The plant patchouli (Pogostemon cablin., family Lamiaceae); is an essential aromatic herb, native to tropical regions of Asia (Wang et al., 2019). Patchouli oil (PO) is commercially utilized in the perfumery, cosmetic, and aromatherapy industries worldwide. Besides being used in cosmetics, it is used as an antidepressant, antiphlogistic, antiseptic, aphrodisiac, astringent, diuretic, febrifuge, and fungicide (Miyazawa et al., 2000; Bunrathep et al., 2006). The major component of patchouli essential oil are patchoulene, caryophyllene, pogostol, guaiene, patchoulol, and α-patchoulene (Zhou et al., 2011; Liu et al., 2015). Patchoulol and α- patchoulene are responsible for maintaining the quality of PO (Ramya et al., 2013; Pandey et al., 2021). The potential application of PO in pharmacology and agriculture has led to an enormous global demand. The estimated global patchouli oil production is 800 tonnes/year, about 60% of which is produced by Indonesia (Tahir et al., 2019). Patchouli cultivation in India is approximately 600 ha, producing 20 tonnes of oil/annum (Choudhri et al., 2023).

Global climatic changes impose a wide range of stresses over crop cultivation and production (Chakraborty et al., 2014; Roychowdhury, 2014; Roychowdhury et al., 2020). The cultivation of shade-loving patchouli faces significant constraints due to its susceptibility to various biotic stresses (pests, fungi, viruses, and root-knot nematodes) and abiotic stresses (Singh et al., 2009; Bhau et al., 2016; Suhesti et al., 2022). In Southeast Asia, patchouli cultivation dominantly occurs in dry land therefore, P. cablin faces drought stress which limits its rapid growth and productivity (Suhesti et al., 2022). These stresses induce excess ethylene synthesis, resulting in senescence, abscission, and leaf yellowing ultimately reducing its essential oil yield (Glick, 2014). Until now, among all the major varieties of patchouli, none of them are showing improved drought tolerance capability. Consequently, preference has been given to developing varieties resistant/tolerant to drought stress with high biomass and better PO quality (Suhesti et al., 2022). Moreover, various conventional propagation methods cannot be practiced for genetic modification of P. cablin-resistant varieties because of the low transformation efficiency. Therefore, biotechnological applications could be successful in performing Agrobacterium tumefaciens- mediated genetic transformation with specific genes (Roychowdhury and Tah, 2013; Roychowdhury et al., 2023). The genetic manipulation provides a new dimension to develop superior patchouli drought resistant lines to meet the industrial demand of PO by cultivating in all seasons (Paul et al., 2012).

Recently, biotechnological techniques have been developed for microbial-plant interactions to utilize beneficial micro-biome characteristics for improving plant quantity and quality (Moon and Ali, 2022; Li et al., 2023a; Li et al., 2023b). One such technique is using ACC deaminase-PGPR for regenerative agriculture, which has shown to be highly advantageous (Glick, 2014; Han et al., 2021). During stress, plants produce excess ethylene called ‘stress ethylene’, which can impair primary metabolism (Singh et al., 2021). Drought stress compromises various physiological and metabolic pathways, resulting in stunted plant growth, interrupted photosynthesis, and abnormal metabolism leading to plant death (Hasanuzzaman et al., 2015; Anumalla et al., 2016; Zhang et al., 2018). As per the literature study, PGPRs that produce ACC deaminase have been found to reduce stress ethylene phytohormone levels under biotic and abiotic stresses (Penrose & Glick, 2003) by catalyzing ACC into α-ketobutyrate and ammonia (Li et al., 2019). It is reported by Glick (2014) that the transformation of the bacterial ACC deaminase gene supports reducing ethylene concentration and improving root permeability along with nitrate availability. Therefore, Agrobacterium-mediated genetic engineering is crucial for decreasing ethylene accumulation without affecting plant growth and development.

To genetically improve P. cablin, an efficient, fast, and highly reproducible regeneration system has been acquired in the present research to transform the ACC deaminase gene for large-scale cultivation in every season. Van (1973) developed the thin cell layer (TCL) technique, a low-cost, fast multiplication, and highly reproducible in-vitro propagation method for large-scale production of genetically stable plants. Either longitudinal (lTCL) or transverse (tTCL) section of ~0.1-5mm thin explants can be used for the propagation and preservation of various significant plants, including endangered ones (Rout et al., 2006; Singh et al., 2012; Zhao et al., 2007). Although regeneration and transformation protocols have been reported in P. cablin (Paul et al., 2010; Paul et al., 2012; Sugimura et al., 2005) from callus and direct regeneration from leaf disc, the transformation parameters were not well-concluded to provide better variety.

By keeping the detailed literature in mind, the present study demonstrates the establishment of an efficient direct regeneration system from leaf, petiole, and internodal transverse thin cell layer (tTCL) explants of patchouli to avoid somaclonal variation for large-scale multiplication in every season. The consequence of fast regeneration provides a smooth platform to address Agrobacterium tumefaciens-mediated genetic transformation with a construct harboring the ACC deaminase gene to analyse the tolerance level of acclimatized greenhouse-grown transgenic P. cablin plants against drought stress.

The commercial demand for patchouli has been increasing due to its tremendous aromatic and medicinal properties. However, the supply of patchouli oil, to meet this global demand, is mainly provided by Indonesia, which massively cultivates versatile patchouli varieties. P. cablin cultivars face drought stress while maintaining healthy patchouli plants, which limits its propagation rate. In this study, a transgenic patchouli drought-resistant variety has been developed that keeps the promise of maintaining propagation rate and yield under drought stress. For the development of transgenic plants an efficient and quick regeneration system was required hence a comprehensive analysis on the basis of culture media, explants, and the effect of different phytohormones was performed (Roychowdhury and Tah, 2011; Roychowdhury et al., 2012; Reddy et al., 2013). During this study plant growth hormone was optimized for better regeneration and explants were compared to achieve a higher multiplication rate under sterile conditions. In our findings, maximum direct shoot organogenesis was obtained from leaf explants on MS medium supplemented with 0.2 mg/L BAP and 0.1 mg/L NAA without callus ( Figures 1E, F ). As per the literature, P. cablin is commonly propagated through leaf discs like most herbaceous plants (Paul et al., 2010; Swamy and Sinniah, 2016). An analysis of previously documented studies, MS medium supplemented with BAP showed considerable efficacy in shoot regeneration but much less than meta-topolin (Lalthafamkimi et al., 2021; Kumaraswamy and Anuradha, 2010; Kukreja et al., 1990; Jin et al., 2014). Sun et al. (2009) have also reported that cytokinins are known for shoot organogenesis. However, Sales and Butardo (2014) revealed that PGRs BAP and NAA are significant causes of clonal variability among micro-propagated plants. The presence of NAA combination with BAP in MS medium has been reported to improve shoot induction frequency from petiole explant in Pelargonium graveolens (Singh et al., 2017) and Mentha piperita (Sarwar et al., 2009).

Van (1973) has introduced the thin cell layer (TCL) technique which is an economical, rapid, and thoroughly reproducible in-vitro propagation method for the up-scale production of genetically stable plants. The recent study is the first contribution to support in vitro multiplication of P. cablin using tTCL explants. The histological analysis of direct shoot regeneration using tTCL was validated to hold the strategy of rapid propagation (15-20 days) as compared to other explants (25-35 days) on a more suitable MS medium supplemented with 0.2 mg/L BAP and 0.1 mg/L NAA ( Figures 6B, C ) and half-strength MS medium was found to be efficient for rooting. Previously, as per Tripathi et al. (2018) report, it was observed that vascular zone cells have a high tendency to generate new shoot buds and the regeneration capacity of explants depends on nutrient transport across media to tTCL, which has more exposed cells on media as compared to conventional large-size explants. Similar studies have also been reported in Talinum triangulare, Bacteris gasipaes, and Dendrobium candidum (Steinmacher et al., 2007; Zhao et al., 2007; Swarna and Ravindhran, 2013). According to the literature, TCL explants have a more significant reproducing tendency than conventional explants (Da Silva and Dobránszki, 2015). Moreover, Raomai et al. (2015) observed that the developed rhizomes of endangered Paris polyphylla using tTCL basal stem explant showed significant enhancement of secondary metabolite production and mass propagation system.

Recent advancement in R&D technologies provides insight into microbial-plant interactions that help us to enhance crop yield and quality through vast biotechnological approaches such as genetic manipulation (Mamgain et al., 2013; Roychowdhury et al., 2013; Khan et al., 2014; Pathak et al., 2022). In a drought state, the plant produces stress ethylene that causes various physiological and metabolic damages in plants (Zhang et al., 2018). Literature confirmed that ACC deaminase has the capacity to reduce stress ethylene levels under abiotic stresses (Penrose and Glick, 2003) by degrading ACC into α-ketobutyrate and ammonia (Li et al., 2019). Generally, for the production of putative transgenic P. cablin plants having ACC deaminase gene, Agrobacterium-mediated genetic transformation protocol establishment is required for the highest transformation frequency. The present study revealed OD₆₀₀ 0.6, AS 200µM, 30mg/L kanamycin, and an infection time of 5 min. is optimum to achieve the maximum transformation rate ( Figures 2A–D ). Kanamycin, used as a selection marker, has been well studied to optimize its concentration for versatile crops. Earlier Paul et al. (2012) has been reported a lower 20mg/L and Sugimura et al. (2005) reported a higher 100mg/L concentration of kanamycin for indirect regenerated putative transformed shoot selection.

Hence, the plant has innate natural resistance against kanamycin; therefore, sensitivity towards kanamycin varies among plant tissues and species of plants (Colby and Meredith, 1990). In literature, other parameters such as bacterial optical density and infection time were studied for optimum heterologous transgene expression. Dutt and Grosser (2009) recommended maximum transformation frequency on lower optical cell density. Moreover, several studies have also suggested that higher OD₆₀₀ values reduce transformation frequency (Saini and Jaiwal, 2007). In addition, similar studies were shown better transformation frequency in less than 30 min. in other plants (Khan et al., 2015; Benazir et al., 2013), unlike (Gupta and Rahman, 2015; Singh et al., 2017). From these observations, we can conclude that variation in infection time to get maximum transformation frequency depends on tissues, species, and plants.

Stachel et al. (1985), reported that AS is a potent phenolic signaling molecule that is generally secreted by wounded plant tissues and helps in the transfer of T-DNA from Ti- plasmid by inducing a signaling cascade of all Vir genes after binding to the VirA protein of bacterial cell. Though every plant has less than the threshold value of endogenous phenolic molecules for efficient transformation, the external addition of AS in the co-cultivation medium fulfills the demand for signaling molecules to obtain optimum transformation frequency. Our study reported 200µM AS limit was best suited for transgenic development whereas in other reports 150µM of AS is optimum for patchouli leaf transformation (Paul et al., 2012), Adverse effect on transformation frequency of plants has also been reported in other species with an increment of AS concentration (Subramanyam et al., 2013; Singh et al., 2017). The study showed optimized transformation protocol was able to produce putative transgenic patchouli plants harboring the ACC deaminse gene with durability 52.0 ± 0.8% ( Figure 3I ) after hardening and acclimatization. The putatively transformed CIM-Samarth lines were screened at first by PCR for the existence of transgene and selection marker ( Figures 7A, B ). Similar studies have also been reported in Ocimum gratissimum, Tagetes erecta, and Pelargonium graveolens (Gupta and Rahman, 2015; Khan et al., 2015; Singh et al., 2017). In this research, ( Figure 7C ) signifies that PT4 showed a high relative expression of ACC deaminase transgene among other best-propagating transgenic lines. The variation in transgene expression among transgenic lines could be due to the constitutive CaMV 35S promoter activity influenced by several regulatory systems (Benfey and Chua, 1990).

Sustainable results were significant in building a homology model of our target gene and checking in silico docking affinity of ligand ACC with receptor ACC deaminase. We found that the Ramachandran plot of the model ( Figure 8 ) showed 94.6% (539) of amino acid residues fell in the most favorable region. Similar data that validate the build model have also been mentioned by Pramanik et al. (2017). The best conformation of our docking result revealed four H-bonds between ACC and surrounding amino acids Cys-196, Val-198, Thr-199, and Gly-200 of the receptor ( Figures 4C-F ) These studies were equivalent to other literature (Vijesh et al., 2013; Jasim et al., 2015). Additionally, as per Singh and Kashyap et al. (2012), few other amino acids that are conserved in other ACC deaminase sequences interact with ACC.

In this investigation, we have created a severe drought stress experiment where our best transgenic patchouli line PT4 confirmed by different molecular analyses was able to survive without compromising other physiological parameters such as RWC, chlorophyll content, and ROS production ( Figures 9 , 5 ). This result correlates with previous reports indicating that under-stress plants develop mechanisms to maintain water retention and transpiration rate ratio (Merah, 2001; Soltys-Kalina et al., 2016). Our study mentioned ~75% of RWC in the transgenic line as opposed to to the control plant’s ~58% in severe stress conditions. However, an earlier study of Pelargonium graveolens RWC of transgenic was 88% in severe stress (Singh et al., 2021). Additionally it has been analyzed that the result depends on various properties of plants such as plant age, species, growth condition, and pot size. Elevated RWC in transgenic lines after severe watered stress over control plants becomes significant in drought conditions. Chlorophyll, a photosynthetic pigment, has a significant role in photosynthesis therefore variation in chlorophyll content may damage the plant photosystem. In our result, chlorophyll content decreased to ~2.3 fold in WT compared to transgenic plants that showed a ~1.6 fold decrease. This denotes that suitable transgenic lines can deal with drought stress without hampering the photosynthetic system. Similar strategies by Afridi et al. (2019); Han et al. (2017), and Bahieldin et al. (2005) have also suggested the same relationship between chlorophyll content and the rate of photosynthesis. Literature surveys suggest, during various biochemical reactions, the ROS produced in different organelles has an important role in growth, development, and function as a defense system in abiotic stress. Moreover, if excess ROS cannot detoxify by antioxidants, it leads to oxidative stress (Apel and Hirt, 2004; Tripathy and Oelmüller, 2012), which causes versatile cellular damage and may even cause the death of the plant. In our finding, DAB and NBT histochemical analysis of transgenic P. cablin leaves were showing less magnified brown and blue spots respectively compared to WT under severe stress conditions ( Figure 5 ) That indicated less ROS (H₂O₂, O₂ ^-) production in transgenic plants expressing ACC deaminase gene. Similar research has also been reported on Petunia hybrida and Pelargonium graveolens (Singh et al., 2021; Naing et al., 2022).

Our research is novel in establishing an efficient direct regeneration and genetic transformation protocol using tTCL in only 15-20 days, which effectively enhances Agrobacterium-mediated genetic transformation frequency in Pogostemon cablin. tTCL sections regenerated faster than leaf and petiole explants, which improves micropropagation by reducing transgenic development time. The comprehensive study is the first report of ACC deaminase integration into the genome for developing transgenic patchouli to deal with drought stress, one of the significant problems in the propagation of aromatic plant ‘patchouli’. There is no report on the physiological parameters RWC, H₂O₂, and chlorophyll content of transgenic patchouli to support improved drought tolerance than wild-type plants. In our work, an in-silico study revealed better ligand interaction with the active site amino acid of the receptor. The regeneration and transformation protocol provides a platform for reverse genetics and helps in the modulation of metabolic pathways to enhance secondary metabolites and PO yield. Furthermore, the heterologous expression of ACC deaminase will support the management of PO global demand and develop a superior drought-tolerant variety of P. cablin plants.

The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.

ZW: Conceptualisation, Methodology, Visualization, Investigation, Data curation, Formal analysis, Writing-original draft, Writing- review & editing, Insilico data analysis, Validation. KK: Writing-review & editing, Formal analysis, Methodology, Insilico data analysis, conceptualisation, Validation. PS: review & editing, SEM analysis, provide ACC deaminase construct. LR: Conceptualisation, Investigation, Methodology, Supervision, Writing and editing. All authors contributed to the article and approved the submitted version