The seeds are reported to show non-deep physiological dormancy, which can be reduced by chilling, leaching, priming, GA3 and KNO3 pre-treatment. The germination was also observed to be higher at 35ºC than at lower temperatures, that is 20ºC to 30ºC (Parihar et al. 2016). Seed treatment with 5% NaCl resulted in higher rates of germination than scarification with sand or water soaking.
L. inermis have been high in demand due to medicinal and commercial uses (Ponugoti 2018). The present study describes an attempt to develop an in vitro protocol for mass multiplication of L. inermis by manipulating the nutrient salts, growth regulators and culture conditions. Rapid shoot multiplication from cotyledonary node regions of Lawsonia inermis L. (Moharanna et al. 2017) was seen (96.6%), on MS medium, supplemented with 8.88µM Benzyladenine and 2.68µM naphthalene acetic acid. The quality and size of shoots improved on medium with 4.44µM of Benzyladenine. Apart from the cotyledonary nodes, apical shoot buds and meristems were also be used for efficient shoot proliferation (Singh et al. 2012). Rout et al. (2001) used MS medium with 0.25 mg/l 6-benzylaminopurine, 0.25 mg/l Kinetin and 0.5 mg/l ascorbic acid for mass propagation of Lawsonia inermis L. It was concluded that axillary meristems served to be better explants in the propagation of Lawsonia inermis L. Nodes from mature plants could be also used on MS medium with 6-Benzylaminopurine (3.33 µM) (Singh et al. 2012). But with an increase in the amount of benzyladenine or Kinetin, the number of shoots gradually decreased (Singh, et al, 2016). An efficient system of regeneration was established using the nodal segments of Lawsonia inermis L. (Ram and Shekhawat 2011). Murashige and Skoog (MS) medium, with hormonal concentration of 0.25 mg/l 6-Benzylaminopurine, 0.25 mg/l Kinetin, 0.1 mg/l indole-3-acetic acid and 150 mg/l ammonium sulphate showed best response. About 95% of the shoots were rooted successfully in soilrite after treating the shoots with 300 mg/l indole-3-butyric acid and 100 mg/l 2-napthoxy acetic acid. In our analysis, the percentage response to shooting was better (100%), regenerating more healthy shoots with less hormonal combinations [(BAP-4 mg/L: 8 shoots); (BAP 2 mg/L + Kn 2 mg/L: 12 shoots)]. Efficient rooting was seen in MS basal medium, devoid of any hormones. These findings further validate the low cost of maintenance of the plant.
Gene ontology data of the two transcript sets show distinct variation in the number of transcripts. In vitro plant set of Lawsonia inermis show a higher accumulation of transcripts than in vivo plants. A similar increase in the transcript number was observed in infected Vigna mungo as compared to the control, non-infected sample (Ganguli et al. 2016). Results show that cellular processes, single cell organism process and metabolic processes are abundant in the plant, indicating the plant’s role in operating the intricate machinery. Response to stimulus can be seen as a dominant function, which might suggest that the plant is prompt in responding to abiotic as well biotic stresses (Fig. 4).
Biochemical assays have shown henna plant to be a rich source of important components, giving the plant the commercial and medicinal importance (Badhai et al. 2013) (Ojewunmi et al. 2014) (Endrini et al. 2007) (Song et al, 2017). These observations can further be validated by the pathway enrichment analysis performed in this study. Phenylpropanoid biosynthesis pathway, along with the enzyme PAL (Phenylalanine ammonialyase) were reported to be instrumental in combating drought stress, as revealed in drought tolerant cultivars of foxtail millet. (Yu et al. 2020) In our analysis we have identified the over representation of phenylpropanoid biosynthesis pathway in in vitro plants, nearly 11% more than in vivo plants. This enzyme PAL plays an important role in contributing to the phenolics and flavonoid content of the plant. The various important medicinal properties of Henna plants (radical scavenging, antimicrobial, anticancer, antiulcer, anti-diabetic), are due to the high phenolic and flavonoid content, the production of which depends largely on the expression of this particular enzyme. The accumulation of flavonoids can also be correlated to the high pigment content in Henna. Similar observations were reported in the ornamental plant of Syringa oblate, with a high accumulation of flavonoids (Zheng, et al. 2015). An enriched expression of flavonoid biosynthesis pathway can also be a result of stress adaptation, reported in wheat subjected to drought conditions (Dalal, et al. 2018). It was interesting to observe that transcript abundance of flavonoid pathway remained the same for in vivo and in vitro Henna plants, showing that, tissue culture conditions did not affect the flavonoid pathway in any way. Apart from defense and stress, flavonoid and anthocyanins also help in the protection of chlorophyll, as observed in light stress induced leaves of Cornussto lonifera (Field et al. 2001). Phenols and terpenoid pathways have also been reported to have been elevated during the successful micropropagation of Nardostachys jatasamani, an important endangered medicinal plant (Dhiman et al. 2020). Presence of pathways related to volatile terpenoids, monoterpenoid, limonene and pinene, were seen to be responsible for the scent in Cinnamomum camphora (Hao, et al, 2020). Our pathway analysis of henna showed an enriched abundance of terpenoid biosynthesis pathway. Comparative transcriptome analysis between Ocimum basilicum and Ocimum sanctum, reported phenylpropanoid and terpenoid pathways to be abundant (Rastogi et al. 2014). Drought affected white grapes similarly showed a heightened expression of these pathways (Savio et al. 2016). Terpenoids, were also reported to have protective action against diabetic complications, detected in Zingiber officinale (Saraswat et al. 2010). In our analysis, we have also observed an enriched expression of pathways related to synthesis of terpenoids, in in vitro plants of Henna, about 7.14%. Steroid hormone biosynthesis pathway, exclusively found in in vitro Henna plants, can be held responsible to help the plant cope with stress.
We have been able to identify the increased expression of HSP 70, HSP 40/ DnaJ in Lawsonia under in vitro condition which has been previously reported to be associated with stress responses (Kampinga and Craig 2010). The presence of “ethylene overproduction protein”, “abscisic acid receptor”, “protein ethylene insensitive”, “TIFY 6B-like proteins” and “Ninja-like protein” suggests that the plant is adept in dealing with abiotic stresses, reported by Thireult, et al, (2015) in Arabidopsis. The presence of “mate efflux protein” and “TRANSPARENT TESTA 12-like proteins” calls for the xenobiotic transporter activity of the plant, as previously proposed by Omote et al. (2006), in plants, bacteria and even in mammals. TPR repeat containing thioredoxin also helps plant to adapt to changes in hydration levels and osmolarity. In vitro plants can be seen to express exclusive proteins and domains like “thaumatin domain”, “TIFY 10-like”, “SAM domain” and various proteins which help to nullify the effect of free radicals (Damme et al. 2002). The plants also show presence of cellulose synthase, callose synthase and cell wall biogenesis proteins, which indicate the capability of plant to synthesize cell wall elements, aiding in responses to wounding. Apart from a large number of serine threonine protein kinases, aiding in pathogen identification and defense responses, (Afzal et al. 2008) MAPK signaling proteins can be detected, aiding in immunity (Zhang and Klessig 2001). Several transcription factors were seen to be well represented, among in vivoandin vitro plants of Lawsonia inermis, like WRKY, WD40, LURP1 related, calcium calmodulin binding proteins, which has pronounced role in pathogen defense (Pandey and Somssich 2009). Chromatin remodeling proteins found in the plant, like topless related protein, Tubby like F-box protein, histone deacetylase, could affect transcription of the genes (Wang et al. 2015). Chloride, sulphate, magnesium, proton transporters and transition metal transporters, were responsible for maintaining the electrochemical balance in cells, without harming the plant (Hall and William, 2003). Transcript abundance of ABC transporters were also observed, which might help the plant sequester toxic compounds, byproducts of chlorophyll degradation, and other unwanted compounds into vacuoles, as previously reported by Walter et al. (2015), in wheat. Ubiquitin E3 ligases, ubiquitin conjugation factors, found to be more enriched in in vitro plants, aiding in hormonal signaling, plant’s response to stress, and also, in regulating the protein content within the plant cell (Sharma et al. 2016).
Gene families encoding various enzymes like, cytochrome P450, phenylalanine ammonium lyase, prephenatedehydratase, tropinonereductase, polyamine oxidase and serine carboxypeptidase, were observed, instrumental in increasing the secondary metabolic pool, aiding in defense responses. Gene families encoding enzymes related to the production of phenols were observed to be very well represented in in vitro plants of Henna. This huge array of gene families encoding specific enzymes, further validates the rich secondary metabolic pool for in vitro plants. (Table 6, Fig. 5)