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Increased production of azadirachtin from an improved

method of androgenic cultures of a medicinal tree
Azadirachta indica A. Juss.
a a
Priyanka Srivastava & Rakhi Chaturvedi
a
Department of Biotechnology; Indian Institute of Technology; Assam, India
Published online: 01 Jul 2011.

To cite this article: Priyanka Srivastava & Rakhi Chaturvedi (2011) Increased production of azadirachtin from an improved
method of androgenic cultures of a medicinal tree Azadirachta indica A. Juss., Plant Signaling & Behavior, 6:7, 974-981, DOI:
10.4161/psb.6.7.15503

To link to this article: http://dx.doi.org/10.4161/psb.6.7.15503

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 Plant Signaling & Behavior 6:7 974-981; July 2011; ©2011 Landes Bioscience

Increased production of azadirachtin

from an improved method of androgenic cultures
of a medicinal tree Azadirachta indica A. Juss
Priyanka Srivastava† and Rakhi Chaturvedi*
Department of Biotechnology; Indian Institute of Technology; Guwahati, Assam, India

†
Current address: Division of Biomedical Sciences; School of Bio Sciences and Technology; Vellore Institute of Technology University; Vellore, Tamil Nadu, India

Key words: anther culture, Azadirachta indica, azadirachtin, haploid plants, RP-HPLC

Abbreviations: 2,4-D, 2,4-dichlorophenoxyacetic acid; BAP, 6-benzylaminopurine; CH, casein hydrolysate; GA 3, gibberellic acid;
IAA, 3-indole acetic acid; Kinetin, 6-furfuryladenine; RP-HPLC, reverse phase high pressure liquid chromatography; MS, mass
spectroscopy
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Present report is the first direct evidence of azadirachtin production in androgenic haploid cultures of Azadirachta indica,
a woody medicinal tree. Anther cultures at early-late-uninucleate stage of microspores were established on MS medium
with BAP (5 μM), 2,4-D (1 μM) and NAA (1 μM) containing 12% sucrose. The calli, induced, were further multiplied on

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2,4-D and Kinetin media. Shoots, differentiated on BAP (2.2 μM) + NAA (0.05 μM) medium, were elongated on MS + BAP
(0.5 μM) and multiplied on MS + BAP (1 μM) + CH (250 mg/l). Thereafter, the shoots were rooted on ¼ MS + IBA (0.5 μM).
Cytological analysis of the calli and regenerants have confirmed their haploid status with the chromosome number as
2n = x = 12. The haploid cell lines and leaves from in vitro grown plantlets were analyzed for azadirachtin by RP-HPLC and
mass spectroscopy. Maximum azadirachtin (728.41 μg/g DW) was detected in calli supporting best shoot proliferation

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while least (49 μg/g DW) was observed in an undifferentiated line from maintenance medium. This study has brought us
a step closer to develop genetically pure lines that could serve as new and attractive alternative ways of homogeneous
controlled production of high value compounds, round the year, independent of geographical and climatic barrier.

reaching 50 kilograms per year.5 Of the fruit yield, only about

Introduction 10% is attributed to seed kernels (after removing the seed coat)
and desired biologically active compounds comprise only 10
Azadirachta indica A. Juss or Neem (family Meliaceae) is a valu- grams per kilogram of kernel weight. Based on this estimate, an
able tropical forest tree which has multiple medicinal, agrochem- adult Neem tree produces only about 20 grams of pesticide com-
ical and economic benefits due to several bioactive ingredients pounds in a season.
present in various parts of the tree. The most notable is azadi- In this context, the perspective of raising in vitro haploid
rachtin, a tetranortriterpenoid, which is chiefly present in the plants, thus, offers scores of foreseeable advantages, like short-
seed kernels.1 Its demand has been on rise in industries due to its ening of breeding period, production of homozygous diploid
immediate application as an ecofriendly, biodegradable biopes- (double-haploid) lines in a single step through chromosomal
ticide and various other significant bioactivities.2,3 The benefits doubling and isolation of valuable recessive traits at sporophytic
claimed in the papers are not fully utilized because of certain level which otherwise, remain accumulated and unexpressed in
practical limitations. The bottlenecks, with regard to extraction natural heterozygous diploid population. Since the plant cells
and availability of azadirachtin-related limonoids, lie in unavail- are totipotent in nature, consequently, the cultured cells will also
ability of genetically pure lines due to out-breeding nature of the contain genetic information for the production of bioactive com-
plant and long reproductive cycle. Improvement by conventional pounds with added potential of increasing yield by manipulat-
methods is also restricted because of the same reasons. Further, ing the culture conditions.6 As cell cultures have a higher rate
the availability of seeds is limited; the reproductive stage in Neem of metabolism than intact plants due to fast proliferation of cell
begins at about three to five years of age, they become fully repro- biomass, thus, production of bioactive compounds can take place
ductive after ten.4 From this time on, the tree yields an average of within a short cultivation time of 4–5 weeks, in comparison to,
about 20 kilograms of fruit per year, with maximum production conventional way of isolation of compounds from intact plants.

*Correspondence to: Rakhi Chaturvedi; Email: [email protected] or [email protected]

Submitted: 02/23/11; Revised: 03/16/10; Accepted: 03/17/11
DOI: 10.4161/psb.6.7.15503

974 Plant Signaling & Behavior Volume 6 Issue 6

 research paper

The motivation to work along this line, in the present study, clear patterns of callusing were seen on the responding medium:
arose from the fact that to date the available reports on azadi- in one, the anther walls burst open and pulled apart due to pres-
rachtin production have utilized only diploid tissues like seeds, sure of callusing microspores, resulting in the release of shiny,
roots, leaves, etc. from heterozygous plants that result in incon- globular masses emerged from within the anthers (Fig. 1D and
sistency and heterogeneity in chemical makeup of the plant. The E). In a few other cases, callus was developed at the base from
haploid cells or tissues of Neem were not explored earlier for anther walls. Only the former cultures were utilized for further
metabolite production. It is noteworthy that genetically pure lines experiments and for data calculation. The callusing started after
of strictly cross-pollinating species, like Neem, are highly desir- four weeks, and by the eighth week excellent callus growth was
able to increase the efficiency of selection and for homogeneous, observed.
constant production of metabolites. The conventional methods The above combination of growth regulators consisting of
to produce homozygous diploid plants is lengthy and laborious 12% sucrose was also tested with B5 and NLN basal media.
requiring 7–8 recurrent cycles of inbreeding, which is impossible While on MS medium callus growth was excellent and the calli
in the case of cross-breeding trees like Neem. However, with in were fresh, soft, friable and cream, those on NLN medium were
vitro haploids, homozygous diploids can be produced in a sin- dark brown, friable with very little growth and mostly the calli
gle step, thus, cutting down the breeding period to almost half. originated from the anther walls. There was no response on B5
The present report, therefore, aimed at developing an improved medium. Among the temperature pretreatments given to cul-
method of androgenesis and evaluating their feasibility of azadi- tured anthers, best response was observed in control at 25°C
rachtin production. All physical and chemical parameters that temperature, followed by one day pre-treatments at 12°C and
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influence the establishment of anther cultures in vitro are also 4°C temperatures (Table 3). Temperatures higher than 25°C
discussed. were found to have adverse effect on callusing. Therefore, fur-
ther experiments on callus induction were conducted with MS
Results and Discussion medium and the cultures were continuously incubated at 25°C

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in dark. Although regular attempts have been made to induce
Callus induction and multiplication. Flower buds of 2 mm androgenesis in woody trees, success rate is limited, chiefly due
size (Fig. 1A) were collected from an elite Neem tree growing at to their recalcitrance in culture. Factors that affect androgenesis
Guwahati (Northeast) India. The anthers bearing early-to-late have been described elaborately by Atanassov et al. Out of the
uninucleate stages of microspores (Fig. 1B and C) were cultured various factors, genotype, environmental conditions, physiologi-

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on medium to raise androgenic haploids of Neem. At the uni-
nucleate stage, the wall of microspores is thin and it is easy to
divert their mode of development from gametophytic to sporo-
phytic. Androgenesis in Neem was earlier reported by Chaturvedi
cal status of the donor plant, developmental stage of microspores
(early-to-late uninucleate stages being most crucial), pretreatment
of anthers, culture media, carbon source and its concentrations
and culture conditions play a vital role in induction of androgen-
et al.7 from a tree growing at Delhi (North), India. However, as esis. Higher sugar concentration during the induction phase is
heterozygosity due to cross pollination and also the environmental shown to have suppressive effect on the division of somatic cells,
conditions of the two regions have great influence on the physiol- therefore, division from haploid cells (microspores) are favored.
ogy of parent plant,8,9 consequently, nutritional requirements of Dark incubation, in most cases has been found essential in the
the two plants may vary under the in vitro conditions. This is in initial periods of induction in cultures. Likewise, anthers from
agreement with the present observation where the anthers taken the first flush of flowering in the season are suggested to be more
from a plant growing in Guwahati, India, needed significant mod- responsive than those borne later.8
ifications at various steps during androgenic plant development The calli that emerged from inside burst anthers were detached
under in vitro conditions, compared to the one, developed by from the parental tissues and transferred to fresh medium with
Chaturvedi et al.7 from the plant growing at Delhi, India. A com- a reduced sucrose concentration of 3% and 16/8 h photoperiod
parative account to these studies has been tabulated in Table 1. regime. All experiments from now onwards related to regenera-
Of the various growth regulator combinations tested, MS tion and chemical analysis had been conducted with these micro-
medium supplemented with 2,4-D (1 μM), NAA (1 μM) and spore derived calli.
BAP (5 μM) induced maximum callusing. The percent anther The induction medium did not support sustained callus
response and the degree of callusing varied with the sucrose growth even after 20 weeks. However, inclusion of one auxin (2,4-
concentrations. At its various concentrations, 12% sucrose was D) and one cytokinin (Kinetin) in the MS medium supported
observed to be significantly better (p < 0.05) for callus induc- sustained callus growth. In terms of biomass, best response was
tion, followed by 9%, 6% and 3%. Sucrose concentrations observed on MS + 2,4-D (1 μM) + Kinetin (10 μM) followed by
above 12% were found to be inhibitory (Table 2). Decline in MS + 2,4-D (0.5 μM) + Kinetin (4.5 μM) and MS + 2,4-D (0.5
response above this concentration may be explained by disturbed μM), in a single growth cycle of 8 weeks. The calli obtained on
osmoticum of the medium. On MS (with 12% Sucrose) + 2,4-D these combinations were cream, soft, friable and moderate to fast
(1 μM), NAA (1 μM) and BAP (5 μM), more than 85% anther growing. The calli were allowed to multiply on these three media
cultures responded for callusing. Compare to this, Chaturvedi et for more than 2 years.
al.7 obtained callus induction response on the best treatment con- Shoot regeneration, elongation and micropropagation. Calli
sisting of 9% sucrose in the medium. In the present study, two maintained on above three maintenance media were utilized

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Downloaded by [New York University] at 12:59 04 June 2015

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Figure 1. Establishment of anther cultures on MS + 2,4-D (1 μM) + NAA (1 μM) + BAP (5 μM); Growth Period: 8 weeks. (A) Flower buds of 2 mm size
bearing correct stage of microspores (x2.2X). (B) An isolated anther at culture, with uninucleate microspores (x70X). (C) Uninucleate microspores
stained with acetocarmine; bar represents 5 μm. (D and E) 8-week-old anther at culture where the anther walls pulled apart with the pressure of
callusing microspores (x55X). (F) 8-week-old callus cultures on multiplication medium, MS + 2,4-D (0.5 μM) + Kinetin (4.5 μM) (x1.2X). (G) 6-week-old
cultures on MS + BAP (2.2 μM) + NAA (0.05 μM), showing shoot regeneration (x1.2X). (H) Histological section of a regenerating callus showing vascular
strands and distinct tracheary elements; bar represents 200 μm. (I) Scanning Electron Micrograph of callus showing well developed nodular structures
(x200X). (J) GA3 (3 μM) pretreated shoot on elongation medium MS + BAP (0.5 μM), after 6 weeks of culture (x1X). (K) A shoot rooted on ¼ MS + IBA (0.5
μM), after 6 weeks (x1X). (L) Cytology of shoot-tip from in vitro haploid plantlet, stained with 2% aceto-carmine, showing haploid number of chromo-
somes (2n = x = 12); bar represents 10 μm. (M) Cytology of root-tip from seedling, stained with 1% aceto-orcein showing diploid number of chromo-
somes (2n = 2x = 24); bar represents 20 μm.

for regeneration experiments. MS + 2,4-D (0.5 μM) + Kinetin first and later compact nodular structures was differentiated into
(4.5 μM) served as the best source (Fig. 1F), as it gave maxi- shoot-buds after 6 weeks. On an average 27.8% cultures formed
mum percentage of regenerating cultures. Shoot regeneration was 4.5 shoot-buds per explant on this combination. However, con-
achieved on two combinations: MS + BAP (2.5 μM) + IAA (5 siderably better shoot-bud proliferation frequency was achieved
μM) + CH (500 mg/l) and MS + BAP (2.2 μM) + NAA (0.05 in the latter medium. In this case, two step protocol was followed
μM). The callus in the former medium, turned bright-green where the calli from multiplication medium were first transferred

976 Plant Signaling & Behavior Volume 6 Issue 7

 Table 1. Comparative account of a previously existing and new improved method of androgenic cultures of Azadirachta indica A. Juss
Parameters/ Previously existing method New improved method
S. No.
Comparison of study (Chaturvedi et al. 2003) (present study)
Flower buds collected from an adult Neem tree Flower buds collected from an adult Neem tree growing in
1. Material Used
growing in Delhi, North India. Guwahati, North-east India.
Emphasized the requirement of 9% sucrose con- In the present investigation, the best callus induction
Sucrose concentration
2. centration in the induction medium during estab- response was obtained in the induction medium with 12%
in induction medium
lishment phase of Neem anther cultures. sucrose concentration.
Effect of temperature pretreatments (cold and heat) were
Anther culture pre- studied. The best response was observed in control at 25°C
3. No studies performed.
treatment temperature, followed by one day pre-treatments at 12°C
and 4°C temperatures; heat treatments had adverse effect.
In the present case, calli from multiplication medium were
Calli from multiplication medium were directly first incubated in a combination of one auxin (NAA) and one
4. Culture medium transferred to regeneration medium that con- cytokinins (BAP) at higher concentration to induce shoot-
sisted of only cytokinin (BAP). buds in the regeneration medium which consisted of ten
times reduced concentration of BAP+NAA.
Maximum shoot regeneration frequency obtained Maximum shoot regeneration frequency obtained was
Shoot regeneration
5. was 75%, with cultures forming an average of 4.5 98.5% with an average of 8.5 shoot-buds/ explant on MS +
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frequency
shoot-buds/explant on MS + BAP (5 µM). BAP (2.2 µM) + NAA (0.05 µM).
Shoots were elongated at ten times lower concen- In the present study, shoot elongation required a pretreat-
Shoot elongation
6. tration of regeneration medium consisting of BAP ment of GA3 (3 µM) for two weeks followed by transfer of
medium
at 0.5 µM. shoots to elongation medium MS + BAP (0.5 µM).

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An improved method is established to raise androgenic
Anther cultures established and androgenic plants
7. Outcome of study plants. Additionally, androgenic cultures and plantlets were
produced.
characterized with respect to azadirachtin production.
Reason for differ- 1. Different environmental conditions of the two regions, Delhi and Guwahati, where the experimental trees were
ent culture medium growing, thus, affected the physiology of the parent plant.

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requirements for the
8.
same explants taken
from two different 2. The tree is highly heterozygous in nature due to strict cross pollination. As a result, the plants growing side by
trees side also show variation due to genetic reasons.

Table 2. Effect of sucrose concentration on callus induction from 1), whereas in the present case it had to be compounded with
anther cultures an auxin (IAA/NAA). Histological sections of regenerating calli
Total anthers Anthers Percent revealed that before the onset of shoot-buds, well organized vas-
Sucrose (g/l) cular strands with tracheary elements were developed (Fig. 1H)
cultured responded response(*)
30 (control) 480 193.82 40.38ab within the calli. These conducting strands later gave rise to nod-
ules as was evident by scanning electron micrographs of the calli
60 480 252.96 52.7b
(Fig. 1I).
90 480 299.28 62.35ab
Since the shoots did not grow much on regeneration medium,
120 480 409.92 85.4a therefore, small shoots were excised, pretreated with 3 μM GA 3
150 480 168 35c for 14 days and transferred to lower BAP concentration at 0.5
180 480 24 5d μM. With this method, on an average, shoots attained a length
Growth Period: 8 weeks. Control: MS + 2,4-D (1 µM) + NAA (1 µM) + BAP of 4.5 cm with four nodes per shoot after 6 weeks (Fig. 1J).
(5 µM) with 3% Sucrose. (*) Those marked with the same letters are not Further shoot multiplication was achieved on MS + BAP (1.0
significantly different at p < 0.05 according to Duncan’s multiple range μM) + CH (250 mg/l) via axillary shoot proliferation by cultur-
test. ing single nodal segments. Here shoots grew 6 cm long with six
nodes per shoot in 6 weeks. The number of propagules obtained
to high BAP (22 μM) and low NAA (0.5 μM) supplemented at the end of a multiplication cycle was taken to be the rate of
medium for 12 weeks, and then subsequently transferred to 10 shoot multiplication. Hence, adopting this method, six-fold
times lower concentration of BAP (2.2 μM) + NAA (0.05 μM). shoot multiplication was achieved every 6 weeks.
With this two step method, the shoot-bud regeneration fre- Rooting of shoots and ploidy analysis. For rooting, 3 cm long
quency was remarkably improved where 98.5% cultures formed terminal portions of 6-week-old elongated shoots were cut and
an average of 8.5 shoot-buds per explant, after 6 weeks (Fig. transferred to quarter (1/4) strength MS medium supplemented
1G). Compare to this, Chaturvedi et al.7 could be able to obtain with IBA (0.5 μM). Remaining part of the shoot was utilized for
regeneration in 75% cultures with an average of 4.5 shoot-buds multiplication by nodal segment culture. On ¼ MS + IBA (0.5
on the best medium consisted of only cytokinin (BAP) (Table μM), root initiation occurred in three weeks directly from the

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 Table 3. Effect of temperature pretreatments at different duration on callusing from anthers cultured on MS (with 12% sucrose) + 2,4-D (1 µM) + NAA
(1 µM) + BAP (5 µM); Growth Period: 8 weeks
Temperature (°C) Duration (days) Total anthers cultured Anthers responded Percent response
25 (control) - 480 409.92 85.4
1 120 94.44 78.7
4 7 120 67.92 56.6
14 120 51.24 42.7
1 120 98.28 81.9
12 7 120 68.64 57.2
14 120 56.76 47.3
1 120 58.18 48.48
32 7 120 60.00 50.0
14 120 74.11 61.76
1 120 66.32 55.27
40 7 120 0 0
14 120 0 0
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Two-way ANOVA to study the effect of different temperatures and duration of pretreatment on anther cultures.

Summary Count Sum Average Variance

Row 1 4 264.35 66.0875 278.7176

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Row 2 4 163.8 40.95 755.93
Row 3 4 151.76 37.94 705.7011
Column 1 3 178 59.33333 329.6033
Column 2 3 186.4 62.13333 317.5433

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Column 3 3 160.24 53.41333 52.82773
Column 4 3 55.27 18.42333 1018.258
Source of Variation SS df MS F p-value F crit
Treatment length 1910.981 2 955.4905 3.758117 0.087476 5.143253
Different temperatures 3695.563 3 1231.854 4.8451-05 0.048185 4.757063
Error 1525.483 6 254.2472
Total 7132.027 11

basal cut end of the shoots and after six weeks 100% cultures cells in haploid state and tendency to undergo autodiploidization
developed an average of 10 roots per shoot (Fig. 1K). through endomitosis to regain their normal status. As a result,
Cytological analysis of callus cells in induction, multiplication the frequency of diploid cells in culture increases with age.10,12
and regeneration media, and shoot-tips from haploid plantlets Identification of azadirachtin by HPLC and mass spectrom-
revealed that majority of the cells were in haploid state (2n = x = 12) etry. The standard and sample extracts, prepared by following the
(Fig. 1L). However, by the time entire plantlets were developed protocol as described in materials and methods, showed elution of
on ¼ MS + IBA (0.5 μM), 40% of the plants maintained their azadirachtin, a tetranortriterpenoid, at 6.39 min in HPLC (Fig.
haploid status while the rest were diploids (2n = 2x = 24) or aneu- 2A). Calibration curve for azadirachtin standard showed good lin-
ploids (2n = 2x - 2 = 22) or (2n = 2x - 1 = 21) which may arose earity at tested concentrations (0.0625 mg/ml to 1 mg/ml) with a
from haploid cells either spontaneously or due to manipulation correlation coefficient (R 2) of 0.9889. The equation generated from
of cultures. The mitotic preparation from root-tips of seedlings the curve by external standard method (Fig. 2A) was used to calcu-
showed the diploid number of chromosomes in Neem is (2n = late amount of compounds present in crude sample. For precision,
2x = 24) (Fig. 1M). It has been established by earlier workers the standard samples at same concentration were analyzed at least
that ploidy levels, lower than the usual ploidy level of a species is five times within the same day and the RSD value obtained was
quite unstable. In long term cultures, diploidization in haploid 3.99%. Similarly, for interday variability, where same concentra-
calli is much more common than the occurrence of tetraploids in tion of the standard compound was run thrice at one day interval,
diploid lines.11 The present study is also in agreement with this the value was 3.78%. From the standard equation obtained, the
finding as majority of the cells maintained their haploid status amount of azadirachtin in different callus lines was calculated and
for a long period but gradually, by the time of plantlet formation, listed in Table 4. Presence of azadirachtin was confirmed in all the
this frequency reduced. It may be attributed to the unstability of callus lines tested. However, it was observed that redifferentiated

978 Plant Signaling & Behavior Volume 6 Issue 7

 similar m/z fragments in both stan-
dard compound and sample fractions
has further confirmed the presence of
azadirachtin.
Assessment of haploid (n) cell lines
and their derivatives, as source of aza-
dirachtin has been done for the first
time in Neem, in the present study.
Till date, the available reports on aza-
dirachtin and related limonoid pro-
duction have utilized cultures from
different somatic parts (heterozygous
diploids) like leaves,13-18 bark,13 stem17
and seeds (embryos).18,19 However, as
is the case with the metabolite con-
tent under in situ conditions, the same
limitation is posed if cultures are estab-
lished from seeds or other somatic tis-
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sues owing to the variability inflicted

upon by prevalent heterozygosity in
the genus due to cross pollination. The
yield range of azadirachtin, obtained

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in the present study, from haploid
lines is much higher (49–728.41 μg/g)
than most of these reports. In available
reports, while the recorded values of
azadirachtin in leaf derived callus was

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bark, embryo and stem cultures yielded
44 μg/g DW, 4–8 μg/g DW and 2.7
μg/g DW azadirachtin, respectively.
Moreover, variability in content due to
heterozygosity is the major issue that
needs to be addressed in cases where
somatic parts are utilized.

Materials and Methods

Figure 2. HPLC chromatogram of standard azadirachtin. (A) A structure of Azadirachtin, a tetranor- Establishment of cultures and callus
triterpenoid (left side). Right side of figure shows Azadirachtin peaks eluted at 6.39 min in HPLC. The induction. Healthy flowering twigs of
table below the peak is showing the data generated from azadirachtin standard. (B) Mass spectro- Neem were collected from a mature
metric profile of standard azadirachtin and HPLC eluted sample fraction.
Neem tree growing near the campus of
IIT-Guwahati, during the months of
calli on MS + BAP (2.2 μM) + NAA (0.05 μM), possessed high- April-May between 5:30 a.m. to 6:30 a.m. The stage of micro-
est azadirachtin content (728.41 μg/g DW) while the least was spore development was checked with acetocarmine squashes.
obtained from dedifferentiated line, maintained on MS + 2,4-D (1 Two mm size flower buds were surface sterilized with 0.1% mer-
μM) + Kinetin (10 μM) (49 μg/g DW). In vitro grown haploid curic chloride solution for 7 min inside the laminar-air-flow cabi-
leaves contained 700 μg/g DW of azadirachtin (Table 4). net (Saveer Biotech, India) and rinsed thrice with sterile distilled
The azadirachtin fractions eluted from HPLC of samples were water. The buds were dissected and twenty anthers, from two
collected and the fragment characteristics were compared by buds, were cultured in 60 mm x 15 mm pre-sterilized, disposable
mass spectra with that of standard azadirachtin procured from Petriplates containing 10 ml of medium with or without various
Sigma, Aldrich (Fig. 2B). Spectra were obtained in full scan combinations and concentrations of growth regulators (2,4-D,
mode. Base peak of m/z 685 resulted due to loss of two water NAA, BAP, IAA, Kinetin) and sealed with parafilm (Pechiney,
molecules [MH+ - 2H2O], m/z 703 formed due to the loss of USA). The best responding combination was checked at vari-
one water molecule [MH+ - H2O] and m/z 743 corresponded ous sucrose concentrations, 3–18%. Three different basal media
to the formation of sodium adduct [MH+ + Na+]. Presence of MS,20 B5,21 and NLN,22 were also tested with best sucrose and

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 Table 4. Evaluation of androgenic lines of Neem for azadirachtin production
Material Medium Amount of azadirachtin (µg/g DW)
Dedifferentiated callus MS + 2,4-D (0.5 µM) 288 ± 0.32
Dedifferentiated callus MS + 2,4-D (0.5 µM) + Kinetin (4.5 µM) 79 ± 0.3
Dedifferentiated callus MS + 2,4-D (1 µM) + Kinetin (10 µM) 49 ± 0.2
Redifferentiated callus MS + BAP (2.5 µM) + IAA (5 µM) + CH + (500 mg/l) 411.38 ± 0.3
Redifferentiated callus MS + BAP (2.2 µM) + NAA (0.05 µM) 728.41 ± 0.4
In vitro leaves (haploids) BAP (0.5 µM) 700 ± 0.52
Control I (Leaves parent plant) - 5435.6 ± 4.0
Control II (Seeds parent plant) - 6800 ± 4.1

growth regulator combinations. Anthers from each treatment Ploidy analysis. For ploidy analysis, calli from induction, mul-
were given a temperature pretreatment of 4°C, 12°C, 32°C and tiplication and regeneration media, root/shoot-tips from in vitro
40°C for 1, 7 and 14 days, respectively, to induce calli. All the raised plantlets and roots tips from seedlings were excised and
cultures were kept in dark until 8 weeks, for callus induction. pretreated with 0.02% 8-hydroxyquinoline (BDH, India) at 4°C
Callus multiplication and shoot regeneration. The calli for 4 h. This was followed by fixation in modified Carnoy’s fluid
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obtained from induction media were further multiplied and containing absolute alcohol, chloroform, glacial acetic acid and
maintained on media containing completely different sets of methanol (7:3:1:1) for 48 h. The fixed material was warmed in a
auxin (2,4-D) or/and cytokinin (Kinetin). From now on calli mixture of nine drops of 1% aceto-orcein (or) 2% aceto-carmine
were maintained in diffused light (1,000–1,600 lux) under 16 h and one drop of 1 N HCl, squashed and observed under Nikon

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photoperiod. For experiments on shoot organogenesis, calli (ca. epifluorescence microscope. The cells showing a good separation
0.2 g) from maintenance media were utilized and grown on MS of chromosomes were photographed.
medium supplemented with cytokinins, BAP in combination Selection of haploid lines and preparation of extract. The
with auxins like NAA or IAA and additives like CH to obtain androgenic calli from different media, leaves from in vitro devel-
shoot regeneration from callus. oped haploid plantlets and leaves (control I) and seeds (control

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Histological studies. For histological sections, regenerating
calli were sampled and wax sections were cut to trace the devel-
oping vascular strands in the calli. Material was fixed in FAA
(5:5:90 v/v/v Formalin: Acetic acid: 70% Ethanol) for 48 h and
II) from adult parent plant were utilized to check azadirachtin
production. The calli cultures were broadly categorized into
dedifferentiated (non-regenerating) and redifferentiated (regen-
erating) lines. These Calli were harvested, washed with distilled
stored in 70% alcohol. After passing through the tertiary-butyl water and filtered under vacuum. Thereafter, washed callus
alcohol (TBA) series for dehydration, the material was infiltrated lines, leaves and seed samples were dried in an oven at 30°C
with paraffin wax (melting point 60°C, E. Merck, Germany) ± 2°C until a constant weight was achieved. The dried sam-
and finally, embedded in pure paraffin wax. Paraffin blocks were ples were soaked in methanol (analytical grade, Merck, India)
mounted on wooden stubs and 8–10 μm thick sections were cut overnight followed by sonication for 45 min at 35% amplitude
using Rotary Microtome (Leica, Germany), attached with a steel (pulser 5 s on/off cycle). The extract was centrifuged at 10,000
knife. The sections were mounted on microslides, dewaxed and rpm for 15 min and was further fractionated by adding water in
double stained with safranin (1%) and astra-blue (1%). 60:40 ratio followed by 50:50 dichloromethane (Merck, India).
Scanning electron microscopy. Nodulated calli from shoot Dichloromethane fractions were pooled, dried in a rotary evapo-
regeneration media were fixed in 2.5% glutaraldehyde and dehy- rator (Buchi R-200, Japan) and further analysed for presence
drated through a graded alcohol series. After drying, the samples of azadirachtin. Extraction from seeds consisted of an addi-
were sputter-coated with gold and observed under a scanning tional step of defattening with hexane prior to extraction with
electron microscope (Leo 1430vp, Carl Zeiss, Germany). methanol.
Shoot elongation, multiplication and rooting. Individual Preparation of azadirachtin standard. Stock solution of stan-
shoots of 0.5 cm length were detached from calli and given a dard azadirachtin (Sigma-Aldrich, USA) was prepared in metha-
14 days pre-treatment of GA 3 at 3 μM concentration before nol (HPLC grade, Merck, India) at a concentration of 1 mg/ml
transferring them to a lower concentration of BAP at 0.5 μM and stored at -20°C. Calibration curve was generated by external
for elongation. After attaining sufficient length, further shoot standard method. The stock solution was serially diluted to five
multiplication was achieved on MS medium supplemented with different concentrations and each concentration was run at least
BAP (1.0 μM) and CH (250 mg/l) by employing single node seg- thrice to check the repeatability of results.
ments. The number of propagules obtained at the end of a mul- Linearity of developed method was checked by running the
tiplication cycle was regarded as the rate of shoot multiplication. standard compound at five different concentrations. Precision
For rooting, individual shoots, measuring 3 cm, with 3–4 of developed assay was evaluated by running same concentra-
nodes, were excised and cultured on ¼ MS (major salts reduced tion of standard compounds at least four times on the same day
to quarter strength) medium supplemented with IBA at 0.5 μM. (intraday) and thrice at one day interval (interday). The values

980 Plant Signaling & Behavior Volume 6 Issue 7

 were calculated in terms of percent relative standard deviation in the subcultures of callus was expressed as percent response.
(% RSD) which is calculated as: (Standard Deviation/Mean) x Data were subjected to one-way or two-way analysis of variance
100. (ANOVA) and means were calculated using Duncan’s multiple
High performance liquid chromatography. Quantitative range test by SPSS 16.0 software. p-values less than 0.05 were
estimation of azadirachtin was carried out on Varian Prostar considered statistically significant. Arcsine transformation was
HPLC system (Varian, USA) equipped with a binary pump, UV done for the percent values before proceeding for ANOVA. For
detector and a 20 μl injection loop. Hypersil BDS RP-C18 col- azadirachtin estimation, all results are an average of three sepa-
umn (Thermo, USA) of dimensions 250 x 4.6 mm was used with rate analysis. Results are represented as mean ± SD.
methanol:water (90:10) (v/v) as the mobile phase at a flow rate
of 0.5 ml/min. The eluted samples were detected at 214 nm. The Conclusions
identification of azadirachtin was done by comparing its reten-
tion time with authentic standard (Sigma, USA). The crude and In our search for novel sources, we believe that haploid lines
standard samples were filtered through 0.22 μm PVDF mem- could be a valuable source of consistent qualitative and quanti-
brane filters (Millipore, USA) prior to analysis and aliquots of 20 tative homogeneous metabolite production. These lines, to our
μl of clean solution were injected into the HPLC system. System knowledge, were never earlier assessed for azadirachtin related
suitability tests were performed by checking linearity and preci- metabolite production. This study has brought us a step closer
sion of the developed assay. to obtain genetically pure lines (homozygous diploid) of Neem
Mass spectroscopy. MS detection was carried out on Waters and to diminish variations which are prevalent in the genus
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Quadrapole-Tof Premier mass spectrometer with micro channel due to heterozygosity. As the conventional methods to produce
plate detector (Waters, USA). Samples were analyzed in positive homozygous diploid plants is lengthy and laborious, requiring
mode with a probe temperature of 400°C and a source block 7–8 recurrent cycles of inbreeding which is impossible incase of
temperature of 150°C. The source was operated with a corona cross-breeding trees like Neem. With in vitro haploids, homozy-

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pin voltage of 3.50 kV and a cone voltage of 25 V. The MS data gous diploids can be produced in a single step, cutting down the
were obtained in full scan mode (mass range 100–1,000 amu). breeding period to almost half. Our goal is to employ double-
A comparison of mass spectra of the standard compound with haploid lines (homozygous diploid) for identification and puri-
that of sample isolated from HPLC, confirmed the presence of fication of compounds in bulk, round the year independent of
azadirachtin. region and vagaries of climate, by employing superior lines.

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Statistical analysis. For culture initiation and induction
responses, 20 anthers were placed in one Petridish with 24 dishes
for each treatment. Data were collected as number of responding
anthers relative to total number of anthers cultured. Regeneration
Acknowledgements
The authors thank Department of Science and Technology
(DST), New Delhi, India, for financial assistance.
9. Pattnaik SJ, Rao NDR, Chary P. Ecomorphometric 16. Kearney ML, Allan EJ, Hooker JE, Mordue AJ.
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