J. CYTOL. GENET. VOL.

17 (NS): 7–14 (2016)

IN VITRO SHOOT REGENERATION FROM NODAL EXPLANTS OF VITEX

AGNUS-CASTUS L.
L. RAJANNA, P. VIJAY RAGHAVAN* AND G. S. SHAILAJA SHARMA
Department of Botany, Bangalore University, Jnanabharathi, Bengaluru 560 056
*For correspondence. Email: vijayragh@gmail.com

(Received 27 November 2015, revised accepted 28 February 2016)

SUMMARY

An efficient micropropagation protocol has been standardized for the shoot

regeneration from the nodal explants of Vitex agnus-castus, an important aromatic
medicinal plant. Nodal segments were cultured on MS medium supplemented with
various concentrations of plant growth regulators. Among them, MS + 2.5 mg/l IBA
or MS + 3.0 mg/l IBA and MS + 2.0 mg/l IAA + 1.0 mg/l KN and MS + 2.0 mg/
l NAA + 1.0 mg/l KN were found to be the best for inducing multiple shoots under
single hormone and with different combinations respectively. Under these hormonal
treatments, the regenerated plantlets were able to develop rooting in the same media
and were successfully hardened and planted in the field within 2 months from the date
of inception of the experiment.

Keywords: Vitex agnus-castus, nodal explants, micropropagation.

INTRODUCTION

Vitex agnus-castus, commonly called “Chaste tree” or “Vitagnus” is an important aromatic

medicinal plant. V. agnus-castus a deciduous shrub native to European, Mediterranean and Central
Asian countries, has long, finger-shaped leaves and displays fragrant blue-violet flowers in
midsummer and bears purple black berries that are yellowish inside, resembling peppercorn with
an aromatic odour. Upon ripening, the berry is picked and allowed to dry. The berries were used
by monks during the Middle Ages to suppress sexual desire; hence, its common names – monk’s
pepper and chaste tree. The twigs of this shrub are very flexible and were used for furniture in
ancient times. References to V. agnus-castus go back to more than 2000 y, describing it as a
healing herb. Ancient Egyptians, Greeks and Romans used it for a variety of health problems. In
400 BC, Hippocrates recommended chaste tree for injuries and inflammation. Later, Greek botanist
Dioscorides recommended it specifically for inflammation of the womb and lactation (Artz 2007,
Roemheld-hamm 2005, Sarkar 2009).

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MS medium used for shoot induction contained full strength of MS inorganic salts and vitamins, 3% su crose
(w/v) and 0.8% agar (w/v). The medium was fortified with either single hormone (2,4-D or IAA or IBA or NAA or
BAP or KN) or combination of hormones (one of the auxins with one of the cytokinins). Medium withou t hormone
was used as control. In both the cases the pH of the medium was adjusted to 5.8 before autoclaving for 15 min.
Explants were cultured in either 25 × 150 mm borosil test tubes with 15 ml medium in each tube havin g single explant
or cultured in 250 ml glass bottles with 35 ml medium in each bottle consisting of 3 explants. For in vitro shoot
induction and root induction, the cultures were incubated at 25 ± 2° C with 16 h illumination under light intensity
of 3000 lux provided by cool florescent lamps.

The shoot induction experiment was carried out with either 12 replicates (test tubes) with each replicate having
1 explant or with 6 replicates (bottles) with each replicate having 3 explants. The experiments wer e repeated thrice.
The shoot length and number of shoots were recorded after 45 d of inoculation. The data was subject ed for mean
length of shoot, mean number of shoots per explant and percentage of shoot and root induction and sh oots survival
rate after hardening. The photographs were taken using Nikon coolpix S6700 camera.

OBSERVATIONS

Multiple shoots of V. agnus-castus were proficiently produced from nodal explants. The
concentrations of auxins and cytokinins used in the present investigation ranges from 0.5 to 4.0
mg/l and the results are presented in Table 1. They were used either singly or in combinations.
The nodal explants were able to produce multiple shoots on MS media fortified with all the tested
auxins and BAP. However, no response was observed when KN was used singly. Among the
auxins, when 2,4-D was added with MS at the concentrations of 0.5 to 1.5 mg/l it did not trigger
shoot initiation. However, in MS + 2 to 3.5 mg/l (2,4-D), shoot emergence (0.5 to 1.5 cm in
length) has been observed in 72.2% of the cultures. In case of IAA, maximum of 6 shoots
developed at 4 mg/l and in case of IBA and NAA, at the same concentration, highest number of
shoots (8) have developed.
The most effective combinations were MS + 2.0 mg/l IBA + 1.0 mg/l BAP, MS + 3.5 mg/
l IBA + 3.0 mg/l BAP, MS + 3.5 mg/l IBA + 1.5 mg/l BAP, MS + 2.0 mg/l IAA + 1.0 mg/l BAP,
MS + 2.0 mg/l IAA + 1.0 mg/l KN, MS + 2.0 mg/l 2,4-D + 1.0 mg/l KN and MS + 2.0 mg/l
NAA + 1.0 mg/l KN. The nodal explants of V. agnus-castus showed initial shooting responses just
after 3 d of innoculation in presence of these combinations and also produced roots in the same
media. However, the growth rate, number of multiple shoots produced and the duration required
for rooting by the explants varied in presence of different combinations. In case of combinations
involving IBA and BAP, the highest number of 15 multiple shoots were produced, however, in case
of MS + IAA + BAP, only 3 multiple shoots were seen. In contrast, when 1.0 mg/l KN was used
with IAA, as many as 40 multiple shoots developed showing faster growth rate. Similar responses
were observed when 2,4-D and NAA are combined with 1 mg/l of KN
Regenerated shoots obtained from explants on MS + 2.0 mg/l IBA + 1.0 mg/l BAP exhibited
slow growth rate and were ready for hardening after 65 d. The nodal explants of V. agnus-castus

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RAJANNA ET AL.:

TABLE 1: Effect of auxins and cytokinin on shoot induction in V. agnus-castus.

Hormones (mg/l) Shoot induction No. of shoots induced Shoot length in cm
used with MS (%) per explant (Mean ± SE) (Mean ± SE)
2,4-D
Up to 1.5 Nil
2 38.89 0.33 ± 0.21 0.56 ± 0.04
2.5 72.22 2.0 ± 0.0 1.11 ± 0.05
3 33.33 1.33 ± 0.21 1.05 ± 0.07
3.5 16.67 0.33 ± 0.21 0.58 ± 0.05
4 Nil
IAA
0.5 Nil
1 16.67 0.33 ± 0.21 0.5 ± 0.05
1.5 33.33 0.66 ± 0.42 0.73 ± 0.06
2 77.78 1.66 ± 0.21 1.6 ± 0.06
2.5 77.78 3 ± 0.25 2.35 ± 0.08
3 83.33 3.5 ± 0.22 3.7 ± 0.09
3.5 94.44 4.8 ± 0.16 5.33 ± 0.07
4 77.78 6.0 ± 0.0 3.1 ± 0.13
IBA
0.5 72.22 1 ± 0.36 1.55 ± 0.09
1 77.78 1.66 ± 0.21 1.78 ± 0.07
1.5 83.33 2.33 ± 0.33 2.63 ± 0.07
2 94.44 4.0 ± 0.0 3.15 ± 0.07
2.5 100.00 5.16 ± 0.3 5.55 ± 0.16
3 100.0 8.16 ± 0.16 3.78 ± 0.10
3.5 83.33 10.0 ± 0.0 2.41 ± 0.15
4 77.78 8.83 ± 0.65 1.65 ± 0.10
NAA
Up to 2 Nil
2.5 50.00 3.66 ± 0.33 1.65 ± 0.09
3 77.78 4.33 ± 0.21 2.75 ± 0.12
3.5 83.33 5.5 ± 0.34 4.33 ± 0.14
4 72.22 6.0 ± 0.0 3.11 ± 0.10
BAP
Up to 0.5 Nil
1 33.33 1.66 ± 0.21 1.81 ± 0.19
1.5 38.89 3.66 ± 0.33 2.65 ± 0.26
2 61.11 5.83 ± 0.30 2.98 ± 0.29
2.5 100.00 8.0 ± 0.0 3.35 ± 0.09
3 100.00 6.0 ± 0.0 3.76 ± 0.12
3.5 77.78 6.16 ± 0.47 2.41 ± 0.07
4 38.89 5.66 ± 0.21 1.6 ± 0.05

produced 13–15 multiple shoots and rooted on the same media after 35 and 25 d, and were ready
for hardening after 48 and 35 d in presence of MS + 3.5 mg/l IBA + 3.0 mg/l BAP and MS +
3.5 mg/l IBA + 1.5 mg/l BAP respectively. MS + 2.0 mg/l IAA + 1.0 mg/l BAP influenced the
explants to produce 2–3 multiple shoots, which rooted in the same media after 50 d and were
ready for hardening after 70 d. In case of MS + 2.0 mg/l 2,4-D + 1.0 mg/l KN the explant
produced 35–40 multiple shoots, rooted in the same media after 35 d and were ready for hardening
at about 48 d after inoculation. The explants showed faster growth rate in presence of the
combinations MS + 2.0 mg/l IAA + 1.0 mg/l KN and MS + 2.0 mg/l NAA + 1.0 mg/l KN. Under
the influence of these combinations the explants produced large number of multiple shoots

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RAJAN NA ET AL.:

used alone with MS did not trigger the shoot initiation in the explants. However, BAP was found
to enhance shoot initiation. MS media fortified with 2.5 mg/l BAP and 3.0 mg/l BAP exhibited
100% shoot initiation with 8 and 6 multiple shoots respectively. In the literature, there are several
examples of BAP being used as potential hormone for the induction of single and multiple shoots.
BAP’s ability of bud breaking and single shoot and multiple shoot induction has been demonstrated
in Litchi chinensis, Gymnema sylvestre, Aristolochia tagala (Khan & Ahmed 2005, Manonmani &
Francisca 2012, Rajanna & Sharma 2015). However, in the present study on the effect of BAP
on shoot induction on VAC the explants took longer period (10 d) for shoot initiation when
compared to explants grown on MS fortified with 2.5 or 3.0 mg/l IBA (3 d).

However, among the tested auxins, explants responded to all the auxins but showed very
less response to 2,4-D when compared to IAA, NAA or IBA. In case of 2,4-D, the explants
showed only shoot initiation but did not grow beyond 1.11 cm (MS + 2.5 mg/l 2,4-D), also the
explants took longer period (more than 25 d) for initiation of shoots. In case of MS media
fortified with different concentrations of IAA, MS + 3.5 mg/l IAA and MS + 4 mg/l IAA showed
better results by producing 4.8 and 6 multiple shoots respectively and in case of MS supplemented
with various concentrations of NAA, MS + 3.5 mg/l NAA and MS + 4 mg/l NAA gave better
results by producing 5.5 and 6 multiple shoots respectively. However, percentage of shoot
induction was less in MS fortified with IAA or NAA when compared to MS with IBA. The best
media composition with single hormone concentration was found to be MS + 2.5 mg/l IBA and
MS + 3 mg/l IBA. In both of these media compositions shoot initiation was observed by third
day after inoculation. They also showed high rate of (100%) shoot induction. The developed
shoots showed root initiation on the same media after 30 d. The phenomenon of bud breaking
and shoot elongation with auxins (IAA, NAA and IBA) alone indicates that shoot regeneration is
not essentially a function of cytokinin activity and has been demonstrated in Dalbergia sissoo and
Glycine wightii (Datta & Datta 1983, Pandey & Bansal 1992). These are some of the very few
examples found in the literature where auxin alone is used for shoot induction.

Among the combinations used, two best combinations observed are MS + 2.0 mg/l IAA +
1.0 mg/l KN and MS + 2.0 mg/l NAA + 1.0 mg/l KN. The explants inoculated onto these
combinations exhibited preeminent responses by producing maximum number of multiple shoots
(35–40) which showed rooting in the same after 25 d and were ready for hardening after 35 d.
Such synergistic effect of IAA and KN on growth parameters has been demonstrated in pepper,
D. sissoo, faba bean etc. (Agrawal et al. 1988 Dawra et al. 1984, Sadak et al. 2013) and that of
NAA and KN has been demonstrated in Zephyranthes and Nicotiana spp. (Philomena et al. 1987,
Schaeffer & Smith 1963). In all the cases (either MS media fortified with single hormone or
combination of hormones) all the regenerated rooted plantlets were successfully hardened and
showed 100% survival rate when exposed to natural environmental conditions.

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 MICROPROPAGATION OF VITEX AGNUS-CASTUS

Attempts have been made previously by Chamandoosti (2007) and Balaraju et al. (2009) to
develop protocols for the in vitro regeneration of V. agnus-castus. Chamandoosti (2007) has used
seeds to obtain seedlings which are then used as explants to obtain the in vitro shoots. This
method involved mechanical treatment of seeds to remove seed coat which were then inoculated
onto media to obtain seedlings which were in turn excised and cultured in vitro to regenerate
shoots. This method is an indirect and time consuming process when compared to in vitro culture
of nodal explants to obtain regenerated plantlets. Balaraju et al., (2009) used apical and nodal
explants to obtain in vitro regenerated shoots. They have observed a maximum of 94.5%
regeneration, 7.7 multiple shoots and have used different media for shoot induction and root
development. Nevertheless, in the present study, 100% regeneration with maximum of 8.16
multiple shoots (under single hormone treatment) and 35–40 multiple shoots (under the treatment
of combination of hormones) were obtained which were able to develop roots in the same media
and were successfully hardened after 35 d of innoculation.

In the present study, an efficient protocol for the micropropagation of a highly important
medicinal plant V. agnus-castus has been developed in which a large number of multiple shoots
could be regenerated which could develop roots in the same media, could be hardened and exposed
to natural environmental conditions in a very short duration of just one and a half to 2 months
from the date of inoculation.

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