MSc

The study entitled “Enhancement of phenyl propanoid glycosides in
Artanema sesamoides Benth (vathomvaretti) by hairy root induction” was
conducted at the Department of Plant Biotechnology, College of Agriculture,
Vellayani and Aromatic and Medicinal Plants Research Station, Odakkali during
2016-2018. The objective of the study was to scale up the production of phenyl
propanoid glycosides in Artanema sesamoides Benth by inducing hairy roots
using Agrobacterium rhizogenes.
The explants for the Agrobacterium rhizogenes mediated transformation
were obtained from the in vitro raised seedlings of A. sesamoides. Seeds collected
from AMPRS, Odakkali were germinated in basal Murashige and Skoog (MS)
and half MS media. Maximum seed germination was observed in basal MS
medium with 94 per cent seed germination. Hundred per cent multiple shoot
induction was observed in basal MS medium and in MS medium supplemented
with Benzyladenine (BA) 0.1 mg L-1 using node as explant. Maximum number of
shoots (21 nos) per explant was seen with BA 0.1 mg L-1. Addition of BA at >0.2
mg L-1 resulted in green undifferentiated mass of cells.
Callus induction was tried in explants like node, leaf and root in basal MS
medium supplemented with combinations of growth regulators like BA, NAA,
IAA and 2,4-D. Node and root produced calli in combinations of BA with NAA
or IAA and their response was better compared to leaf. MS medium supplemented
with 2, 4-D or BA did not produce any callus.
Hairy root induction in A. sesamoides was attempted with A. rhizogenes
strains, MTCC 2364, MTCC 532 and ATCC 15834. A. sesamoides callus was
found insensitive to cefotaxime (bacteriostatic agent) upto 500 mg L-1. Among the
different explants tried for hairy root induction, calli infected with MTCC 2364
(Infection for 15 and 20 minutes and co-cultivation period of 48 h) and MTCC
532 (Infection for 15, 20 and 30 minutes and co-cultivation period of 48 h)
showed 100% survival on MS medium supplemented with cefotaxime (200 mg L-
1
). They also showed regeneration to produce shoots and roots in two weeks. No
hairy root formation was observed in any of the treatments. The in vitro roots
obtained were analysed for the presence of rol gene using PCR and no
amplification was found.
Phenyl propanoid glycosides were quantified using HPLC with the help of
class LC 10 software (Shimadzu, Japan) with PDA detector. Peaks related to
acteoside, artanemoside A, isoacteoside, leucoseptoside A and martynoside were
identified and quantified from roots of field grown (in vivo) plants, in vitro roots
and callus. Callus showed the highest quantity of all the five phenyl propanoid
glycosides, followed by in vivo roots and in vitro roots.
Economics analysis showed that the cost of production per gram dry
weight of the callus was found to be less compared to that of in vivo roots. Since
phenyl propanoid glycosides content in the callus is more than the in vivo root,
large scale production of the compound is economically feasible in in vitro
conditions.
In the present study, in vitro multiplication of A. sesamoides is reported for
the first time. Hairy roots could not be produced in this study, which may be due
to the non virulence of the A. rhizogenes strains used or the presence of inhibitory
substances in the explant, which needs further investigation. Increased phenyl
propanoid glycosides content in callus indicates the scope of using an in vitro
system for the continuous production of these compounds.