Two and a Bud 58:80-86, 2011
RESEARCH PAPER
Crop diversification for tea plantation
multiplication of elite cultivars
In vitro approach for mass
M.P. Borthakur, D.K. Sarmah, J. Hazarika and S.c. Nath
Department of Medicinal Aromatic and Economic Plants, North East Institute of Science & Technology, Jorhat-785006,
Assam, India
ABSTRACT
Crop diversification is intended to give a wider choice in production and expansion of a variety of crops with value
addition. It proves to be of paramount importance in meeting the challenges of economic return by minimizing the
risk of crop failure that might result from the vagaries of climate or any other associated factor.
Looking at the crop diversification
Chlorophytum arundinaceum Baker, Curcuma aromatica Salisb, HOl1lalomella
aromatica SchoU and Plumbago sps., viz. P. rosea Linn. and P. zeylanica Linn., locally known as Bon Haldi, Safed
Musli, Sugandh Mantri, Agyachit and Chitrak respectively may be selected to grow in the tea gardens.
However, these plant species are difficult to propagate and Tissue Culture
broaden the genetic base of the species for mass utilization programme.
Technique
has been the sole means to
Shoot multiplication and plant regeneration were achieved from freshly sprouted shoots cultured on selected basal
media. For axenic establishment followed by subsequent experiments, Murashige and Skoog (Murashige and Skoog,
t 962) basal media was selected for all the plant species. Although bud-break was dependent on 6-benzylaminopurine
(BAP) supply, the synergistic combination of BAP (0.5-3.0 mg/I) and Ii - Napthalene acetic acid (NAA) 0.1-1.0 mg/
I had favoured to induce optimum frequency of shoot formation even with simultaneous rooting in C. aromatica anlt
and Plumbago sps. an additional step for root induction was required
H. aromatica. In case of C. arundinaceum
where indole 3-butyric acid (IBA) was found to be the best root inducing hormone. Subculturing of shoot enabled
continuous production of healthy shoots with similar frequency. Micropropagated
plants established in garden soil
were found uniform and identical to the donor plants with respect to their growth characteristics.
Thus the
micropropagation
procedure could be a useful tool for raising stock for plantation of genetically homogenous
materials for field cultivation.
INTRODUCTION
high demand either in medicinal or in perfumery industry
can be considered for cultivation in the non-tea areas of tea
Implementation of diversified crop system is a productive
way to maximize production and profit. Considering the
fact, tea, the biggest agro-industry of Assam, has also been
embarked on a diversification plan to retain its profitability
and market share looking at options of alternative cropping.
Presently, some of the tea companies are in the process of
expanding its product portfolio by launching new products
of tea, and also taking steps to grow various income
generating medicinal and aromatic herbal plants.
gardens. However, the nature of diversification differs
across region due to existence of wide heterogeneity in agroclimatic and socio-economic environments.
In this paper we have cited a few high valued medicinal
and aromatic plants of North East India, viz. Chlorophytum
arundinaceum
Baker, Curcuma aromatica
Salisb,
Homalomena aromatica Schott and Plumbago sps., viz. P
rosea Linn and P. zeylanica Linn, locally known as Bon
Haldi, Safed Musli, Sugandh Mantri, Agyachit and Chitrak
respectively which will be very much suitable for cultivation
in tea growing soil of Assam.
Thus, considering the present world scenario of plant
diversification, many economically important plants having
80
Normal propagatjon of the plant species through seed is
difficult due to their very poor seed setting and germination
rate and vegetative propagation of rhizome, small cuttings
are also not adequate for mass plantation. Propagation by
seeds does not even maintain homogeneous population,
resulting in great variability in its important composition.
Under such circumstances Plant Tissue Culture Technique
has been considered as the sole mean of multiplication for
mass cultivation programme.
MATERIALS
AND METHODS
1. Plant species
1.1. Ch/orophytum arundinaceum
The plant C. arundinaceum Baker (Liliaceae), commonly
known as Safed Musli, has high m~dicinal value in
Ayurvedic medicine due to its rich saponin contents (Tandon
and Shukla, 1992) (Plate 1). The plant grows in the wide
range of climatic variation and prefers the sandy loam soil
for growth. On an average this crop gives an yield of2030 quintals of wet musli per acre.
Plate 1. Chlorophytum arundinaceum
planting onwards. Average fresh yield of rhizome is about
20,000 kg/ha.
1.2. Curcuma aromatica
C. aromatica (Zingiberaceae), locally known as Ban Haldi,
has got vast ethnobotanical value, already known in India
as tonic, carminative, antidote to snake bite and astringent
(Plate 2). C. aromatica is given for curing indigestion,
rheumatism, dysentery and also for healing wounds and
fractured bones. It also helps in blood circulation to remove
blood stasis, treating cancer (Thippeswamy and Bharathi,
2006) and also for antitumor activity (Shi et al., 1981).
1.4. Plumbago sps .
P rosea L. and P. zeylanica L. (Plumbaginaceae) are rich
in root alkaloid plumbagin, (Plate 4). The alkaloid contains
broad spectrum important etabolites, such as anticancer
(Krishnaswamy and Purushothaman,
1980; Devi et al.,
1994), antimicrobial (Didry et aI., 1994), lieshmanicidal
(Kayser et al., 2000) and insecticidal (Kubo et al., 1983).
It prefers clay loam soils rich in organic matter (humus),
acidic soil pH and partial shade. Plantation can be raised
by planting rhizomes and 12 q of rhizomes is required for
planting 1 ha area. The crop should be harvested only after
2 years of growth in the field and about 200 kg of nydrodistilled oil (on dry weight ba~is) can be produced from 1
ha of the crop.
1.3. Homa/omena aromatica
H. aromatica,
Schott (Araceae),
locally called as
sugandhmantri, is a low volume high cost rhizomatous
aromatic perennial herb (Plate 3). It is indigenous to
northeastern region. The m3jor component of the rhizome
is reported as 62.1 % Linalool (Singh et al , 2000). It likes
warm and humid climate with high rainfall (2000-3000 mm)
and prefers sandy-loam to clay-loam soil of pH 5-6. Fresh
rhizome with active buds of 3-4 cm. size can be used as
seed materials. It is more profitable to grow as inter-crop
with some plantation or other tall perennial crops. The
harvesting of the plant can be started trom yd year of
Plr.te 2. Curcuma aromatica
81
•
Plate 3. Homalomena
aromatica
It is also considered as antifeedant, insecticidal, growth
regulatory and sterilant on insect pests (Gujar, 1990) etc.
The plant prefers well drained/deep sandy loam to clayey
loam soil with high organic content. In natural habitats the
plant prefers moist soil with high organic content 'and
partially shaded conditions.
Plate 4. Plumbago (a) P. zeylanica (b) P. rosea
2.
Preparation
of plant materials
and culture
conditions
At the onset of monsoon freshly sprouted vegetative buds
from field growing plants and storage rhizome collected
from experimental garden of NEIST, 10rhat, Assam were
used for establishment of axenic cultures for all the species.
Explants were washed thoroughly in 5% Teepol solution
for 20 minutes and then washed several times in single
distilled water. They were then disinfected by (0.1 %) w/v
aqueous mercuric chloride solution followed by three rinses
in sterile distilled water and implanted in sterilized media.
Treating the explants with an anti-fungal, viz. Mancozeb
and an anti-bacteria! solution Gentamycine (0.1 % each) for
30 minutes prior to the surface sterilization was needed
specially when explants were isolated from underground
rhizome source.
3. Axillary, adventitious
shoot formation
and rooting:
Axillary or adventitious shoots initiated from primary
culture of Plumbago species, viz. rosea and zeylanica were
separated and cultured on MS media containing BAP 0.5,
1.0, 2.5 and 3.0 (mg/I) for determining the optimal plant
growth regulator
(PGR) regime.
In case of C.
arundinaceum, along with BAP, Gibberlic acid (GAJ) 1.0
mg/! was also added to the medium. Based on earlier reports
on Zingiberaceae,
(Nayak, 2002), BAP alone or in
combination with a-Napthalene acetic acid (NAA) were
tried for C. aromatica and in case of H. aromatica, besides
these combinations, Kinetin (Kn) and 2-isopentyladenine
(2ip) were also attempted. Frequency of shoot initiation
and whole plants produced in each treatment recorded after
four weeks of growth.
Due to shortage of suitable explant material, preliminary
experiment was conducted with a single cytokinin 6benzylaminopurine (BAP) 0.5 - 3.0 mg/l incorporated to
the Murashige and Skoog (Murashige and Skoog, 1962)
basal medium as a nutritional support. Shoots were
multiplied by subculturing at 3-4 weeks intervals by
separating adventitious shoots or by node cutting when
internodes were elongated. The pH of the medium was
adjusted to 5.6 before autoclaving. The cultures were
incubated at 23 ± 2°C with a 16-h photoperiod under
fluorescent light (40 flmol m-2 I-I). Explants developing
shoot clumps from primary culture were used for further
shoot multiplication experiment.
As per earlier reports (Verma et al., 2002; Samantaray and
Maiti, 2011) root induction experiments were conducted
by taking single auxins, viz. indole 3 - butyric acid (lBA)
0.5 - 3.0 mg/I in case of both the Plumbago species and
also for C. arundinaceum. In case of C. aromatica and H.
aromatica no additional step was adopted for root induction.
Rooting responses in terms of rooting frequency, number
and length of roots per explant were evaluated after 4-6
weeks of culture.
82
4. Recovery of intact plants and acclimatization
medium even after more than a year. Rooting performance
responded highly to the presence of IBA in the medium.
The highest frequency (60.4%) root induction along with
satisfactory
root numbers
was obtained
on MS
supplemented with IBA (3.0 mg/I) in case of P rosea.
However, MS medium supplemented with IBA (0.5 mg/I)
showed rooting more than 94% with induction of maximum
root numbers in case of P zeylanica. Optimum root lengths
(4.5 - 4.6 cm.) were recorded from both the species of
Plumbago. Effect of different concentrations of IBA tested
for rooting of the shoots for Plumbago species are depicted
in (Fig. 4).
Rooted plantlets were transferred gradually to hormone free
MS, half strength ofMS and quarter strength ofMS liquid
medium for an interval of 15 days each and then ultimately
to filtered water for another 10 days allowing further growth
and elongation. Thereafter based on hardening, plantlets
were transferred to soil containing 1: 1mixture ofautoclaved
sand and garden soil. They were watered every alternate
day with tap water and kept in net house. Based on new
shoot growth planlets were transferred to soil and allowed
to grow normally.
RESULTS AND DISCUSSIONS
In BAP fortified media
aromatic was maximum
2ip added media showed
whereas shoots induced
Different explants after being placed in MS basal media
supplemented with BAP alone, expanded from basal to the
middle part followed by development of adventitious buds
within 3 weeks of culture of C. aromatica, H aromatica
and C. arundinaceum. In Plumbago species, multiple shoot
formation started to be evident after 12-15 days arising from
axillary buds grown at the basal ends and at the nodes of
the main axis. For further proliferation of shoot buds,
different growth regulator combinations added to culture
media were tested separately.
for shoot
(Plate 8.).
abnormal
from Kin
multiplication of H.
Shoots induced from
curled leafy structure
added media showed
Among the various ranges of BAP (0.5, 1.0,2.5 and 3.0
mg/l) tested separately
shoof proliferation
of C.
arundinaceum had been observed more or less in all tested
bigger leaf morphology.
Addition of NAA to these
combinations also did not show satisfactory performance,
whereas incorporation ofNAA (0.1 mg/I) in BAP augmented
media showed an overall positive response on shoot
proliferation along with simultaneous induction of roots
(Fig. 5.). The result showed a synergistic combination of
auxin and cytokinin for promotion of both shoot and root
regenerations. This response was in support with the well
known rhizogenic effect of auxins (Haissig et al., 1992).
Moreover, root induction was also recorded from the
treatment devoid of any exogenous growth regulators
(control set).
media (Fig. I). However, MS medium supplemented with
BAP (1.0 mg/I) showed maximum shoot proliferation from
the implanted primary shoot cluster (Plate 5). Similar type
of observation was reported earlier from C. borivilianum
by Purohit et al. (1994). Addition of GAl (1.0 mg/I) to this
BAP incorporated media showed a very well developed
growth of shoots accompanied with increase in shoot
number. Low level of IBA (0.5 mg/I) was found to be the
best in respect of root number initiation and root length
(Fig. 2).
Single cytokinin BAP (5.0 mg/I) alone or in combination
with auxin NAA and BAP at varied ratios like ( I: I, 1:2
and 1:3 ) were tested separately for subculturing of shoots.
Similar to H. aromatica, a synergistic action of auxin and
cytokinin for induction of both shoot and root were recorded
(Fig. 6). Unlike the earlier report ofNayak (2002), higher
range ofBAP alone was not found to be suitable for healthy
shoot initiation or simultaneous rooting. Considering all
the factors like maximum number of shoot and root
initiations along with satisfactory shoot and root lengths,
1:2 ratio of NAA and BAP was selected for optimum
regeneration of whole plant (Plate 9).
Primary shoot cultured of both the Plumbago species, rosea
and zeylanica, had initiated growth of axillary shoots in all
the treatmaents. However, BAP level tested for both the
Plumbago species produced a significant response upon
numbers and shoots formed per explant and also showed
influence on production of multiple shoots along with
development of shoot length (Fig. 3). For P rosea shoot
proliferation occurred more or less in all BAP fortified
media (Plate 6). However, higher concentration of BAP
(3.0 mg/I) was found to be suitable for optimal shoot
initiation (10 - 18 nos.). In case of P zeylanica also, shoot
proliferation was observed from almost all the treatments
(Plate 7). But, in this case frequency of multiple shoot
formation as well as shoot elongation was found to be
optimum at BAP (1.0 mg/I). These proliferation rates did
not decrease in the successive subcultures of individual
shoots or clum of shoots (3-5 shoots/clump)
After emergence of roots, plantlets were transferred to
hormone free MS liquid basal medium which favoured
further development of rooted shoots. The plantlets were
propped up in the liquid medium with the help of filter
paper strips. After one month, plants were hardened
gradually by elimination of MS basal to 1/2 strength of MS
and then ~ strength ofMS medium, ultimately on the filter
water and successfully acclimatized in net house conditions.
Hardened plants were transferred to pots containing garden
soil and sand mixture (I: I) and also transferred directly to
the soil. Survival rate of plants in the field after
transplantation was recorded as 70-85%.
on the same
83
Plates (5-9).
shoots formation of Chlorophytum arundinaceum, Plumbago rosea, Plumbago zeylanica, Homalomena
aromarica and Curcuma aromatica respectively.
Multiple
84
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formation of Chlorophytum arundinaceum
Fig. 1.
Fig. 2.
Rooting
Root length
__
of regenerated
Root numbers
of Chlorophytum
shoots
arundinaceum
o
I
Plumbago roseo
_% Respondingexplants
_Shoot
Fig. 3.
,qti,••lb~~).
Plumbago zeylanica
_
~~
-
multiple shoots
• lMaH<>lIlj,l tll n
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7JiI;t: :~.J
R('~l()l'\dl '-llpIM'IU' _
<lllMlt;th(cm)
No~of ''OOh
length (em)
Fig. 4.
Effects of different nutrient compositions on shoot
formations of Plumbago rosea and P. zeylanica
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No. of shoots _
---Shoot
Fig.
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No, of leaves _
Media composition
No, of roots
c:::::J Multiple Shoot _
___ Shoot length
5.- ..-Effects·~f-different-·nutrient
..compositions-on
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Root nllmbers
->- Root length
Root length
shoot
Fig. 6.
85
Effects of different nutrient compositions
formation of Curcuma aromatica
on shoot
Murashige, Tand Skoog F. (1962). A revised medium for
rapid growth and bioassays with tobacco tissue
cultures. Physiol. Plant. 15:495-7.
Tissue Culture techniques have been increasingly applied
for micropropagation and conservation of economically
useful plants especially for those where vegetative mode
of propagation is a constraint. Simultaneously the developed
techniques can be exploited for those plants where the roots/
rhizomes or the whole plants are used in drug or perfumery
industry. The present work has demonstrated simple and
reproducible methods for in vitro multiplication of a few
underutilized but promising plant species for their mass
multiplication
aiming at bulking up of materials for
economic return.
Nayak, S., (2002). In vitro multiplication of micro rhizome
induction in Curcuma aromatica Salisb. Plant
Growth Regul. 32:41-7.
Purohit,
S. D., Dave, A. and Kukda, G. (1994).
Micropropagation of safed musl i (Chlorophytum
borivilianum),
a rare Indian herb. Plant Cell,
Tissue and Organ Cult. 39:93-6.
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86