Somatic embryogenesis in Leptadenia reticulata (Retz.) Wight and Arn along with assessment of shoot and callus cultures for HPTLC fingerprint and quantification of p-coumaric acid

Abstract

Leptadenia reticulata (Retz.) Wight and Arn is an important medicinal plant of Asclepiadaceae family. In the present study regeneration was attempted using leaf and nodal explants in Murashige and Skoog’s (MS) medium fortified with sucrose (3%) and different cytokinins like 6-benzyladenine (BA), kinetin (Kn) and adenine sulphate (AdSO 4 ) in 5–20\xa0µM concentration range and auxins like indole-3-acetic acid (IAA), 1-naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D) in 0.1–1.0\xa0µM concentration range. Through leaf explants total 21.70\u2009±\u20092.06 somatic embryos were recorded in 100% cultures in BA (15\xa0µM) with AdSO 4 (15\xa0µM). Further these embryos were transferred to static medium augmented with gibberellic acid (GA 3 , 1\xa0µM) facilitated development of plantlets within 8 weeks. Inoculating nodal explants in BA (10\xa0µM) with AdSO 4 (10\xa0µM) formed total 5.10\u2009±\u20090.33 shoots (100% response). Callus and shoot cultures derived through leaf and nodal explants respectively were further qualitatively and quantitatively analyzed for their biosynthetic potential. High-performance thin layer chromatography (HPTLC) fingerprints of the samples confirmed that the in vitro shoots showed almost similar banding patterns in comparison with in vivo shoots in terms of number of peaks and band on TLC plate, whereas in callus samples the banding pattern was differed. Further quantification of p -coumaric acid revealed that in in vivo and in vitro shoots it was in LOD (10\xa0µg) range but out of LOQ (100\xa0µg). Whereas it was quantified 78.08\u2009±\u20092.20\xa0mg\xa0g −1 of dry weight (DW) in 6 weeks old callus cultures obtained from BA (20\xa0µM)\u2009+\u2009NAA (0.5\xa0µM) fortified medium, which indicate that it can become a chemical marker for L . reticulata . This protocol can be utilized for true-to-type plant regeneration as well as conservation of this threatened medicinal plant. Chemical fingerprint can be utilized for authentication of L . reticulata and callus cultures as an alternative source to wild plants for production of p -coumaric acid. SEs were efficiently developed in L. reticulata . In vitro cultures were able to synthesize metabolites but callus is prominent source for p -coumaric acid. Hence it can be alternative to wild plants.