Optimization of factors affecting Agrobacterium -mediated hairy root induction in Vitex negundo L. (Lamiaceae)

: Vitex negundo L. is an aromatic, woody, blooming shrub in the Lamiaceae family which can grow into a small tree. Traditionally, V. negundo root has been used to cure diabetes, colic, boils, leprosy, and rheumatism. Keeping the importance of its roots in mind, an attempt has been taken for development of a protocol for efficient hairy root proliferation system. The Agrobacterium rhizogenes strains (MTCC 532 and MTCC 2364) were used for hairy root induction. For A. rhizogenes infection, both in vitro and in vivo leaves as well as internodes were used as explant. In vitro leaves and internodal explants were obtained by the inoculation of matured nodal segments on the optimum medium [MS + 2.0 mg/L N 6 -Benzylaminopurine (BAP)] with c.a. 91.6% shoot regeneration and an average of 8.1 shoots per explants. In vitro leaf showed best hairy root induction followed by in vitro internode on ½ MS medium augmented with acetosyringone. Highest transformation efficiency was achieved using MTCC 2364 strain, while no transformation was observed in MTCC 532 strain. Different factors affecting transformation including co-cultivation period, infection time and optical density (O.D.) value were standardized. The highest efficacy, 88.8% hairy root induction was observed in in vitro leaves infected by MTCC 2364 for 60 minutes infection time with an O.D. value of 0.29 maintained over a 44-48 hours of co-cultivation period. The prescribed protocol may be used as a reference for development of industrial scale hairy root production for bioactive compound located in root of V. negundo.


INTRODUCTION
Vitex negundo L. (Lamiaceae) is an aromatic, woody and blooming shrub found mainly in India, Ceylon and China.The plant is also found at an altitude of 1500 m in the outer Himalayas (Usha et al., 2007).The term "Vitex" is originated from the Latin word 'vieo', meaning "to weave together", because of the flexible quality of the twigs and stems of the plant (Ahuja et al., 2015).V. negundo is usually an agroforestry plant and also used as fence.Besides, the plant is a traditional medicinal plant that has also been adopted in modern medicine.From root to fruit, the plant contains a variety of secondary metabolites that enhance the plant's ability to provide matchless medical benefits (Singh et al., 2016).Leaf is aromatic, used as tonic, and a vermifuge.The secondary metabolites present in the root juice (i.e.Dibutyl malate, Ajmalicine, Bruceantin, Beta carotene, Stearoyldelicone etc.) are expectorant, used as tonic for diuretic property and also used to treat colic, worms, rheumatism, dyspepsia, leprosy and boils (Gade et al., 2023;Ghani et al., 1998).Fruit is helpful for cerebrospinal, anxiety and emmenagogue therapies; dried fruit is a vermifuge, and blossoms are employed in cold and astringent treatments (Nadkarni et al., 2002).Scrofulous ulcers and sinuses are treated with floral oil of this plant.V. negundo has also been found as an antimicrobial (Dewade et al., 2010) and anti-inflammatory drugs (Chawla et al., 1992).Powdered seeds are used to cure spermatorrhoea and can be utilized as an aphrodisiac when mixed with milk and dried Zingiber officinale (Yunos et al., 2005).
Due to its immense medicinal property the plant plays an important role in pharmaceutical industry.Further secondary metabolite profiling of the V. negundo roots depicted about the presence of vitexin, isovitexin, negundin-A, negundin-B, acetyl oleanolic acid, sitosterol, alkaloids, phenols, flavonoids, glycosidic irridoids, tannins and terpene (Ladda & Magdum, 2012).Biotechnological intervention is useful for development of various efficient strategies to explore the therapeutic potential of V. negundo.Production of aerial parts like leaf, fruit, seed and flower has been already achieved by a number of workers (Vishwanathan & Basavaraju, 2010;Rana & Rana, 2014;Kamal et al., 2022) through in vitro regeneration of shoot using different explants like node and internode.But for large scale in vitro root production in V. negundo has not yet been reported in spite of being the source of most of the active compounds of pharmaceutical importance.A. rhizogenes interacts with plant cells through a defined segment of its large root inducing plasmid DNA (pRi T-DNA), which is conjugationally transferred into the plant genome and expressed.This results in the development of a tumors "hairy root" disease, distinguished by localized adventitious root proliferation at the infection site, and hiding two auxin biosynthetic genes (iaaM, iaaH).Under in-vitro culture, these genetically modified roots exhibit a rapid rate of growth along with abundant lateral branching, negative geotropism, cytogenetic stability, and sustained growth and biosynthetic potential in the medium without the need for growth regulator input (Sahu et al., 2015).In this context, in vitro root cultures in particular hairy root production could be the most appropriate alternative method which will be useful for secondary metabolites production in order to fulfill the requirement of drug designing and manufacturing.The advantages of production of secondary metabolites through hairy root culture also includefast growth, low doubling time, ease of maintenance of hairy roots, and their ability to synthesize a wide range of chemical compounds which could be a continuous source for the production of valuable secondary metabolites.To successfully induce and establish hairy root cultures of V. negundo using A. rhizogenes, various factors impacting genetic transformation must be meticulously evaluated and standardized.These include explant source and type, culture matrix, bacterial strains, bacterial cell density (by O.D. "Optical Density"), acetosyringone pre-culture, method of infection, infection time and duration of co-cultivation.Therefore, for the first time, efforts were made to optimize different factors to maximize the efficiency of the transformation and to develop a reproducible protocol for hairy root production in V. negundo which could serve as a feasible and sustainable source of pharmaceutically important phytochemicals.

In vitro Shoot Proliferation for Agrobacterium rhizogenes Infection
The Vitex negundo species was collected from Athagarh, Cuttack, Odisha, India and established in the experimental garden of Department of Botany, Ravenshaw University, Cuttack, Odisha (Figure 1 a).The V. negundo nodal explants were surface sterilized by washing with running tap water for 30 minutes, then treating with 2% Teepol (Reckitt Benckiser Ltd., India) for 10 minutes followed by 2% Bavistin (Antracol fungicide, Bayer, Crystal Crop Protection Ltd., India) treatment for 10 minutes.Finally, the explants were treated with 0.1% HgCl2 (Merck, India) for 7 minutes as the last step of surface sterilization.For multiple shoot proliferation, the nodal explants were inoculated on different strengths of Murashige and Skoog's (1962) (MS) medium i.e., MS, ½ MS, ¼ MS, ⅛ MS either alone or, MS medium augmented with various concentrations (0.5 to 3.0 mg/L) of Benzylaminopurine (BAP; Hi Media, India).The welldeveloped in vitro shoots (Figure 1 b) were served as a source of in vitro explants (Leaf and Internode) for A. rhizogenes infection.

Maintenance of Bacterial Culture
The A. rhizogenes cultures MTCC 532 and MTCC 2364 (CSIR-IMTECH, MTCC, Chandigarh) were revived in nutrient broth (1.3 g/100 mL; Hi media, India) with a pH of 7.4±0.2.For bacterial growth, the temperature was set at 26±1°C for 24 to 48 hours inside incubator (Shaker and Incubator, N. Biotek, NB 205 QF).The O.D (Optical Density) of both bacterial cultures, MTCC 2364 and MTCC 532, maintained in between 0.02 and 0.8.after 24 to 48 hours of incubation were used for hairy root induction.After revival of both the strain in nutrient broth, the cultures were stored and maintained on Nutrient agar medium (1.3 g/100 mL nutrient broth and 1.8 g/100 mL bacteriological grade agar (Himedia, India) inside a refrigerator at 4-5°C for future use (Figure 1 e and f).

Agrobacterium Infection and Hairy Root Induction 2.3.1. Transformation and hairy root induction in internodes
For transformation and hairy root induction experiment, both in vitro and in vivo internodes were used.In vitro shoots from a 45 to 60-days-old culture were cut into small pieces (1.0 -2.0 cm) and the excised in vitro internodes were punctured at one end.The punctured side was dipped in the bacterial suspension for different infection time 15-75 minutes.After the infection, the internodes were soaked with sterile tissue paper and inoculated on ½ MS and half MS + acetosyringone (19.6 mg/mL; Hi-Media; Figure 1 c) media by the other side of the piercing.These ½ MS media were gelled with 0.6% agar and pH was adjusted to 5.8±0.01.The co-cultivation time was optimized as 44-48 hours inside an incubator at 26°C.After 44-48 hours of co-cultivation, the internodes were transferred into a new flask with ½ MS + acetosyringone medium and kept in a culture room at 24°C under dark condition for hairy root induction.Agrobacterium infection in the in vivo internode was carried out, after surface sterilization, in the same way as in vitro internodes.The procedure of surface sterilization of the in vivo internode explant was carried out following the same procedure mentioned earlier for mature nodal explants.

Transformation and hairy root induction in leaves
Forty-five to sixty days old axenic shoot cultures were taken as the source for in vitro leaves.The leaves were cut into small segments (0.5 cm width ×1 cm length) and a sterile needle was used to pierce the mid rib and entire surface of the leaves.The pierced leaves were dipped in the bacterial suspension for 15-75 minutes.The leaves were dried with sterile tissue paper before being inoculated on ½ MS medium (Hi-Media, India; pH 5.8±0.01) and ½ MS + acetosyringone (Stock :196 mg/ 10 mL i.e., 0.1 M, Hi-Media, India; pH 5.8±0.01; Figure 1 d) media gelled with 0.6% agar.The co-cultivation time was optimized between 44-48 hours at 26°C inside an incubator.After 44-48 hours of co-cultivation, the leaves were transferred to fresh flasks containing ½ MS + acetosyringone (0.1 M) medium and kept in dark inside a culture room at 24°C.The in vivo leaves, after following the surface sterilization procedure were subjected to A. rhizogenes infection in the same way as in vitro leaves.

Statistical Analysis
All values are presented as the mean with three biological replicates.Mean values within column with different superscript alphabets are significantly different.Data were analyzed by analysis of variance (ANOVA) using Duncan's multiple range test (p < 0.05).

FINDINGS 3.1. In vitro Shoot Proliferation from in vivo Nodal Explants
Out of different strengths of MS basal media tried for in vitro shoot multiplication from in vivo nodal explants, the full-strength MS was found better in terms of shoot proliferation percentage, shoot number and shoot length whereas all these parameters were enhanced after the addition of BAP in different concentrations into the full-strength MS.The shoot regeneration from the nodal segments was observed after 7 days of inoculation of explants.Highest percentage of shoot regeneration (91.6) with highest number (8.1) of shoots with 8.33 cm average shoot length were recorded on MS medium augmented with BAP (2.0 mg/L) after 40-50 days of inoculation (Table 1).

Induction of Hairy Roots
For Agrobacterium transformation, we tested four different explant types, including in vivo (leaf and internode) and in vitro (leaf and internode).Further, two different media types, including ½ MS without acetosyringone and ½ MS with acetosyringone, were employed for the inoculation after Agrobacterium infection.There was no rooting tendency in the two sets of control group i.e., in-vivo and in-vitro (internodes and leaves) explants of ½ MS and ½ MS with BAP medium, which were not infected by A. rhizogenes.However, only the in vitro (leaf and internode) inoculated on the ½ MS with acetosyringone medium showed positive response for hairy root induction.

Hairy root induction from internode
The A. rhizogenes strains MTCC 2364 and MTCC 532 were subjected for hairy root induction from both in vivo (mature) internode and in vitro internode, among which in vitro internode showed hairy root induction with MTCC 2364 strain.Different infection time (15, 30, 45, 60 and 75 minutes), O.D. values (0.042, 0.375 and 0.572) with co-cultivation time (44-48 hours) were examined for hairy root induction and the results were depicted in Table 2.The initiation of hairy root was observed after 15 days of infection whereas on the 30 th day of observation i.e., with 30 minutes infection time 2.27 root numbers (1.66 cm), with 45 minutes infection time 1.5 root numbers (1.25 cm), 0.66 root numbers (0.83 cm) root length and with 60 minutes infection time 2.90 root numbers (2.92 cm) were observed.The rest of the explants of in vitro internode did not show any hairy root induction rather eventually all these explants became brown and died after few days of infection (Table 2).Overall, at O.D. value 0.042 with 60 minutes infection time and 44-48 hours co-cultivation period exhibited best results for hairy root induction (Table 2; Figure 1 g).Another strain MTCC 532 was not found effective with any type of explants tested for hairy root induction.It is noteworthy to explain that during co-cultivation period the explant inoculated on ½ MS medium fortified with acetosyringone only got success in hairy root transformation but explant inoculated on ½ MS without any addition of acetosyringone failed to respond.

Hairy root induction from leaves
The A. rhizogenes strains MTCC 2364 and MTCC 532 were also used for infection of both matured in vivo leaves and in vitro leaves, among which in vitro leaves showed hairy root induction with MTCC 2364 strain.The other strain MTCC 532 was not found effective for hairy root induction in both in vivo and in vitro leaf.Like the case of internode, during cocultivation period the leaf explant inoculated on ½ MS medium fortified with acetosyringone only resulted in production of hairy roots but ½ MS without supplemented with acetosyringone could not be able to develop a single hairy root from either type of leaves.Different infection time (15, 30, 45, 60 and 75 minutes) at different O.D. (0.086, 0.290 and 0.375) with cocultivation period of 44-48 hours were examined for hairy root induction (Table 3).The initiation of hairy root was observed from midrib and side cut of infected in vitro leaves after 15 days of infection (Figure 1 h).Overall, at O.D. value 0.290 with 45 minutes infection time and 44-48 hours co-cultivation period using MTCC 2364 strain exhibited best results (approx.89%, 3.1 hairy roots with a length of 2.94 cm) for hairy root induction (Table 2).The in vivo leaves failed to show any hairy root induction, rather they eventually became brown in colour and became necrotic (Table 3).2019) (Rauwolfia serpentina) also used these two types of strains for hairy root induction.However, in contrast to our study, in all these experiments, although MTCC 2364 was found to be effective for hairy root induction but the strain MTCC 532 was more effective in term of percentage of hairy root induction, biomass, number and length of hairy root.On the other hand, MTCC 532 also works effectively for hairy root cultures of Plumbago rosea, Rubia tinctorum, Arachis hypogaea, and Withania somnifera (Brijwal & Tamta, 2015).Different types of explants, in vivo leaf, internode, in vitro leaf and internode were used in this experiment, out of which, only in vitro leaf and in vitro internode explants responded to the induction of hairy roots.Of the two in vitro explants, the in vitro leaf performed better than the in vitro internode.Jesudass et al. (2020) (Cucumis anguria) also reported about the susceptibility of in vitro explant towards the infection of Agrobacterium strain which agrees with this result.Contrary to our result, Bhagat et al. (2019) got success in hairy root transformation in R. serpentina in vivo leaf explants.Swain et al. (2010)  The O.D. value plays a crucial role for transformation and hairy root induction.Multiple investigations have reported that the A. rhizogenes and their densities have an impact on the rate of hairy root induction during transformation (Kumar et al., 2006;Shahabzadeh et al., 2014).All strains considerably enhanced the transformation frequency after identifying the best inducing O.D. (Majumdar et al., 2011).In our investigation, the best O.D. value at O.D600 of the MTCC 2364 is 0.29 and 0.042 for the hairy root induction in in vitro leaf and internode respectively.Like our experiment different workers reported about different O.D. values of A. rhizogenes for optimum result for transformation.In R. serpentina, both MTCC 532 and MTCC 2364 showed 31% and 24% hairy root induction respectively at 0.6 optical densities of A. rhizogenes culture (Bhagat et al., 2019).According to the findings of Mahendran et al. (2022), different strains had different optimal induction concentrations.Further, lower bacterial concentrations resulted in lower transformation frequencies, whereas bacterial densities above the O.D. value 1.5 resulted in lower transformation rates due to severe necrosis.A higher cell density increased the T-DNA adherence to host cells, which resulted in browning of explants and a decrease in the frequency of hairy root induction.
In this experiment, we have tried some variable time (i.e., 40-44, 44-48, 48-52, 52-56 hours) of co-cultivation period whereas, the effective co-cultivation duration was standardised as 44-48 hours for hairy root induction.In corroboration to our result, Giri et al. (2001) as well as Brijwal and Tamta, (2015) also suggested about the 48 hours of co-cultivation duration as beneficial for hairy root induction for Artemisia annua and Berberis aristate respectively.Further, 48 hours of co-cultivation time was suggested by Srinivasan et al. (2023) for hairy root development in Aerva javanica by five different bacterial strains (ATCC 15834, R1000, LBA 9204, MTCC 2364, and MTCC 532).
Different infection times like 15, 30, 45, 60, and 75 minutes were studied and found that 60 minutes of infection time gave the best results for our explants; however, increasing the infection time up to 75 minutes in both in vitro leaf and in vitro internode decreased the rate of hairy root induction.The finding of Orlikowska et al. (1995) in the explants of safflower cv. and Kumar et al. (2023) in Plumbago zeylanica 'Centennial' reported a similar trend like our experiment.In Carthamus tinctorius, 30 minutes infection time was found suitable for higher transformation efficiency however, increasing the infection time up to 45 min caused a drop in effectiveness, which may be related to the explants' hypersensitive reaction to bacteria.Gelvin (2000) explained that acetosyringone enhances the frequency of transformation of infected explants by activating vir genes.According to Brijwal & Tamta (2015), acetosyringone in co-cultivation media not only enhanced explant transformation but also reduced the time needed for hairy root induction in contrast with acetosyringone free medium.They reported that in Berberis aristata, acetosyringone supplemented medium showed 72.22% transformation frequency whereas, acetosyringone free medium showed 61.11%.In this present hairy root experiment, ½ MS supplemented with acetosyringone (AS; 19.6 mg/mL) media effectively induces hairy roots whereas no hairy roots were observed from any type of the explants inoculated on ½ MS without addition of acetosyringone.Bhagat et al. (2019) and Moola et al. (2022) also suggested about the addition of acetosyringone in 125 µM for Rauwolfia serpentina and 100 µM for Celastrus paniculatus respectively for hairy root induction.However, Godwin et al. (1991) found that differences in transformation efficiency at various acetosyringone concentrations are significantly influenced by plant genotype and bacterial strains.
A protocol for hairy root transformation of V. negundo using A. rhizogenes strains MTCC 2364 was established.The in vitro was found as most suitable explant for hairy root induction.However, this is a preliminary work and further research is required for molecular conformation analysis of transformed root, hairy root biomass enhancement, parameters optimization for production and enhancement of bioactive secondary metabolites of particular interest as well as designing of bioreactor for up-scaling of hairy roots of V. negundo.As V. negundo is a significant medicinal plant, hairy root cultures might be considered as a useful system for large-scale production of important secondary metabolites in general and those are located in the root in particular.
pooled from 03 explants per flask, 02 flasks per replication and the experiment repeated 03 times (3*2*3=18).Each value is the mean from 3 replications.Mean values showed by different letters are significantly different at p ˂ 0.05 (DMRT).

Figure 1 .
Figure 1.a: In-vivo Vitex negundo plant in the garden, b: In-vitro shoot culture in MS with BAP (2 mg/L) media, c: In-vitro internode infected by MTCC 2364 before hairy root induction, d: In-vitro leaves infected by MTCC 2364 before hairy root induction, e: MTCC 2364 grow in petriplate, f: MTCC 532 grow in petriplate, g: Hairy root induction from in-vitro internodes infected by MTCC 2364, h: Hairy root induction from in-vitro leaves infected by MTCC 2364.4.DISCUSSION and CONCLUSION Out of the two strains namely, MTCC 2364 and MTCC 532 employed in this hairy root induction experiment, the MTCC 2364 strain of A. rhizogenes was found effective for hairy roots induction, while the MTCC 532 strain was found ineffective in our experiment.Similar to this study, other researchers Muthiah et al. (2016) (Bacopa monnieri), Bathoju et al. (2017) (Chlorophytum borivilianum), and Bhagat et al. (2019) (Rauwolfia serpentina) also used these two types of strains for hairy root induction.However, in contrast to our study, in all these experiments, although MTCC 2364 was found to be effective for hairy root induction but the

Table 1 .
In vitro shoot proliferation from in vivo nodal explant of Vitex negundo.
ab Data pooled from 02 explants per flask, 04 flasks per replication and the experiments were repeated 03 times (2×4×3 =24).Each value is the mean from 3 replications.Mean values showed by different letters are significantly different at p ˂ 0.05 (DMRT).

Table 2 .
Factors affect hairy root induction in in vitro internode infected by MTCC 2364.
Data pooled from 03 explants per flask, 02 flasks per replication and the experiment repeated 03 times (3*2*3=18).Each value is the mean from 3 replications.Mean values showed by different letters are significantly different at p ˂ 0.05 (DMRT).

Table 3 .
Factors influence hairy root induction in in vitro leaves infected by MTCC 2364.
also proposed that stem internodes are significantly more sensitive than leaf explants, irrespective of the Agrobacterium strains used.But many of the researchers like Sajjalaguddam et al. (2016) (Abrus precatorius) and Bathoju et al. (2017) (Chlorophytum borivilianum) in vitro explants like germinated seedling and shoot base respectively for hairy root induction experiment.