Murraya koenigii

The present study was undertaken in curry leaf plant (Murraya koenigii), one of the most important aromatic and medical plants in the world to explore the best DNA isolation protocol. Presence of abundant quantity of secondary metabolites in curry leaf plant resulted in poor quality and quantity of isolated genomic DNA. Thus, two DNA extraction protocols namely CTAB (Cetyl Trimethyl Ammonium bromide) modified method and Dellaporta modified method were evaluated for extraction of DNA. The quantity and quality of the DNA extracted was compared using UV-spectrophotometer and agarose gel electrophoresis. CTAB modified method was found to exhibit consistently positive results in terms of both quality and quantity of DNA (A260/280) as compared to Dellaporta method. The isolated DNA was highly intact and devoid of shearing. Further, this method was explored to different species of Rutaceae family, giving significant DNA yield.

Murraya koenigii (L.) Spreng, commonly known locally as “curry patta” or “mitha neem” in India, is a valuable medicinal plant known for its biochemical and aromatic properties. This paper presents a protocol for the rapid and high frequency shoot regeneration from nodal and inter-node explants from matured plant and epicotyl, cotyledons, cotyledonary node (embryonic axis), juvenile leaf, hypocotyl and root segment of in vitro derived seedling via axillary and adventitious shoot formation of M. koenigii. Adventitious regeneration, which is a pre-requisite in most genetic transformation studies using Agrobacterium and ballistics, needs to be developed as a protocol for micropropagation of M. koenigii. Cytokinins benzylaminopurine (BAP), Kinetin (Kin), adenine sulphate (ADS) and indole-3-acetic acid (IAA) were used for multiple shoot induction. Addition of an auxin along with cytokinin improved the shoot production capacity. Shoot buds could be initiated from all the explants tested, with epicotyl explants producing the highest average number of shoots/explant. N6-benzyle adenine (BA), kinetin, adenine sulphate (ADS) and indole-3-acetic acid (IAA) in combination were the most effective PGRs for shoot induction.

Present study was carried out to standardize a protocol for high efficiency in vitro adventitious shoot regeneration from Murraya koenigii using cotyledon and hypocotyl explants. Adventitious regeneration, which is a pre-requisite in most genetic transformation studies using Agrobacterium and ballistics, needs to be developed as a protocol for micropropagation of M. koenigii. Direct adventitious shoot proliferation was achieved from intact seedling on Murashige and Skoog (MS) medium supplemented with various concentrations of 6-benzyleaminopurine (BAP) 2.64 µM to 22.21 µM, Kinetin 2.34 to 13.96 µM and Adenine sulphate (ADS) 40.72 to 244.39 μM to induce in vitro multiple shoots. Percentage response of cotyledon explants was 95.00 ± 0.58 which was significantly higher than the response of hypocotyl explants (76.2 ± 0.06) explant in the MS basal medium supplemented with 12.95 µM BAP, 8.98 µM Kinetin and 152.74 µM ADS. The 35-40 mm elongated shoots were cultured to MS basal medium augmented with different concentrations of indole-3-butyric acid (IBA). The maximum percentage, 84.8 ± 0.13 of rooting was achieved on MS basal medium containing 17.26 µM IBA. In-vitro plantlets regenerated from cotyledon and hypocotyl explants were hardened for four weeks in a green house. The hardened plantlets were transferred to field conditions. Eighty percent hardened plantlets were successfully survived under natural conditions

In the present study a highly efficient and reproducible protocol was established to regenerate direct somatic embryogenesis from hypocotyl segments (HYP) of Murraya koenigii. The surface sterilized seeds were inoculated on half strength Murashige and Skoog (MS) basal medium for germination. Explants were obtained from 60 days old axenic seedlings of Murraya koenigii and cultured on MS basal medium supplemented with different concentrations of 6-benzyleaminopurine (BAP) 0.56 to 8.89 μM and thidiazuron (TDZ) 0.48 to 9.37μM. The globular embryos originated from cut ends and entire surface of the hypocotyl explants within 35-40 days. The highest rate of conversion of torpedo, heart and cotyledonary stages from globular stage was obtained in MS medium supplemented with 7.98 μM TDZ. The matured somatic embryos were transferred to the MS basal medium without Plant Growth Regulators (PGRs). Highest 81% of the matured embryos were germinated on transfer to ½ MS basal medium without PGR, where they grew for a further 4-5 weeks.

Rapid shoot proliferation was established by adventitious shoot formation from inter-node segments. Shoots were directly regenerated in vitro without callusing. The concentration of plant growth regulators (PGRs) in MS medium exhibited a discrete role in the efficacy of adventitious shoot induction. Maximum number of shoot (82.02 ± 0.08) regeneration was achieved on modified MS medium supplemented with BAP 13.01 µM, Kinetin 8.87 µM and ADS 136.52 µM. Rooting of in vitro shoots occurred in three to four weeks on transfer to MS medium containing IBA (19.68 µM). About 88 % of in vitro-raised plantlets were survived under field conditions. The protocol development in present study for rapid adventitious shoot regeneration from inter-node segments of Murraya koenigii could be helpful in carrying out various genetic modifications in this economically important medicinal plant