MSc

The study entitled “Elicitation of phenylpropanoid glycoside biosynthesis and
expression profiling of key acteoside biosynthetic genes in Artanema sesamoides Benth
(vathomvaretti)” was carried out during 2019-2021, at the Department of Plant
Biotechnology, College of Agriculture, Vellayani. The objective was to enhance the
phenylpropanoid glycosides synthesis in the callus culture of Artanema sesamoides
using elicitors like salicylic acid, yeast extract and pectin and to study their effect on the
expression of acteoside biosynthetic genes PAL (phenylalanine ammonia-lyase) and HCT
(Hydroxycinnamoyl-CoA Shikimate).
In vitro raised seedlings of A. sesamoides were used for the establishment of
callus culture in MS medium supplemented with 0.5 mgL-1 BA and 0.5 mgL-1 NAA.
Two-week old callus cultures were transferred to liquid MS medium with 0.5 mgL-1
NAA and 0.5 mgL-1 BA and stabilized for 10 days. Elicitors viz., salicylic acid (SA 40
and 100μM), yeast extract (YE 1 and 2 gL-1
) and pectin (100 and 200 mgL-1
) were
added to the suspension culture and incubatedin an orbital shaker (120 rpm) at 24°C for
3 weeks. After elicitation, callus was harvested, dried and finely powdered for analysis.
The spent medium was also analysed for the PPGs.
Phenylpropanoid glycosides (PPGs) were extracted using n-butanol and the
extracts were evaporated to get the crude residues. The total yield of the crude residue of
phenylpropanoid glycosides was maximum (44.88 mgg-1
) in the suspension culture
treated with pectin (200 mgL-1
) and this treatment showed maximum residue (41.6 mgg-1
)
in the callus also. While the additionof pectin increased the accumulation of PPGs in the
callus, the addition of SA and YE increased their exudation in the medium.
Six important PPGs such as acteosides, artanemoside, isoacteoside,
leucosceptoside, martynoside and plantainoside were identified in the butanol extracts
using HPLC. Phenylpropanoid glycosides showed maximum absorbance at 330 nm and
the peaks appeared within the retention time of 40 to 75 min. All the elicitors increased
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the synthesis of plantinoside. Treatment with SA (40µM) enhanced the content of
martynoside (3.032%) and plantainoside (45.444%) in the callus by 10 and 50 folds
respectively.
In this study, the acteoside content was found to be more in the medium than the
callus. Treatment with (SA 40µM) and YE (2 gL-1
) was found to increase the acteoside
content to a maximum of 10 to 17 folds in the medium. Up to 34.4% increase in the
artanemoside content was observed in the medium with the elicitation by Pectin (100
mgL-1
). Isoacteoside content was increased to 17.3% with elicitation by SA (100 μM).
Real-time PCR was performed to find the effect of elicitors on the expression of
acteoside biosynthesis genes such as PAL and HCT in the callus after 24h and 48h of
elicitation. The quality of cDNA was confirmed by PCR using β-ACTIN gene-specific
primers. The Cq values obtained in RT-qPCR for each gene was further analysed and
relative expression values were obtained using 2-ΔΔCq (Livak) method with ACTIN as the
reference gene.
After 24h of elicitation, 83-fold increase in the expression of PAL gene was
observed in the callus treated with SA (40 μM). Elicitation with YE (1 gL-1
) and Pectin
((100 mgL-1
) showed 8 and 2.3-fold increase in the expression of PAL gene after 24h.
The expression of both PAL and HCT genes was increased up to 2-folds in the callus
treated with SA (100 μM), pectin (100 mgL-1
) and YE (1 gL-1
). After 48h of elicitation,
the expression of PAL and HCT genes was decreased drastically in most of the
treatments.
The study shows a possible use of yeast extract, salicylic acid and pectin for the
in vitro production of phenylpropanoid glycosides from Artanema sesamoides.