Direct Organogenesis of Seaside Heliotrope (Heliotropium crassavicum) Using Stem Explants
Heliotropium crassavicum L. is a sand binder salt marsh
herb with enormous traditional value and widely found in South Asia America
and Europe. In the direct method of regeneration from stem explants, we observed
the maximum number of shoot regeneration after four weeks culture of MS elongation
medium with 2.0 mg L-1 of 2, 4-D (17.27±0.51). It was clear
that MS medium with 2.0 mg mL-1 2, 4-D alone suitable for shoot multiplication
as well as shoot elongation then compared to other combination of auxin and
cytokinin. In vitro shoots were excised from shoot clumps and transferred
to rooting medium containing 2, 4-dichlorophenoxy acetic acid (0.5-3.0 mg L-1).
The maximum number of root regeneration (6.4±0.416) and root length (6.08±0.07)
were observed in MS rooting medium fortified with 2.5 mg L-1 of 2,
4-D after 2 weeks of culture. 85% of in vitro raised plantlets with well-developed
shoots and roots were transferred to ex vivo conditions into polythene
bag containing sterile compost with ratio (v/v/v) of organic fertilizer: sand:
peat (1: 2: 2; 3: 1:0 or 2: 2: 1). Sixty five percent of acclimated plants were
transferred to the pots under full sun where they grew well without any detectable
Received: December 14, 2012;
Accepted: January 28, 2013;
Published: April 18, 2013
Heliotropium curassavicum is a species of heliotrope that is native
to much of the South Asia, Europe and America, be found on other continents
as an introduced species. It is known several common names, such as Seaside
Heliotrope, Salt Heliotrope, Monkey Tail and Quail Plant (Wolff,
1977). Wild Heliotrope belongs to the Boraginaceae family also it is a low,
spreading perennial herb which can take from a prostrate creeper with succulent
leaves and small white flowers. It is often found in salty or alkaline places
like dry streambeds (McAuley, 2007). Traditionally, it
has been used for various external applications to ulcers, wounds, local inflammation,
ringworm, rheumatism, urticaria (Kirtikar and Basu, 1998)
and recently for cancer and diabetes (Sharma et al.,
2009). Numerous techniques are available for rapid and extensive multiplication
of elite and desirable plant species through in vitro conditions. Among
these the Clonal multiplication become a major subject of investigation through
plant tissue culture techniques; because investigators are developing interest
on improvement of the biomass production of available resources of fast growing
trees (Cavusoglu et al., 2011). Medicinally
important Coastal plants Citrullus colocynthis (Satyavani
et al., 2011) Ruta graveolens (Gurudeeban
et al., 2012) and Aegle marmelos (Ramanathan
et al., 2011) are also, micro propagated through direct and indirect
organogenesis. Therefore, our present study focused to regenerate Heliotropium
curassavicum through direct regeneration method from stem explants; it is
the best and first report to our knowledge on large scale multiplication in
a short period of time for conservation of medicinally important species Heliotropium
MATERIALS AND METHODS
Source of explants: Heliotropium curassavicum were freshly collected
from Parangipettai (Southeast coast of Tamil Nadu, Lat 11°26N;
Log 79°46 E) India during
November 2010 and kept under shade net (50%) house environment. The specimen
was certified by Botanical Survey of India (BSI) Coimbatore and by the herbaria
of C.A.S.in Marine Biology, Annamalai University, Parangipettai, Tamil Nadu,
India (Voucher No. Aucasmb 63/2010).
Surface sterilization: The stem was used as explant material for plantlet
regeneration. The explants were washed in running tap water containing one drop
of Tween 20 for 10 min and were rinsed three times in sterile distilled water.
The stem explants were surface sterilized with ethyl alcohol for 1-5 min followed
by three times rinsed with distilled water. Finally, the explants exposed to
HgCl2 for 3 min. The explants were then washed 4 times with sterile
distilled water to remove traces of HgCl2. The experimental chemicals
were purchased from Hi-Media, Mumbai.
Media and incubation conditions: The stem explants used were planted
for direct regeneration on MS medium Murashige and Skoog (1962).
The media used for all purposes were supplemented with 3% (w/v) sucrose and
solidified with 0.8% (w/v) agar. The pH of media was adjusted to 5.7 before
autoclaving for 15 min at 121°C. The pH of the medium was adjusted with
0.1 N NaOH or 0.1 N HCl. Cultures were maintained in a growth chamber at 28°C±2°C
with 16 h light/8 h darkness. The stem explants were excised with the help of
sterile forceps and surgical blade. The stem explants were cut into 0.5 to 1.0
cm sized segments. The explants were cultured on different concentrations (1.0,
2.0 and 3.0 mg L-1) of auxin (2,4-D and NAA) and 1.0 and 2.0 of cytokinin
(6-BA) for respective shoot and root regeneration.
Micro propagation through direct regeneration techniques: The plantlet
regenerate directly from the stem explants without undergoes for callus induction.
0.5 to 1.0 cm long stem explants cultured on MS elongation media containing
various concentrations (0.5 and 1.0 mg L-1) and combinations of auxin
and cytokinin such as 2,4-dichlorophenoxy acetic acid (2,4-D), Napthyl Acetic
Acid (NAA) and 6-Benzyl Adenine (6-BA) for shoot regeneration. Directly regenerative
shoots 2 to 3 cm long with 5 to 6 leaves were induced to root in vitro
by culture on agar-solidified MS rooting medium. Elongated and healthy regenerated
multiple shoots from the elongation medium were rooted on of MS rooting medium
fortified with 0.25-2.5 mg L-1 of 2,4-D for root development. The
cultures were regularly subculture on fresh medium at two weeks interval and
observation was recorded.
Rooting and acclimatization condition: After two weeks, shoots that
had produced roots were recorded and transferred to sterile compost with ratio
(v/v/v) of organic fertilizer: sand: peat (1:2:2; 3:1:0 or 2:2:1) in polythene
bag a greenhouse. The plantlets after 10-15 days in green house; they were then
placed in the normal environment for 1 h and assessed for sighs of wilting.
The exposure was increased daily until the plants established fully under normal
environment conditions, they then transferred into the field for normal growth.
Statistical analysis: Visual observation of culture was made every week.
Data on shoot proliferation and root induction were recorded after three weeks
of inoculation and used for calculation. For each treatment 15 explants were
used for shoot proliferation and 15 shoots were used for rooting. All the treatments
were repeated thrice. Significance of the treatment effects was determined using
analysis of variance (ANOVA, p≤0.05) and comparison between mean values of
treatments were made by Tukeys
RESULTS AND DISCUSSION
In the present study, revealed that cumulative work on plant tissue culture
of H. curassavicum through direct method of regeneration from stem explant,
it mainly depends on donor tissue and influenced by type of growth regulator
and their concentration in the nutrient medium. Adventitious shoot formation
is a reliable technique for clonal propagation as it prevents somaclonal variations
in the cultures. In the present study, we observed the maximum number of shoot
regeneration after four weeks culture of MS elongation medium with 2.0 mg L-1
of 2,4-D (17.27±0.51), then compared to other combination of hormones
(Table 1). From the results, it was cleared that MS medium
with 2.0 mg mL-1 2, 4-D alone suitable for shoot multiplication as
well as shoot elongation. Then compared to other combination of auxin and cytokinin
(1.0 to 2.0 of 2, 4-D with 0.5 mg mL-1 of 6-BA and NAA). Then the
above mentioned elongation medium was regularly sub cultured with 2.0 mg L-1
of 2, 4-D. Ramanathan et al. (2011) regenerated
a medicinally important spiny tree (Aegle marmelos), they used MS media
with Kn (1.5 mg L-1)+0.5 mg L-1 2,4-D with NAA(1.0 mL
L-1) for maximum shoot formation. Among the auxins, 2, 4-dichlorophenoxy
acetic acid is most efficient and widely used in tissue culture media. In our
findings, the 2, 4-D alone plays an efficient role in the root regeneration
with 2.5 mg L-1 in MS rooting medium. Jeyachandran
et al. (2010) proved that the plant can be grown in single hormone
up to in vitro flowering and rooting. In vitro shoots were excised
from shoot clumps and transferred to rooting medium containing 2, 4-dichlorophenoxy
acetic acid (0.5-3.0 mg L-1) shown in Table 2.
Among these, the maximum number of root regeneration (6.4±0.5) and root
length (6.08±0.07) were observed in MS rooting medium fortified with
2.5 mg L-1 of 2, 4-D after 2 weeks of culture.
Figure 1 shows the In vitro direct plantlet regeneration
of H. curassavicum from stem explants. However, this being the last
stage of in vitro culture, it is important to transform the plant from
heterotrophic to autotrophic mode of nutrition, the supply of exogenous sugar
should be reduced at this time.
|| In vitro direct plantlet regeneration of H. curassavicum
from stem explants: (a) Stem explant inoculation, (a and b) Shoot induction
and Shoot elongation in MS elongation medium with 2,4-D (2.0 mg L-1)
and (c) In vitro rooting of regenerated micro shoots in MS rooting
medium with 2.5 mg L-1 of 2,4-D
||Ex vivo transplantation of H. curassavicum plantlets
under environment shade net house in polythene bag containing sterile compost
with ratio (v/v/v) of organic fertilizer: sand: peat (1: 2: 2; 3: 1:0 or
2: 2:1) in polythene bag a greenhouse
The rooting response differed according to different concentration and combinations
of hormones (Satyavani et al., 2011). Eighty
five percent of in vitro raised plantlets with well-developed shoots
and roots were transferred to ex vivo conditions. During this period
of acclimation the shoots elongated, leaves expanded and turned deep green looking
good and healthier (Afrobz et al., 2009). Ex
vivo transplantation of H. curassavicum plantlets under environment
shade net house in polythene bag containing sterile compost with ratio (v/v/v)
of organic fertilizer: sand: peat (1: 2: 2; 3: 1:0 or 2: 2: 1) in polythene
bag a greenhouse shown in Fig. 2. Acclimatized after which
the successfully sixty five percent of acclimated plants were transferred to
the pots under full sun where they grew well without any detectable phenotypic
In conclusion, the high production cost of nursery plants and the time required
for restored plants to complete their life cycle are commonly considered the
barriers to successful propagation, to overcome this problem our study provided
the simple protocol with a low cost manner.
||Effect of plant growth regulators (2, 4-D and 6-BA) on shoot
|Effect of 2, 4-D and 6-BA on shoot regeneration from stem
explants of H. curassavicum Mean±SD elongation medium after
four weeks of culture
||Effect of 2, 4-D on root induction from in vitro raised
shoots of H. curassavicum
|Effect of 2, 4-D on root induction from in vitro raised
shoots of H. curassavicum after 4 weeks of culture. Values are represented
Also it will useful for production of secondary metabolites, conservation of
medicinally important endangered coastal flora, synthesis bio medically potential
metallic nanoparticles and stress tolerant studies etc.
The authors are gratefully acknowledged to the Director and Dean, Faculty of
Marine Sciences, Annamalai University, India for providing all support during
the study period.
Cavusoglu, A., Z. Ipekci-Altas, K. Bajrovic, N. Gozukirmizi and A. Zehir, 2011.
Direct and indirect plant regeneration from various explants of eastern cottonwood clones (Populus deltoids
Bartram ex Marsh. with tissue culture. Afr. J. Biotechnol., 10: 3216-3221.Direct Link |
Afrobz, F., S. Hassan, L.S. Bari, R. Sultana and N. Begum et al
., 2009. In vitro
shoot proliferation and plant regeneration of Physalis minima
L. a perennial medicinal herb. Bangladesh J. Sci. Ind. Res., 44: 453-456..
Gurudeeban, S., K. Satyavani, T. Ramanathan and T. Balasubramanian, 2012.
Effect of antioxidant and anti-aggregating properties of micro-propagated plantlets of Ruta graveolens
. Asian J. Biotech., 11: 1497-1504..Direct Link |
Jeyachandran, R., X. Baskaran and L. Cindrella, 2010. In vitro
direct regeneration of nodal explant of Justicia prostrate
Gamble. Int. J. Biol. Technol., 1: 90-93.
Kirtikar, K.R. and B.D. Basu, 1998.
Indian Medicinal Plants. 2nd Edn., International Book Distributors, Dehradun, India, ISBN: B-0007B1B64, pp: 804-806
McAuley, M., 2007.
Plant of the Month: Wildflowers of the Santa Monica Mountains. Santa Monica Mountains Trails Council, Agoura Hills, California
Murashige, T. and F. Skoog, 1962.
A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol. Plant., 15: 473-497.CrossRef | Direct Link |
Ramanathan, T., K. Satyavani and S. Gurudeeban, 2011. In vitro
plantlet regeneration of gum-bearing tree Aegle marmelos
. E-Int. Scientific Res. J., 3: 47-50.
Satyavani, K., T. Ramanathan and S. Gurudeeban, 2011.
Effect of plant growth regulators on callus induction and plantlet regeneration of bitter apple (Citrullus colocynthis
) from stem explant. Asian J. Biotechnol., 3: 246-253.CrossRef | Direct Link |
Sharma, R.A. B. Singh, D. Singh and P. Chandrawat, 2009.
Ethnomedicinal, pharmacological properties and chemistry of some medicinal plants of Boraginaceae
in India. J. Med. Plants Res., 3: 1153-1175.Direct Link |
Wolff, K.C.F., 1977.
Thia Generationis, 3rd Edn., Habae ad Salam.