full issue - Association of Biotechnology and Pharmacy

Current Trends in Biotechnology and Pharmacy
Vol. 7 (1) 511-517 January 2013, ISSN 0973-8916 (Print), 2230-7303 (Online)
513
8.08 ± 0.81 shoot buds with average 2.71 ± 0.32
cm lengths (Table 1) followed by 18-20 days old
seedling derived cotyledons. In addition,
cotyledons procured from 14-15 days old
seedlings formed adventitious shoot buds early.
Cotyledons of 10> days old seedlings were too
juvenile to form shoot buds and it is likely that
the cotyledons may absorb nutrients and growth
regulators from the medium and then form a few
shoot buds at late. On the other hand, cotyledons
of 20< days old seedlings were somehow beyond
of juvenility and produced few shoots. This study
revealed that cotyledons excised from 14-15 days
old seedlings were suitable for direct adventitious
shoot organogenesis in A. mangium. In Dalbergia
sissoo, 8-10 days old in vitro seedling derived
cotyledons produced adventitious shoots directly
on MS medium (14).
Cotyledon explants lacking of the
embryonal axis showed varied responses in
terms of direct organogenesis, depending upon
the concentration and combination of growth
regulators (Table. 2). When BA was used as sole
growth regulator, maximum number of shoots
(3.87 ± 0.67) as well as longest shoots (2.32 ±
0.96) was found at 2.0 µM BA containing medium.
Incorporation of auxin along with BA to the
medium enhanced the frequency of shoot bud
differentiation considerably. NAA was found to
be more appropriate auxin in combination with
BA for direct shoot proliferation from cotyledons
while IBA showed less activity. In this experiment
2.0 µM BA + 1.0 µM NAA was found as optimum
growth regulator combination for direct shoot
organogenesis, where 10.88 ± 0.35 shoots with
3.90 ± 0.23 cm length were produced at a faster
rate (Fig. 1b and c). In all the cases, adventitious
shoot buds were developed only from the
proximal end of cotyledons and no bud was
formed at the distal part as this part was also
wounded during culture. Findings of this
experiment are supported by a previous report
on Dalbergia sissoo (14), where 0.26 µM NAA
along with 4.44 µM BA enhanced shoot bud
differentiation directly from the proximal region
of cotyledons. In another reports on Leucaena
leucocephala (16), it has been demonstrated that
cotyledons produce adventitious shoots directly
from the proximal end with forming callus at the
base of cotyledons when transfer them from
a pre-induction medium having high BA
concentration to low BA containing medium. In
addition, no shoot bud formation was found from
the distal part of cotyledons of L. leucocephala.
During this experiment, when cotyledons placed
in adaxial (upper surface facing down) orientation
on medium surface more adventitious shoot buds
were produced from the cut margin of cotyledons
(Data not shown). In tomato (18), cotyledons
produced greater number as well as longest
shoots when placed in abaxial (lower surface
Table 1. Effect of seedling age on direct shoot development from cotyledon explants cultured on
MS medium supplemented with 2.0 µM BA + 1.0 µM NAA.
Age of % of explant Days to sprout Average No. Average length
seedlings produced shoots adventitious of shoots/ of shoots/
(days) shoot bud culture culture (cm)
10> 41.66 ± 4.48 c 15.29 ± 0.58 ab 1.66 ± 0.65 c 1.10 ± 0.40 b
14-15 100.00 ± 0.00 a 10.71 ± 0.92 c 8.08 ± 0.81 a 2.71 ± 0.32 a
18-20 98.33 ± 1.66 a 12.18 ± 0.39 bc 5.75 ± 0.59 b 1.55 ± 0.14 b
22< 83.33 ± 3.76 b 16.12 ± 0.37 a 4.66 ± 0.82 b 1.25 ± 0.20 b
Values represent means ± standard error of 12 explants per treatment in three repeated experiments.
The different letters within column show significant differences by ANOVA and Duncan’s multiple
range tests at p