Importance of Biochemistry in

Agriculture & Plant Sciences

Chapter 1

Tissue Culture-Based Micro-Cloning of

Shorea Tumbuggaia Roxb.--A Globally
Endangered Indian Medicinal Tree
Shastri Prasad Shukla
Department of Horticulture, College of Agriculture & Environmental Sciences, Adigrat University, Adigrat
Ethiopia (Africa).
Email: [email protected]
Abstract

The genus Shorea of Dipterocarpaceae family is represented by 196

rainforest tree species. Four species of Shorea i. e, Shorea assamica, S. robusta,
S. roxburghii and S. tumbuggaia have been documented in India. Of these, Shorea
tumbuggaia Roxb. commonly known as green dammer tree, is an endemic,
globally endangered, semi-evergreen tree, restricted to the Southern Eastern
Ghats parts of Chittor, Cuddapah and Nellore districts of Andhra Pradesh and
North Arcot and Chengalpattu districts of Tamil Nadu in India. In addition to its
timber-yielding potential, the tree is also known for its medicinal properties as
an external stimulant and a substitute for Abietis; Resina and Pix Burgundica of
European pharmacopoeias. The plant extract is used as a cure for ear-aches in
children. The bark is reported as having anti ulcer activity and leaves are used in
the treatment of dysentery. The establishment of the seedlings of this tree is very
low due to seed dormancy and heavy attack with an unidentified Bruchid beetle
insect pests at the pre-or post-dispersal stage of seed resulting in its dwindling
population. The present study, therefore, highlights the urgent need for tissue
culture interventions in S. tumbuggaia propagation for raising quality disease-
free propagules for its conservation. Collection tour to South Peninsular region
of India (Tirumula Hills, Tirupati, Andhra Pradesh and nearby areas) were
conducted for the purpose of collection of plant materials. Stem cuttings and
young seedlings of S. tumbuggaia were collected and established in the glass-
 Importance of Biochemistry in Agriculture & Plant Sciences

house at Plant Tissue Culture Laboratory, CSIR-Central Institute of Medicinal

and Aromatic Plants (CIMAP), Lucknow. Nodal explants excised from fresh
sprouts have been cultured in vitro for mass propagation.

An in vitro plant tissue culture approach provides a feasible alternative

Desai GS

for extensive mass manufacturing and conservation of elite and critically

endangered medicinal plant species. The presented work describes a proficient
in vitro seed germination and proliferation procedure for the conservation of
an endangered high trade medicinal tree, Shorea tumbuggaia Roxb. Given the
precarious position of this species, effective conservation is highly indispensable
for its continued existence, primarily for ecological reasons and also for forestry
purposes as this tree species is a valuable source of timber and oleoresin. In
the present study, over 90% in vitro seed germination was achieved. Multiple
shoot formation was established from seedlings with a maximum of 4.60 ±
0.30 multiple shoots per explant using MS (Murashige and Skoog) medium
supplemented with a combination of 6.66μM 6-benzylaminopurine and 0.454
μM thidiazuron. Rooting of multiple shoot cultures of 85% was achieved on
half strength MS medium supplemented with 2.24μM indole 3-butyric acid
(IBA) with 6.48 ± 0.58 roots per plantlet. Rooted plantlets showed more than
70% acclimatization in potting conditions and were established successfully in
a greenhouse, with morphological similarities to mother plants. Clonal fidelity
assessment of micropropagated plants with ten inter simple sequence repeat
(ISSR) primers reproduced a total of 24 monomorphic bands. The presented
work represents the first report of in vitro proliferation, conservation, and clonal
fidelity assessment of tissue culture generated S. tumbuggaia plantlets.
Keywords: Shorea tumbuggaia; Tissue culture; In vitro proliferation; in vitro conservation; clonal fidelity;
ISSR
1. Introduction
The genus Shorea of Dipterocarpaceae family is represented by 196 rainforest tree
species [13,25]. Of these, Shorea tumbuggaia Roxb. commonly known as green dammer, is a
globally endangered, semi-evergreen tree, restricted to the Southern Eastern Ghats of Chittor,
Cuddapah and Nellore districts of Andhra Pradesh and North Arcot and Chengalpattu districts
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of Tamil Nadu in India [6]. This resinous tree attains the maximum height of about 20-30m
with a maximum width of 150-190 cm. The trunk of the plant is used as flag poles for temples.
In addition to its timber-yielding potential, the tree is also known for its medicinal properties
as an external stimulant and a substitute for Abietis; Resina and Pix Burgundica of European
pharmacopoeias [34]. The plant extract is also used as a cure for ear aches and leaf juice as
ear drops in children [27]. Methanolic extract of leaves of this plant has been reported to have
antinociceptive and anti-inflammatory activity [14] whereas the bark is a rich source of anti-
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 Importance of Biochemistry in Agriculture & Plant Sciences

ulcer molecules [23]. Leaves are also used in the treatment of dysentery and stem resin is used
for making incense sticks. The oleoresin which is exuded from the stem bark of Shorea can be
used to cure hydrosis and alexiteric [36]. Besides, antidiabetic and antioxidant activity of S.
tumbuggaia is also reported in alloxan-induced diabetic rats and revealed that this plant had
anti hyperglycemic and hypolipidemic actions [25].

Conventionally, S. tumbuggaia is a self-pollinating plant and propagated by seeds.

The extensive exploitation of this plant, habitat degradation, and other abiotic interferences
including low seedling germination rate, incapability of seedlings to contend with neighbor
vegetative populations, high rates of seed and flower abortion, seed predation by insect pests
at the pre- or post-dispersal stage, less availability of viable seeds, and non-annual ephemeral
flowering, has brought S. tumbuggaia to the IUCN list of endangered medicinal tree plant
species [9]. For the ecosystem restoration and reforestation, efficient propagation methods are
required to generate the plant material in natural habitats where populations are most depleted.
Therefore, immediate attention towards maintained cultivation and conservation are required
to guarantee the unremitting supply of good quality plant material [3,11,24,29]. Plant tissue
culture-based in vitro proliferation strategies have been used for the propagation of a large
number of threatened plants in ex-situ conservation programs [30,31]. In vitro plant tissue
culture-based micropropagation provides several significant advantages over conventional
vegetative propagation. It is used to reduce the high cost of rapid production and establishment
of good quality plant material resulting from limited stock plant material supply and inefficient
traditional vegetative propagation [19]. Members of Dipterocarpaceae are generally considered
non-amenable to in vitro tissue culture. To date, there has been only one report of tissue culture
of Shorea genus with limited success reported [35].

In the present study, optimized procedures were developed for efficient in vitro culturing
for seed germination, proliferation, and rooting followed by greenhouse acclimatization of S.
tumbuggaia using shoot tips derived from aseptically grown seedlings. This appears to be the
first report on the procedure for in vitro proliferation and conservation of S. tumbuggaia. This
practice can be employed for the purpose of conservation using large-scale propagation for
forestation and ecosystem restoration programs. The concern for the genetic fidelity of the
progeny developed from in vitro conserved grown seedlings is highly required to check the
relatedness of progeny with mother plants, so the clonal fidelity of in vitro raised plantlets was
also evaluated for the first time using ISSR markers.

2. Materials and Methods

2.1. Collection of Plant Material and In Vitro Seed Germination

Healthy and young hanging matured fruits (with dry wings and hard endocarp; Figure:1a,
b) were collected in the fruiting season (April and May) from the scattered population of
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 Importance of Biochemistry in Agriculture & Plant Sciences

S. tumbuggaia, from different locations of Tirumala Hills, Tirupati, Andhra Pradesh, India
(Latitudes 13° 40′59.7° N, Longitude 79° 20′ 49.9°E and altitude 853 M above sea level).
For in vitro seed germination, the hard and stony endocarp of mature fruits was first removed
(Figure:1c). Seeds were washed thoroughly with 5% (v/v) Tween®20 solution (Sigma-
Aldrich®, St Louis, MO) and 70% (v/v) Savlon®(Jhonson and Jhonson, New Delhi, India)
for 10 min followed by a 5 min treatment of antifungal Bavistin ® (5% (w/v); BASF

India Ltd., Mumbai, India) for pre-sterilization, followed by rinsing 4 to 5 times with
autoclaved distilled water to remove any residue from earlier treatments. Final sterilization was
done with 0.1% (w/v) HgCl2 solution (Sigma-Aldrich®) for 3 to 4 min, followed by rinsing 4
to 5 times with sterile double distilled water under aseptic conditions. After sterilization, seeds
were blot dried and cultured in culture tubes (50 mL; Borosil, New Delhi, India) containing
seed germination medium composed of half strength MS (Murashige and Skoog medium [20],
HiMedia ®, Mumbai, India) fortified with 3% (w/v)sucrose (HiMedia®) and 0.8% (w/v) agar
(plant tissue culture grade, HiMedia®). The pH of all medium combinations was adjusted
to 5.8 with 0.1 N HCl (HiMedia®) or 0.1NNaOH (HiMedia®) before autoclaving at 121°C
(103.4 kPa) for 15 min. Ascorbic acid at 100 mgL−1 (Sigma-Aldrich®) and 0.02% (w/v)
activated charcoal (HiMedia®) were also added to the germination medium to prevent the
leaching of phenolics from the germinated seeds. The percentage of seed germination was
recorded 4 wk after the seeds were placed on the seed germination medium. Fifty seeds were
used for the germination treatment and the experiment was performed three times.

2.2. Multiple Shoot Induction and Proliferation

The apex of in vitro germinated seedlings was removed using a sterilized blade. Shoot
tips (1.0 –1.5 cm) were separated from 4-wk-old seedlings and initially cultured for 2 wk
on half strength MS medium fortified with 3% (w/v) sucrose (Hi Media ®) and 0.8%(w/v)
agar (plant tissue culture grade, Hi Media®), and then shifted to full strength MS medium
fortified with 3% (w/v) sucrose (Hi Media®) and 0.8% (w/v) agar (plant tissue culture
grade, Hi Media®) having different concentrations (2.27–13.6μM) of 6-Benzylaminopurine
(BAP) (Sigma-Aldrich ®),2.32–13.92μM Kinetin (KN) (Sigma-Aldrich®) and 2.27–13.6μM
Thidiazuron (TDZ) (Sigma-Aldrich®), either individually or in double combinations to obtain
multiple shoot cultures. Morphogenic responses like shoot multiplication response, number
of shoots per explant, length of shoots, rooting response, number of roots per explant, and
length of roots were measured during the course of study. The number of explants responding
and the number of shoot buds induced per responsive explants were recorded every 2 d for
the initial 2 wk and then at weekly intervals. Induction of roots in the multiple shoot cultures
was optimized by culturing well established healthy multiple shoots on half and full strength
MS medium, alone or supplemented with 0.224–4.48μM indole 3-butyric acid (IBA) (Sigma-
Aldrich®), 0.285–5.70μMindole 3-acetic acid (IAA) (Sigma-Aldrich®), or 0.268–5.36μM
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 Importance of Biochemistry in Agriculture & Plant Sciences

1-Naphthalene acetic acid (NAA) (Sigma-Aldrich®). All cultures were maintained at 26 ±

2°C under a 16 light photoperiod using cool white fluorescent tubes (Philips, Gurgram, India)
of 48 μmol m−2s−1 intensity.

2.3. Acclimatization and Establishment of Plantlets in Soil

In vitro rooted plantlets were carefully removed from the culture vessel and gently
rinsed with sterile distilled water to remove adhered agar. Rooted plantlets were submerged
in Bavistin® (5%, w/v) solution for 5 min and transplanted in pots containing an autoclaved
mixture of 1:1:2 proportion of garden soil, vermin compost (Cocogarden ®, Dindigul, India),
and vermiculite (Keltech Energies Ltd., Bangaluru, India). The potted plantlets were maintained
in the greenhouse for acclimatization and were covered with a translucent polythene bag to
retain humidity. Plants were supplemented with half strength liquid MS medium lacking
sucrose biweekly. The polythene bag was gradually opened and fully removed after 4 wk of
acclimatization and then plants were transferred to earthen pots with garden soil and allowed
to grow under greenhouse conditions for further development.

2.4. DNA Isolation and Genetic Fidelity Analysis by ISSR

The genomic DNA from the leaves of one mother plant and nine randomly selected in
vitro grown plants derived from different seeds collected from this same mother plant was
isolated using the CTAB method [15]. DNA quality and yield were analyzed and measured by
0.8% (w/v) agarose gel (Sigma-Aldrich®) electrophoresis and Nanodrop™ (ND1000, Thermo
Fisher Scientific ®, Waltham, MA) methodology according to manufacturer’s protocol. For
ISSR analysis, 10 universal UBC primers (University of British Columbia, Vancouver, Canada)
were randomly selected and tested. The PCR amplification was carried out using a reaction
mixture of 25μl volume containing 2.5μl of polymerase chain reaction buffer (10X), 1.0μl
of MgCl2(25 mM),1.0μ l of dNTPs(100mM),0.2μl of DNA Taq polymerase from DNA Taq
polymerase Kit (Sigma-Aldrich ®), and 1.0μl primer, 2.0μl of template DNA (40 ng), and
17.3μl of milli-Q water. PCR amplification was programmed for initial DNA denaturation
at 95°C for 5.0 min (one cycle), followed by 45 cycles of denaturation at 94°C for 1 min,
annealing at 48°C for 1 min and extension at 72°C for 2 min, and a final extension at 72°C
for 10 min (one cycle). PCR amplifications using ISSR primers were performed at least three
times and only the reproducible PCR products were scored. Amplified products were resolved
on 1.5% (w/v) agarose gel (Sigma-Aldrich®).The gels were stained with Nancy 520 (Sigma-
Aldrich®) and documented using (Nighthawk™, pdi Inc., New York). 1 kb DNA ladder (1 kb
RTU, BR Biochem, New Delhi, India) was used as the molecular standard for the identification
appropriate bands amplified with ISSR markers. The amplified bands were visualized under
UV light and imaged using a Gel Documentation System (XR-Quantity One®, BioRad®
Laboratories, Hercules, CA).
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 Importance of Biochemistry in Agriculture & Plant Sciences

2.5. Experimental Design and Statistical Analysis

The in vitro proliferation experiments were performed in a completely randomized

design (CRD), performed three times with a minimum of 50 seeds and 20 healthy shoots
for each treatment. Observations were made after 7 d interval for shoot multiplication and
rooting experiments. Data obtained from seed germination, shoot multiplication and rooting
experiments are represented as a mean ± standard error (SE). Statistical differences between
results for plant growth regulators used for multiplication and rooting were evaluated by two-
way analysis of variance (ANOVA) and post hoc Tukey’s HSD test using SPSS V. 17.0, and the
values at p<0.05 were considered statistically significant. For the genetic stability and clonal
fidelity analysis study, clearly visible, and reproducible bands within the size range of 250 to
1500 bp were scored. The genetic association of mother plants with in vitro raised plants was
assessed by calculating the Jaccard similarity coefficient [10] for pairwise comparisons based
on the quantity of shared bands produced by the primers using the NTSYS-PC (Numerical
Taxonomy System), version 2.1.

3. Results and Discussion

3.1. Seed Germination and Shoot Multiplication

For the optimization of the shoot bud multiplication medium, germinated seeds
(Figure:1d) were used. In the present study, a greater than 90% seed germination rate of S.
tumbuggaia was achieved using half strength MS medium lacking plant growth regulators as
earlier reported [2]. Leaching of phenolics into the culture medium posed a crucial problem
during the early stage of the seedling establishment. Therefore, to minimize the lethal
browning of explants caused by phenolics compounds, antioxidant ascorbic acid (100 mg l−1)
and phenolics absorbent activated charcoal (0.02% w/w) were added to the seed germination
medium. However, the addition of this antioxidant and phenolics absorbent had little effect
over browning of seeds but effectively reduced the leaching of phenolics in culture medium.
In vitro seed germination in Dipterocarpaceae was earlier reported by few workers [18,32,
33] who studied seedling germination in four genera of Dipterocarpaceae using BAP as a
key regulator. However, in vitro seed germination and establishment protocols using half
strength MS medium devoid of plant growth regulators were found to be a considerably
improvement and less time consuming for Dipterocarpaceae [12,21,33]. The effectiveness
of cytokinins (BAP, TDZ, and KN) alone and/or in combination was assessed for axillary
shoot bud induction, shoot elongation, and proliferation from shoot tip explants cultured on
half strength MS medium (Figure:1e,f). The concentration and combinations of cytokinins
used influenced the average number of shoots per explants as well as the mean shoot length.
Different concentrations of BAP, TDZ, and KN when used alone and/or in combination were
found to be significant different for shoot multiplication, numbers of shoots per explant, and
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 Importance of Biochemistry in Agriculture & Plant Sciences

shoot length response (Tables 1 and 2). Individually, supplementation of BAP (4.44 μM), TDZ
(6.81μM), and KN (13.92μM) resulted in a maximum of 75.6, 66.6, and 55% multiplication
response, respectively (Table 1). However, when these plant growth regulators were tested
in double combinations, 6.66μM BAP+0.45μM TDZ and 13.3μM BAP+0.54μM NAA, the
double combinations induced a maximum of 89.5 and 73.8% multiplication response with 4.60
± 0.30 shoots per explant and 4.80 ± 0.44 cm shoot length and 4.25 ± 0.77 shoots per explant
and 4.41 ± 0.41 cm shoot length, respectively (Figure:1g; Table 2). Similar results have been
reported in several studies performed with Bougainvillea glabra and Catha edulis [8, 34]. In
the study done by Jain and Chaturvedi [12] with Shorea robusta, BAP, when used alone, was
found effective for in vitro proliferation of shoot cultures. Whereas, few workers reported that
IAA enhances in vitro proliferation of Shorea roxburghii, but with limited success [21, 32]. In
these studies, BAP alone at 4.44μM induced a 75.6% multiplication response, however, it was
found less effective when compared to BAP (6.66μM) and TDZ (0.45μM) used in combination
with 89.5% multiplication response observed in the present study.

4. Rooting and Acclimatization

The frequency of root induction together with the number of roots per shoot and root
length were recorded after 7d of culture and were significantly affected by the hormone
concentration used (Table 3). When the shoot cultures were placed on full strength MS medium
devoid of any plant growth regulator, the rooting response was not obtained. The addition of
different concentrations of auxins influenced the rate of root induction along with the number
of roots per shoot. Of the plant growth regulators tested, IBA was found to be more effective
for root induction compared to IAA and NAA. The fortification of half strength MS medium
with IBA at concentrations of 0.22 and 0.44 μM increased the root induction rate up to 75%
with 5.11 to 5.81 roots per shoot, respectively. Similarly, IBA at concentrations of 0.22 and
0.44 μM produced 5.10 to 6.10 cm root length, respectively. Increasing IBA concentration
(0.672 to 2.02) only induced swelling in the lower region of shoot cultures and failed to induce
a rooting response when supplemented in either half and full strength MS medium. An IBA
concentration of 2.24μM was found the most suitable for root induction, with an 85% rooting
response, 6.48 ± 0.58 roots per shoot, and 6.55 ± 0.36 cm root length within a 30-d culture
period (Figure:1h). Supplementation of IAA failed to provide a root induction response
except at 2.85μM concentration with half strength MS medium, which induced a 50% rooting
response with 6.06 ± 0.67 roots per shoot and 4.23 ± 0.16 cm root length response. The addition
of NAA also did not provide an effective rooting response and resulted in only 30 to 50% root
induction.

Based on these observations, IBA was selected as a suitable rooting plant growth regulator
for S. tumbuggaia. Results obtained from earlier studies carried out with S. robusta, Shorea
leprosula and Dipterocarpus alatus and Dipterocarpus intricatus supports these findings,
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 Importance of Biochemistry in Agriculture & Plant Sciences

where a higher concentration of IBA alone and in combination with IAA was associated with
an increased rate of root induction and establishment [1,12,18].The observations presented
in these studies also support and indicate a considerably better effectiveness of lower NAA
concentrations for root induction.

4.1. Genetic Fidelity Testing of Micropropagated Plants

The rooted plantlets generated from seedlings of S. tumbuggaia appeared morphologically

similar to the plantlets obtained from the explant collection site. To authenticate the retention
of genetic stability of in vitro generated multiple shoot cultures, ISSR molecular marker-
based genetic assessment was performed. The ISSR marker assessment technique for genetic
assessment provides a benefit over the dependency on morphological and chemical traits to
confirm the genetic fidelity of in vitro raised plants [7,22]. The ISSR technique produces more
stable products than Random Amplification of Polymorphic DNA (RAPD) although it is as
simple as RAPD in operations. Thus, ISSR is generally used to study population genetics,
taxonomy, and phylogeny in several plant species [38,39]. A total of nine randomly selected
multiple shoot cultures derived from different seeds collected from the same mother plant with
the mother plant were subjected to ISSR profiling with10 UBC-ISSR primers. Results obtained
from ISSR profiling showed that in vitro raised plantlets and mother plants have very little
genetic disparity after in vitro proliferation. A total of 24 monomorphic and 6 polymorphic
bands were scored ranging from 250 to 1500 bp, with an average of three bands per primer
(Table 4; Figure. 2). Studies completed with Ochreinauclea missionis (4), Ceropegia spiralis
(6), Moringa peregrine (16), and Ceropegia evansii (5) showed similar results, where in vitro
raised plants were found to be similar to their mother plant.

These results indicate true to type character of the regenerated plantlets. However,
the possibility of genetic variations arising during the in vitro process cannot be ruled out
because tissue culture techniques are known to induce somaclonal variations in regenerated or
micropropagated plants, due to stress induced by high concentrations of growth regulators and
the fast rate of sub-culturing. Therefore, testing of clonal fidelity of regenerants remains one
of the most important prerequisites for plant tissue culture generated plants [17] especially in
trees and woody shrubs with long rotation cycles. These results indicate the genetic stability
and homogeneity among the generated progenies.

5. Conclusion

The present study constitutes the first report wherein an efficient in vitro propagation
protocol developed in S. tumbuggaia was utilized for greenhouse establishment and
conservation of this highly endangered tree species. It is pertinent to highlight that the members
of Dipterocarpaceae are generally considered non-amenable to in vitro tissue culture and
therefore, there are only a few reports on tissue culture of S. roxburghii and S. robusta [12,
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 Importance of Biochemistry in Agriculture & Plant Sciences

21, 32, 35] with limited success. These limited in vitro studies with genus Shorea also make
it difficult to understand the different aspects of in vitro proliferation and conservation and
therefore, more extensive research work is needed. During the collection of seeds, there were
very few trees of S. tumbuggaia in the Tirumala Hills, which is supposed to be the biggest
genetic source of this rare species [2]. Unfortunately, viable attempts were not made for in
vitro establishment and proliferation of this endangered tree. In light of the obtained results, the
conclusion was made that half strength MS medium without plant growth regulator provides
efficient seed germination. The maximum explant regeneration, shoot buds per explants and
shoot length, was obtained when shoot tips were cultured on MS medium supplemented with
6.66 μM BAP and 0.45μM TDZ. IBA at a 2.24μM in MS medium was found to be effective
for root induction and elongation. Furthermore, the ISSR primers used in the study produced
more monomorphic bands compared to polymorphic bands in the regenerated plants. Thus, the
present study generated valuable information with regard to the efficacy of ISSR primers to
analyze the genetic stability of regenerated plants of S. tumbuggaia that can be used to screen
in vitro produced and conserved plants. The established procedure can be effectively used for
seed-based in vitro shoot proliferation and conservation of elite genotypes and to provide high
quality plants for field cultivation and commercial farming with minimal risk of producing
somaclonal variants.
6. Figures

Figure 1. In vitro micropropagation and greenhouse establishment of Shorea tumbuggaia Roxb. (a) Healthy, young hanging winged
fruits on plant. (b) Mature fruits. (c)Seeds removed after breaking the hard seed coat. (d–f) Germinating seeds (on half strength
MS medium; (d) 4th day, (e)10thday, (f) 15thday).g Multiple shoot induction and axillary bud proliferation (on MS +6.66μMBAP +
0.45μM TDZ). (h)Root induction (after 14th day on half
MS +2.24μM IBA). (i) Mature plantlets with roots (2 mo old). (j) Greenhouse established plantlets. MS medium (Murashige and
Skoog 1962) without plant growth regulators. IBA indole acetic acid, TDZ thidiazuron.
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 Importance of Biochemistry in Agriculture & Plant Sciences

Figure 2. Amplified DNA bands generated from ISSR primer-based profiling of in vitro raised Shorea tumbuggaia Roxb. plantlets
after seven sub-culture passages. (a)UBC-818. (b)UBC-808. (c) UBC-822.M molecular marker (1 kb DNA, RTU, Biochem Life
Sciences, India), MP mother plant. Lanes 1–9 are randomly selected in vitro grown plants. Pink arrow represents band size of DNA
marker; yellow arrow represents polymorphic bands.

7. Tables
Table 1: Effect of different plant growth regulators on multiple shoot induction in Shorea tumbuggaia

Morphogenic responses (Mean ± SE)

PGR concentration
(μM) Multiplication
No. of shoots/explantx Shoots length (cm)x
response (%)x

BAP 2.22 50.3 ± 1.21bc 3.08 ± 0.19bcd 3.36 ± 0.15a

BAP 4.44 75.6 ± 1.92j 4.19 ± 0.29fg 4.66 ± 0.37c

BAP 6.66 68.3 ± 1.23ij 3.88 ± 0.35def 4.08 ± 0.48abc

BAP 8.88 67.3 ± 1.32hi 4.66 ± 0.22g 4.50 ± 0.48c

BAP 11.1 60.3 ± 1.54efgh 3.97 ± 0.28efg 4.41 ± 0.39c

BAP 13.3 62.6 ± 1.44fghi 3.58 ± 0.26ef 4.41 ± 0.51abc

TDZ 2.27 ns ns ns

TDZ 4.54 58.3 ± 1.22efg 2.08 ± 0.19a 4.66 ± 0.18c

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 Importance of Biochemistry in Agriculture & Plant Sciences

TDZ 6.81 66.6 ± 1.45ghi 4.05 ± 0.35fg 4.08 ± 0.26abc

TDZ 9.08 58.3 ± 1.43cdef 3.16 ± 0.29cde 4.66 ± 0.31c

4.50 ± 0.41 c
TDZ 11.35 60.3 ± 1.67efg 3.66 ± 0.22def

TDZ 13.6 48.3 ± 1.47b 3.91 ± 0.28efg 4.41 ± 0.37bc

KN 2.32 50.0 ± 1.49bc ns ns

KN 4.64 50.0 ± 1.49ac ns ns

KN 6.96 44.6 ± 1.33ab ns ns

KN 9.28 50.3 ± 1.39c 2.93 ± 0.25abc 4.16 ± 0.39abc

KN 11.6 50.3 ± 1.39bc 2.71 ± 0.14abc 4.83 ± 0.50c

2.58 ± 0.19ab
KN 13.92 55.0 ± 1.32cde 3.58 ± 0.25ab
Analysis of variance (ANOVA)

Mean sum of squares

Source of DF
variation Multiplication
No. of shoots/explant Shoots length (cm)
response (%)

Treatments 18 1369.8* 28.32* 23.1*

Error 36 5.164 0.056 0.084

Means (±SE) within a column followed by the same letter are not significantly different at p≤0.05 PGR plant growth
regulators, IBA indole butyric acid, IAA indole acetic acid, NAA naphthalene acetic acid, ns no response, ½MS half
strength, MS full strength MS medium (Murashige and Skoog 1962)x Data were scored after three passages (2 wk each)
on MS medium with given plant growth regulator concentrations*p<0.05

Table 2: Effect of different hormonal combination on multiple shoot induction in Shorea tumbuggaia
Morphogenic responses (Mean ± SE)
PGR concentration (μM)
Multiplication Average number of
Shoots length (cm)x
response (%)x shoots/explantx

BAP 2.22 + TDZ 0.454 64.4 ± 1.2ab 1.41 ± 0.12ab 3.56 ± 0.44ab

BAP 4.44 + TDZ 0.454 65.5 ± 1.6ab 2.83 ± 0.53bcd 4.91 ± 0.34c

BAP 6.66 + TDZ 0.454 89.5 ± 2.1c 4.60 ± 0.30fg 4.80 ± 0.44c

BAP 8.88 + TDZ 0.454 75.0 ± 1.9bc 2.34 ± 0.36abc 4.05 ± 0.56abc

BAP 11.1 + TDZ 0.454 58.3 ± 1.5ab 1.25 ± 0.19a 4.75 ± 0.51c

BAP 13.3 + TDZ 0.454 60.0 ± 1.3ab 1.34 ± 0.15a 3.65 ± 0.28ab

BAP 2.22 + NAA 0.536 56.5 ± 1.2ab 3.26 ± 0.53cdef 4.66 ± 0.38c

BAP 4.44 + NAA 0.536 65.0 ± 1.5ab 4.19 ± 0.49defg 4.33 ± 0.29bc

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 Importance of Biochemistry in Agriculture & Plant Sciences

BAP 6.66 + NAA 0.536 60.0 ± 1.5ab 4.16 ± 0.36defg 4.08 ± 0.33abc

BAP 8.88 + NAA 0.536 55.6 ± 1.8ab 3.83 ± 0.32defg 3.16 ± 0.20a

BAP 11.1 + NAA 0.536 55.6 ± 1.4ab 4.33 ± 0.72efg 3.83 ± 0.36ab

BAP 13.3 + NAA 0.536 73.8 ± 1.9bc 4.25 ± 0.77efg 4.41 ± 0.41bc

BAP 2.22 + KN 0.464 ns ns ns

BAP 4.44 + KN 0.464 48.3 ± 1.1ab 2.12 ± 0.32abc ns

BAP 6.66 + KN 0.464 46.3 ± 1.1bab 3.75 ± 0.17cde ns

BAP 8.88 + KN 0.464 56.6 ± 1.4bab 4.83 ± 0.20g 4.25 ± 0.30bc

BAP 11.1 + KN 0.464 70.6 ± 1.6bab 4.66 ± 0.22cg 3.91 ± 0.33abc

4.75 ± 0.21fg 4.16 ± 0.32bc

BAP 13.3 + KN 0.464 68.3 ± 1.5ab
Analysis of variance (ANOVA)

Mean sum of squares

Source of DF
Multiplication
variation Average number of
response (%) Shoots length (cm)
shoots/explant

Treatments 18 48.193* 4.819* 6.562*

Error 36 3.744 0.187 0.084

Means (±SE) within a column followed by the same letter are not significantly different at
p≤0.05 PGR plant growth regulators, BAP 6-benzylaminopurine, TDZ thidiazuron, NAA
naphthalene acetic acid, KN kinetin, ns no response, x Data were scored after three passages (2 wk each) on MS medium
with given plant growth regulator concentrations, *p<0.05

Table 3: Effect of different auxins on in vitro rooting of micropropagated shoot cultures of Shorea tumbuggaia
Morphogenic responses (Mean ± SE)
PGR concentration
Medium used Rooting No. of roots/
(μM) Root length (cm)x
response (%)x shootx

½ MS No PGR 48.0 ± 1.12ef 3.12 ± 0.12ab 4.62 ± 0.25bc

MS No PGR ns ns ns

½MS IBA0.224 40.0±1.11c 5.11 ± 0.22ef 5.10 ± 0.20cd

½ MS IBA 0.448 75.0 ± 1.21h 5.81 ± 0.45gh 6.10 ± 0.10ef

½ MS IBA 2.24 85.5 ± 1.29i 6.48 ± 0.58i 6.55 ± 0.36f

½ MS IBA 4.48 46.6 ± 1.12de 4.17 ± 0.13cd 5.40 ± 0.32d

MS IBA 0.224 55.0 ± 1.23g 5.06 ± 0.31efg 5.27 ± 0.25d

MS IBA 0.448 56.6 ± 1.35g 5.22 ± 0.41efg 6.43 ± 0.38f

MS IBA 2.24 55.0 ± 1.39g 4.61 ± 0.25de 4.49 ± 0.13bc

12
 Importance of Biochemistry in Agriculture & Plant Sciences

MS IBA 4.48 40.0 ± 1.19c 3.68 ± 0.26bc 5.45 ± 0.10d

½ MS IAA 0.285 ns ns ns
½ MS IAA 0.570 ns ns ns

½ MS IAA 2.85 50.0 ± 1.30ef 6.08 ± 0.67hi 4.23 ± 0.16b

½ MS IAA 5.70 ns ns ns
MS IAA 0.285 ns ns ns

MS IAA 0.570 ns ns ns

MS IAA 2.85 ns ns ns

MS IAA 5.70 ns ns ns

½ MS NAA 0.268 45.0 ± 1.24d 2.68 ± 0.26a 5.45 ± 0.19d

½ MS NAA 0.536 35.0 ± 1.09b 5.67 ± 0.23fgh 4.45 ± 0.16bc

½ MS NAA 2.63 30.0 ± 1.03a 3.08 ± 0.24ab 3.45 ± 0.16a

½ MS NAA 5.36 ns ns ns
MS NAA 0.268 50.3 ± 1.21 f
5.51 ± 0.15 fgh
6.35 ± 0.24f
MS NAA 0.536 40.0 ± 1.12c 4.32 ± 0.28d 5.74 ± 0.23de
MS NAA 2.63 39.3 ± 1.05c 3.45 ± 0.56b 4.26 ± 0.14b
MS NAA 5.36 ns ns ns
Analysis of variance (ANOVA)

Mean sum of squares

Source of
DF Rooting
variation
response No. of roots/shoot Root length (cm)
(%)
Treatments 26 2174.715* 18.419* 21.453*

Error 52 12.439 0.056 0.040

Means (±SE) within a column followed by the same letter are not significantly different at p≤0.05 PGR plant growth
regulators, IBA indole butyric acid, IAA indole acetic acid, NAA naphthalene acetic acid, ns no response, ½MS half
strength, MS full strength MS medium (Murashige and Skoog 1962)x Data were scored after three passages (2 wk each)
on MS medium with given plant growth regulator concentrations*p<0.05

Table 4. Primer sequences and number of scored monomorphic and polymorphic bands produced by ISSR markers in
in vitro raised Shorea tumbuggaia
Primer used Primers
Annealing No. of No. of polymorphic Band range
scored per sequences
temperature (°C) monomorphic bands scored per (bp)
plant (5′—3′)
bands plant

UBC 807 (AG)8T 44.6 3 1 350–1000

UBC 808 (AG)8C 47.1 2 0 1500–1700
UBC 809 (AG)8G 47.1 3 1 250–1000
UBC 810 (GA)8T 44.6 2 1 500–1500
UBC 811 (GA)8C 47.1 2 0 250-1500
UBC 812 (GA)8A 44.6 3 1 350-1000
UBC 814 (CT)8A 44.6 2 1 500–1000

13
 Importance of Biochemistry in Agriculture & Plant Sciences

UBC 815 (CT)8G 44.6 2 1 350–1500

UBC 818 (CA)8G 44.6 2 0 750–1500
UBC 822 (TC)8A 46.1 3 1 250–750
Average bands per primer 3.0

8. Acknowledgements

The author gratefully acknowledges the Director, CSIR-CIMAP, Lucknow for providing
the facilities to carry out this work. SPS gratefully acknowledges Science and Engineering
Research Board (SERB) Department of Science and Technology (DST), Government of India,
New Delhi, for awarding DST FAST TRACK PROJECT (SR/FT/LS-78/2011). SPS is also
thankful to Professors (Drs.) K. Madhav Chetty and N. Savithramma, the Department of
Botany, Sri Venkateshwara University, Tirupati (A.P.) for identification of the plant reported in
the present study.

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