgenic and non-transformed plants after one year of establishment on respective selection media according to Dellaporta et al. (1983). Isolated DNA was analyzed by PCR for rolA, rolB, rolC, rolD, nptII and hptII genes using gene-specific primers (Beck et al. 1982;Diouf et al. 1995;Sevón et al. 1997; 1 3 Wang et al. 2001;Christensen et al. 2008). Plasmid DNA isolated from the corresponding bacteria following standard alkali lysis protocol (Sambrook and Russel 2001) was used as positive control, whereas genomic DNA from non-transformed plants was used as negative control. To eliminate the false positive result due to bacterial contamination, virD1 gene-specific primers (Alpizar et al. 2008) were used. PCR amplicons were resolved by 1.2 % (w/v) agarose gel electrophoresis with a 100 bp plus DNA ladder (Thermo Scientific, USA) and visualized by ethidium bromide staining under UV light. Documentation was done using Gel Doc™ EZ Imager (BioRad). For each transgenic plant, ten replicates per clone were used and the experiment was repeated thrice.
Expression of transgenes at the transcription level was analyzed by reverse transcriptase polymerase chain reaction (RT-PCR). Total RNA was extracted from fresh tissues of 4-weeks-old transgenic and non-transformed plants using HiPurA™ Plant and Fungal RNA Miniprep Purification Kit (HIMEDIA) following the manufacturer's protocol. RT-PCR reactions were performed as described earlier (Majumdar et al. 2011;Paul et al. 2015).

Maintenance of regenerated transgenic plants
Axenic rolA-transgenic, nptII-transgenic, rolB-transgenic and Ri-transformed plants were maintained on respective selection media for over three years with regular subculture at 8-weeks interval.

Study of in vitro morphogenic potential in explants derived from transgenic plants on basal medium
Excised leaves (~ 0.95 cm), internodes (~ 0.90 cm) and root (1.5-2.0 cm) from 4-weeks-old different transgenic and nontransformed plants were cultured on MS basal medium as reported earlier (Sarkar and Jha 2017). After four weeks, morphogenic responses from different explants were evaluated. For leaf and internode explants, number of shoot buds/ micro shoots induced per explant and number of roots induced per explant were recorded whereas shoot bud development and/or root tip growth were examined for root explants. For each experiment, 30 explants of each type per transgenic clone and non-transformed plant were used and the experiment was repeated thrice.

Analysis of morphology of transgenic plants maintained in vitro
Morphological characterization of different transgenic plants was done based on morphological descriptors reported earlier for Ri-transformed plants (Majumdar et al. 2011). Shoot tips (~ 2 cm) were cultured in culture tubes (15 × 2.5 cm) containing 20 ml MS containing kanamycin (100 mg l − 1 ) for rolA-transgenic and nptII-transgenic plants, MS with hygromycin (15 mg l − 1 ) for rolB-transgenic plants and MS basal medium for Ri-transformed plants under 16/8 h photoperiod. One shoot tip was cultured per culture tube. Shoot tips from non-transformed plants were similarly cultured on MS basal medium (control). After six weeks, the plants were harvested, washed, blotted dry and morphological data were taken. For each experiment, 30 explants were used per transgenic clone and non-transformed plant and the experiment was repeated thrice.

Study of auxin sensitization response and effect of exogenous cytokinins on in vitro morphogenesis in explants derived from transgenic plants
Leaves, internodes and root segments from 4-weeks-old different transgenic and non-transformed plants were cultured on MS media fortified with 0.01, 0.05 and 1.0 mg l − 1 of filter-sterilized auxin i.e. IAA (indole-3-acetic acid) and cytokinins i.e. 6-benzylaminopurine (BA) and kinetin (KN). After four weeks, number of roots induced per explant in auxin-supplemented media and number of shoot buds/micro shoots induced per explant in cytokinin-supplemented media were recorded for leaf and internode explants whereas shoot bud development and/or root tip growth were examined for root explants in all hormone-treated media. For each hormone concentration, 30 explants of each type per transgenic clone and non-transformed plant were used and the experiment was repeated thrice.

Statistical analysis
All experiments were randomized and were repeated at least thrice. Data were examined by one-way analysis of variance (ANOVA) to detect significant differences (p ≤ 0.05) in the mean (Sokal and Rohlf 1987). A post hoc mean separation was performed by Tukey B test at the same 5 % probability level using SPSS software (version 22.0). Variability in the data was expressed as the mean ± standard deviation (SD).

A. tumefaciens-mediated transformation and establishment of rolA-transgenic and rolB-transgenic plants on selection media
Direct shoot organogenesis was observed from wound sites of explants infected with A. tumefaciens harbouring rolA and nptII genes and A. tumefaciens harbouring nptII gene (nptII control) within four weeks, whereas control explants showed no organogenesis and necrosed within 6-8 weeks on MS medium containing cefotaxime (500 mg l − 1 ) and kanamycin (100 mg l − 1 ) ( Fig. 1a-f). The frequency of explants infected with A. tumefaciens harbouring rolA and nptII genes showing shoot bud induction was recorded 7 % (leaf) and 4 % (internode). The frequency of leaf explants infected with A. tumefaciens harbouring nptII gene showing shoot bud induction was 6 % whereas infected internode explants showed no shoot organogenesis and necrosed. The rolAtransgenic and nptII-transgenic plants were established and maintained on MS medium containing 100 mg l − 1 kanamycin for over three years with regular sub-culture at 4-weeks interval.
Direct shoot organogenesis was observed from wound sites of explants infected with A. tumefaciens harbouring rolB and hptII genes within four weeks, whereas explants infected with A. tumefaciens harbouring hptII gene (hptII control) and uninfected control explants showed no shoot organogenesis and necrosed within 6-8 weeks on MS medium containing cefotaxime (500 mg l -1 ) and hygromycin (15 mg l -1 ) (Fig. 1 g-l). The frequency of infected leaf explants showing shoot bud development was 5 % whereas internode explants did not respond and necrosed. The rolBtransgenic plants were finally established and maintained on MS medium containing 15 mg l -1 hygromycin for over three years with regular sub-culture at 4-weeks interval. However, hptII-transgenic plants could not be established in the present study.

Establishment of Ri-transformed root culture and development of Ri-transformed plants following infection with A. rhizogenes strain A4
Roots were induced from wound sites of infected leaf and internode explants (30 %) within four weeks on MS medium containing cefotaxime (500 mg l − 1 ). The roots induced were thin, light-green and fast-growing with lateral branches. Each putatively transformed primary root induced at each wound site was maintained as root line. Shoot buds (5 per explant) were regenerated spontaneously in Ri-transformed root lines (60 %) within 10 days when excised and cultured on the same medium. Micro shoots regenerated from shoot buds induced from different Ri-transformed root lines were maintained as separate clones.
Roots induced from uninfected control explants necrosed within four weeks on MS medium containing cefotaxime (500 mg l − 1 ). Shoots were induced from excised roots of non-transformed plants on MS medium containing BA (0.01 mg l − 1 ) within four weeks and were used as control.

Confirmation of transformation
PCR and RT-PCR analysis showed integration and expression of rolA and nptII genes in rolA-transgenic plant clones, nptII gene in nptII-transgenic plant clones, rolB and hptII genes in rolB-transgenic plant clones and rolA, rolB, rolC, rolD genes in Ri-transformed plant clones (Fig. S1). The amplified products were of expected size and identical to respective positive controls. Absence of virD1 gene amplification in transgenic plants confirmed no bacterial contamination. No amplification was observed in the genomic DNA of non-transformed plants.

Comparative morphology of transgenic plants harbouring rolA, rolB and Ri T-DNA genes
In the present study, rolA-transgenic, rolB-transgenic and Ritransformed plants of B. monnieri displayed notable changes in vegetative morphology compared to respective controls.
The rolA-transgenic plants were shorter with reduced internode length, higher number of nodes/plant and leaves/ plant than nptII-transgenic and non-transformed plants (Fig. 2). The number of roots/plant and root length were also increased in rolA-transgenic plants. However, rolAtransgenic plants were similar to nptII-transgenic and nontransformed plants in leaf length and shape (oblanceolate) with no axillary shoot formation (Fig. S2). The morphological characteristics of nptII-transgenic plants were similar to that of non-transformed plants with respect to shoot and internode length, number of nodes/plant, leaves/plant, roots/ plant and leaf morphology. However, roots induced from nptII-transgenic plants were shorter than the roots induced from non-transformed plants.
The rolB-transformants were dwarf due to decreased shoot and internode length, with greater number of nodes/ plant and leaves/plant than non-transformed plants (Fig. 2). Leaves of rolB-transgenic plants were smaller due to alteration in leaf shape (roundish) (Fig. S2b). These transgenic plants produced higher number of roots/plant and longer roots. Axillary shoots were also noted in rolB-transgenic plant clones unlike in non-transformed plants.
Insertion of Ri T-DNA had a pleiotropic effect producing highly aberrant Ri-transformed plants with reduced internode and shoot length, increased number of nodes/plant and leaves/plant than non-transformed plants (Fig. 2). Leaves were also smaller and round in shape (Fig. S2b). Ri-transformed plants had increased rooting and the roots were long. Reduced apical dominance in these plants resulted in the formation of axillary shoots.
Therefore, each of rolA and rolB genes seemed to cause phenotypic alteration in B. monnieri, although the degree varied. Additionally, both rolB-transgenic and Ri-transformed plants showed characteristic morphological changes associated with A. rhizogenes-mediated transformation. The morphology of rolB-transgenic plants was found to be more or less similar to Ri-transformed plants, suggesting that rolB gene has a pronounced effect in altering plant phenotype in this species.

Organogenesis from leaf, internode and root explants derived from rolA-transgenic plants
In the present study, excised leaf and internode explants of rolA-transgenic and nptII-transgenic plants showed shoot and root organogenesis within two weeks on MS basal medium as observed in explants of non-transformed plants (Fig. 3). However, after four weeks, the rates of shoot and root organogenesis were enhanced significantly in explants of rolA-transgenic plants than explants of nptII-transgenic and non-transformed plants (Fig. 4). The number of shoot buds/micro shoots induced per explant was 1.5-fold higher in both leaf and internode explants of rolA-transgenic plants than nptII-transgenic explants whereas it was 1.6-fold (leaf) and 1.4-fold (internode) higher than non-transformed explants. The number of roots induced per explant was 2.0fold and 1.7-fold greater in leaf explants of rolA-transgenic plants than explants of nptII-transgenic and non-transformed plants respectively. But, internode explants of rolA-transgenic plants were found to be less responsive. No significant differences in the rate of shoot and root organogenesis was observed between leaf and internode explants of nptII-transgenic and non-transformed plants. Excised root explants of rolA-transgenic, nptII-transgenic and non-transformed plants showed no morphogenic response and eventually died within six-eight weeks on MS basal medium (Fig. 3).

Organogenesis from leaf, internode and root explants derived from rolB-transgenic plants
Shoot and root organogenesis were observed from both leaf and internode explants of rolB-transgenic plants within two weeks on MS basal medium similar to explants of non-transformed plants (Fig. 3). However, after four weeks, shoot organogenic potential was 2.4-fold and 1.6-fold higher in 1 3 leaf and internode explants of rolB-transgenic plants than explants of non-transformed plants respectively (Fig. 4a). The rolB-expressing explants exhibited 3.6-fold (leaf) and 1.4-fold (internode) increase in root organogenesis compared to explants of non-transformed plants (Fig. 4b). Excised root explants of rolB-transgenic plants showed no morphogenesis and necrosed within six-eight weeks on MS basal medium (Fig. 3).

Organogenesis from leaf, internode and root explants derived from Ri-transformed plants
Leaf and internode explants of Ri-transformed plants developed adventitious shoots and roots within two weeks on MS basal medium similar to explants of non-transformed plants (Fig. 3). However, after four weeks, shoot organogenesis was significantly enhanced in leaf (5.2-fold) and internode (3.3-fold) explants of Ri-transformed plants compared to explants of non-transformed (Fig. 4a). Root regeneration potential was also enhanced in Ri-transformed leaf (4.1-fold) and internode (1.9-fold) explants than non-transformed ones (Fig. 4b). Root explants of Ri-transformed plants showed shoot regeneration and extensive root tip growth (4-6 cm) along with primary laterals formation within four weeks on MS basal medium (Fig. 3).

Discussion
Ri-transformed roots and Ri-transformed plants were established in B. monnieri following A. rhizogenes infection to demonstrate the effect of T-DNA genes on growth, biomass accumulation and bacosides production in transformed tissues (Majumdar et al. 2011;Largia et al. 2016). Attempts were also made on A. tumefaciens-mediated transformation where hygromycin/ kanamycin resistant transgenic B. monnieri plants were produced (Majumdar et al. 2012;Yadav et al. 2014). But, to our knowledge, transgenic B. monnieri plants harbouring individual rol genes have not been developed. Therefore, the present study reports for the first time on successful establishment of rolA-transgenic and rolBtransgenic plants in B. monnieri through A. tumefaciens mediated transformation. Ri-transformed plants harbouring Ri T-DNA were also regenerated from A. rhizogenes induced Ri-transformed roots to investigate the effects associated with integration and expression of rol genes, individually or synergistically, on plant morphology and morphogenesis in vitro.
In the present study, rolA and rolB genes, individually, causes shortening of internodes with increased number of nodes, leaves and roots per plant in B. monnieri. Additionally, rolB-transgenic plants exhibited certain transgenic characters due to which their morphology closely resembled Ri-transformed plants. This result indicates that rolB gene is more potent in causing developmental abnormalities in B. monnieri as reported earlier in other species (Bettini et al. 2016;Kodahl et al. 2016). The abnormal phenotypic traits caused by individual rol genes became exaggerated in Ri-transformed plants, probably due to the combined expression of rol genes since each of these genes is associated with specific phenotypic alterations (Sarkar et al. 2018).
The inherent morphogenic ability of excised leaf and internode explants of B. monnieri on MS basal medium was demonstrated earlier (Sarkar and Jha 2017) and was again confirmed in the present study. Enhanced shoot and root formation in explants of rolA-transgenic, rolB-transgenic and Ri-transformed plants therefore indicate a positive correlation between rol genes and organogenic competence of this plant species. According to Skoog and Miller (1957), plant organogenesis can be controlled by auxin/ cytokinin ratio. Decades-long study of A. rhizogenes-plant pathogenic interaction has revealed that rol genes of Ri T-DNA perturb endogenous hormone metabolism/sensitivity, primarily auxin and cytokinin, in transformed cells (Delbarre et al. 1994;Faiss et al. 1996) which could explain enhanced morphogenesis in transgenic explants. However, maximum morphogenic responses in Ri-transformed explants were probably due to the synergistic action of rol genes on endogenous auxin/cytokinin ratio. In addition to this, growth of root tips and shoot regeneration in Ri-transformed root explants, but not in rolA transgenic, rolB-transgenic and non-transformed roots denoted that rolA or rolB gene alone is not sufficient for morphogenesis in excised B. monnieri roots.
The fact that rol genes affect hormone-controlled morphogenesis in transformed cells became more prominent when exogenous auxin and cytokinin were added to the culture medium in the present study. Enhanced rhizogenesis in explants derived from rolA-transgenic, rolB-transgenic and Ri-transformed plants of B. monnieri in presence of auxin is supposed to be the consequence of increased auxin sensitivity in rol-transgenic cells. This also explains the formation of lateral branching and callus induction in rolA-transgenic and rolB-transgenic root explants and root tip growth in Ritransformed root explants in IAA-supplemented medium. According to van Altvorst et al. (1992), root formation in leaf explants of RolA, RolB and RolABC tomato plants in NAA-supplemented medium but not in control could be due to enhanced auxin sensitivity of rol-expressing tissues. Furthermore, inhibition of root formation by oligogalacturonides in rolB-transgenic tobacco leaf explants and protoplasts by inhibiting auxin-dependent rolB gene expression makes the idea of involvement of rolB gene in auxin signaling pathway more apparent (Bellincampi et al. 1996).
Enhanced shoot organogenesis in explants derived from rolA-transgenic and rolB-transgenic plants of B. monnieri in presence of cytokinin also describes the possibility of increased cytokinin sensitivity rendered by rolA and rolB genes in the present study. Altamura et al. (1998) also predicted the presence of a positive interaction between rolB gene and exogenous cytokinin due to the promotion of shoot formation in rolB-transgenic tobacco TCLs and leaf explants in presence of very low cytokinin concentrations although the mechanism is unclear. Later on, rolB is hypothesized to cause meristem formation triggered by increased auxin sensitivity in transformed cells which can develop into different types of organs depending on local hormone level and cell's organogenic competency (Altamura 2004). Recently, Bulgakov et al. (2018) showed promising results in the modification of both auxin/cytokinin signaling pathways by rolB-transformed cells of Arabidopsis thaliana Columbia (Col-0). The rolC gene is also found to affect shoot and root organogenesis in presence of cytokinin and auxin respectively in D. caryophyllus leaf explants (Casanova et al. 2003). Therefore interaction of rol genes, in combination, with hormonal metabolism/sensitivity offers a plausible explanation for maximum morphogenic response in explants of Ri-transformed plants of B. monnieri in hormone-supplemented media. However, further investigations are needed to clarify expression level of rol genes, integration sites, their copy number and interaction of these genes with other genes involved in morphogenic pathway to establish the exact correlation between rol genes and morphogenesis.

Conclusions
In conclusion, the present study demonstrated successful establishment of rolA-transgenic and rolB-transgenic plants of B. monnieri for the first time through direct shoot organogenesis following A. tumefaciens mediated transformation. Ri-transformed plants harbouring Ri T-DNA were spontaneously regenerated from Ri-transformed roots induced by A. rhizogenes infection. Respective transgenes were integrated and expressed in all transgenic plants, confirming their transgenic nature. The rol genes, either singly or in combination, played significant role in substantial increase in shoot and root organogenic potential on hormone unsupplemented medium by interacting positively with inherent morphogenic potential of this species. Each of the rol genes is found to be associated with the alteration of plant morphology in B. monnieri, although their degree varied. Furthermore, enhanced morphogenic response in transgenic explants containing rolA or rolB or Ri T-DNA genes even in presence of low concentrations of exogenous auxin and cytokinin might therefore be a manifestation of increased auxin and cytokinin sensitivity respectively although the exact mode of action of these genes needs further investigations.