¹Department of Botany, Ravenshaw University, Cuttack-753003, Odisha, India.
²ICAR-National Rice Research Institute (ICAR-NRRI), Cuttack-753006, Odisha, India

*Corresponding author:Arpita Moharana, ICAR-National Rice Research Institute (ICAR-NRRI), Cuttack-753006, Odisha, India. E-mail Id: arpiarpita22k@gmail.com

Copyright: © Moharana A. 2025. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Article Information: Submission: 03/01/2025; Accepted: 05/02/2025; Published: 07/02/2025

In this study, a protocol for hairy root production for bioactive compound enhancement of Lawsonia inermis through intervention of Agrobacterium rhizogenes was attempted. Two types of explants, leaf (both in vivo and in vitro) and internode (both in vivo and in vitro) were taken for hairy root transformation by two Agrobacterium rhizogenes strains (MTCC 532 and MTCC 2364). Among leaf and internode, better transformation observed in leaf than internode where as in vitro leaf was found most suitable explant for optimum transformation than in vivo leaf explant. Better transformation was achieved by MTCC 2364 than MTCC 532 in all leaf and internodal explants. Highest 83.3% hairy root induction was noticed in in-vitro leaves infected by MTCC 2364 for 40 minutes infection time with an O.D. value 0.6 and co-cultivation time 24-26 hours. Root emergence in in vitro leaf started within 15-17 days and highest c.a.9.2 roots were observed. The in vivo leaf was found to develop hairy roots within 20-23 days from the day of infection with 73.3% transformation efficiency infected by MTCC 2364 and developed c.a 8.4 roots.

Keywords:Agrobacterium rhizogenes strains (MTCC 532 and MTCC2364); In vivo leaf and internode; In vitro leaf and internode; Secondary metabolites

Lawsonia inermis L. (syn. Lawsonia alba Lam. or Lawsoniaruba L.), commonly known as Henna or Mehendi [1,2] is a monotypic genus of family Lythraceae. India’s ancient history discusses its many applications and significant importance in Ayurvedic or natural herbal remedies. Apart from cosmetic use, Mehendi leaves are used as a prophylactic against diarrhea, skin diseases, renal lithiase, and gastric problems. The flowers have also medicinal properties, used as refrigerant, soporific, febrifuge, cardio tonic, as an emmenagogue and applied against bruises. The seed powder is effective for dysentery, liver disorders, and associated problems. Besides the aerial plant part, the root plays an immensely significant role in the treatment of various diseases. Root has astringent properties and is used for sore eyes, bruises, and boils of children’s heads. The root of this plant is helpful in the treatment of hysteria, gonorrhea, herpes, tumor, nervous disorders, and improves the liver and kidney [3-5]. Most importantly, in Odisha and in particular Koraput district and Kandhamal district, the root has also been used for the treatment of jaundice by common people, tribal people as well as local traditional healers [6,7]. They used root paste of L. inermis along with raw rice water for treatment of jaundice. Besides, pharmacologically the methanol extract of roots of L. inermis was most effective as an abortant [3] ethanolic root extract is effective as an antitumor, antiproliferative and has been used to improve the hepatic and renal function.[8,9] In addition, the presence of important secondary metabolites in the aerial part (in vivo and in vitro) as well as in root (in vivo and in vitro) has been proven by High Performance Thin Layer Chromatography (HPTLC; Moharana et al., 2018b) [10]. Due to the immense significant traditional and pharmacological properties of L. inermis root, its popularization as herbal medicine and extraction of secondary metabolites for pharmacological uses is necessary. For pharmaceutical commercialization, large scale production of L. inermis root is a prerequisite.

In this context, in vitro root cultures in particular hairy root production could be the most appropriate alternative method. Biotechnological intervention through Agrobacterium rhizogenes can be used as a strategy for a sustainable industrial scale root production. A. rhizogenes (now known as Rhizobium rhizogenes), a soil-dwelling Gram-negative phytopathogenic bacteria when infect the plant caused “hairy root” disease. As a consequence, uncontrolled root growth at the site of injury and infection of the plant is found. The additional advantages of the production of secondary metabolites through in vitro hairy root culture includes fast growth, low doubling time, ease of maintenance of hairy roots, and genetic stability [11]. The biosynthetic ability of “hairy root culture” to produce secondary metabolites is often equal to or greater than mother plant. [12-14] Interestingly, hairy root has also been proven to synthesize those compounds which are known to accumulate in aerial part only. [15,16,14]

In short, L. innermis is a traditional medicinal plant of which root is a most important part attributing many pharmacological activities. Therefore, an increase in root biomass without uprooting the plant is a prerequisite for industrial-scale production of secondary metabolites particularly located in the root. So, in this part of work attempt has been taken to increase root biomass through hairy root cultures of L. inermis using A. rhizogenes for production and enhancement of secondary metabolites located in the root. Apart from that, factors influencing genetic transformation including explant source and type, culture matrix, bacterial strains, bacterial cell density, method of infection, and co-cultivation period were evaluated and standardized to maximize the efficiency of the transformation and hairy root production in L. S.

For Agrobacterium infection experiment, two types of explants i.e., leaf and internode (mature and axenic) were taken. Young, fresh leaves and internodes were collected from a six years old L. inermis plant (Figure1A) present in campus of Ravenshaw University. The plant specimen which is used as the mature source (in vivo) of explants was identified and deposited in the herbarium of Botanical Survey, Odisha (voucher specimen number 2539/CBT). The mature leaves and internodal explants were kept under running tap water separately in a beaker with constant shaking of about 30 min for removal of external adherents like dust and dirt. Then, both types of explants were washed for 5 min with 1 % (v/v) aqueous solution of Teepol (Reckilt Benckiser Ltd., India), followed by five times rinsing with double distilled water. Then a 0.1 % (w/v) aqueous solution of mercuric chloride (HgCl2, Hi-Media, India) was used for surface sterilization (3 min for leaves and 4 min for internodes), followed by five-six rinsing of sterile double distilled water [5]. After rinse, both leaves and internodal explants were ready for Agrobacterium infection.

For axenic (in vitro) internode and leaf, the in vitro regenerated plantlets maintained in our laboratory (Figure 1B) were used as a source of explant. The in vitro shoot culture was established by inoculation of

in vivo nodal explant on Murashige and Skoog, medium (MS, 1962) supplemented with BA (1.0mg/ml) and subsequent elongation on MS medium devoid of any plant growth regulators (PGRs) [5].

Two types of strains of Agrobacterium rhizogenes, MTCC 532 and MTCC 2364, were procured from IMTECH (Institute of Microbial Technology), Chandigarh, India and revived in nutrient broth (Hi- Media). For bacterial growth, the temperature was set at 26±1°C for 44 to 48 hours inside the incubator. The broth cultures were stored at 4 °C for the Agrobacterium transformation experiment. For explant infection, a 100μl aliquot of bacterial suspension was added into 50 ml of liquid medium in a 150-ml Erlenmeyer flask and incubated in an incubator shaker at 26±1°C. Prior to bacterial inoculation, the OD of both the bacterial suspensions of both the strains was adjusted to 0.6 (at 660 nm).

For the transformation and hairy root induction experiment, thein vivo internodal segments were excised (1.5-2.0cm) from both sides to eliminate the damaged tissue that were affected during the process of surface sterilization. The axenic internodes were excised (1.5-2.0cm) from in vitro regenerated shoots. Then both types of internodes were punctured on one tip side end, followed by the dipping in the bacterial suspension for 10-60 minutes to allow the infection. Likewise, leaves were excised from in vitro shoot culture,

punctured around the midrib and dipped in the bacterial suspension for 10-60 minutes for Agrobacterium infection.

Both leaves (in vivo and in vitro) and internodes (in vivo and in vitro) were taken out of the bacterial suspension and rinsed with sterile distilled water, followed by drying with sterile tissue paper. Internodes were inoculated by the other side of the piercing and leaves were by their dorsal side up on solid half MS (agar 0.7 %, Hi- Media; pH 5.8±0.01). The co-cultivation time was optimized between 24-26 hours inside an incubator at 26±1°C. After co-cultivation, explants were transferred to fresh flask with solid half MS (agar 0.7 %, pH 5.8±0.01) and kept inside a dark culture room at 25±1°C for hairy root induction. One set of controls was maintained by the inoculation of both types of leaves and internode separately on solid half MS medium without exposure to bacterial suspension.

After hairy root induction from both in vivo leaf and in vitro leaf, individual transformed roots (1.5–2.0 cm) were excised and transferred to a conical flask (Borosil, India) containing 100ml of liquid ½ MS medium without antibiotics incubated inside a dark culture room at 25±1°C. At one week interval, the hairy roots were transferred into a freshly prepared liquid ½ MS medium without antibiotics in the same conditions for root biomass enhancement.

For hairy root development, each treatment of the experiment, consisted of 5 replicates (culture flasks) and two explants/flask was the experimental unit. Each experiment was repeated thrice at a 5 days interval. Mean value was taken from three biological replications. The percentage of explant showing hairy root induction, number of roots/explants, and root length were recorded after 45 days by visual observations. 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). Explants with hairy roots were photographed by a Canon DSLR P3000 camera and uploaded to the computer by inserting the memory card into computer.

For Agrobacterium transformation, both mature and axenic leaves, were taken into consideration. No hairy root development was observed in the control set of the experiment. Early rooting response and higher root regeneration frequency were observed in in vitro leaves than in vivo leaves [Table 1]. The hairy root development in both types of leaves was observed mostly from the cut ends of the petiole and midrib. Root induction occurred in 15-17 days in in vitro leaf whereas, 20–23 days were required for rhizogenesis of in vivo leaf

[Table 1] infected by MTCC 2364. The in vitro leaves resulted in the highest 8.5 (Figure 1C) hairy roots whereas, mature leaf developed the highest 7.2 (Figure 1D) hairy roots. Bacterial contamination was found more in in vivo explants, where as in vitro explants was devoid of such types of contamination.

Among the two MTCC 532 and MTCC 2364 A. rhizogenes strains used for hairy root formation, MTCC 2364 found much better in hairy root induction irrespective of explant types. The microbial strain MTCC 532, resulted in a lower percentage of root regeneration with few numbers of roots whereas, MTCC 2364 showed a higher transformation frequency in both in vivo (73.3) and in vitro (83.3) leaf. Apart from that, a comparatively higher number of days are required for rhizogenesis in the case of MTCC 532 than MTCC 2364 irrespective of explant types [Table 1].

Out of different infection time periods, 40 min. was found to be more effective in terms of the highest number of hairy root regeneration both in in vivo (8.4) and in vitro (9.2) leaves, whereas much lower infection time (10 mins.) did not show any response in any type of leaf explant rather the leaves eventually became brown and died after a few days of infection. Leaf explants infected for more time (60 min.) found excess bacterial growth around them with no hairy root [Table 1].

The O.D. values for both the strains were optimized (data not shown) to 0.6 at 660 nm and co-cultivation time as 24-26 hours (data not shown) for optimum transformation in both types of leaves.

Out of in vivo and in vitro internodes, higher transformation by Agrobacterium was observed in in vitro internodes than in vivo internodes. The in vitro internodal explant showed an early rooting response than the in vivo explant. The hairy root development in both types of internodes was observed mostly from the piercing cut end. Root induction occurred in 20-25 days on in vitro internodes whereas, 25–30 days were required for root emergence on internodes from mature explants (in vivo) (Table 2) infected by MTCC 2364. The in vitro and in vivo internodes infected by MTCC 532 showed root emergence in 25-30 and 30-35 respectively. The in vitro internodes resulted in the highest 3.1 (Figure 1E) hairy roots whereas, mature internodes developed the highest 4.8 (Figure.1F) hairy roots. Both types of internodal explants were found devoid of bacterial contamination. No hairy root development was observed in the control set of the experiment.

Among MTCC 532 and MTCC 2364 A. rhizogenes strains used for hairy root induction, MTCC 2364 found much better in hairy root development irrespective of explant types. The microbial strain MTCC 532, resulted in a lower percentage of root regeneration with a few numbers of roots whereas MTCC 2364 showed higher transformation frequency in both in vivo and in vitro internodes. Apart from that, a greater number of days are required for rhizogenesis in the case of MTCC 532 than MTCC 2364 irrespective of explant types [Table 2].

Out of different infection time periods, 40 min. was found to be more effective in terms of highest number of hairy root regenerations both in in vivo (3.1) and in vitro (4.9) internodes whereas much lower infection time (10 min.) did not show any response in any type of internodal explants rather, the internodes became brown and died after a few days of infection. The higher percentage of transformation frequency was also recorded with 40 min. of infection time in both in vivo (53.3 %) and in vitro (60 %) internodal explants. Internodal explants infected for more time (60 min.) found excess bacterial growth around them with no hairy root [Table 2].

The O.D. values for both the strains were optimized (data not shown) to 0.6 at 660 nm and co-cultivation time as 24-26 hours (data not shown) for optimum transformation in both types of internodes.

Optimum hairy root induction was resulted by the infection of MTCC 2364 in leaf explant in terms of percentage of explant response (73.3 % in in vivo and 83.3 %in vitro) and number (8.4 in in vivo and 9.2 in in vitro) of hairy root regeneration as a result of transformation. So, the further experiment was carried out by taking hairy roots developed from both types of leaves. Actively growing individual roots (hairy roots; 1.5-2.0 cm) from transformed leaves, were excised and transferred to liquid ½MS medium. After 2 weeks, the roots in liquid medium showed a slight elongation as well as regeneration of secondary roots from the primary root (Figure 1 G),(Figure 1H)

Out of the two strains, MTCC 532 and MTCC 2364 were selected for the hairy root induction experiment, the MTCC 2364 strain of A. rhizogenes was found more effective for hairy root induction in comparison to MTCC 532. These two strains were also previously used by Brijwal and Tamta, 2015[17] (Berberis aristate),Bathojuet al., 2017[18] (Chlorophytum borivilianum) for hairy root induction. However, out of these two strains, MTCC 532 was reported by them as more effective in terms of percentage of hairy root induction, biomass, number, and length of hairy root than MTCC 2364, which is contrary to the result of this present study. At the same time, Vishwakarma et al., 2017[14] and Mahakuret al., 2024 [19] reported MTCC 2364 is more effective in hairy root formation than MTCC 532 in Mucuna pruriensand Vitex negundorespectively, which is similar to the result of the present work. On the contrary, Deore and Kide, 2015 [20] (Chlorophytum species) found MTCC 2364 as totally ineffective for hairy root development. Except for these two bacterial strains, Bakkali et al. [15] (1997) successfully developed hairy root from the in vitro leaf of L. innermis by the transformation of Agrobacterium rhizogenes NCIB 8196.

The types of explants influenced the hairy root production so, with a view to determie the suitable explant for optimum Agrobacterium transformation, different types of explants i.e., leaf (in vivo and in vitro) and internode (in vivo and in vitro) were used, out of which, both in vivo and in vitro leaf explants were more responsive than both types of internodal explants. Whereas, in vitro leaf showed better transformation percentage as well as more number root development than the in vivo leaf. Contrary to this result, internodal explant of Clitoriaternatea reported as better for Agrobacterium transformation than the leaf [21] (Swain et al., 2012). Most of the researchers, including Thilipet al., 2015[22] (Withaniasomnifera) and Jesudasset al., 2020[23] (Cucumis anguria) suggested the use of in vitro or axenic explant for hairy root induction, which is similar to this part of the experiment. Preference of in vitro explants may be to avoid the crucial step of surface sterilization which is time consuming and has a chance of microbial contamination. But Swain et al., 2012 [21] (Clitoriaternatea), Srinivasan et al., 2023[23](Aerva javanica) also got success in regeneration of transformed roots from in vivo explants.

Agrobacterium cell density is a fundamental factor influencing genetic transformation system (Kumar et al., 2006; Binka et al., 2012; Shahabzadeh et al., 2014; Asande et al., 2020). [24-27] In this part of the experiment, the suitable O.D. value of the MTCC 2364 was found to 0.6 for the hairy root induction in all types of explants. Swain et al., 2012[21] (Clitoriaternatea) suggested a 0.6 O.D. value as suitable for optimum hairy root production for A. rhizogenesA4T which corroborates the result of this part of the research work. But it is a fact that for perfect transformation the optical densities of Agrobacterium suspension cultures ranged from 0.1 to 1.0 depends on the genotype, Agrobacterium strain, and plant species (Asandeet al., 2020).[27] Higher Agrobacterium density can cause uncontrolled growth of bacterial cells thus limiting the explant survivility and subsequent reduction in transformation efficiency. Like-wise, lower cell density cannot induce transformation.

The transfer of T-DNA to the plant genome from Agrobacterium during transformation process is time-dependent and therefore, transformation efficiency depends on both time of infection and cocultivation duration (Markandan et al., 2015; Asandeet al., 2020). [27,28]Apart from that, A. rhizogenes strains are different in their virulence, which leads to the different development rates of hairy root (Giri et al., 2001).[29]Variation in the infection durations had an influence on the transfer of T-DNA from Agrobacterium to plant cells of L. inermis from which, 40-min of infection time was recommended for successful transformation. Furthermore, the hairy root induction with 20- and 30-min infection periods resulted in significantly lower infection rates and number of roots. Due to inadequate bacterial infection duration, a shorter infection period like 10 min. was found ineffective. A higher rate of infectivity was not found when the infection duration crossed 40 min. Rather, 60 min infection time showed overgrowth of bacteria leading to explant contamination followed by necrosis. Therefore, 40 min was determined to be the optimal time for infection for L. inermis. Similar type of observation was reported by Srinivasan et al. (2023) where, he suggested 20 min. as the optimum infection period for hairy root formation, but lower than 20 min. and higher than 20 min. are not preferable. Effect of infection time on transformation frequency using A. rhizogenes also proven to be dependent on plant species. Five minutes of infection to wounded explants was effective in inducing hairy roots in Linum mucronatum (Samadi et al., 2012)[30]and in Agastache foeniculum (Nouroziet al., 2016),[31]whereas in Silybum marianum (Rahnama et al., 2008)[32]and in Fagopyrum tataricum (Thweet al., 2016),[33-37]10 min of infection time was found effective for optimum transformation. Adding to that, 20 min. in Artemisia annua (Giri et al., 2001)[29]and one hour in Berberis aristata(Brijwal and Tamta, 2015)[17] were required for maximum hairy root formation.

To give a step forward for production and enhancement of root specific important phytochemicals, induction and establishment of hairy root in L. inermis by by Agrobacterium mediated transformation was carried out in which, in vitro leaf was found as the most suitable explant for hairy root induction by A. rhizogenes MTCC 2364 with O.D 0.6 and infection time 40 min. with co-cultivation time 24-26 hours. Factors impacting genetic transformation including explant source and type, culture matrix, bacterial strains, bacterial cell density, method of infection, and co-cultivation period were evaluated and standardised to maximize the efficiency of the transformation and hairy root production in L. inermis.

However, this part of the research work remains as a preliminary part of the work. Further research is under process i.e., enhancement of transformation efficiency, root number, and length. Molecular validation of transformed roots by PCR amplification, hairy root biomass enhancement, parameter optimization for production and enhancement of important secondary metabolites and phytochemical validation of secondary metabolites in both transformed root and roots from the mother plant will be accomplish. This part of the work might be a path forward for optimization of industrial scale production of roots biomass or bioreactor design aiming in the production of secondary metabolites, particularly accumulated in root, for drug manipulation to combat the antibiotic-resistance human pathogens, as L. inermis holds the status of a multipurpose medicinal plant.

Declaration of Conflicting Interests and Ethics: The author declares no conflict of interest

Funding support: The author declares that she has not taken any funding support to carry out this part of research work.

Authorship contribution statement: The Author designed, carried out the whole experiment, analyzed the data and wrote the entire manuscript

The author is grateful to Department of Botany, Ravenshaw University, Cuttack-753003 for providing necessary laboratory facilities. AM is highly obliged to Dr. Durga Prasad Barik and Dr. Soumendra Kumar Naik for their support, guidance, and providing the bacterial strains in carry out the experiment.