Surface sterilization
In the tissue culture process, various surface sterilants are being used for explant surface sterilization to get rid of contamination which includes different concentrations of ethanol, calcium hypochlorite, mercuric chloride, hydrogen peroxide, sodium hypochlorite, bromine water and silver nitrate [da Silva, 2016; Tyagi, 2011]. Again, plant preservative mixtures (0.1mL/L to 1mL/L) can be used along with the culture media to reduce airborne contaminations in the culture media [Plant Cell Technology 1998]. From the experiment of explant surface sterilization using 0.1% mercuric chloride, 2 minutes and 3 minutes treatment showed maximum explant survival rate after 21 days of culture initiation. 2 minutes treatment with 0.1% mercuric chloride showed 75% explant survival and 25% explant contamination and 3-minute treatment with 0.1% mercuric chloride also showed 83% explant survival but only 8.33% explants got contaminated and 8.33% explants got damaged after 21 days of culture initiation. 4 minutes treatment resulted in 50% explant survival and another 50% explants got damaged. 1-minute treatment showed a minimum (46%) explant survival rate and explants without mercuric chloride treatment resulted in 100% explant contamination after 21 days of culture initiation. Previously it was reported that for explant surface sterilization of L. antipoda (L.) 10% NaOCl treatment for 10 minute and 0.15% bavistin treatment showed most effective result (Jabir et al, 2016).
Explant response to different concentrations of Benzyl Aminopurine (BA) and Naphthaleneacetic acid (NAA):
The use of different plant parts for culture also may require different media components for growth (Murashige, T., & Skoog, F. 1962). Also, the response may vary due to environmental conditions and growth regulators used. From the experiment, it was observed that explants responded differently in different media with or without any growth regulators in the culture media. Shoot proliferation and multiplication were observed best in the combinations of BAP and NAA (Fig I). The highest average numbers of shoots per explant were observed in BM2 media (MS + 1 mg/L BAP + 0.2 mg/L NAA) i.e., an average of 33 shoots per explant and the lowest shoot proliferation and multiplication resulted in the BM6 media (MS + 5mg/L BAP) i.e., an average of 14 shoots per explant. The control media (without any PGRs) formed an average of 17 shoots per explant. Explants in the media BM1, BM3, BM4 and BM5 showed moderate shoot proliferation and multiplication (Table I).
Ex vitro rooting
All the in vitro cultured shoots of Lindernia pusila formed roots after being treated in IBA solution and survived in a normal environment but the rates of explant forming roots varied in different concentrations of IBA. 1mg/L and 2mg/L IBA treatment resulted in a maximum rooting rate per explant in the shoots of in vitro multiplied L. pusila.
Random Amplified Polymorphic DNA (RAPD) assay:
Molecular markers are a very essential tool for DNA fingerprinting, genetic study, molecular breeding and germplasm characterization. They can also be used to identify somaclonal variation in tissue culture, and transgenic plant production (Soniya et al. 2001). RAPD is one of those tools which are very effective for the detection of somaclones in tissue cultured plants as large number of samples can be quickly analysed and many loci can be sampled with numerous markers (Soniya et al. 2001). RAPD is one of the most widely used dominant molecular marker technique which is initially used to detect polymorphism. It is effective and sensitive assay which has the capability to identify DNA damage, mutations. Therefore this technique can be used to study genotoxicity and carcinogenesis [Tawar, 2008]. The RAPD assay using 14 RAPD primers formed a total of 355 DNA bands in three genomic DNA samples of wild, micro propagated (non-hardened) and micro propagated (hardened) Lindernia pusila plant. Out of 14 primer, 7 primers (OPC07, OPC08, OPA04, OPA13, OPA12, OPC01, OPC04) formed 178 similar DNA bands which had no variations or can be said as common in the parental genotypes. The other 7 (OPC 02, OPC 05, OPC 09, OPA 01, OPA2, OPC 03 and OPC 06) primers formed 177 polymorphic DNA bands showing variations in the DNA bands in the tissue cultured plants from the wild (parental) plants (Fig II). The highest number of polymorphic DNA bands (14) were observed in the genome of micro propagated plants (hardened and non-hardened) by the OPC 08 primer and the lowest number of polymorphic DNA bands were observed by OPC 04 (3 & 1) primer in the genome of hardened micro propagated plant.
Comparative in-vitro antioxidant tests of wild and tissue cultured extract of L. pusilla
Total Phenol Content
The total Phenol Content of the Dried extracts of L. pusilla were determined using FCR method described by Maheswari et al. (2011) and determined using the regression curve (y = 2.21x + 0.0856, R² = 0.994) and expressed as mg of gallic acid equivalents (GAE). In this present study, the total phenolic content in of wild and tissue cultured plant extract of L. pusilla were varied, the wild extract showed 54.7 ± 5.3mg and the tissue cultured extract showed 61 ± 3.4 mg GAE/g powder weight.
Total flavonoid content
The total flavonoid content in L. pusilla extract was determined by the regression curve (y = 1.446x − 0.0728, R2 = 0.9966) expressed as mg of quercetin equivalents (QE). The total flavonoid content determined using method described by Chang et al. (2002) was 24 ± 7.7 mg QE/g in the tissue cultured extract and 20 ± 7 mg/g in the wild extract of L. pusilla.
Total antioxidant capacity
The total antioxidant capacity of the L. pusilla extract was determined using ammonium phosphomolybdate method described by Shah et al. (2013) and determined using the regression curve (y = 1.7089x − 0.0556R² = 0.9922) and found 114.7 ± 7.2 mg ascorbic acid equivalent (AEE/g) of dried extracts in tissue cultured extract and 94 ± 7.2 mg AEE/g in wild extract.
Comparative quantitative HPLC analysis for detection of gallic acid and quercetin
Gallic acid and quercetin estimation method was developed and validated. Tuning of mobile phase was done to standardize the HPLC protocol. Prior to injection, the system was stabilized to bring the mobile phase into equilibrium. It is an isocratic elution of 30% (v/v) HPLC-grade water and 70% (v/v) methanol. These variables were found to be an effective mobile phase for gallic acid and quercetin separation at a flow rate of 1 mL/min utilizing a Waters RP-18 column The quantity of gallic acid and quercetin in wild and micropropagated Lindernia pusilla was satisfactorily measured using this technique. The standard curve (Figure III) and chromatograms of quercetin and gallic acid measured from prepared extract of L. pusilla are listed in table below. Gallic acid and quercetin had retention times of 2.6 and 4.42 minutes, respectively. From the study the gallic acid and quercetin content of tissue cultured methanolic extracts of L. pusilla were comparatively higher than the wild plant (Table II).
Comparative GC-MS compound analysis in tissue cultured and wild extracts of L. pusilla
Medicinal plants are the resources of new drug inventions of various diseases for the bioactive compounds found in the medicinal plants. Study of the plant extracts using GC-MS mostly reveal the availability of important bioactive compounds in the plant. In the present study the bioactive compounds found in wild and tissue cultured plant extracts of L. pusilla were studied using GC-MS analysis. GC-MS chromatogram of aqueous methanolic extract of L. pusilla were shown in the figure IV. The compound search was selected by virtue of retention time (RT), molecular formula, molecular weight, Peak area % (concentration). From the wild extract 8 chemical compounds are identified and from tissue cultured extract the 9 compounds were identified and presented in the Table III.
The compound Vigabatrin found in the wild extract is used for treatment of infantile spasm and refractory complex partial seizures (Singh R & Carson RP 2023), Panaxydol identified in tissue cultured and wild extract induces apoptosis in cancer cells through EGFR activation and ER stress to inhibit tumor growth in mouse model (Kim et al, 2016), another bioactive compound found in wild sample Malic acid found in wild extract is widely used in food industry as sweetness agent in fruit juice, carbonated juice, and other baverges. Anthracene found in wild extract is widely used as UV tracer in printed wiring board.