MSc

The study entitled “Somatic embryogenesis in black pepper (Piper nigrum
L.)” was conducted at the Department of Plant Biotechnology, College of
Agriculture, Vellayani, Thiruvananthapuram during 2015-2017, with an objective
to standardise a protocol for somatic embryogenesis in black pepper (Piper
nigrum L.) var. Panniyur 5.
Regeneration through somatic embryogenesis is the most appealing
technology in in vitro propagation since it produces large number of somatic
embryos from single explants, and also acts as a suitable system for crop
improvement through genetic modification. Earlier studies reported that
regeneration in black pepper is highly influenced by genotypes. Somatic
embryogenesis mediated protocol is not yet reported in any of the Panniyur
varieties.
In the present study, explants (shoot tip, node and leaf) from field grown
and in vitro raised seedlings were used. Systemic contaminants and polyphenols
are the major problems associated with in vitro culture of black pepper.
Pretreatments of the explants taken from the field grown plants with 250 mg L-1
ascorbic acid for 50 minutes and 50 mg L-1 of PVP for 1 hour reduced the
phenolic exudation. Dipping explants in 300 mg L-1 of cefotaxime for 1 hour and
addition of 15 mg L-1 of CuSO4 in the medium were found to be effective in
controlling the systemic contaminants.
Percentage of callus induction varied among type of explants as well as the
medium used. Leaf tissue from in vitro raised plants was found to be most responsive to
the callus induction (68.75% in 40 days) in MS medium supplemented with1.5 mg L-1 of
picloram (Pic). The same concentration of Pic in SH medium produced 57.4% callus
induction from leaf. Shoot tip and node taken from in vitro raised raised seedling showed
the highest callus induction in MS medium supplemented with mg L-1 of Pic (57.14% and
42.85% respectively). Among the explants collected from the field grown plants, the
maximum callus induction occurred in leaves (37%). Calli obtained from leaf, shoot tip
and node did not show embryogenesis in any of the 50 treatments and the calli turned
black and dried.
As there was no response on the somatic embryo induction from shoot tip ,stem
node and leaf, somatic embryogenesis was tried in fully ripened seeds of Panniyur-5.Out
of 48 treatments tried (MS and SH media, with different combination of 2,4-
D,BA,IAA,NAA,TDZ and Pic), only seven treatments responded. In 46% of the seeds,
somatic embryos emerged directly from the micropylar region of the seed (SH with 1.5 or
3% sucrose and half strength SH with 3% sucrose) and callus induction was occurred in
the rest. The calli initiated from seeds inoculated in MS and SH media supplemented with
1.5 and 2 mg L-1 Pic and 3% sucrose produced somatic embryos in the same medium.
Somatic embryogenesis was early in SH medium (40 days) compared to MS medium (60
days).
The somatic embryos produced from the calli, failed to produce secondary
embryos in any of the 42 treatments tried, however, they germinated to produce plantlets.
Somatic embryos directly formed (SH with 3% sucrose) from the seed showed secondary
embryo formation from the root pole of the primary embryo (20%) in the same medium
itself. The primary embryos formed in SH medium with 1.5% sucrose produced the
maximum (51.4%) secondary embryos in semi solid SH medium with 1.5% sucrose
medium. Secondary formed in SH+3.5% sucrose got regenerated in to plantlets in the
same medium. Secondary embryos from semi solid SH medium were regenerated only
when transferred to liquid SH medium with different concentration of sucrose (1.5% and
3.5%) with continuous shaking (110 rpm). Plantlets regeneration was early (48.73 days)
in liquid SHmedium with 3.5% sucrose.
Somatic
embryogenesis
was
confirmed
by
visualizing
the
different
developmental stages of the somatic embryos viz. globular, heart, torpedo and
cotyledonary stages under the stereo microscope. Histological studies of the embryos
confirmed their origin from micropylar tissue and not from zygotic tissue. It showed the
presence of secondary embryos from primary embryos as well as the emergence of
globular structure from the callus.
The present study was successful in developing a somatic embryogenesis
mediated regeneration protocol in black pepper var. Panniyur 5 which can be used for the
in vitro propagation and genetic modification based crop improvement programmes