In vitro Plant Regeneration from Leaf Explants of Artemisia vulgaris L . – A Medicinal Herb

A reliable protocol for callus induction and regeneration were developed for leaf explants of Artemisia vulgaris L. Percent of callus induction and regeneration were higher in the young leaves. MS medium containing 1.0 mgl BAP and 3.0 mgl NAA is the optimum concentration for induction of callus. So produced callus was subcultured on Murashige – Skoog (MS) medium with 1.0 mgl 6-benzylaminopurine (BAP), 3.0 mgl gibberellic acid (GA3) produced the highest mean number of shoots (35.85±0.81) per explant. Half strength of MS was found to be the best for rooting, however, addition of IAA (0.5-1.0 mgl) was found essential to induce longer roots. More than 84% of the rooted plants were established in polycups after hardening.


Introduction
Artemisia vulgaris Linn, an important perennial medicinal herb belongs to the family Asteraceae.The plant is aromatic, shrubby 0.6-2.4mhigh and pubescent.The plant has a hot, sharp and pungent taste.It is considered to be a valuable stomachic, deobstruent and antispasmodic.The expressed juice is used in diseases of children.The leaves and shoot tips are administered in nervous and spasmodic affections connected with debility, in asthma and diseases of the brain.It shows antispasmodic and anthelmintic activity (Kirtiker and Basu, 1935).

Plant material
Leaf explants from 6-month-old mature plant of Artemisia vulgaris L. were used to initiate in vitro cultures.The leaf segments were washed with 5% (w/v) bavistin, 10% (w/v) antibiotic and 5% (w/v) Tween 20 by continuous shaking for 20 min followed by washing in tap water, then rinsed three to five times with double distilled water.

Shoot induction and elongation
Shoots were induced by transferring the leaf calli on MS medium containing different concentrations and combinations of BAP (0.5-1.0 mgl -1 ) and GA 3 (0.5-3.0 mgl -1 ).Elongation of shoots were also observed on the same medium.

Rooting and Acclimatization
Microshoots were excised from the parent cultures and transferred onto half strength MS medium supplemented with different concentrations and combinations of IBA, IAA and NAA for root induction.The rooted shoots were gently removed from the culture vessels, washed under running tap water and transferred to polycups containing sand:soil:vermiculite (1:1:1) in the greenhouse conditions for acclimatization.

Combination of BAP and GA 3
The effect of GA 3 on the embryogenic frequency became significant after two weeks in the induction medium and caused a noticeable rise in the intensity and number of embryos and shoots in Artemisia vulgaris.There are many reports that GA 3 enhances the number of somatic embryos and shoots from the calli of Santalum album L. (Lakshmi et al., 1979); Rumex acetosella L. (Culafic et al., 1987); Mentha piperita (Ghanti et al., 2004) and Phyllanthus amarus (Chitra et al., 2009),

Rooting and Acclimatization
The shoots formed in the calli derived from leaf segments were transferred onto half strength MS medium supplemented with different concentrations and combinations of IAA, NAA and IBA for root induction.Data were recorded for percentage of shoots forming roots, number of roots/shoots and root length.Root induction occurred in 10-12 days of culturing with highest root induction (87%) on MS medium containing 0.5 mgl -1 (IAA) (Table3 & Fig. 1D).Root formed in IAA were thick, long and white.The role of IAA as an effective root inducing auxin had also been in Amygdalus communis L. (Akbas et al., 2009) and Centaurium erythraea (Piatczak and Wysokinska, 2003).
The plantlets having sufficient root and shoot system were taken out from the culture vessels and were washed under running tap water to remove the agar attached to shoots.The plantlets were transferred to polycups (Fig. 1E) containing autoclaved sand: soil: vermiculite (1:1:1) mixture in the greenhouse condition.They were transferred to the field condition.
In conclusion, the above protocol describes rapid callus induction from leaf explants, which can ensure a stable supply of this medicinally oil yielding plant irrespective of any seasonal variation and may serve as a better source for biological active compounds.

Table 1 .
Callogenesis from leaf explants of Artemisia vulgaris L. at different concentrations of BAP and NAA.
*Mean of 12 replicates per treatment in three repeated experiments

Table 2 .
Effect of growth regulator on in vitro shoot induction from leaf explants of Artemisia vulgaris L.

Table 3 .
Effect of different concentrations of IAA on rooting of in vitro shoots of Artemisia vulgaris L.
*Mean of 12 replicates per treatment in three repeated experiments