Effect of Amino Acids and Growth Regulators on Indirect Organogenesis in Artemisia vulgaris L.
S. Pradeep Kumar
B.D. Ranjitha Kumari
Artemisia vulgaris L. (mugwort) belongs to the family Asteraceae and is a tall aromatic perennial herb. Mugwort contains volatile oils, sesquiterpene lactones and flavonoids used for insecticidal, antimicrobial and antiparasitical properties. All parts of the plant are used for antihelmintic, antiseptic, antispasmodic, carminative, cholagogue, digestive, expectorant, nervine, purgative and stimulant. This study describes the effect of amino acids and growth regulators on callus induction, multiple shoot and root induction using cotyledonary explants. Murashige and Skoog medium supplemented with B5 vitamins containing 2, 4D, NAA and cysteine combination was found better response for callus induction. Maximum number of multiple shoots (95.1%) per explants after 20 days of culture, with combination of BA, TDZ and tyrosine was found better response in the medium. NAA, AgNO3 and glutamine combination produced maximum number (98.7%) of roots per explants in the medium. Plants produced from de novo regeneration on excised tissues will be useful for crop improvement through genetic engineering and cell culture techniques.
Artemisia vulgaris L. (mugwort) belongs to the family Asteraceae and
is a tall aromatic perennial herb. Mugwort contains volatile oils, sesquiterpene
lactones and flavonoids used for insecticidal, antimicrobial and antiparasitical
properties. In traditional medicine, this plant is being widely used for the
treatment of diabetes, epilepsy, depression insomnia and anxiety stress (Walter
et al., 2003).
All parts of the plant are antihelmintic, antiseptic, antispasmodic, carminative,
cholagogue, digestive, expectorant, nervine, purgative and stimulant. The essential
oils of the plant were reported to exhibit 90% mosquito repellency against Aedes
aegypti, a mosquito that transmits yellow fever (Ram and
Mehrotra, 1995). A paste or powder of the leaves is applied over skin diseases
(Kapoor, 2000). In recent years, there has been an increased
interest in in vitro techniques, which offers powerful tools for germplasm
conservation and the mass multiplication of many threatened plant species (Murch
et al., 2006). In vitro micropropagation using shoot tips
(Arditti and Ernst, 1993), stem nodal segments (Nayak
et al., 1997) and root tips (Park et al.,
2003) has been successfully used for propagation of a number of orchids
either for conservation or for commercial production. Direct regeneration of
multiple shoots without an intermediate callus phase shortens the duration for
regeneration and reduces the likelihood of incidence of Somaclonal variation
(Polonca et al., 2004). Dual phase or liquid
medium overlay culture technique is a novel approach in plant tissue culture
which improves the rate of regeneration and development (Visure,
1985; Thomson et al., 2007; Pullman
and Skryabina, 2007; Sim et al., 2007). Such
a technique involves the use of an agar solidified medium overlaid with a liquid
medium fraction. This technique has been shown to have certain advantage over
the use of conventional agar solidified medium or purely liquid medium. Induction
of multiple shoots in a monopodial orchid hybrid (Aerides vandarum Reichb.f
x Vanda stangeana Reichb.f) using thidiazuron and analysis of their genetic
stability (Kishor and Devi, 2009). Besides development
of a novel micropropagation system, an ef?cient growth regulator capable of
inducing tremendous organogenesis from different explants is necessary. Thidiazuron
(TDZ: N-phenyl-N0-[(1, 2, 3-thidiazol-5-yl)urea]), a non-purine cytokinin compound,
has been shown to exhibit a stronger effect than N6-benzyladenine (BA) on
in vitro morphogenesis of a wide range of crops (Malik
and Saxena, 1992; Nayak et al., 1997; Vinocur
et al., 2000; Park et al., 2003; Sujatha
and Kumari, 2007).
Exogenously added amino acids play an important role in plant tissue culture
but culture media of existing regeneration protocols are scarcely supplemented
with amino acids. Effect of various amino acids on shoot regeneration of sugarcane
Sacchrum officinarum L. (Asad et al., 2009).
Specific media components involving amino acids have been found to play an important
role on tissue culture systems of certain species (Benson,
2000). Amino acids have been used as organic nitrogen source in in vitro
cultures of several species as alfalfa, maize, sorghum, pineapple, rice and
other monocots to enhance somatic embryogenesis and regeneration (Skokut
et al., 1985; Claparols et al., 1993;
Rao et al., 1995; Hamasaki
et al., 2005; Grewal et al., 2006).
It has been suggested that positive effect of organic nitrogen, in comparison
to that of inorganic sources is associated to enhanced mobility of the former
at a lower energy cost than the later (Kim and Moon, 2007).
Therefore, the objective of the present study was to conduct detailed and systematic
studies on three different amino acids to determine optimum amino acid concentration
to develop the most efficient Artemisia vulgaris regeneration system.
Three different amino acids, glutamine, cysteine and tyrosine were evaluated
for their ability to induce callus, multiple shoots and root induction in Artemisia
vulgaris. To our knowledge, this is the first report in which a number of
different amino acids have been compared for their effects on Artemisia vulgaris
MATERIALS AND METHODS
Plant Material, Culture Medium and Culture Conditions
Artemisia vulgaris L. seeds were collected from Johnnys selected
seeds, USA located at Winslow, Maine during the year July 2007-2008. In vitro
seed germination was carried out and seed sterilization processes were described
previously by Sujatha and Kumari (2007). Surface sterilized
seeds were inoculated Murashige and Skoog (1962) germination
(MSG) medium. Cultures were initially incubated in darkness for 5-7 days at
a temperature of 23°C to facilitate germination. Later, they were transferred
to photoperiodic conditions and maintained for another 28-30 days for seedling
In Direct Organogenesis and Plantlet Regeneration
Calli were induced from cotyledonary nodal explants excised from 30-d-old
seedlings grown on MS medium containing B5 vitamins, 2% (w/v) sucrose, 0.8%
(w/v) Difco Bacto Agar (Hi-media, Mumbai, India) and supplemented with various
concentrations (0, 0.5, 1.0, 2.0 and 3.0 mg L-1) of each of 2, 4-dichlorophenoxyacetic
acid (2, 4-D), α-Naphthalene acetic acid (NAA) and cysteine (cys). With
controls without additional amino acids, all the experiments were repeated three
times and completely randomized design experiment was conducted with three replicates
per treatment. The frequency of explant forming embryogenic callus, color and
texture were recorded after 6-9 weeks of culture. After 5-7 days of callus initiation,
the primary tiny calli of explants were separated aseptically from the source
of explants, so that no contact of parental tissue remained and set them again
on the same medium for proliferation of calli without root-shoot differentiation.
Repeated sub-culturing was done after every two weeks for maintenance and proliferation
of the calli. The quantitative measurement of callus growth was estimated in
terms of percentage of callus.
Multiple Shoot Induction
Cotyledonary nodal explants cuttings (1 cm long) were inserted either vertically,
with 2±3 mm of the cutting inserted into the medium and the apical 7±8
mm protruding, or horizontally on the surface of the culture medium, containing
B5 vitamins with various concentrations (0, 0.5, 1.0, 2.0 and 3.0 mg L-1)
BA, TDZ and Tyrosine (Tys) combination with controls without additional amino
acids. At the end of the incubation period, the number of shoots which developed
was recorded separately for each cutting end. All Plant Growth Regulators (PGRs)
were purchased from Sigma (St. Louis, MO, USA). The pH of the medium was adjusted
to 5.7 with 1 N NaOH or HCl and autoclaved at 121°C and 1.05 kg cm-2
pressure for 20 min.
Rooting and Transplantation
To induce rooting, individual elongated shoots (5-7 mm) in length were excised
and Cultured for 60 day on 10 mL of semi-solid medium containing different types
of auxins and amino acid viz., α-Naphthalene acetic acid (NAA) (0.5-3.0
mg L-1) silver nitrate (AgNO3) (0.5-1.5 mg L-1)
and glutamine (Gln) (0.5-1.0 mg L-1) individually. One set of the
cultures were inoculated in basal MS medium without the addition of amino acid
and kept as control. The regenerated plants with well developed sufficient root
system were ready for transfer to soil. When the plantlets remained in culture
they were brought out of the controlled environment of growth room and were
kept in the room temperature for 2-3 days to bring then in the contact of normal
temperature. The plantlets were then rescued very carefully from the culture
tubes. Agar attached to the root was washed gently under running tap water.
Immediately after that they were transplanted to small pots containing sterilized
ground soil, sands and cow dung in the ratio of 1:2:1. The pots with plantlets
were kept in shade place and necessary cultured management was undertaken for
good growth and development of the plant. After 7 days, the pots with plants
were transferred to direct sunlight and after 15-25 days plantlets were finally
transferred in new pots.
Slide write software was used for graphical presentation of data and test
of significance was carried out using analysis of variance. Treatment means
were separated using the Duncan's Multiple Range Test (PROC GLM, SAS Institute,
Data were tested by ANOVA and level of significance (Gomez
and Gomez, 1976).
Callus was induced from cotyledonary explants on MS medium containing 2,
4-D, NAA and cysteine (cys) combination (1.5+1.0+0.5 mg L-1) was
found most effective for callus induction when compared with 2, 4-D and NAA
(2.0+1.0 mg L-1) (Table 1). It produced 72.4, 83.3,
96.7 and 90.6% callus respectively in 2, 4-D, NAA and Cysteine combination,
when compare to 2, 4-D, NAA produced (67.8, 76.6, 89.3 and 83.1%) (Fig.
1a, b). The nature of cotyledonary nodal explants derived
calli were yellows compact but fragile and tends to be very dry with increasing
concentration of 2, 4-D.
Effect of Growth Regulators on Indirect Multiple Shoot Induction
The effect of growth regulators on shoot formation at the basal end of Cotyledonary
nodal explant cuttings inserted in the medium supplement with BA, TDZ and Tyrosine
(Tys) combination (2.0+1.0+0.5 mg L-1) was found most effective for
development of multiple shoots when compared to BA, TDZ in the medium (Fig.
2a-f). It produced (70.8, 79.4, 97.7 and 88.6%) of multiple
shoots, respectively. Where as BAA and TDZ produced (59.5, 66.8, 79.9 and 74.5%)
(Fig. 3a, b). Similar result was also reported
in Artemisia vulgaris L. by Sujatha and Kumari (2007)
but it is in stem and node explants. Frequency of shoot induction was observed
to be more in 2-3 weeks of incubation. In three different modes of Amino acids
supplementation the experiment was carried out (Table 2).
Rooting and Establishment in Soil
A proportion of the regenerated shoots rooted without the addition of an
auxins to the medium (Table 2). The rooting frequency was
higher in the medium supplemented with NAA+AgNO3+Glutamine (2.0+1.0+0.5
mg L-1) combination when compare to NAA+AgNO3 (2.0+1.0
mg L-1) in the medium.
|| Effect of growth regulators on callus induction of in
vitro raised Cotyledonary Nodal Explants of A. vulgaris L.
|Evaluation was made after 2 weeks of culture. Treatment means
followed by different letters are significantly different from each other
at 5% levels of significance (p≤0.05) according to DMRT
induction percentage of growth response |
||Effect of various combinations of growth regulators and amino
acids on in Direct Organogenesis in Artemisia vulgaris L. (a) In
vitro seed germination, (b) fragile callus induction, (c) compact callus
induction, (d) indirect multiple shoot, (e) rooting from cotyledonary node
and (f) elongated shoots with roots
||Effect of various concentrations (mg L-1) and combinations
of growth regulators and amino acids on regeneration of shoot and root induction
from cotyledonary nodal explants
|Evaluation was made after 3 weeks of culture Treatment means
followed by different letters are significantly different from each other
at 5% levels of significance (p≤0.05) according to DMRT
b) Multiple shoot induction percentage of growth |
b) Rooting induction percentage of growth response |
The percentage of rooting was high in NAA+AgNO3+Glutamine (89.2+91.8+98.7+93.1%)
(Fig. 4a, b) combination, when compare to
NAA+AgNO3 (77.3+81.5+85.3+80.6%) in the medium. A control group was
also maintained. Plantlets significantly developed lengthy roots and root induction
was strengthened within 15 days of culture. After 2 weeks, plantlets developed
primary and secondary root system (Fig. 2). Frequency of rhizogenesis
was almost 98%.
The successfully rooted plantlets were transferred to plastic cups containing
sterile garden soil, farmyard soil and sand (2:1:1) for hardening plantlets
were maintained in the culture room (25±1°C) conditions initially
for 4 weeks and after transferred to normal laboratory conditions and maintained
for about 1 week. Finally the plantlets were transferred to Botanical Evaluation
Garden and maintained. The survival rate decreased to 98.3 and 80.7%, respectively
after 5-6 weeks. There was no detectable variation among the acclimatized plants
with respect to morphological and growth characteristics. All the micropropagated
plants were free from external defects.
Amino acids have been found critical to induce somatic embryogenesis in plant
tissue culture medium. In orchard grass, embryos formed on amino acid containing
medium showed high percentage of conversion and considerably less incidence
of precocious germination (Trigiano et al., 1992).
In vitro regeneration of multiple shoots directly from well developed
seedlings of monopodial vandaceous orchids is a novel technique for their clonal
propagation. Such a technique was reported earlier by Thomas
and Michael (2007) and Kishor and Sharma (2008) using
agar solidified medium. The main effect of BAP, TDZ and Tyrosine on Indirect
regeneration in Artemisia vulgaris multiple shoot production was evaluated
after 2 weeks, when compare to previous study conducted by Kishor
and Devi (2009) and also Sujatha and Kumari (2007).
It was observed that type of amino acids and amount used in the medium had significant
effect on the induction of multiple shoot. Tyrosine promoted maximum shoots
production among the tested amino acids at 0.5 mg L-1, while other
non-amino acid treatments induced fewer shoots and the number of shoots developed
increases with concentration up to at least 3.0 mg L-1.
The few reports in which this pathway of regeneration has presumably been studied
show a similar response (Goh et al., 1995) reported
that some direct shoot regeneration occurred in the upper cut end of epicotyl
segments of Citrus grandis on basal medium without added hormones. The
number of multiple shoots induced by the present technique was approximately
twice more than that obtained by Thomas and Michael (2007)
for the monopodial orchid Rhyncho- stylis retusa (8.4 shoots per seedling) when
it was cultured on agar solidified half-strength MS medium supplemented with
4.0 L M (2 mg L-1) TDZ. So far, as the effectiveness of TDZ over
benzyladenine (BA) in the induction of multiple shoot is concerned, Sujatha
and Kumari (2007) obtained a two-fold increase in average number of shoots
by 4.5 μmo L-1 (2 mg L-1) TDZ over 4.4 μmo L-1
(2 mg L-1) BA on multiple shoot induction in Artemisia vulgaris
in vitro but these results were in stem and nodal explants only. Vinocur
et al. (2000) also reported the superiority of TDZ over BA on shoot
regeneration of root explants of Populus tremula and found that TDZ had exerted
a 10-fold increase in the number of regenerated shoots.
Maximum percentage of root formation (Fig. 2) from cotyledonary
nodal (98.7%) was observed on medium supplemented with NAA+AgNO3+Glutamine.
This result is in agreement with the findings of Dhar et
al. (2000) on Pittosporum napaulensis and Agrawal
and Sardar (2006) on Cassia angustifolia, where IBA was found better than
NAA and IAA to induce the formation of maximum number of roots. The promotion
of rooting from callus by IBA has also been reported in many other plant species
(Saritha et al., 2002; Soniya
and Das, 2002; Soniya and Sujitha, 2006). The overall
objective of the current study was to develop a system for the mass propagation
and aseptic growth of A.vulgaris plantlets derived from intact seedling
system can be a prolific tissue for the biochemical characterization of medicinally
active components and for the selection and cloning of superior individual genotypes.
Plants produced from de novo regeneration on excised tissues will be
useful for crop improvement through genetic engineering and cell culture techniques.
These potential use of amino acids in promoting regeneration in other plant
species could be applied in commercial propagation of elite cultivars.
I wish to thank anonymous reviewer for their comments on the manuscript and similarly I am highly grateful to Dr. B.D. Ranjitha Kumari, for her support and guidance to the work and manuscript.
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