ORIGINAL PAPER

Localisation of Storage Reserves in Developing Seeds of Pongamiapinnata (L.) Pierre, a Potential Agroforestry Tree

H. R. Pavithra • B. K. Chandrashekar Sagar •

K. T. Prasanna • M. B. Shivanna •

Balakrishna Gowda

Received: 24 April 2013 / Revised: 12 August 2013 / Accepted: 14 August 2013 / Published online: 1 September 2013

� AOCS 2013

Abstract Pongamia pinnata is an important oil yielding

perennial tree species. The aim of the present study was to

document the histological and ultrastructural change that is

occurring during pongamia seed development. The seeds

were sampled at five stages of development at intervals of

3 weeks starting from 30 weeks after flowering up to

42 weeks. The seed development was followed micro-

scopically using toluidine blue staining. The seed coat was

made up of an external layer of palisade cells, an internal

layer of hourglass cells followed by a parenchymatous cell

layer and aleurone cell layer. The seed reserve compounds

such as polysaccharides, proteins and starch showed dis-

tinct histochemical characterisation. Lignin was mainly

found in the seed coat cell layers, while polysaccharides,

proteins and starch granules in the cotyledon cells. The

ultrastructural studies showed marked cellular changes

during the seed development. The cell size varied from 9.4

to 78 lm during the seed development. The number of oil

bodies per cell ranged from 200 to 300 at 42 weeks after

flowering. Protein storage vacuoles were observed during

the later stages of seed development. The plastids con-

tained electron-dense starch granules. The seeds harvested

after 42 weeks after flowering had maximum physiological

maturity with high oil content and other seed reserve

materials. This basic knowledge on pongamia seed devel-

opment could invariably be used for further understanding

of biochemical changes that might be involved in the

biosynthetic pathway of oil.

Keywords Histology � Histochemistry � Pongamia

pinnata � Reserve material � Seed development �Ultrastructure

Abbreviations

PSV Protein storage vacuole

WAF Weeks after flowering

Introduction

Pongamia pinnata (L.) Pierre [family: Fabaceae; Syn.

Pongamia glabra Vent., Derris indica (Lam.) Bennett and

Millettia pinnata (L.) Panigrahi], locally called honge,

pongam or karanj, is an indigenous tree in the Indian

subcontinent. It is one of the most suitable tree species for

the production of biofuel, a sustainable substitute for fossil

fuel [1]. Pongamia is a multipurpose tree species well

known for its timber and medicine [2] and its application in

agriculture and insecticide [3]. Because of its versatility,

the species has been introduced and cultivated in Australia,

the United States of America and China [4]. The plant

starts producing fruits at the age of 6–7 years and the fruits

are harvested during February–April. Pongamia oil consists

of karanjin, karanjone and diketone pongamol [5].

Electronic supplementary material The online version of thisarticle (doi:10.1007/s11746-013-2335-8) contains supplementarymaterial, which is available to authorized users.

H. R. Pavithra � K. T. Prasanna � B. Gowda (&)

Biofuel Park, Department of Forestry and Environmental

Sciences, University of Agricultural Sciences, GKVK,

Bangalore 560065, Karnataka, India

e-mail: gowdabk@yahoo.com

H. R. Pavithra � M. B. Shivanna

Department of Applied Botany, School of Biosciences,

Kuvempu University, Jnana Sahyadri, Shankaraghatta,

Shimoga 577451, Karnataka, India

B. K. C. Sagar

Department of Neuropathology, National Institute of Mental

Health and Neurosciences, Bangalore 560029, Karnataka, India

123

J Am Oil Chem Soc (2013) 90:1927–1935

DOI 10.1007/s11746-013-2335-8

The seeds contain proteins, carbohydrates, starch, lipids

and mineral ions that are stored as the primary reserve food

material and are used during seed germination and seedling

growth [6]. Lipids contribute up to 80 % of the total dry

matter and are an important storage component in oilseed

tree species. Lipids occur as oil bodies in the cotyledonary

tissues [7] and carbohydrates are stored in starch granules

or cell wall thickenings in the cotyledonary cells [8] while

proteins are accumulated as protein bodies [9]. The process

of seed maturation encompasses a series of morphological,

physical, physiological and biochemical changes that occur

following egg fertilisation to seed maturity. Seed devel-

opment phases are well understood in Phaseolus vulgaris

[10], Vicia faba [11] and Vitis vinifera [12] including

delineation of sub-cellular storage reserve compartments.

Pongamia is considered to be the most important non-

edible oil yielding tree with ecological, economical and

nutritional value as detoxified animal feed in recent years;

therefore the histochemical, ultrastructural, biochemical

and molecular processes underlying the seed development

require greater understanding. The information on the

developmental stages of pongamia seed and the determi-

nation of the proper stage for seed harvesting is lacking in

the literature. In a previous study, an ideal time for har-

vesting of mature pongamia fruits was identified as

42 weeks after flowering (WAF) when the fruits ripen and

possess high oil, oleic acid and karanjin contents [13]. The

purpose of the present study was to provide histological,

histochemical and ultrastructural evidence for identifying

the exact harvesting period when high reserve accumula-

tion and oil content coincides with the physiological

maturity of pongamia seed.

Materials and Methods

Identification of Trees for Collection of Seed Samples

Ten 20-year-old pongamia trees at the flowering stage in

the campus of Gandhi Krishi Vignana Kendra, University

of Agricultural Sciences, Bengaluru, India were identified

and marked for collecting the seed samples. The charac-

teristics of the trees were five to ten sub-branches, a cir-

cular canopy with drooping branches. The inflorescence

was tagged a few days before the anthesis of flowers

(unfolding of standard petal indicated flower opening) [14].

Throughout the duration of flowering the inflorescences

were monitored and on the third day closed flowers without

corolla (completion of pollination) were tagged. The tag-

ged pods of all the marked trees were harvested at an

interval of 3 weeks starting from 30 WAF until 42 WAF

and pods at each specific developmental stage were pooled

separately. The pods (both single and two seeded) were

studied for their morphological characteristics. The indi-

vidual pod samples at specific stages were stored in sealed

polythene bags at -20 �C. The seeds at different devel-

opmental stages were separated from pods manually and

were subjected to histochemical and ultrastructural studies.

Histology and Histochemistry of Developing Seeds

Cotyledons with the seed coat (1 9 1 cm) were fixed in

Carnoy’s fixative solution (60:30:10; ethyl alcohol:chlo-

roform:acetic acid) for 24 h, dehydrated in a gradient

ethanol and butanol series and embedded in wax (9:1;

paraffin:beeswax). The tissue blocks were prepared and cut

into 12-lm thick sections using a rotary microtome (Erma,

Japan). Toluidine blue O was used to study the seed mor-

phology [15]. The histochemical stains used were periodic

acid/Schiff (PAS) reagent for insoluble polysaccharides

[16], mercuric bromophenol blue staining for proteins [17]

and Lugol’s reagent for starch [18]. Fresh sections of the

cotyledon were used for staining oil droplets by Sudan red

7B (Sigma Aldrich, India) [19]. All sections were mounted

on glass slides and observed under a microscope at 409

magnification (Labomed, India) coupled with a camera

(Canon Power Shot A95, USA).

Ultrastructure of Developing Seeds

Cotyledons were separated from the pods, segmented

(1 9 1 mm) and fixed (3 % glutaraldehyde in 0.1 M

sodium phosphate buffer (pH 7.2) for 24 h) at room tem-

perature and rinsed in phosphate buffer (pH 7.2) twice for

15 min (to remove the fixative). The specimens was post-

fixed in 1 % osmium tetroxide for 2 h at 4 �C and rinsed in

phosphate buffer two times for 15 min (post fixation). The

samples were dehydrated in a graded ethanol series (70, 80,

90 and 100 %) at room temperature for 1 h in each and

cleared with propylene oxide two times for 15 min each at

room temperature. The material was then embedded in an

epoxy resin (Taab, UK). Ultra-thin sections (60 nm) were

obtained with a glass knife (Leica MZ6, EMUC 6) and

sections were double stained with uranyl acetate followed

by lead citrate. The sections were observed with a trans-

mission electron microscope (Technai 12, Netherlands).

Results and Discussion

The pongamia trees started producing flowers during

March at the study site. The flowering lasted till May. The

pod setting, an elaborate process was initiated during June–

July and completed during January–February. The pods

were flat and thin initially but were fully formed by

August–September. During this period the embryo is

1928 J Am Oil Chem Soc (2013) 90:1927–1935

123

highly immature with minimum size. The embryo starts

developing from 30 WAF onwards during October until it

attains physiological maturity. The pods appear green

during June to January and half brown during January–

February and completely dried pods turned brown.

In the present work, the histological, histochemical and

ultrastructure of pongamia seed were determined at the five

intervals (30, 33, 36, 39, 42 WAF) such as the early green

immature pod stage, half brown pod stage and late dark

brown pod stage. In pongamia seeds, energy resources

stored in cotyledons help in seedling development, the

proteins in the seed are also source of food for animals in

the form of detoxified oil cake and the oil is a substitute for

petroleum fuel. Generally, the accumulation of each seed

storage material varies depending on the seed development

stage. The initial seed development is characterised by a

slow cell mass accumulation which is followed by matu-

ration phase with continuous increase in dry matter and

accompanied by reserve material accumulation. The pre-

vious study on pongamia showed that pod length and

breadth did not vary much however the pod thickness, seed

length, breadth and thickness increased from 30 to 42

WAF. The oil content increased with seed maturity and the

oleic acid content remained high at the end of pod maturity

while karanjin content varied significantly across different

stages of pongamia seed development [13]. Localisation of

seed storage reserves in pongamia showed a marked

compartmentation similar to that observed in Phaseolus

vulgaris [10]. The third phase is characterised by seed

dehydration with maximum physiological maturity.

Microscopic Analysis of Developing Seed

The meta-chromatic reagent used for staining the different

stages of seed development revealed the differential

staining ability of the seed parts. The longitudinal sections

of the seed revealed the presence of different zones

evolving from the integuments that surround the ovule

before fertilisation (Supplemental file 1).

At 30 WAF, the seeds appeared green. The developing

embryo at 30 WAF was compact and completely attached to

the seed coat which is represented by a thin layer of cuticle.

The palisade cells were thin with less secondary thickenings

and the hourglass cells had not yet formed. The inner

parenchyma cells were present below the hourglass cells. A

thin layer of aleurone cells was seen below the parenchyma

cell layer. The vascular bundles are represented by long

tapering tracheids that were formed in the parenchyma

region of the seed coat near the hilum (Supplemental file 2).

The parenchyma cells of the cotyledon are irregular in shape

and loosely arranged with a few and many secretory cells at

the adaxial and abaxial regions, respectively.

At 33 WAF, the palisade cells were thickened and

highly compact. Thin layer of hourglass cells with a

thickness of one to two cell layers were observed. The

inner parenchyma cells increased in number and were

loosely arranged. The aleurone cell layer was clearly vis-

ible with the remains of outer endosperm layer. The

interspace between the seed coat and cotyledon was clearly

visible. The vascular bundles extended laterally into the

parenchyma region of the seed coat. A thin outer epidermal

layer of the cotyledon was visible. The cotyledonary

parenchyma cells were irregularly shaped and the cell size

increased gradually. The walls of secretory cells were

thickened in the developing cotyledon.

At 36 WAF, a thin layer of deeply staining cuticle was

observed. The palisade cell layer was unevenly arranged,

with a few cells having heavy secondary thickenings in

their cell wall. The wall of hourglass cell started to thicken.

The parenchyma cells increased in number and turned

spherical. The inner aleurone cell layer was deformed

along with the remains of outer endosperm layer. The

interspace between the seed coat and cotyledonary cells

widened as compared to the previous stage. The tracheid

bar appeared slightly distinct in the parenchyma region of

the seed coat. The outer epidermis of cotyledon cells was

uneven. The number of cotyledonary parenchyma cells

increased as compared to the previous stage. The paren-

chyma cells enlarged and attained polygonal shape. The

cell wall of cotyledonary cells was slightly thickened. The

number of secretory cells gradually increased in the

developing cotyledon.

At 39 WAF, the palisade cell layer as well as the cell

wall of hourglass cell layer was increasingly thickened.

The parenchyma cells assumed a spherical shape and were

arranged in the seed coat matrix; the cells increased in

number and size as the seed matured. The aleurone cell

layer was prominent with a predominant increase in the

interspace between the seed coat and the cotyledon, as

compared to the previous stage. The tracheid bar was

clearly distinguishable in the parenchyma region of the

seed coat. The outer epidermis of the cotyledon was also

highly prominent. The number of secretory cells increased

at this stage of seed development.

At 42 WAF, the seed coat was deformed and plasmol-

ysed as the seed moisture content decreased. The cell wall

thickening of the palisade cell layer reached maximum.

The hourglass cell layer was not clearly visible. The seed

coat parenchyma cells were densely packed and the aleu-

rone cell layer was deformed. The interspace now was

extended between the seed coat and cotyledonary tissue.

The vascular bundle at the hilum region of the seed coat

was distinct. The outer epidermal layer of the cotyledon

was prominent. The cotyledonary parenchyma cells were

J Am Oil Chem Soc (2013) 90:1927–1935 1929

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densely packed with regular shape. The number of secre-

tory cells increased at this stage of seed development.

During 30–42 WAF, the pongamia seed coat undergoes

a sequence of changes, from a single entity to multiple cell

layers which persisted for varying time interval. This per-

iod of development corresponded to intensive cellular

division and differentiation which result in increase in the

size of seed. The thickness of the seed coat increases ini-

tially and is attached to the developing cotyledonary tissue.

The secondary thickenings of palisade cell layer of the seed

coat varied during the development of seed at 36 and 39

WAF. The palisade and hourglass layers become thick

walled and prominent at seed maturity (42 WAF). Similar

developmental changes in secondary thickenings of the

palisade cell layer have been reported in soybean seed coat

[20]. The aleurone cell layer in the seed coat is visible

during the 30–33 WAF, but as the seed matured, the

aleurone cell layer was compressed and resulted in a

remnant due to loss of moisture. The aleurone cell layer is

known to synthesise, transport and secrete nutrients into the

developing seed [21].

The surface of the pongamia seed coat is a typical

rugose–foveate type [22] at the time of maturity (42 WAF).

The mature seed consisted of an outer seed coat layer, an

interspace region between the seed coat and cotyledon and

inner cotyledon cells. The seed coat is made up of an outer

cuticle layer. This layer is followed by the palisade layer

(macrosclereids) with radially elongated cells. Hourglass

cells are composed of thick walled osteosclereids. These

layers comprise the outer integument layer. The layer

adjacent to this is parenchymatous layer formed of six to

eight layers of thin-walled cells which are loosely arranged.

This layer comprises the inner integument layer. The next

layer is the aleurone cell layer which is comprised of the

degenerated intact outer endosperm layer. This was fol-

lowed by an interspace between the seed coat and cotyle-

don parenchymatous cells with secretory cells. The general

structure of pongamia seed including the structure of seed

coat and cotyledon corresponded to that of soybean [20]. A

variety of differentiation pattern is seen in the cell layers of

seed coat. This could be indicative of the fact that the fate

of the cell layer undoubtedly arises from the functional

requirement during the seed development. In general,

secretory cells are present in many parts of plants including

cotyledons for the production of secondary metabolites [23,

24]. In pongamia, the secretory cells are present in the

cotyledon of the developing seed. These secretory cells

might synthesise karanjin and other flavonoids in pongamia

and provide a defense against fungal and bacterial decay. A

similar kind of secretory cell secreting secondary metabo-

lites has been reported in Azadirachta indica [25] and

Caesalpinia peltophoroides [26]. In legume seeds, the

developing pod walls and seed coat are transient reserves

of assimilates and other nutrients for transportation into the

developing cotyledons [27, 28]. In pongamia seed devel-

opment, the single vascular bundle (tracheid bar) was

observed in the hilum region which was similar to that in

Medicago truncatula [29].

Histochemistry of Developing Seeds

The histochemical staining of the developing pongamia

seeds is detailed in Table 1, the cotyledon cells stained

positive irrespective of the developing stage of the seed

(Supplemental file 3).

Lignin

The toluidine blue stain caused an intense to very intense

bluish green staining of the seed coat and cotyledon. At 30

WAF, a light bluish green colour was visible on the cell

walls of the palisade cells. The cotyledon cells stained light

blue. At 33 WAF, the cell wall of the palisade cell layer

stained intensely. At 36 WAF, the palisade cell layer

stained intensely as compared to the previous stage. The

colour intensity of the seed coat layer increased up to the

end of 42 WAF. The colour intensity of cotyledonary cells

showed little variation although there was a slight variation

in the number, shape and size of cells. The palisade layer of

the seed coat responded the most to toluidine blue staining.

Table 1 Histochemical staining of developing Pongamia pinnata seeds from 30 to 42 WAF

Biochemical compound Stain/reagent Seed coata Cotyledon cells

1 2 3 4 5

Lignin Toluidine blue O ? ??? ?? ? ?? ?

Insoluble polysaccharides Periodic acid/schiff reagent - ?? ?? ? ? ???

Proteins Mercuric bromophenol blue - - - - - ???

Starch Lugol’s reagent - - - - - ???

Lipid Sudan red 7B - - - - - ???

?, presence; -, absence; the number of signs signify the staining intensitya 1, cuticle cell layer; 2, palisade cell layer; 3, hourglass cell layer; 4, parenchyma cell layer; 5, aleurone cell layer

1930 J Am Oil Chem Soc (2013) 90:1927–1935

123

Polysaccharides

The cuticle stained intensely with a reddish purple colour.

At 30 WAF, the cell walls of the seed coat tissue were

slightly coloured and as the seeds matured from 33 to 42

WAF, they stained intensely. The parenchyma cells of the

cotyledon were lightly stained. The reddish purple colour

of the cotyledon cell content remained constant until har-

vest. The polysaccharide granules increased from 33 to 42

WAF. The granules were equally distributed across the

cotyledonary tissue from the abaxial region to the adaxial

region.

Proteins

The proteins in the cotyledon stained dark blue. The

parenchyma cells of the seed coat and cotyledons stained

lightly at 30 WAF. The staining intensity of the cytoplasm

of the parenchymatous cells increased from 33 to 42 WAF

during seed development. The protein granules were highly

condensed at the time of seed harvest. The protein granules

were uniformly distributed across the developing cotyledon.

Starch

The starch granules in the parenchyma cells of cotyledon

stained dark purplish blue. The seed coat stained light

brown. The cotyledon cells showed lightly stained starch

granules at 30 WAF. The starch granules were deeply

stained as the seed matured from 33 WAF to 42 WAF. At

the time of maturity, the starch granules were distributed

throughout the cotyledonary cells.

Lipids

The lipid granules in the form of oil droplets in the

parenchyma cells of cotyledon stained red. The cotyledon

cells showed lightly stained oil droplets at 30 WAF. The

number of oil droplets increased as the seeds matured from

32 to 42 WAF. At the time of seed maturity, the oil

droplets were distributed throughout the cotyledonary cells.

Lignin is the secondary cell wall component in the pal-

isade cell layer of the seed coat. Proteins, insoluble poly-

saccharides, starch and oil bodies are located mainly in the

cotyledonary parenchyma cells. According to Taiz and

Zeiger [30] lignin is associated mainly with the hemicel-

lulose in seed coat layer ensuring the protective survival of

the offspring by maintaining an environment around the

embryo during the extreme conditions. As the moisture

content of developing seed is reduced to 14 % at the time of

physiological maturity (42 WAF) [13], the seed coat cells

shrink with the progress of seed maturity and the seed

surface becomes rough and brown. The browning of the

seed coat could be attributed to condensation of tannins as

reported in other legume seeds [31]. The secondary wall

thickenings of palisade and hourglass cells resulted from

lignin impregnation causing a reduction in cell lumen.

Lignin in the cell wall renders the seed hard and strengthens

the seed coat with definitive structure. It has been reported

earlier that at different seed moisture content levels, the

hydrophobic lignin content in palisade layer of seed coat

makes it impermeable and hard-seeded and with decrease in

moisture content the seed becomes harder [32]. The lignin

content in seed coat might decide the moisture content

requirement of seeds. Pongamia seed requires 10 to 14 %

moisture content for effective germination. Although seed

coat confers impermeability to the mature seed, physical

dormancy is not seen in pongamia since the seed coat is thin

whereas physiological dormancy has been reported [33].

Therefore seeds collected after 42 WAF confers physio-

logical maturity with maximum germination. The seed

reserves like insoluble polysaccharides, proteins, starch

granules and oil bodies ensure the survival of the seedlings

during the initial growth [30, 34]. At 30 WAF, the palisade

cells were light blue in colour due to thin cell wall and very

few insoluble polysaccharides were seen. The increase in

polysaccharide content might be necessary to promote

growth by cell division during 33–42 WAF. In legume

seeds, the capacity of cotyledons to accumulate dry matter

could be dependent on the final cell number [35]. In

pongamia, the cotyledons stored starch but in relatively low

quantities, however they accumulated lipids mainly in the

form of oil bodies. Therefore it is recommended to harvest

the seeds after 42 WAF with complete accumulation of

lipids, proteins and fully developed cotyledons.

Ultrastructure of Developing Seeds

The TEM analysis of the developing pongamia seeds

revealed a series of changes as the seed matured (Figs. 1, 2).

At 30 WAF, the walls of the cotyledon cells were poorly

developed. There were a few plastids at the early stage with

yet to differentiate thylakoid, as well as starch granules,

few oil bodies. The cells were highly vacuolated with a

small electron-dense region. The cellularisation of cotyle-

don was complete at the end of 30 WAF.

At 33 WAF, the cotyledon was completely cellularised

and cells were irregular in shape and the size ranged from

9.4 to 27 lm; the cell walls were thickened. A prominent

nucleus was visible at this stage of cotyledon development.

The plastids were elongated, with a few developing into

mature structures with the embedded starch. The formation

of protein bodies was initiated. The fragmentation of big

vacuoles into small ones was succeeded by the protein

deposition. The cytoplasm of cotyledon cells started

accumulating a few oil bodies.

J Am Oil Chem Soc (2013) 90:1927–1935 1931

123

At 36 WAF, the cell size ranged from 30.3 to 44.6 lm.

The wall thickening increased with the cell attaining a reg-

ular polygonal shape. The electron-dense nucleus was

observed between the oil bodies in the cytoplasm. The

number of plastids ranged from 10 to 14 with dense starch

granules. Most of the plastids were oval in shape. The

spherical oil bodies were localised around the angular crys-

talloid protein granules in the cytoplasm. The membrane of

Fig. 1 Transmission electron micrographs of developing Pongamia

pinnata seed (early stages) showing the distribution and structure of

reserve materials. a, b Portion of cotyledon cell at 30 WAF showing

cell wall (CW), plastids (PL), starch (S), unfragmented vacuole (V),

oil bodies (OB), protein granules (P) (bar = 2 lm). c, d Cotyledon

cell at 33 WAF showing developing cell wall, many plastids with

starch granules, oil bodies, protein granules (bar = 10 and 2 lm). e,

f Cotyledon cell at 36 WAF showing cell wall, protein storage

vacuoles (PSV), plastids, starch granules, fragmented vacuoles (FV),

numerous oil bodies with oil body membrane (OBM) embedded in the

cytoplasm and nucleus (N) (bar = 10 and 1 lm)

1932 J Am Oil Chem Soc (2013) 90:1927–1935

123

the oil bodies was protein dense and the thickness ranged

from 0.04 to 0.06 lm. The oil body size ranged from 0.7 to

1.3 lm and the number increased up to 50–60.

At 39 WAF, the cell wall of the cotyledonary cells was

undulated and the thickness increased; the cell size ranged

from 51.4 to 70 lm. The number of plastids remained

constant but the thylakoid membrane integrity changed

slightly. At this stage, of the two kinds of vacuoles

observed in the parenchyma cells, one contained more or

less granular particles that were dispersed throughout and

the other was a homogenous electron-dense material which

is localised at the periphery of the vacuole. These vacuoles

represented different stages of the formation of protein

bodies. The fragmented vacuole developed into a protein

storage vacuole (PSV) in the cell matrix. The PSV size

ranged from 10.7 to 16.4 lm and electron-dense protein

crystalloids increased in number and size. The spherical oil

bodies were deposited around the PSV in the cytoplasm.

The number of oil bodies increased up to 100–200 per cell

with the size ranging from 0.9 to 1.9 lm; the oil body

membrane thickness ranged from 0.10 to 0.12 lm.

At 42 WAF, the cell wall of the cotyledonary cells was

completely developed and the thickness increased. The cell

size ranged from 70 to 78 lm. A prominent nucleus was

observed at the time of maturity. Starch granules in the

plastids were heavily electron dense and very few plastids

were observed. The uneven shaped oil bodies that were

arranged compactly at the periphery of the cell wall

increased in number ranging from 200 to 300 per cell. The

oil body membrane thickness ranged from 0.14 to 0.24 lm.

The size of the unevenly shaped oil bodies ranged from 0.3

to 1.2 lm. The PSV were highly electron dense, 6-8 in

number and ranged in size from 5.8 to 15.2 lm.

In general, the ultrastructure of pongamia cotyledons

revealed the presence of cells filled with oil and protein

bodies, irregular nuclei, plastids, and mitochondria. The

sub-cellular organelles such as mitochondria and chloro-

plasts were found to disintegrate during the later stages of

seed development. The loss of thylakoid membranes and

chloroplast of developing pongamia seed is coincident with

the onset of summer (January–February). This reduction in

the sub-cellular membrane surface could be the mechanism

Fig. 2 Transmission electron micrographs of developing Pongamia pinnata seed (late dark brown stage) showing the distribution and structure

of reserve materials. g, h Portion of cotyledon cell at 39 WAF showing cell wall (CW), numerous oil bodies (OB) surrounding the protein storage

vacuoles (PSV), protein bodies (PB), dedifferentiated plastids with starch granules (bar = 5 and 1 lm). i, j Cotyledon cell at 42 WAF showing

developed cell wall, numerous oil bodies with thick oil body membrane (OBM), protein storage vacuoles and nucleus (N) (bar = 10 and 1 lm)

J Am Oil Chem Soc (2013) 90:1927–1935 1933

123

to slow down cellular metabolism and prevent physical

damage to the internal membranous system of cell [36].

The most important storage lipids in pongamia seeds are

triglycerides surrounded by a membrane consisting of

phospholipids and embedded oleosin structures called oil

bodies [37]. Since pongamia seeds are exo-endospermous,

the oil bodies are found mainly in the cotyledon cells. The

genesis of oil bodies in the cytoplasm is associated with the

presence of endoplasmic reticulum and plastids. The

diameter of the oil bodies observed in pongamia seed was

at par with the other diverse species [7]. The diameter of

the oil bodies (0.3 to 1.2 lm) was correlated to the high oil

content of the mature seed (36.53 %) [13]. The correlation

between high oil content and small oil bodies was also

reported in Brassica napus [38]. The membrane thickness

of oil bodies increased during 36–42 WAF and correlated

with the extensive moisture loss during this period of seed

development. At 42 WAF, with the decrease in moisture

content, oil bodies tend to experience cytoplasmic com-

pression but following resistance to aggregation, they

remain as individual entities [39]. The increase in the

number of oil bodies coincides with the total oil content of

seed at 42 WAF [13]. At the later stages of seed devel-

opment, the oil bodies accumulate at the periphery of the

cell. It has been suggested that these oil bodies serve as an

immediate reserve during seed germination [36].

Protein bodies arise from the endoplasmic reticulum or

Golgi vesicles and are sequestered into the vacuoles to

form PSV [40]. Phytoferritins, the iron storage protein play

an important role in iron metabolism that is observed in the

early stages of pongamia seed development and very few

plastids are observed at the later stages of seed develop-

ment. The presence of phytoferritins at the early stages and

their absence at subsequent stages of development has been

reported in Myrsine laetevirens [41]. These phytoferritins

have also been reported in other legumes as well as in non-

legume species like Vicia faba [11] and Chenopodium

quinoa [42]. Crystalloid structures are present in the pro-

tein bodies at the later stages of seed development. The

globoids are electron-dense inclusions that mainly consti-

tute mineral reserves such as iron, manganese, magnesium,

potassium and calcium [43]. The average diameter of PSV

of the developed pongamia seed is similar to the PSV of

mature seed of Caesalpinia peltophoroides [26].

In developing pongamia cotyledons, starch grains are

present in the plastids with irregular shapes. Starch gran-

ules within the plastids of developing peanut have also

been reported by [8]. In these plastids, association of starch

grains with thylakoid might indicate that this structure

would result in chloroplast formation during germination.

The electron-dense region within the developing starch

grains of plastids might provide evidence of the location

for enzymatic activity during the biosynthesis of starch.

These starch reserves provide a sugar source during seed

germination.

The harvesting period of oilseeds depends on the

physiological maturity and accumulation of seed reserve

material [44]. Generally, pongamia pods are harvested

when most of them ([80 %) in an inflorescence attain

morphological maturity leading to improper collection.

The premature harvesting of pods might result in reduced

availability of seed reserve material and oil content with

low germination efficiency. Since the cotyledon cells at the

end of 42 WAF showed a higher number of oil bodies

(200–300 per cell) with high protein storage vacuoles and

few starch granules this stage is the most appropriate for

pod harvesting. Hence based on this, the present study

suggested that pongamia seeds could be collected at the

end of 42 WAF when the oil content is high and accu-

mulated with seed reserve material. Such seeds probably

possess maximum seed germination and high seed vigor.

Conclusion

The seeds of pongamia during development show charac-

teristics very similar to the seeds of other legume species.

The present study revealed that the major reserve food

materials in pongamia seeds are lipids and proteins com-

partmentalised in oil bodies and protein storage vacuoles,

respectively in addition to the presence of a small number

of starch granules. This knowledge on pongamia seed

development could be used for further studies on predicting

the ideal time of pod harvesting with respect to biochem-

ical changes and enzymes involved in the biosynthetic

pathway of oil. Research needs to be undertaken in pong-

amia to define the role of pod wall development and its role

in nutrient transfer to the developing seed.

Acknowledgments The authors acknowledge the financial support

and facilities received from the Department of Agriculture, Karnataka

State Biofuel Development Board, Government of Karnataka and The

University of Agricultural Sciences, GKVK, Bengaluru and the

Department of studies in Applied Botany, School of Biosciences,

Kuvempu University, Shimoga and the Department of Neuropathol-

ogy, National Institute of Mental Health and Neurosciences, Ben-

galuru. Sincere thanks to Dr. Chandrika K. and Mr. Rajesh Kumar for

their help with histochemical studies and TEM analysis.

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