SUMMARY

The present work on Boerhaavia diffusa has been undertaken with the sole motive
of understanding the floral biology and the reproductive strategies of the plant. The
plant is of immense importance because of its medicinal value.
Boerhaavia diffusa (Nyctaginacea) is an important medicinal plant much
used in Ayurveda and Unani medicines and other traditional medicines in many
parts of the world. The importance of this plant can be gauzed from the fact that
there are almost forty regional names for it the world over (http://www.raintree.com/ervatostao.htm). The plant commonly known in India as Punarnava (and
also, Shothagni, Rakta punarnava) in Ayurveda because the top of the plant dies
during hot summers and puts forth fresh shoots after rains and is believed to be a
rejuvenator. Punarnava works well in combination with guduci, haritaki, devadara
and guggulu, the mixture of these powders given with cows urine, alleviates
anasarca, ascites, anaemia, worm infestation and diabetes. It is also a very good
expectorant and used as a cure in jaundice (Anonymous, 2001). The active
principle is a body of alkaloid called as punarnavine. The higher active principle of
alkaloid is found in dry conditions.
Medicinal plants constitute an important group of non-wood forest
products. Approximately 80% of the world population uses plants as a source of
medicine for healthcare (Anonymous, 1998). Of the 250,000 plant species known
in the world, the World Health Organization has listed around 21,000 species
which are used for medicinal purposes. According to an EXIM Bank report the
value of medicinal plants related trade in India is estimated to the order of Rs. 5.5
billion per year, while the world trade is over US$ 60 billion per year, and is
growing at the rate of 7% per annum. The annual export of these plants is valued at
Rs. 1200 million (http://www.eximbankindia.com/old/press031113.html, 2003).
Boerhaavia diffusa is such an important medicinal plant that almost 2000 Metric
tonnes of the whole plant or its roots are collected annually (Anonymous, 2000).

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So much is the collection pressure on this plant that it has almost become extinct
from the campus of Banaras Hindu University, Varanasi and the authorities had to
put a total ban on its collection (Singh, 2007).
The present study revealed that the plant, Boerhaavia diffusa is in dynamic
equilibrium with its environment, whether it is in terms of production of flowers or
germination of pollen grains or formation of healthy seeds. The plant is a creeping
herb which puts forth fresh flush of shoots year after year in the month of March,
which eventually die at the end of the growing season in November. The root stock
with several dormant shoot buds on the crown survives the severe winter. Over the
years the root stock grows deeper into the ground increasing in girth and
accumulating a large number of secondary metabolites. The new shoots that are
formed at the beginning of the season bear at their terminals young buds arranged
in an umbel. The new shoots that are either green or pink bear unequal leaves in an
opposite decausset arrangement. A temperature around 35 oC appears to be the
most optimum for maximising flower production. Extremes of temperatures on
either side (24 oC and 42 oC) adversely affect flowering density.
In B. diffusa (present work) the parianth of the flower varies in colour from
light pink to dark pink to almost maroon. The flowers are hermaphrodite with three
stamens and a pistil. The filament of the stamen is richly pink and the pistil is of
pastel pink. The plant has a penchant for unequal sizes of the same type of
structures. Like the unequal size of leaves opposite each other, the two lobes of the
anther are also of unequal size. And so are the pollens contained by them. The
large lobe has bigger and small lobe has smaller pollen grains. The anther in B.
diffusa (present study) is bisporangiate with a delimiting epidermis beneath which
are present a single layer each of the endothecium, middle layer and the tapetum.
Bisporangiate anthers are encountered in many families and plant species. In B.
diffusa (present study) the pollen grains are 59-76 m across with an exine
architecture consisting of spines and pores. The spines give the pollen the

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stickiness and help in adhering to the stigma surface, which is made of loose cells
of papillae. The pollen grains are a rich source of carbohydrates, lipids and
proteins.
The disc shaped stigma of B. diffusa is designed as a launching pad with
incurved peripheral rim is also rich in polysaccharides, lipids and proteins (present
study). Ants frequent the flower to collect the pollen and the stigma heads and help
in transferring the pollen to the stigma in B. diffusa (present study).
Histological and SEM studies of the stigma and style of B. diffusa (present
study) has revealed that the stigma is the dry type. The outer epidermis is made up
of a large number of papillae which are rich in polysaccharides, lipids and proteins.
Beneath the epidermis are present elongated cells which have a rich supply of
vasculature and mucilage cells. The peripheral rim is also several layers thick and
has mucilage cells. The style has an outer epidermis with elongated cells making
up the rest of the body. Vascular supply and mucilage cells were noted in the style
as well. Stigma is a site of hectic enzyme activity was demonstrated by localization
and quantification of four enzymes, peroxidases, polyphenol oxidase, acid
phosphatase and esterase. The former two were active 2-4 h after pollination while
the later two were active between 4-6 h after pollination.
Being self-compatible B. diffusa (present work) are compulsively
autogamous, though they tolerate geitenogamy within the remet but not in the
genet. The large size of pollen grains and their low numbers are supportive to the
fact that the plants display obligate autogamy. The peripheral rim of the stigma is
the naturally receptive region as the pollen grains land directly there when the
anthers dehisce. Just about 10 % of the pollen grains produced by an anther (130
pollen grains) are able to effect successful fertilization.
There is a single ovule per ovary in B. diffusa (present work). A minimum
of one to a maximum of nine pollen tubes have been found (present work) within
the styles of B. diffusa and consequent fruit set has also been observed. It was
observed in the present study that the number of pollen grains on the stigma
surface also varied from 2 to 12, which also resulted in fruit set. However, per cent
fruit set varied considerably depending on the number of pollen found on the
stigma surface (present work). B. diffusa produces about 130 pollen per anther and
there are three stamens producing a total of about 400 pollen grains. This number

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is more than sufficient and could be the reason of compulsive autogamy. The spare
pollen are available to the ants.
The stigma surface stained intensely for all the four enzymes, peroxidase,
polyphenol oxidase, acid phosphatase, and esterase. Freshly collected stigma had a
convex turgid surface with pastel pink colouration. Treatment of the stigma for
specific enzymes gave colour typical of that enzyme. (a) Peroxidase was found to
be abundantly present when tested by both TBA and guaiacol method. ROS/H2O2
is the in vivo substrate for peroxidase and its abundance throughout the stigma was
indicated by the appearance of blue colour on treatment with TMB (3,39,5,59tetramethylbenzidine-HCl). Treatment with guaiacol gave orange/red-brown
colouration to the periphery to start with which latter spread to the entire stigma
surface, typical for peroxidase; (b) Acid phosphatase was found to be present on
the stigma surface. Stigma when treated with sodium -glycerophosphatate gave a
blackish-brown colour indicating presence of acid phosphatase. Acid phosphatase
activity could be observed only on the periphery of the stigma. The centre of
stigma did not give the coloured reaction for acid phosphatase; (c) Esterase was
also present throughout the stigma surface and was indicated by brown colour on
treatment with 1-napthyl acetate; (e) Polyphenol oxidase breaks down o-catechol
and gave a typical brown colour to the entire stigma surface In B. diffusa (present
study) although anthesis of flowers is at 4 AM but the stigma is receptive two
hours later, an hour before the anthers dehisce. The stigma continues to be
receptive for almost six hours. However, in majority of the flowers pollination has
been observed to be over by 8-9 AM. The ants are generally active around this
time. Chronological mapping of the various events of the floral biology done
during this study indicate that within 2-3 h of pollination the pollen tube reaches
the ovule and effects fertilization. The ovule is anatropous, unitegmic with large
nucellus.
The activity of the four enzymes fell in two categories depending on when
they reach their peak activity. Peroxidase and polyphenol oxidase reach their peak
activity in the early hours after pollination while, acid phosphatase and esterase
peak towards the later hours after pollination. Peroxidase activity showed a single
sharp peak two hours after pollination (50000 moles min-1), thereafter, its activity
declined as suddenly. It is interesting to note that peroxidase activity was zero at

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the time of anthesis when pollination had not occurred. Only after pollination does
the peroxidase activity showed a sudden burst. Polyphenol oxidase was active
between 1 and 4 h after pollination during which time the enzyme activity peaked
and stabilized around 8000 moles min-1. Thereafter, there was a sharp decline in
the enzyme activity. It was seen stigmas maintain reasonably high levels of
polyphenol oxidase activity even in the just opened unpollinated flowers. Acid
phosphatase and esterase peak quite late after pollination has occurred probably as
response to some internal stimuli. As the flower approaches maturity the activity of
acid phosphatase increases although just opened unpollinated flowers show an
activity of about 5-7 moles min-1. The enzyme shows significant activity in the
stigma and peaks at 40 moles min-1, six hours after pollination then declines
sharply to the level equivalent to that in just opened flowers. Esterase activity in
just opened unpollinated flowers is maintained at reasonably high levels of 200
moles min-1. The enzyme reaches peak activity of 1200 moles min-1 six hours
after pollination, thereafter, its activity comes to the ground state equivalent to that
in just opened flowers.
The present studies on B. diffusa have revealed that the embryo sac is an
elongated structure being slightly broader at the micropylar side than that towards
the chalazal side. Within the embryo sac two polar cells, an egg and antipodal cells
were clearly visible. The entry of pollen tube is through the centre of the
micropylar opening. It is not clear whether the pollen tube bursts in the synergids
or elsewhere as appropriate sections showing this particular event could not be
obtained. However, the path traversed by the pollen tube was clear and it appears
the pollen tube enters quite deep into the embryo sac and the pollen tube bursts
somewhere near the egg. Just before the sperm fuses with the polar cells or the egg
it was observed that the polar cells move close towards the egg.
In the present study two stages were identified that clearly showed a sperm
and the egg to be lying close to one another and the second where the two polar
cells were fusing. The zygote, its early divisions forming the 2, 4, 6, 8, 16, 32 and
64 celled stages, towards the formation of the globular stage, early heart shaped
stages and finally transformation into the mature dicotyledonary stage could be
traced in B. diffusa in the present study.

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Although, B. diffusa (present study) puts in so much effort in the formation

of seeds but it was found that more than 50 % of these seeds are aborted. Detailed
experimentation has shown that the plant is not able to allocate sufficient resources
for the development of all the seeds that are formed and only those that happen to
be better sinks are able to survive and develop into healthy seeds. Environmental
factors such as temperature, water availability and edaphic factors such as nutrition
availability play a major role in the successful conversion of all events of
fertilization into healthy seeds. Supplemental nutritional experiments corroborate
this finding. Plants irrigated with Hoaglands nutrient solution resulted in
production of as high as 90 to 99 % healthy seeds. Net seed set in a plant is the
number of ovules per fruit or pod, the number of fruits (pods) per plant and the
frequency of embryo abortions.
In vitro pollen germination studies in B. diffusa conducted during the
present work have yielded interesting results. The pollen germination is greatly
influenced by temperature. The pollen required the presence of sucrose (10 %),
minerals as per Brewbaker and Kwack (Brewbaker and Kwack, 1964) medium and
pH of 7.5. Under these conditions the most optimum temperature that supported
maximal pollen germination was found to be 35 oC. Higher or lower temperatures
also supported good pollen germination if the pH was increased to 10 and the
sucrose concentration was reduced to 6 % especially when temperatures were low.
A new medium modified from Brewbaker and Kwack composition was developed
that supported 54 % pollen germination compared to 43 % in the standard
Brewbaker and Kwack medium. It was realised that sucrose may not always be the
best source of carbohydrate in the germination medium. Mannitol yielded
consistent pollen germination on a wide range of concentrations. Fructose proved
to be best of the carbohydrates as it supported almost 75 % pollen germination.
That osmoticum alone might be sufficient to push pollen tube growth was proved
when

79

pollen

germination

was

achieved

in

the

presence

of

Polyvenylpyrrolidon. Interestingly, 2,4-D had a hormesis effect on pollen

germination as the lowest concentration 10-4 M yielded the best germination
percentage (57 %), which slowly decreased as the concentration increased. This
was achieved in the absence of sucrose. With sucrose 2,4-D or Kn did not support
good germination. Stigma extracts from Boerhaavia itself supported reasonably

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good germination of pollen when diluted with Brewbaker and Kwack medium
without sucrose. However, the inflorescence extract from Spilanthus acmella
proved to support much better pollen germination over a large of concentration
giving 56 % germination. Salicylic acid in combination with 6 % sucrose supports
as much as 60 % pollen germination. Mathematical modelling could be used for
optimizing pollen germination conditions was demonstrated when optimal
conditions generated by Neural Network and Genetic Algorithm proved to support
germination as high as 83 %.
The morphological, chemical, genomic and metabolomic studies of the
trichomes of Boerhaavia diffusa L. in the present work contribute to a better
understanding of their functional and ecological significance. Trichomes were
distributed on vegetative parts such as the stem and leaf, the floral parts such as
bract, parianth and ovary and the fruit of B. diffusa. It was observed that the
density of trichomes was much greater in the apical parts of the stem and in young
leaves. The ovaries displayed much denser trichome distribution which decreased
with the formation and maturation of the fruit. Plenty of variation in the types of
trichomes could be observed in B. diffusa. There were both secretory or glandular
and non-secretory or non-glandular trichomes. The vegetative parts, the parianth
and bract had both the types but the ovary and fruit displayed only the secretory
types. Non-secretory branched trichomes were observed on the parianth. The
secretory or glandular trichomes could be distinguished on the basis of the head
cell which was either cylindrical or spherical. On this basis the secretory trichomes
were Glandular Cylindrical Trichomes (GCTs) or Glandular Spherical Trichomes
(GSTs). GCTs were ubiquitous in their presence and comparatively less influenced
by the environmental factors. The GSTs were restricted to the ovaries and the
fruits only and their presence was highly influenced by environmental factors.
These were abundant during the relatively cooler months.

The GCTs and GSTs

could be either short or long depending on the number of stalk cells and their
dimensions.
Histological and SEM studies of the trichomes of B. diffusa also revealed
that they have a distinct foot made up of 2 to 4 basal cells embedded in the
epidermis, 2 to 4 stalk cells of varying dimensions and a secretory or non-secretory
head cell (present study). The present study in B. diffusa revealed that trichomes

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were more abundant on the younger regions of the stem and leaf and on the
ovaries. As the fruit develops and matures the number of trichomes decrease. Since
B. diffusa grows in comparatively harsh abiotic conditions the younger parts
require more protection from desiccation, UV irradiation and insect attack and as
the parts mature the numbers of trichomes reduce. In B. diffusa more number of
trichomes on ovaries indicated that they require greater protection as compared to
the fruit.
The secretions which accumulate in the heads of the GCTs and GSTs on the
fruits of B. diffusa stain positively for lipophilic substances (present study).
Phenols have been found in the glandular trichomes of B. diffusa (present study).
The present study showed the presence polysaccharides in both the GCTs and
GSTs of fruit and GCTs of the stem and leaf. However, pectin was present only in
the cuticle of the trichomes of all parts studied. In B. diffusa as well the detachment
of the secretory sheath from trichome head cells may be occurring along a zone
possibly containing pectins as revealed by the staining of the outer cuticle by
Ruthenium Red.
Only GCTs and GSTs on fruit of B. diffusa showed the presence of proteins
(present study). In B. diffusa both the GCTs and GSTs of the fruits and GCTs in
the stem and leaf showed the presence of terpenes (present study). Pectin did not
form part of the cellular contents and was observed to be present in the cuticle of
both types of trichomes in all the three parts of B. diffusa studied in the present
work. The secretion from head cell in glandular trichomes of B. diffusa was noted
in the present study to be by the rupture of the cuticle in regions of greater
fragility.
mRNA isolated in the present study from the GCTs and GSTs of B.diffusa
were found to be about 200-300 bp long. After plating and random selection, 300
cDNA clones were sequenced and BLAST in NCBI and trichOME database.
Functional annotation was assigned to 300 randomly chosen ESTs. Out of these
ESTs about 25-30% had no predicted functions (no database hit, or a match to a
gene with unknown function). Most of the ESTs corresponded to one lipid transfer
protein gene that was the most highly expressed gene represented in the trichome
library. Senescence-specific cysteine protease was found to be present in the ESTs
isolated from B. diffusa in the present study.

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WRKY transcription factor, Syntaxin, LEA (late embryogenesis-abundant)

genes were also found in B. diffusa trichomes (present study). WRKY geneencoded transcriptional regulators were found in the GSTs of B. diffusa (present
study). WRKY appears to be involved in various physiological and developmental
programs, including biotic and abiotic stress responses. These proteins may reflect
the exposed positions of the trichome on the exterior of the plant. Syntaxin was
found in GCTs of B. diffusa (present study) and could be contributing to plant
resistance against bacteria and secretion of pathogenesis-related protein. In the
same way Cysteine protease, found in the GCTs of B. diffusa (present study) may
play an important role in proteolysis and nitrogen remobilization during the
senescence process. In B. diffusa about 1 % of ESTs were found of Sesquiterpene
synthase enzyme (present study). This enzyme plays an important role in the
biosynthesis of Sesquiterpenoids and they are thought to be prominent in plant
fungal interactions.
The NCBI BLAST revealed the presence of Chalcone synthase in GSTs of
B. diffusa (present study) and was represented by 20-25 ESTs. This suggests that it
is reasonably well represented within the trichomes of B. diffusa (present study).
This is a key enzyme in the flavonoid biosynthesis, catalyzing the condensation of
one molecule of 4-coumaroyl-CoA with three molecules of malonyl-CoA to form
naringenin chalcone, which is the central intermediate in the biosynthesis of
flavonols, flavones, isoflavonoids, and anthocyanins. Another 5-6 % of ESTs were
found of flavonol 6-hydroxylase in GSTs of B. diffusa (present study). Flavonoids
are plant secondary metabolites that are widespread throughout the plant kingdom.
They act as UV light scavengers to protect against oxidative damage, as
antimicrobial compounds to defend against pathogens, and as pigments in fruits,
flowers, and seeds, where they have a function in attracting pollinators and seed
dispersersal to facilitate reproduction. Small GTP binding proteins found in the
trichomes of B. diffusa (present study) play a critical role in cytokinin biosynthesis
and/or metabolism; as a defence signal transducer by sensitizing the woundperception system.
Seed maturation proteins found in the ESTs of B. diffusa (present study)
are associated with the activation of a variety of genes encoding storage proteins
and various hydrophilic, late-embryogenesis-abundant (LEA) proteins that

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possibly function as desiccation protectants. Lipoxygenase present in GSTs of B.

diffusa (present study) and in some other plant species may be involved in
Jasmonic Acid (JA) biosynthesis.
The present studies reveal that the trichomes of B. diffusa are a rich source
of a large number of metabolites. That these metabolites are produced within the
trichomes is suggested by the presence of an array of important enzymes involved
in the biosynthesis of flavonoids and terpenes. Isolation of ESTs related to these
and many more enzymes suggest transcriptional and translation activities within
the trichomes. Presence of many of the above referred metabolites/intermediates
and enzyme complexes are suggestive of the fact that trichomes of B. diffusa are
involved in imparting biotic and abiotic protection to the plant.
The in vitro morphogenetic and secondary metabolite studies on B. diffusa
undertaken in the present work yielded interesting results. The results threw up
more questions than answers. The results may apparently appear to be unique but
have many parallels in the literature, which are discussed in the following
paragraphs.
B. diffusa has a unique morphology and biochemistry. It is a trailing herb
which exhibits anomalous secondary growth and is rich in a large number of
secondary metabolites, chief among them are the flavonoids and isoflavonoids.
Three types of explants from three sources were used to establish in vitro cultures.
The internodes and leaf explants were obtained from both mature plants growing
in field and juvenile seedlings raised in vitro. The third explant was the juvenile
tissue of early torpedo stage embryos. All the three explants upon culture in
Murashige and Skoog medium developed callus in the presence of the auxin 2,4-D.
The quantity of 2,4-D was very critical and callus initiation could be obtained only
on lower concentrations of 0.5-1.5 mg/l. Beyond 1.5 mg/l the auxin was
detrimental to the survival of explants itself. Of the lower concentrations profuse
callusing was observed only in 0.5 mg/l. Though 1 and 1.5 mg/l 2,4-D did yield a
reasonably good callus but the callus could not survive subsequent subcultures.
The callus obtained on 0.5 mg/l 2,4-D was creamish, compact to friable and
appeared to have morphogenetic potential. The callus when transferred to basal
medium, died almost within 10 days, without resulting in either somatic embryos
or any kind of regeneration. Addition of cytokinins such as kinetin or BAP at

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lower concentrations of 0.5 mg/l did increase the life of callus but for only six
weeks. Two other cytokinins, 2iP and TDZ that were also tested could not support
callus growth beyond one week. In none of the kinetin containing media the callus
formed somatic embryos.
The second attempt was to obtained de novo regeneration from the callus.
Consequently, the callus was transferred to several media compositions. The callus
was

transferred

to

several

combinations

of

NAA+Kn,

NAA+BAP,

NAA+BAP+TDZ, NAA+2iP, and IAA+BAP totalling about 100 media

compositions. However, no regeneration could be obtained in any of the media
compositions. In the present study various growth regulators were used both to
initiate callus and for regeneration. However, no in vitro organogenesis or somatic
embryogenesis was obtained in the present work. In the present work presence of
an auxin such as 2,4-D or NAA, that too at lower concentration (0.5 mg/l) was an
absolute requirement for callus survival and growth. Even a slight increase in the
auxin concentration to 1.5 mg/l was detrimental for callus growth. The callus could
not survive in either kinetin or BAP alone without the auxins. At higher
concentrations of the auxins (2,4-D 1.5-3 mg/l; NAA 1-5 mg/l) and cytokinin
(BAP 1-5 mg/l; Kn 1-2 mg/l; 2iP 2-25 mg/l; TDZ 0.2-1.5 mg/l) the callus failed to
survive beyond two weeks and in some cases died within a week. Another major
problem with the callus of B. diffusa was that even in the combination of the auxin
and cytokinin that supported the best callus growth (1 mg/l 2,4-D+ 0.5 mg/l BAP)
the callus did not survive beyond six subcultures. During this period the callus was
gradually becoming brown and the quality of healthy callus reduced in each
subculture. The results obtained in the present work are not in agreement with
previous reports on regeneration in B. diffusa mentioned above. As a matter of
fact, the concentrations of auxin and cytokinins used in the earlier reports of
regeneration in B. diffusa proved to be detrimental for callus growth in the present
work. One possible reason could be genotypic variations in B. diffusa.
A striking result of the present study was that an auxin in the culture media
was essential for proper growth of the callus. The callus was sensitive to both
absence and high concentrations of auxin, slight increase in auxin 2,4-D or NAA to
1.5 mg/l adversely affected callus growth. A possible explanation could be found
in the flavonoid accumulation of the callus cells. Flavonoid such as naringenin are

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known to be polar auxin transport inhibitors of auxin. Present studies in B. diffusa

have shown the accumulation of different types of flavonoids such as quercetin,
myrcetin, and kaempferol in the callus so an auxin-flavonoid interaction cannot be
ruled out.
The major problem as stated above besides non-regeneratibility of callus,
has been the long term maintenance of healthy and actively growing callus in B.
diffusa cultures in the present work. To improve and sustained callus growth in the
present study several adjuvants such as silver nitrate (anti-ethylene agent), ascorbic
acid (an anti-oxidant agent), casein hydrosylate (complex source of reduced
nitrogen), proline (an amino acid), and activated charcoal (an absorbant) were
supplemented to the medium along with various growth regulators but to no avail.
In some of the experiments in the present work MS salts were used at half strength
to reduce callus browning and improve growth. However, this was not much of
help in improving the callus growth.
The overall growth of callus whether the explant was derived from field or
seedling or embryos was poor. The callus from any of these explants in any of the
several media tried did not survive beyond 5 or 6 subcultures. This was
problematic from the point of view of both conducting regeneration studies and
also for subsequent in vitro secondary metabolite studies. It was essential that the
callus could be maintained for at least 10 to 12 subcultures. This necessitated a
detailed study on growth of callus over a time period, its composition with respect
to types of cells and ultimately developing a strategy of subculture. The following
experiments were conducted to fulfil this need.
(a) Growth of callus as affected by size of callus at the time of inoculation was
measured. Changes in fresh and dry weights of callus of different sizes maintained
over a period of time was taken as a parameter of growth,
(b) The callus was analyzed for the type of cells it was made up of at the end of a
growth period of 30 days,
(c) Spatiotemporal location of different types of cells in callus was done by cutting
thin free-hand sections,
(d)Change in the numerical composition of cells at regular intervals was noted as
the callus grew over a 30-day period,

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(e) The callus became brown with the passage of time in cultures. Browning is
associated with peroxidase activity. In the cultures, therefore, change in peroxidase
activity was quantified at regular intervals over a period of 30 days, and
(f) Developing a strategy for subculture.
The observations are given below.
(a) Influence of size of callus: All experiments were performed using callus
initiated from MS+0.5 mg/l 2,4-D and cultured on MS+1mg/l 2,4-D+0.5 mg/l
BAP.
The size of callus definitely influenced fresh and dry weights taken as a
parameter of growth. When a 5 mm callus was used the maximum weight
increment was achieved by 15th day. Thereafter, no increment could be observed.
The maximum dry weight increment was achieved by the 10th day, after which it
became constant. By doubling the size of callus to 10 mm fresh and dry weight
increment continued till the 28th day. Thereafter, a plateau was achieved for the
fresh weight but the dry weight slightly decreased. A further increase in callus size
to 15 mm and 20 mm resulted in an almost linear graph with no plateau indicating
a steady increase in fresh weight even on the 25th day. The maximum dry weight
increment was only till 28th day, thereafter, it slightly dipped.
It was concluded from this study that 15-20 mm callus was the most
appropriate size for subculture and this was adopted for all future studies.
(b) Cell types in the callus: Callus growing on the medium MS+1 mg/l 2,4-D and
0.5 mg/l BAP was used at the end of 30-day growth period. The study threw up
interesting observations. In a total of 3400 cells counted almost 2450 i.e., 72 %
cells were nucleated. 1120 or 46 % of these nucleated cells were small and
isodiametric with a centrally placed nucleus and the remaining 56 % of these
nucleated cells i.e., 1325 cells were elongated but, with sparse cytoplasm and
nucleus present on one side. The remaining 960 or 28 % of the total cells were
elongated but enucleated, as no nuclear staining with acetocarmine was seen. The
only cells that appeared to be meristematic were the small isodiametric cells.
(c) Spatiotemporal location of cell types: Thin vertical sections of the actively
growing callus were observed in microscope. It was seen from that the actively
growing small isodiametric cells are present close to the medium and are about 20
to 30 cell layers thick. It was also seen that the elongated cells are present on the

330

uppermost crust of the callus and are arranged one upon the other in a longitudinal
fashion. There are 3 to 4 or some times more layers of the long cells. In this region
each cell is present on top of the narrow side of the lower cell. It could be that the
small isodiametric cells are elongating vertically giving rise to the elongated cells.
(d)Variation in the numerical composition of cells: It was understood from the
above observations that the small isodiametric cells are meristematic and
proliferate to contribute towards the callus growth. It was important to understand
the change in composition of different cell types in continuously growing callus
cultures at regular time intervals. At the start of the experiment care was taken to
culture the lower portion of callus that contained the maximum numbers of small
cells and elongated cells were removed as far as possible. It was that there was a
continuous increase in the total number of cells starting from 400 cells on day 0 to
720 cells on day 30 till the end of the experiment. However, the distribution of
different types of cells followed a unique pattern. The small cells which were
about 350 on day 0 gradually decreased till day 10 when about 200 cells were left.
There was a sudden spurt in their numbers to about 256 by the end of day15.
Thereafter, there was a steady decline in their numbers and by the end of day 30
only 130 cells were left. Interestingly, the fate of the elongated and enucleated
cells was reverse. About 80 elongated cells were present on day 0 which gradually
increased to almost 5 times resulting in about 400 cells by the end of the 30-day
growth period. The enucleated cells were zero at the beginning of culture period
with no new cells formed even at the end of day 5. However, there was a sudden
spurt and their numbers increased to 150 on day 10 which stayed stable till day 15.
Thereafter, there was a steady increase in enucleated cells to about 250 by the end
of the 30 day culture period. These observations indicated that probably the
elongated and enucleated cells are formed from the small cells, eventually
affecting their number.
However, the net change in the number of different types of cells is not
clear. To understand the net change in different types of cells it was necessary to
know interval to interval increment or decrement in cells. The number of small,
elongated and total cells on day 0 when the cultures were initiated was taken as
zero. At subsequent intervals any increment or decrement in the number of cells
was arrived at by subtracting these results from the results of the previous interval.

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The numbers of enucleated cells was taken as counted because at the start of
experiment their number was zero. The following conclusions were drawn.
The total number of cells showed a steady increase till day 15 thereafter,
there was slight decline with the plateau being achieved on day 25 and 30.The
number of elongated cells steadily increased registering a steep and maximum
increase on day15. Thereafter, there was a gradual decline in their numbers till the
minimum is reached on day 30. It was that on the cell wall of the long cell some
brownish substance was deposited.
(i) The enucleated cells were absent at the beginning of the experiment and till day
5 no new cells were formed. However, on day 10 there was a sudden appearance of
150 enucleated cells which stayed almost stable till day 15. Their numbers further
increased to 185 which stayed stable till day 25; again these increased to 226 cells
by day 30. The enucleated cells are the only ones which were not present in the
beginning but showed a steady increase till the end of the subculture period.
(ii) The balance sheet of small cells was mostly negative throughout the culture
period except on day 15 when an increment of about 50 cells was recorded. It was
that both the elongated and the enucleated cells are continuously increasing and are
being derived from the small cells. So much so that the old stock of small cells and
the new cells formed by its division both contribute towards the formation of the
other two types of cells. The elongated cells and the enucleated cells appear to
have no contribution towards increase in the total number of cells since they do not
divide. Eventually, at the end of the growing period the number of small cells is so
few that it cannot sustain any further growth of callus.
(e) It was observed that the callus turns brown with the passage of time and at the
end of the subculture period the entire callus became brown and died. Since
browning of callus is associated with oxidation of phenolic compounds an increase
in peroxidase activity was predicted. This was confirmed by measuring peroxidase
activity at different time intervals in a continuously growing callus of 30 days. It
was seen that so long as the small cells are in sufficient numbers (day 10) the
peroxidase activity was almost absent, however, as the number of small cells start
to decline and the number of elongated and enucleated cells increase the
peroxidase activity starts to increase and on day 20 the enzyme activity peaks, and
thereafter, by day 25 and 30 its activity declines. It could be seen that on day 20

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cumulative numbers of elongated and enucleated cells are sufficiently large and
only a few small cells are present. Thereafter, there is a rapid slide in the number
of small cells and an increase in number of elongated and enucleated cells.
Peroxidase activity though, was minimum on last two intervals (day 25 and 30) of
cultures but it never becomes zero. The decline in peroxidase activity could be
attributed to increase in enucleated cells and elongated cells which are
continuously becoming brown and dying so much so that the actual number of
cells showing peroxidase activity decline sharply.
(f) The following strategy for subculture was adopted from the above studies
(i) Subculture of callus to fresh medium was done after every two weeks instead of
the earlier 3 or 4 weeks.
(ii) The callus was cleaned from the top of all the elongated, brown and dead
cells.
(iii) Only the lower part of the callus that contained majority of small cells was
transferred to the fresh medium.
Following this procedure carefully the life of callus could be increased to
almost up to 10 to 12 subculture.
I.

In vitro secondary metabolite production

An important prerequisite for getting in vitro secondary metabolite

production is to develop continuously growing callus cultures for several

generations. These continuously growing cultures are used for establishing
suspension cultures so that at a later date appropriate fermentor could be
developed. From time to time it was necessary to analyze the callus cultures for the
presence of secondary metabolites. With this objective in view the following
experiments were designed for the callus of B. diffusa: (a) Growth kinetics of
callus, (b) establishment of suspension cultures, and (c) analysis of callus cells for
secondary metabolite accumulations.
A comparison of various media with respect to fresh and dry weight
increments. As stated previously dry weight increments are a better parameter to
assess cell growth. From this point of view media containing t-cinnamic acid were
much better than any other media. Amongst the various concentrations of t-

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cinnamic acid 10 mg/l elicited the best response and dry matter accumulation at the
end of the culture period was 19 times to that on day 0.
In 500 mg callus about 5x106 cells were counted an important step
successfully establishment of suspension cultures. Till eight subcultures majority
of the suspension was composed of small and large clusters of cells but from the
9th subculture onwards the population of single cells started increasing and in the
10th subculture most of the suspension consisted of single cells. Thereafter, single
cell cultures could be maintained on a routine basis. From time to time the cells
from the suspension culture were stained with acetocarmine and observed in light
microscope. The cells stained with acetocarmine and showed the presence of
nucleus and cytoplasm also stained densely indicating the cells were viable.
The callus produced in vitro by plants that accumulate large quantities of
secondary metabolites tend to become brown at an early stage. Accumulation of
secondary metabolite in the callus cells have been implicated in browning and
early death due to apoptosis of in vitro callus. It was observed in present study as
well, that the callus of B. diffusa becomes brown at an early stage and eventually
dies. Microscopic examination of the cell types revealed that the small
meristematic cells are continuously elongating and becoming enucleated.
Microscopic examination revealed deposition of some substances on the cell wall
of the elongated cells. Presumably these morphological changes and possible
biochemical changes (as revealed by change in colour of the cells and depositions
on cell walls) could be resulting in apoptosis of the callus cells.
The in vitro secondary metabolite studies in B. diffusa in the present work
have revealed the active biosynthesis of alkaloids and flavonoids. Fractionation of
30 g of dry callus of B. diffusa yielded 30 mg of the hexane fraction, 80 mg of
chloroform, 440 mg of ethanol, 2720 mg of aqueous fraction. Preliminary
chemical tests for the ethanol and aqueous fraction indicated the presence of
alkaloids and flavonoids. The spectrophotometer analysis of the samples in UVVIS spectrophotometer showed twin peaks of absorption in UV range which could
be matched and categeorized with the UV absorption peak of flavonoid standards
such as quercetin, kaempferol, myrcetin and luteolin. The samples were further
analysed on HPTLC against these flavonoids standards. It was estimated that the
callus of B. diffusa contained about 0.36 g/l of flavonoids. Of the flavonoids

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identified quercetin was present in maximum (0.95 g/l) followed by kaempferol

(1.5 g/l) and myrcetin was the least (0.95 g/l).
The present study is only a preliminary work towards understanding the
reproductive biology in all its phases, trichomes, in vitro morphogenetic responses,
and in vitro biosynthesis of flavonoids and their identification. The work on
trichomes revealed presence of certain key enzymes such as chalcone synthase,
cytochrome P450, monoxygenase, flavon-6-hydroxylase in the flavonoid
biosynthetic pathway. The present work opens the path for undertaking detailed
study on Boerhaavia diffusa in many directions.

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