The effects of elicitors and precursor on in vitro cultures of Sauropus androgynus for sustainable metabolite production and antioxidant capacity improvement

• Main Author: Wee, Sin Ling
• Published: University of Nottingham 2015
• Subjects: 582.13; QK710 Plant physiology
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.666922

Sauropus androgynus, commonly known as ‘sweet shoot’ is an underutilized crop known for its high nutritive values and medicinal properties. To date, scientific studies assessing the potentially important benefits of sweet shoot for use as medicinal plants are still limited, with only six studies reporting on in vitro propagation and nine articles describing the production of secondary metabolites. The present study revealed that shadehouse-grown plants contained a low yield of bioactive phytochemicals, such as phenolic (61.20 µg/10g FW), flavonoid (193.62 µg/10g FW), naringenin (128.01 µg/10g FW), quercetin (1.56 µg/10g FW) and kaempferol (274.85 µg/10g FW). Moreover, it also had very low antioxidant activity in DPPH (54.03%) and FRAP (397.56 µg/10g FW) assay. The elicitation of cultured tissues is therefore necessary to improve the production of phytochemical compounds and to increase the antioxidant capacity in sweet shoot. In this study, four different types of cultured tissues (in vitro shoot cultures, light-induced callus, dark-induced callus and somatic embryos) were selected to achieve this goal, followed by the extraction of phytochemicals from these cultured tissues treated with elicitors and precursor for better production of phytochemicals. For in vitro shoot induction, nodal explants cultured on semi-solid MS medium supplemented with 2.0 mg/l 6-benzylaminopurine (BAP) and 0.5 mg/l indole-3-acetic acid (IAA) produced the highest number of shoots (7 shoots per explant) with longer shoot length (5.74 cm). For light-induced callus induction, leaf explants grown in illuminated conditions with semi-solid MS medium enriched with 2.0 mg/l α-naphthalene acetic acid (NAA) and 1.0 mg/l kinetin showed good proliferation from the leaf explants (71.67%) with the highest callus fresh weight (4.53 g) and highest callus expansion rate (18.50 cm2). Likewise, leaf explants induced in dark condition on semi-solid MS medium containing similar hormone composition, also displayed the highest callus fresh weight of 4.54 g and callus expansion rate of 13.85 cm2. Three-month-old dark-induced callus were transferred onto liquid MS medium fortified with a different concentration of NAA and kinetin to further induce somatic embryos. After three weeks of callus inoculation, as high as 83.33% of embryogenic cell cultures achieved its maximum density of 5.2 ml in liquid MS medium supplemented with 2.0 mg/l NAA and 1.0 mg/l kinetin. In histodifferentiation medium (liquid MS medium containing 1.0 mg/l NAA and 0.5 mg/l kinetin), a mean number of 15.60, 14.80 and 13.20 embryos per g callus of globular, heart-shaped and torpedo-shaped embryos developed respectively after 9 weeks of embryo induction. The torpedo-shaped embryos were then inoculated into hormone-free MS medium and 90% of these embryos successfully differentiated into cotyledonary embryos after 3 weeks of maturation. These results showed a complete ontogeny of sweet shoot somatic embryo from the globular stage to heart-shaped, torpedo-shaped and cotyledonary stage. After two months of shoot initiation on MS medium supplemented with 2.0 mg/l BAP and 0.5 mg/l IAA, a relatively high percentage (>75%) of shoot proliferation occurred from nodal derived shoot (6.74 shoots per explant), light-induced callus (6.23 shoots per callus) and somatic embryos (6.45 shoots per embryo) of sweet shoot. These well proliferated shoots were then subjected to root initiation in half strength MS medium containing 1.0 mg/l IAA and high percentage of root formation was successfully achieved in 90% of the plantlets after 10 days of culture. For hardening off, the rooted plantlets were transferred to culture jars containing purified water and maintained at ambient conditions for one month. High survival rate (>76.67%) was achieved in perlite:compost mixture (1:1) after one month of acclimatization in shadehouse. To enhance the production of phytochemicals and antioxidant capacity in tissue cultures of sweet shoot, elicitor and precursor treatments served as alternative methods in influencing the biosynthetic pathway for the accumulation of phytochemicals. In this study, shadehouse-grown plants and cultured tissues of sweet shoot were treated individually with different concentrations of methyl jasmonate (MJ), salicylic acid (SA) and phenylalanine (Phe) for a treatment period of 3 weeks. Light-induced callus culture produced the highest amount of phenolic and flavonoid compounds amongst the tested plant samples with those treated with Phe producing the highest antioxidants, followed by MJ and SA. After 3 weeks of Phe treatment at 20 mg/l, the highest levels of total phenolic (246.62 µg/10g FW), total flavonoid (636.26 µg/10g FW), naringenin (12081.05 µg/10g FW), quercetin (134.36 µg/10g FW), kaempferol (11325.13 µg/10g FW) and antioxidant activities (97.35% for DPPH and 5941.66 µg/10g FW for FRAP assay) were detected in light-induced callus cultures of sweet shoot. Since phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS) were the key enzymes for the biosynthesis of phenolics and flavonoids, both enzymatic activities were also measured in light-induced callus cultures treated with Phe. The highest PAL (101.18 mmol CA/g FW) and CHS (14.49 nkat/mg protein) enzymatic activities were also attained in light-induced callus cultures fed with 20 mg/l of Phe at week 3. Light-induced callus cultures treated with Phe produced the highest amounts of phytochemicals, antioxidant capacity and enzymatic activities, and these results were chosen to undergo Pearson’s correlation coefficient analysis, which verified the positive co-relationship seen between all of the above-mentioned parameters. These findings showed that the addition of Phe enhanced the enzymatic activities in the phenylpropanoid pathway and increased the concentrations of phenolic and flavonoid compounds (naringenin, quercetin, kaempferol) which in turn contributed to the increase in antioxidant activities in light-induced callus cultures of sweet shoot. Data from this study showed that sweet shoot has the potential to be developed as a plant-based antioxidant for the pharmaceutical industry. Additionally, this study is the first to report on the complete ontogeny of sweet shoot and the positive effects of elicitation in tissue cultures of sweet shoot.