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An updated overview on Atropa belladonna L

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Abstract

Atropa belladonna L. (Family: Solanaceae; commonly known as belladonna, deadly nightshade, devil’s berries amongst others), a perennial herb (2n=72) is native of Europe, North Africa and Western Asia, possesses a long tradition as one of the classic poisons of antiquity. The species is also the source of atropine alkaloid (dl-hyoscyamine) and is important in the study of autonomic pharmacology. Considering the therapeutic uses of A. belladonna as well as its significant toxic effects (due to tropane alkaloids including scopolamine and hyoscyamine), an overview on all necessary aspects is documented to provide information for further exploration of the species for human benefits.
Datta K. Animesh et al. IRJP 2011, 2 (11), 11-17
INTERNATIONAL RESEARCH JOURNAL OF PHARMACY, 2(11), 2011
INTERNATIONAL RESEARCH JOURNAL OF PHARMACY ISSN 2230 8407
Available online www.irjponline.com Review Article
AN UPDATED OVERVIEW ON ATROPA BELLADONNA L.
Paul Rita2 and Datta K. Animesh1*
1Department of Botany, Cytogenetics and Plant Breeding Section, University of Kalyani, Kalyani, West Bengal, India
2Department of Botany, Charuchandra College, Kolkata- 29, India
Article Received on: 12/09/11 Revised on: 10/10/11 Approved for publication: 03/11/11
*Dr. Animesh K. Datta, Professor in Botany, Department of Botany, Cytogenetics and Plant Breeding Section, University of Kalyani, Kalyani 741235, West Bengal, India
Email: dattaanimesh@gmail.com
ABSTRACT
Atropa belladonna L. (Family: Solanaceae; commonly known as belladonna, deadly nightshade, devils berries amongst others), a perennial herb (2n=72) is native of Europe,
North Africa and Western Asia, possesses a long tradition as one of the classic poisons of antiquity. The species is also the source of atropine alkaloid (dl-hyoscyamine) and is
important in the study of autonomic pharmacology. Considering the therapeutic uses of A. belladonna as well as its significant toxic effects (due to tropane alkaloids including
scopolamine and hyoscyamine), an overview on all necessary aspects is documented to provide information for further exploration of the species for human benefits.
KEY WORDS: Atropa belladonna, Overview.
INTRODUCTION
Atropa belladonna L. (commonly known as belladonna, deadly
nightshade, divale, dwale, banewort, devils berries, naughty mans
cherries, death cherries, beautiful death, devils herb, great morel
and dwayberry1), a perennial herb (Family: Solanaceae) hallowed by
long tradition as one of the classic poisons of antiquity. The name
Atropa is said to be derived from Greek goddess Atropos; while, the
name belladonna means beautiful lady in Italian language1. The
foliage and berries of the species are extremely toxic containing
tropane alkaloids which includes scopolamine and hyoscyamine
causing a bizarre delirium and hallucinations1. The plant species is
the source of alkaloid atropine (C17H23NO3, dl-hyoscyamine) which
has been proven to be a cornerstone in the study of autonomic
pharmacology2. A. belladonna is also significant for use in medicine
and cosmetic1. Considering the significance of the medicinal plant
species an overview on A. belladonna is conducted covering nearly
all essential aspects with an objective to provide unabridged
repository of references to researchers for its effective exploration as
well as utilization in human welfare.
Distribution and Habitat
The plant species is native to Europe, North Africa and Western
Asia1 but not common in England (occurs locally as an apparent
native in open vegetation on calcareous soil, also sub-spontaneously
and as a relic of former cultivation3) and Scotland4. A. belladonna is
a weed species in parts of the world, where it colonizes in areas with
disturbed soils5. The species grows most luxuriantly forming bushy
habit in shade of trees, on wooded hills, on chalk or limestone4;
while, plants exposed to too much sun become stunted6. Growing
under natural conditions the species is more subjected to the attacks
of insects4.
Plant description
A. belladonna1,4 is a branched herbaceous perennial (about 5 ft tall,
rare often it is 6.0 ft in height) with purplish coloured stem, stout,
undivided at the base but dividing a little above the ground into three
to more, rarely 2 or 4 branches each of which branched freely; roots
thick, fleshy, whitish, branched, about 6 inches long or more; leaves
3 to 10 inches long, ovate, dull, darkish green in colour, the lower
leaves solitary, the upper ones in pairs alternately from opposite
sides of the stem, one leaf of each pair much larger than the other,
acute at apex, entire with short petioles, veins prominent in
undersurface and depressed on upper surface; plant glabrous though
soft downy hairs may occur in stem and leaves when they are quite
young; flowers solitary in the axil of the leaves, dark and dingy
purplish colour, tinged with green, about an inch long, pendent, bell
shaped, furrowed, the corolla with 5 large teeth or lobes, slightly
reflexed, flowering time from June to early September; fruits berry
and five-cleft calyx spreads round the base, shining black colour,
full of dark inky juice, sweet and consumed by animals that disperse
seeds, even though the seeds contain toxic alkaloids7.
Cultivation and Harvesting
The species4,6 prefers a well-drained, well-limed soil in full sun or
part shade; however, light, permeable and chalky soil is most
suitable for the crop. The soil should be kept moist all times.
Belladonna is most frequently propagated by seed sown in flats and
the seeds take 4-6 weeks to germinate and when the seedlings are an
inch or so high they are set out 18 to 20 inches apart, watered after
transplantation and shaded for several days. Belladonna may also be
propagated by cuttings of the green branch tips.
Belladonna cultivation in the slope of a hill gives especially good
results in regards of high percentage of alkaloids. The limits of
growth of the species are between 500 and 550 N. latitude and an
altitude of 300 to 600 ft, though it may descend to sea level where
the soil is calcareous with good drainage and adequate shade. The
crop may be appreciably increased by the use of farmyard manure,
or a mixture of nitrate of soda, basic slag and kainit.
First year plants are generally 1.5 feet high and flowers in
September, the leaves and tops are only collected and the plants are
thinned to 2.5 to 3.0 feet apart at the approach of winter to avoid
overcrowding in the following year. In June, the second year plants
are cut to 1 inch above the ground at flowering and in good years a
second crop will be ready for harvesting in September. The roots
may be harvested in autumn of the fourth year. The parts harvested
should be dried quickly in the sun as faded leaves and plant parts
yields small amount of alkaloids.
Yield
Atmospheric conditions show marked influence on the alkaloid
contents of Belladonna, the highest percentage of alkaloid being
yielded in plants grown in sunny and dry seasons. About 0.68%
alkaloid has been reported in Belladonna in the months of May and
June in sunny and dry seasons in contrary to 0.34% in the same
months lacking sunshine. August and September proving very wet
season yields about 0.35 to 0.38% alkaloids. English growers found
excellent results when soil treated with basic slag and the percentage
of total alkaloids in dry leaf and stem from third-year plants
accounted to 0.84. The average crop of fresh herb in the second and
third years is 5 to 6 tons per acre, and 5 tons of fresh leaves and tops
yield 1 ton of dried herb. The yield per acre in the first year of
growth is average about 6 cwt. of dry leaves. The greatest loss of
plants is in wet winters4.
Datta K. Animesh et al. IRJP 2011, 2 (11), 11-17
INTERNATIONAL RESEARCH JOURNAL OF PHARMACY, 2(11), 2011
Chemical constituents
Hartmann et al.8 found a total of 13 alkaloids from roots of A.
belladonna following high resolution GLC and GLC-MS; while, the
above ground parts of the plant revealed 7 alkaloids (hygrines
completely absent). Scopolamine has been reported to be
synthesized in roots only but Norhyoscyamine has been frequently
found in shoots of the plant species. Hyoscyamine N-oxide was
found in various plant parts. Hedges and Herbert9 reported a natural
plant constituent δ-N- Methylornithrine from A. belladonna as a
tropane alkaloid precursor following radioactive [5- 14C and 5- 3H]
feeding of ornithine to the plant. Arraez- Roman et al.10 developed a
rapid and easy CE- electrospray interface (ESI)-TOF-MS procedure
to isolate several important compounds such as tropine,
belladonnine, norhyoscyamine, apoatropine, hyoscyamine, 6β-
hydroxyhyoscyamine and scopolamine from leaf extract of A.
belladonna. Baralle and Gros11 reported the presence of
cuscohygrine (using sodium acetate-2-14C) among other alkaloids.
Van Haga and Reinouts12 reported that cuscohygrine as a normal
constituent alkaloid of A. belladonna. Shvets et al.13 isolated eight
steroidal glycosides tentatively named atroposides A, B, C, D, E, F,
G and H from the methanolic extract of A. belladonna seeds by
Sephadex gel filtration and column chromatography on silica gel
impregnated with silver nitrate. The structure of atroposides A, C, E,
G are elucidated as 3-O-alpha -D-galactopyramoside; 3-O-beta-D-
glucopyranosyl (1-->4)-beta-D-galactopryranoside; 3-O-beta-D-
glucopyranosyl (1-->2)-beta-D-glucopyranosyl (1-->4)-beta-D-
galactopyranoside; and 3-O-alpha-L-rhamnopyranosyl (1-->4)-beta-
D-glucopyranosyl (1 -->2)-beta-D-glucopyranosyl (1-->4)-beta-D-
galactopyranoside.
Alkaloid production and Isolation
Kamada et al.14 reported the presence of atropine and scopolamine
in hairy roots (hairy roots induced by inoculation of stem of sterile
plants of A. belladonna with Agrobacterium rhizogenes) by TLC
and HPLC but their amounts were quantified by GLC, and the
results showed that the amount of two alkaloids in the axenic
cultures was same as those of normal plants grown in the field.
Dimitrov et al.15 suggested an integrated process of extraction
(applying pertraction in a rotating film [RF] contractor) and liquid
membrane isolation (di-isopropyl ether as liquid membrane and
sulfuric acid as a stripping agent) of atropine from roots (87% field,
about 48 times higher than in the native extract. Srivastava and
Chadha16 studied the effect of two surface-active agents (Tween 20
and Tween 80 - 0.2%) on the extraction of belladonna herb by
percolation and mechanical agitation process using 70% alcohol and
water as solvents leading to near complete and expedient extraction
of the drug (Tween 80 gave better result).
Biochemistry
Mino et al.17 made complete amino acid sequences of [2Fe-2S]
ferredoxin from A. belladonna and Hyoscyamus niger by automated
Edman degradation of the entire S-carboxymethylcysteinyl proteins
and of the peptides obtained by enzymatic digestion. The two
ferredoxins exhibited 1-8 differences in their amino acid sequences
compared to other tropane-alkaloid -containing plants (Scopolia
japonica, Datura stramonium, D. metel and D. arborea) and 9-23
differences among the other solanaceous ferredoxins, thereby
suggesting that the tropane-alkaloid containing plants are closely
related taxonomically.
Factors affecting growth and chemical constituents
Salonen and Simola18 suggested that growth and NRA (nitrate
reductase activity) can be stimulated by NH4+ and by proline, by
proline plus ornithine, but not by glutamate, in NO3 containing
medium. Amino acids tested were not inhibiting. Bensaddek et al.19
also reported that nitrate and ammonium concentrations in the
culture medium possess a strong influence (dose dependent) on the
scopolamins/hyoscyamine ratio. Phillipson and Handa20 found
marked fluctuations in N-oxide content in the species during
organogenesis, highest being found in ripe fruit. Sporer et al.21
studied the seasonal variation in total alkaloid content and alkaloid
patterns (by HPLC analysis; examined in June and July from berries
and seeds) in belladonna (hyoscyamine was the main product
examined), and two peaks were found significant: maximal alkaloid
yields were detected at early night and at early morning while in
matured seeds, it was highest in the afternoon. Falk and Doran22
assessed the influence of inoculum morphology on growth of A.
belladonna hairy roots and production of tropane alkaloids and it
was inferred that although hyoscyamine was found to accumulate at
higher concentrations in more mature root tissues (2.1 ± 0.2 mg g-1)
than in the tips (1.1 ± 0.3 mg g-1), hyoscyamine content was
however independent of inoculum morphology. Kanokwaree and
Doran23 suggested that increase in the number of hairy root tips (3 to
9) in shake flasks (50 and 100 ml of medium) reduced growth rates
by 40% in belladonna, thereby indicating the influence of medium
conditioning and oxygen mass transfer on root growth. Lee et al.24
analyzed growth and tropane content in transformed root cultures of
A. belladonna (strain M8) grown in 300 ml flasks and after one
month in culture, the biomass of the transformed roots increased 15
times and reached 5 g dry wt l1; the alkaloid contents were 0.7% for
hyoscyamine, 0.1% for 6β-hydroxyhyoscyamine, 0.02% for
scopolamine and 0.02% for littorine. Out of the seven chemicals
added (individually to 18-day-old cultures) glutathione (5 mM),
chitin (0.1%), chitosan (0.1%) or yeast extract (0.1%) were with no
effect on the release of alkaloids into the medium but treatment with
5 mM H2O2 induced a transient release of tropane alkaloids from
transformed roots and maximum amount was 0.35 mg per flask. Cu2
and Cd2 (5 mM) also improved the excretion of tropane alkaloids
into the medium; however, led to lysis of transformed roots.
Baricevic et al.25 noted the effect of water stress and nitrogen
fertilization on the content of hyoscyamine scopolamine in the roots
of nightshade, and experimental results indicated that maximal
content of alkaloids was achieved with 95% depletion of available
soil water and a nitrogen supply of 1.60 g/pot. Ali26 studied the
effect of putrescine on germination and seedling growth of A.
belladonna under the influence of NaCl and it was found that
presoaking seeds in 102 mM putrescine can alleviate the adverse
effect of NaCl during germination and early growth of the species
but increase alkaloids as well as endogenous putrescine.
Apart from these stage of development27, Gibberellic Acid28, heavy
water29, Tiron30 (a water-soluble radical scavenger) were the factors
amongst others in relation to alkaloid content, growth and
morphology of A. belladonna.
Therapeutic uses
Drops prepared from the belladonna plant are used to dilate pupils of
an eye31,32. A. belladonna is used in traditional treatments for an
assortment of conditions including headache, menstrual symptoms,
peptic ulcer disease, histaminic reaction, inflammation and motion
sickness33 as well as in homeopathic drug1. Belladonna is used as a
sedative, to stop bronchial spasms in asthma and whooping cough
and as a cold and hay fever remedy33. It is also used for Parkinsons
disease, painkiller and ointments of belladonna is applied for join
pain (rheumatism), leg pain (sciatica), and nerve pain (neuralgia)6.
Apart from these, belladonna is also used in plasters, treating
psychiatric disorders (hyperkinesis), excessive sweating
(hyperhidrosis), and hemorrhoid suppositories amongst others6.
Ramoutsaki et al.34 reported belladonna as an analgesic and emetic
against miscellaneous diseases or ailments and as an antidote for
snakebite. Bousta et al.35 reported that A. belladonna possesses
significant neurotropic and protective effects on behavioral and
gastric alterations induced by experimental stress.
Datta K. Animesh et al. IRJP 2011, 2 (11), 11-17
INTERNATIONAL RESEARCH JOURNAL OF PHARMACY, 2(11), 2011
Adulterants
Different plants species like Scopolia japonica (Japanese Belladonna
- rhizome part), Scopolia carniolica (Scopolia - rhizome), Inula
helenium (Elecampane - root), Medicago sativa (Medicago - root),
Althaea officinalis (Marshmallow - root), Arctium lappa (Lappa -
root) are used as adulterants of A. belladonna. (Source: Felter HW,
Lloyd JU. King's American Dispensatory 1898.)
Other uses
Cosmetic industry; however, its use is limited as it may cause minor
visual distortion and other adverse effects1.
Clinical trials
Tita et al.36 used rat model to suggest the ability of A. belladonna
and atropine for urinary retention, and A. belladonna was found to
be more effective. Bettermann et al.37 performed single-blind
placebo-controlled study to investigate the dose-dependent vagolytic
and vagotonic effects (parameters studied : heart rate and
noninvasive arterial finger blood pressure) after a oral administration
(4 hour after) of A. belladonna tincture (ABT : 0.1 mg/ml alkaloid
concentration, atropine / scopolamine = 20:1; doses = day 1: 2ml,
day 2 : placebo, day 3: 5ml, day 4: 1 ml) and found that ABT can be
effectively used to stimulate parasympathetic activity in man. The
mode of vagal activation changes between 2 and 5 ml ABT from
vagotonic to vagolytic. ABT was found to possess no or very little
effect on blood pressure control. Toporcer et al.38 studied the
mechanical properties of skin wounds after A. belladonna
application in rats (24 animals were randomly divided into 3 groups;
two symmetrical skin incisions were given on the back of each
animal and immediately sutured) and wound tensile strength of each
group (Group A plant extract not given, served as control; B
extract given for first two days after surgery; C extract given for
five consecutive days after surgery) was measured 120 hours after
surgery. A. belladonna extract treated groups (B group: 244 ± 48g;
2.09 ± 0.42% of unwounded skin; C group: 254 ± 67g; 2.18 ± 0.58%
of unwounded skin) was significantly higher (P<0.05) than untreated
group (A: 194 ± 31g, 1.67 ± 0.26% of unwound skin), thereby
suggesting a positive effect on aseptic surgical skin wound healing.
Gal et al.39 studied the effect of A. belladonna (AB) on skin wound
healing following biomechanical and histological study in rats and in
vitro study in keratinocytes, 3TC fibroblasts, and human umbilical
vein endothelial cells. Results of in vivo experiments showed that
AB treated wounds shortened the process of inflammation and
accelerated collagen formation as well as significantly increased
wound stiffness as compared to control tissues. In vitro
examination showed that highest AB extract concentration
expressed CK19; in addition all concentrations were stimulatory to
human umbilical vein endothelial cell proliferation. In addition only
the AB extract at the lowest tested concentration increased fibroblast
growth, but higher concentrations decreased cell survival. Bogan et
al.40 reported a case study of 48-year old man (ingested three
handful of Belladonna) who experienced disorientation,
aggressiveness and trachycardia was treated initially with diazepam
(and intravenous infusion of physostigmine and activated charcoal)
and hospitalized. Blood sample analyzed showed that a muscarinic
receptor total binding equivalent to binding of 130 μg/l atropine
(determined by a radio receptor technique).
Toxicology
The root is the most poisonous, the leaves and flowers less so, and
the berries, except to children, least of all4. Lange and Toft41
reported a case where a boy (9 years old) ate 20-25 berries of
belladonna plant and serious atropine poisoning was caused which
commenced with psychosis; however, the boy survived with
intravenous administration of physostigmine Belladonna has been
reported to induce blockage of body nervous system, gastrointestinal
blockage, urinary retention, paralytic ileus, atony, stenosis, fast
heartbeat, constipation, esophageal reflux worse apart from dry
mouth, enlarged pupils, blurred vision, red dry skin, fever, inability
to sweat, hallucination, spasms, mental problem, convulsions, coma
amongst other as a part of toxic effect of the plant species. Southgate
et al.42 reported severe poisoning by deadly nightshade in two adults
who mistakenly ate berries. Atropine levels were reported in urine
and treatment with physostigmine was ineffective. Caksen et al.43
analyzed intoxication of deadly nightshade on 49 children and
observed symptoms and signs were meaningless speech,
tachycardia, mydriasis and flushing but not death.
Diseases of Belladonna
Smith44 reported virus infection in the plant species and it occurs in
fairly high concentration withstanding for 6-11 days in extracted sap
but was inactivated at a temperature between 75 and 800C. Sol et
al.45 reported that A. belladonna mosaic virus is transmitted by
nematodes in sandy soil. Heuss et al.46 described belladonna mottle
virus (belonging to turnip mosaic virus group) as small spherical
virus particles containing 180 protein subunits and arranged in a T=3
icosahedral surface lattice. The top and bottom viral components
crystallize isomorphously in hexagonal space group R3 (a = B = 296
Å, C = 729Å). The insect pest that attack belladonna leaves is so
called fleabeetle and it perforates the leaves to such an extent that
it makes it unfit for sale in a dried state (naphthalene kept in soil
may be useful to keep the beetles off)4.
Cytology
The chromosome number in the species was reported to be 2n=72
(krumbiegel and Schiender47, Gleba et al.48, Babiychuk et al.49).
Cell biology
Bajaj50 grew isolated PMCS (pollen mother cells) aseptically in a
nutrient medium in microculture and found a multicellular structure
giving rise to 6 to 8 microspores which were released at maturity
due to rupturing of thick callose wall caused by inside pressure.
Simola51 studied the developmental changes (4 stages) in the
subcellular structure of the seeds. The young cells contain
spherosomes and the cytoplasm with ribosomes, proplastids and
mitochondria and during ripening the vocuoles of endosperm and
embryo developed into protein bodies with globoid cavities and the
protein mass contains a roundish or crystalline body (stain with
periodic acid Schiff-reagent). The embryo and endosperm of ripe
Atropa seeds were similar containing protein bodies and small
spherosomes.
Hybrid analysis
Krumbiegel and Schieder47 made selection of somatic hybrids after
fusion of protoplasts from Datura innoxia (diploid - 2n=24, and a
tetraploid - 4n=48 chlorophyll deficient mutant) and A. belladonna
(2n=72), and the 13 selected hybrids produced chromosomes at
metaphase varying from 84 to 175 and the chromosomes of either
species were distinguishable from their sizes. On the contrary,
sexual incompatibility exists between the two plant species
(Krumbiegel and Schieder52). Gleba et al.53 successfully produced
intertribal hybrid cell lines of A. belladonna × Nicotiana chinensis
by cloning individual protoplast fusion products and the hybrid
nature of the clone lines (thirteen obtained) in confirmed by
biochemical (studies of amylase isozymes), cytogenetic (size and
morphology of chromosomes) and physiological (peculiarities of
cell growth in vitro) analyses. Results obtained were interpreted
as new evidence for the possibility of using non-sexual hybridization
for the production of such plant hybrids which cannot be obtained by
sexual crossings. Gleba et al.54 made ptotoplast fusion of N. tabacum
(B6S3) crown gall cells and A. belladonna leaf mesophyll to obtain
57 hybrid lines which were verified from biochemical, molecular
and cytogenetical studies. Kushnir et al.55 developed functional
cybrid (mesophyll protoplast fusion) between N. tabacum
(chlorophyll deficient, treptomycin-resistant) and wild A. belladonna
using the polyethylene glycol/high pH/high Ca++/dimethylsulfoxide
method. Three groups of regenerants were identified - (a) nuclear
Datta K. Animesh et al. IRJP 2011, 2 (11), 11-17
INTERNATIONAL RESEARCH JOURNAL OF PHARMACY, 2(11), 2011
hybrids; (b) Atropa plants arising from rare surviving parental
protoplast; (c) Nicotiana/Atropa cybrids possessing tobacco genome
and Atropa plastome and the cybrids were diploid morphologically
and phenotypically similar to tobacco yielding viable seeds. Gleba et
al.48 obtained asymmetric hybrids between Nicotiana
plumbaginifolia (n=10) and A. belladonna (n=36) by gamma-
fusion and cytogenetic analysis of hybrid plants revealed that the
plants possessed multiple (tetra-or hexaploid) sets of N.
plumbaginifolia along with 6 to 29 Atropa chromosomes some of
which were deleted. rDNA genes of both parental species were
present and amplified in majority of the hybrids but chloroplast
DNA was only from Nicotiana parent. However Kushnir et al.56
reported nucleo-cytoplasmic incompatibility in cybrid plants
possessing an Atropa genome and a Nicotiana plastome. Babiychuk
et al. (1992) showed spontaneous extensive chromosome
elimination of A. belladonna in somatic hybrid lines developed
between N. tabacum and A. belladonna (2n=72). All lines (3)
possess 48 large chromosomes similar to N. tabacum and one small
fragment of A. belladonna. Ahuja et al.57 induced intergeneric
somatic hybrid plants between A. belladonna (2n=72) and
Hyoscyamus muticus (2n=28) and the plants varied in chromosome
number (100 to 178) and gross morphology, chromosome
complementation as well as elimination (Atropa) were noted in
hybrid plants.
Mutagenesis
Kaul and Choudhary58 treated seeds of A. belladonna with
nitrosoguanidine (NG- 1 and 2 mM) and ethylene imine (EI- 0.05
and 0.025 %) resulted in significant increase in plant height, tiller
number, leaf number, leaf length, leaf width and alkaloid content. It
was also observed that all the treatments (except NG, 1 mM)
resulted in a considerable increase in variability in alkaloid content
as compared to the control. Abdel-Hady et al.59 carried out
investigation to improve germination percentage and alkaloid
production in Belladonna seeds following treatments with gamma
irradiations (50, 80, 110 and 150 Gy) and gibberellic acid
(20,40,60,80,100 and 120 ppm) and it was found that enhancement
in germination occur upto 150 Gy (irradiation) and 100 ppm GA
treatments and best was recorded at 110 Gy irradiation and 100 ppm
GA. Seed colour (red seeds) and three promising high alkaloid
containing mutants (M-11-1, M-11-2 and M-15-1) were selected.
Molecular genetics
Jung and Tepfer60 induced genetic transformation in A. belladonna
enhanced biomass and tropane alkaloid production by root inducing
(Ri) T-DNA from A. rhizogenes. Borisjuk et al.61 studied the
behavior of ribosomal RNA genes in the process of somatic
hybridization between N. tabacum and A. belladonna. Blot
hybridization of parental species DNAs to 32P-rDNA specific probes
revealed two classes of ribosomal repeats (N. tabacum 11.2 kb,
10.4 kb; A. belladonna 9.4 kb, 10.2 kb) and a new class of
ribosomal DNA repeat absent in parental species was found in
hybrid line NtAb-1. Mathews et al.62 developed transgenic A.
belladonna via A. tumifaciens (C58 C1, harboring the plasmid pGV
3850::1103 containing the coding sequences of neomycin
phosphotransferase; explants cultured overnight with or without
acetosyringone), and Southern hybridization showed the integration
of foreign DNA into the plant genome. Of the progeny tested, 2 out
of 3 showed monogenic dominant segregation for kan super (R).
Kurioka et al.63 reported kanamycin-resistant transgenic by
transforming with a CaMV 35S-rol C chimeric gene (co-introduced
NPT-II) of the Ri plasmid of A. rhizogenes, and the transformed
plants exhibited dramatic promotion of flowering, reduced apical
dominance, pale and lanceolate leaves and smaller flowers. Saito et
al.64 also raised herbicide- resistant A. belladonna using Ri binary
vector (pRi15834 and pARK5) and integration of T-DNAs from
pRi15834 and pARK5 were confirmed by DNA blot hybridization.
The transgenic plants showed resistance towards bialaphos and
phosphinothricin. Yun et al.65 induced transgenic A. belladonna
(hyoscyamine 6 beta-hydroxylase which catalyzes the oxidative
reactions in the pathway leading from hyoscyamine to scopolamine
under the control of the cauliflower mosaic virus 35S promoter) by
the use of an Agrobacterium mediated transformation system.
Jaziri et al.66 obtained transgenic A. belladonna doubly transformed
with different A. rhizogenes (ATCC 15834 and MAFF-03-01724)
strains in hairy root culture. The transformants were tested by the
opine assay and polymerase chain reaction (alkaloid content
intermediate while IAA level decreased in transformed roots).
Yoshimatsu et al.67 induced root cultures (integration of beta-
glucuonidase and neomycin phosphotransferase genes by binary
vector method) from diploid and haploid A. belladonna using Ri
plasmid of A. rhizogenes (strain A4). Except for the rol h and rol c
insertion mutants, the diploid plantlets were with hairy root
syndrome; while, the haploid exhibited dwarfing features. Suzuki et
al.68 isolated hyoscyamine 6β-hydroxylase gene from A. belladonna
and showed that the gene was differentially expressed in the root
pericycle and anthers. Bonhomme et al.69 raised transformants of A.
belladonna hairy root lines via A. tumefaciens (2 series: rol C and
npt-II; rol ABC and npt-II) and were confirmed by PCR analysis.
Possible role of rol C gene in hairy root growth rate and tropane
alkaloid in hairy root culture was studied and found to be enhanced
several folds. Schmitz-Linneweber et al.70 sequenced the plastid
chromosome of A. belladonna (circular DNA of 156, 688 bp) and
compared with the published sequence of N. tabacum to understand
nuclear-plastid incompatibilities and to speciation. It was noted that
(1) promoters as well as translational and replicational signal
elements in both species were well conserved; (2) genes were highly
conserved, with differences residing predominantly in regions of low
functional importance, and (3) RNA editotypes differ between the
species causing rapid reproductive isolation of populations. Fukami
et al.71 induced Salicylic Acid Carboxyl Methyltransferase in hairy
root cultures of the species and the gene was expressed to a high
concentration of exogenously added salicylic acid, expression begins
12h after the exposure and continued over 144h. Nouar et al.72
reported six differentially expressed genes (confirmed by RT-PCR)
in A. belladonna leafy crown gall following Rhodococcus fascians
infection. Kress et al.73 made comparison of the total plastid genome
of tobacco and deadly nightshade and suggests that the sequences in
the loci (nuclear internal transcribed spacer region and plastid trnH-
psbA intergenic spacer) were potential to discriminate the large
number of species for barcoding purposes. Yuan et al.74 inserted a
retroposon in the nuclear gene granule-bound starch synthase I
(GBSSI or waxy), which reveals the extinct diploid ancestor in the
polyploid origin of belladonna, thereby suggesting that retroposons
were promising molecular markers to study polyploid evolution.
Kwon et al.75 studied the expression of A. belladonna salicylic acid
(SA) carboxyl methyltransferase gene, AbSAMT1, encoding S-
adenosyl-L-methionine (SAM): SA carboxyl methyltransferase
(SAMT) in A. belladonna after application of biotic and abiotic
stresses to plants. Results suggested that AbSAMT1 may play a dual
regulation role of distinct signaling in A. belladonna plants, namely
the signaling pathway of the SA-dependent response and also a
jasmonic acid dependent response in local regions.
Biotechnology
Rajbhandary et al.76 initiated root, callus and cell suspension
cultures from seedlings of A. belladonna. Cultured roots and plants
raised from shoots initiated on cultured callus were shown to contain
atropine (hyoscyamine) and hyoscine and cuscohygrine. The
alkaloids were found to be absent from cultured callus and cultured
cell suspensions and from leaves when initiated without roots on
callus. Thomas and Street77 observed organogenesis in cell
suspension culture from excised cultured roots of the species.
Datta K. Animesh et al. IRJP 2011, 2 (11), 11-17
INTERNATIONAL RESEARCH JOURNAL OF PHARMACY, 2(11), 2011
Thomas and Street78 noted the factors (time that the root cultures
were maintained, composition of the medium, subsequent culturing,
elevated amount of ammonium ions) influencing morphogenesis in
excised roots and suspension cultures of Belladonna. Konar et al.79
studied the diversity of morphogenesis in suspension cultures of A.
belladonna. Rashid and Street80 studied the development of haploid
embryoids from anther culture of A. belladonna. Rashid and Street81
further studied growth, embryogenic potentiality and stability of a
haploid cell culture of A. belladonna. Gosch et al.82 isolated
protoplast from actively growing cell-suspensions of A. belladonna,
which divide repeatedly and to undergo embryogenesis eventually
developed into plantlets. Optimum protoplast yield was upto 80% in
4-5h by treating cell suspension with enzyme mixture of cellulose R
10(1%) and macerozyme R10 (0.5%) in 0.6M sorbitol at 300C.
Eapen et al.83 investigated morphogenetic and biosynthetic ability of
tissue cultures established from haploid and diploid plants of the
species. It was observed that haploid tissue regenerated plantlets
much more readily than did the diploids. The regenerants obtained in
vitro were successfully transplanted to soil and grown to flowering
stage (alkaloids in quantities comparable to the plants raised from
seeds). The authors84 established tissue cultures from leaves of one
anther-derived haploid plant of A. belladonna, and the regenerants
obtained from callus cultures were transferred to soil and reared to
maturity. Callus cells (alkaloid content low) and regenerants
(content variable) exhibited variable degree of ploidy. Lorz and
Potrykus85 isolated leaf mesophyll protoplasts of A. belladonna and
when cultured in defined liquid culture media regenerate cell walls,
divide and form calli. Subsequent induction of shoot and root
organogenesis leads to plantlets which grow to maturity after
transfer to soil. Heberle-Bors86 noted the effects of different types of
iron chelates and activated charcoal in anther culture of belladonna
(Nitsch medium). FeEDTA was superior to FeDTPA (Sequestrene
330), FeEGTA, FeEDDHA (Sequestrene 138) or Fe citrate.
Activated charcoal in combination with FeEGTA and FeEDDHA
inhibited haploid formation due to the preferential adsorption of
these iron chelates by activated charcoal. Iron chelates seemed to be
active during the transition from the globular to the heart-shaped
stage of embryogenesis; while, activated charcoal increased the
amount of premitotic pollen after a week of culture. Salonen87
showed glutamate and aspartate derived amino acids were used
alone, or with inorganic N, or in various combinations as N sources
effectively enhance growth for stem callus cultures of A. belladonna
but growth was found to be retarded by arginine, ornithine,
hydroxyproline, lysine, threonine, isoleucine and methionine. Ondrej
and Protiva88 induced crown galls and hairy roots in in vitro
cultivated seedling of A. belladonna by different A. tumifaciens and
A. rhizogenes strains. Only root cultures induced by Ri plasmid A4
synthesized detectable amounts of alkaloids. Nyman and Simola89
showed that suspension cultures of A. belladonna possess limited
capacity to regulate the transport of phenylalanine (precursor of
atropine) into the cells at stationary phase of growth. The rate of
phenylalanine uptake was fastest from 2 to 7 days (increase from 50
to 300% depending on cell line) after the start of the suspension
culture. The enhancement may be inhibited by cyclohexamide as
well as glutamine by repressing the synthesis of phenylalanine.
Simola and Nieminen90 initiated callus cultures (n=156) from one
young root and from stems of 12 plants of A. belladonna. The calli
derived from the same piece of stem showed wide variations in their
growth response on modified Wood and Brauns nutrient medium
and in their alkaloid production. Levels of alkaloids were not
significantly higher in the callus cultures of intact mother plants
possessing a high level of hyoscyamine and scopolamine in seeds,
roots, or leaves than in callus cultures originating from plants with
low alkaloid production. The maximum hyoscyamine content (0.2
0.3g/Kg dry wt.) was usually found between 7th and 9th passage in
stem callus lines. After the 9th passage the alkaloid content decreased
rapidly, and the repression of synthesis may not be prevented by
lower temperature (150C as against 250C) or by lower or higher
auxin level of the medium. The best sterilization protocol in A.
belladonna is that 10 minutes marinating by neutral scour, 30
minutes over-washing by tap water, 6 minutes sterilization by 0.1%
mercuric chloride, rinsing 3 to 5 times by sterile water. Appropriate
medium for inducing the differentiation on bud of the first culture is
MS + 0.7mg/L 6-Benzyl adenine + 0.2mg/L NAA + sucrose 3.0%.
The appropriate medium on step-generation and propagation of bud
is MS + 0.5mg/L 6-BA + 0.2mg/L NAA + sucrose 3.0%; while, for
rooting the best medium recommended was MS + 0.2mg/L IBA +
sucrose 2.0 % (100% rooting). Axillary buds dipped in 0.1%
colchicine induce highest rate (45%) of polyploidy91. Nyman92
labeled U-14C arginine (Arg), ornithine (Orn) and phenylalanine
(Phe) and incorporated into hyoscyamine and scopolamine of both
dissected roots of intact plants and homogenous or aggregating
suspension culture of A. belladonna and found that hygrine,
tropinone and tropanol were present only in roots, and alkaloid
synthesis proceeded as far as to scopolamine thereafter. In the
synthesis of the tropane skeleton, Orn was used more efficiently than
Arg. Phe both in roots and suspension cultures, and the label was
incorporated in both ethanol insoluble compounds (particularly
proteins) and ethanol soluble compounds (phenolics). It has been
reported that in heavily root suspensions neither hyoscyamine nor
scopolamine were found, but traces of tropanol were detected
thereby suggesting that the synthesis of tropane alkaloids seems to
be regulated at the later biosynthetic steps. Hilton and Wilson93
studied growth and the uptake of sucrose and mineral ions by the
transformed root cultures of A. belladonna and other related species
and hybrids (examined during batch culture over 28 days in
modified 14 litre stirred tank reactors containing Gamborgs B5 salt
medium; all cultures completely removed NH4+ and PO43- from the
medium). Each of the culture consumed NO3- than any other ion,
and concomitant uptake of sucrose and release of glucose into the
medium. Roots were found to contain low levels of free sugars and
hyoscyamine production ranged from 115mg to 633mg per reactor.
Aoki et al.94 studied variation of alkaloid productivity among several
clones of hairy roots and regenerated plants. Lee et al.95 showed
enhancement in tropane alkaloid production (1490 mg produced in
30-1 tank) by transformed root cultures of Belladonna. Yang et al.96
studied floral organogenesis and development following SEM
analysis in A. belladonna and in a Solanaceous member (Anisodus
tanguticus). Rothe et al.97 fused pmt (Putrescine N-
methyltransferase) gene from N. tabacum with CaMV 35S promoter
and integrated into A. belladonna genome and the transgenic plants
derived from root cultures showed over expression of the gene.
Expression level of pmt alone is apparently not limiting for tropane
alkaloid formation in A. belladonna. Richter et al.98 reported that the
overexpression of tropinone reductases alters alkaloid composition
in root cultures of the species. It has been found that strong
expression of the tropine-forming reductase enhanced 5-fold
scopolamine and 3-fold hyoscyamine compared with control roots.
CONCLUSION
Present and future researchers interested in the plant species may
think of enhancing the existing gene pool (although the plant species
is perennial but seed propagated) through the methodology of
induced mutagenesis and, if possible, by biotechnological
approaches to screen desirable variants with enriched alkaloid
contents. Furthermore, sustainable cultivation of the crop under
suited climatic conditions is also an essentiality for its effective
utilization in human benefits.
Datta K. Animesh et al. IRJP 2011, 2 (11), 11-17
INTERNATIONAL RESEARCH JOURNAL OF PHARMACY, 2(11), 2011
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... Yalnızca yapraklar ve üst kısımları toplanır ve sonraki yıllarda aşırı dallanmayı engellemek için 75-90 cm yukardan kış aylarında seyreltme yapılır. İkinci yılında bitkiler haziran ayında çiçeklenme zamanı kesilir ve mevsim şartlarının iyi olduğu durumlarda bitki ikinci mahsul için eylül ayına kadar yeniden hasata gelir [46]. Kuru sıvı ekstreler, tentür, merhem, bandaj ve gliserin hazırlıkları şeklinde işlenmek için çiçeklenme zamanında bitkinin tamamı kesilir ve kurutulur. ...
... Mayıs ve hazirandaki güneşli günlerde alkaloid oranı %0,68 olurken, güneş ışığının yetersiz olduğu durumlarda alkaloid oranı %0.34 olarak bildirilmiştir. İngiliz yetiştiriciler toprağa tomas fosfatı uygulamasının üçüncü yıldaki bitkilerde ve kuru yapraklarda %0,84 gibi iyi sonuçlar elde etmişlerdir [46]. Yaprak hasatında kurutma sonucu 7-8 kg yaş yapraktan 1 kg kuru yaprak elde edilir. ...
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Atropa belladonna is a perennial herbaceous plant that is systenatically found in Solanaceae family. The flowers are greenish purple, the leaves are oval, the fruits are black and bright. Deadly nightshade includes important alkaloids suvh as atropine, skololamine, belladonnaine, hypocaine,apoatropine. The most important chemical substance is atropine. İts active ingredient was isolated in 1809 and later classified as alkaloid by 1819. All parts of the plant contain alkaloids although the highest alkaloid ratio is in the green leaves and in the fruites. İt is the desirable that the ratio of several coteche alkaloids is not less than %0,3. Deadly Nightshade is a plant that has been planted in many countries since ancient times due to ıts active ingredients and the synthetically derived derivatives of atropine whis is the main alkaloid have important place in pharmacy. In our country Atropa belladonna has an important place among tropane alkaloids and herbal medicines
... The belladonna plant is one of the most important perennial herbal medicinal plants in the Solanaceae family (Rita and Animesh 2011). The original home of the belladonna plant is Middle and Southern Europe, from which it spread to Middle and Western Asia (Chevalio 2010). ...
... The leaves and fruits of this plant are highly toxic, containing tropane alkaloids, which include scopolamine and hyoscyamine which cause hallucinations and bizarre delirium. The species of plant is the source of the alkaloid atropine that has proven to be the mainstay in the studies of autonomic pharmacology (Lee 2007;Rita and Animesh 2011). Belladonna is important for use in medicine and cosmetics. ...
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The plant produces large numbers of chemical compounds with diverse physiological roles. The plant takes advantage of cheap and available natural resources in its surroundings of air, water, mineral elements, and solar energy to produce these chemical compounds. These compounds are called phytochemicals, in which biotechnologists are concerned with the mass production of economically important compounds. The compounds produced by the plant are generally divided into two groups, primary metabolites and secondary metabolites. In this paper, we will review what researchers have reached in the field of accurate propagation of some important medicinal plants through a presentation of several protocols used to propagate some medicinal plants that aim to mass micropropagation them or expose them to treatments aimed at increasing the production of secondary compounds that have a role when extracted in the manufacture of medicines and pharmaceuticals.
... Atropa belladonna L. is an important herbal plant used by human beings. Modern pharmaceutical science has revealed that A. belladonna plants produce anticholinergic tropane alkaloids (TAs), including hyoscyamine, anisodamine, and scopolamine [1]. Atropine is the racemic mixture of hyoscyamine, and its name derives from A. belladonna, which is widely cultivated to produce atropine or hyoscyamine [2][3][4]. ...
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Atropa belladonna L. is one of the most important herbal plants that produces hyoscyamine or atropine, and it also produces anisodamine and scopolamine. However, the in planta hyoscyamine content is very low, and it is difficult and expensive to independently separate hyoscyamine from the tropane alkaloids in A. belladonna. Therefore, it is vital to develop A. belladonna plants with high yields of hyoscyamine, and without anisodamine and scopolamine. In this study, we generated A. belladonna plants without anisodamine and scopolamine, via the CRISPR/Cas9-based disruption of hyoscyamine 6β-hydroxylase (AbH6H), for the first time. Hyoscyamine production was significantly elevated, while neither anisodamine nor scopolamine were produced, in the A. belladonna plants with homozygous mutations in AbH6H. In summary, new varieties of A. belladonna with high yields of hyoscyamine and without anisodamine and scopolamine have great potential applicability in producing hyoscyamine at a low cost.
... Esters of tropane alkaloids are the most common secondary metabolites of these plants. Tropane alkaloids are highly bioactive and of immense pharmaceutical value due to which they are globally used as active ingredient of a large number of commercially available drug formulations, for example, belladonna, used in the treatment of asthma, whooping cough, motion sickness, and Parkinson's disease (Paul and Datta 2011). Owing to such pharmaceutical significance, the huge commercial demand for plant TAs always remains unmet. ...
Chapter
Tropane alkaloids (TAs) are a special class of alkaloids found naturally in a diverse group of flowering plant families. To date, about 200 TAs are known, the most prominent being hyoscyamine, scopolamine, calystegine, and cocaine. These compounds possess pharmacological properties and are used in medicine as anticholinergic agents and stimulants. Because of their medicinal value, tropane alkaloids have been the subject of study for several years now. Over the years, research has been directed at elucidating the biosynthetic pathways leading to the production of pharmacologically active TAs. The present chapter discusses recent developments in the understanding of TA biosynthesis with emphasis on the genes involved in the TA biosynthetic pathways and the role transcriptome profiling played in their identification. In recent years, mining of the transcriptome data of TA-producing plants, such as Atropa belladonna, has led to a near-complete elucidation of the biosynthesis of hyoscyamine and scopolamine. Advances in gene elucidation made through such studies can be potentially used for metabolic engineering in transgenic plant systems or microbial platforms to sustainably meet the global demand of pharmaceutically important TAs.
... In Atropa belladonna majority of alkaloidal contents are present in ripe fruit and green leaves. It has been used from ancient times in order to treat various human ailments including menstrual disorders, headache, peptic ulcer, inflammation and histaminic reaction [61]. Ultradiluted belladonna concentrations like 1:10 or 1:100 are used in homeopathy and they are recommended for management of all the infectious diseases and illnesses [62]. ...
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Zika virus (ZIKV) is a newly emergent relative of the Flaviviridae family and linked to dengue (DENV) and Chikungunya (CHIVKV). ZIKV is one of the rising pathogens promptly surpassing geographical borders. ZIKV infection was characterized by mild disease with fever, headache, rash, arthralgia and conjunctivitis, with exceptional reports of an association with Guillain–Barre syndrome (GBS) and microcephaly. However, since the end of 2015, an increase in the number of GBS associated cases and an astonishing number of microcephaly in fetus and new-borns in Brazil have been related to ZIKV infection, raising serious worldwide public health concerns. ZIKV is transmitted by the bite of infected female mosquitoes of Aedes species. Clarifying such worrisome relationships is, thus, a current unavoidable goal. Here, we extensively described the current understanding of the effects of ZIKV on heath, clinical manifestation, diagnosis and treatment options based on modern, alternative and complementary medicines regarding the disease.
Chapter
The liver controls the body's internal environment via various physiological and metabolic processes, either independently or together with other organs. Liver disease is defined as an injury to the hepatic cells and tissues, mainly caused by viral infections, toxic compounds, high doses of drugs, and excessive alcohol intake. There are an estimated two million hepatic disease–associated deaths recorded annually worldwide with a diverse etiology. Numerous medicinal plants and their phytochemicals have been reported to display hepatoprotective activity. Among those, bioflavonoids, low-molecular-weight phenolics possessing a basic C6–C3–C6 structure, have been reported to have the ability to treat hepatic diseases. In the current study, we aimed to summarize hepatic diseases and strategies to treat them and added a brief note on the bioflavonoids that have been tested against hepatic diseases.
Chapter
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Chapter
The medicinally important species of the Solanaceae family belong to genera viz., Solanum, Atropa, Datura, Hyoscyamus, Nicotiana, etc. The active phytoalkaloids which are present in them are tropane, pyrrolidine–pyridine, and steroid alkaloids. Some of the pharmacologically potent alkaloids are scopolamine, atropine, hyoscyamine, nicotine, cocaine, solanidine, and solasodine. Tropane alkaloids, their semisynthetic, and synthetic derivatives are used as antispasmodic, antiemetic, mydriatic, and bronchodilator because of anticholinergic actions. Compounds with tropane nuclei bearing diester group on their structure without quaternary ammonium have shown local anesthetic and CNS stimulant actions. Nicotine is a pyrrolidine–pyridine alkaloid that acts on nicotinic receptors. Steroidal alkaloids like solasodine are poisonous and serve as starting material for the synthesis of steroidal hormones. This chapter describes general methods of extraction and isolation of alkaloids. The sources, classification, pharmacological actions, and structure–activity relationship of tropane alkaloids are illustrated with a special emphasis on chemical structure elucidation of atropine.
Chapter
Plant tropane alkaloids are the secondary metabolites known for their immense pharmaceutical importance and have been a sole ingredient of various ancient and modern medicinal drug formulations. These drugs are meant for the treatment of a range of simple to serious ailments like muscle spasms, stomachaches, blood circulatory diseases, and chronic obstructive pulmonary diseases. Members of Solanaceae and other plant families like Erythroxylaceae, Convolvulaceae, and Brassicaceae are the natural repositories of tropane alkaloids and have been exploited globally for their biotechnological production. Hairy root cultures of the various plant genera belonging to these families have proven their worth as a suitable production system for these metabolites and are widely utilized for the biotechnological production of tropane alkaloids. The present chapter explores the hitherto and current status in advancement of using hairy root cultures of TA-producing plants for the biotechnological production of tropane alkaloid. The text focuses on various primary and additional advanced strategies for establishment, optimization, and enhancement of tropane alkaloid yields using hairy root cultures.
Article
Atropa belladonna L. is a perennial plant endemic to Central and Southern Europe and India and is being cultivated worldwide. It is used in the treatment of headache, menstrual symptoms, peptic ulcer, inflammation and motion sickness, bronchial spasms and whooping cough, Parkinson’s disease, antidote for snake bite, and gastric agent. The plant species is considered as extremely toxic due to the presence of alkaloids. The toxic Atropa alkaloids cause delirium, hallucination, tachycardia, mydriasis, dry mouth, flushed skin, urinary retention, vomiting, and anhidrosis in humans. Besides A. belladonna, four other species of Atropa are found: Atropa acuminata in Asia, Atropa baetica in Spain and Morocco, and Atropa caucasica and Atropa komarovii in Russia. Cell culture studies were established in Atropa plant by using various explants of A. belladonna for the enhancement of yield of the tropane alkaloids.
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Behavior of ribosomal RNA genes in the process of somatic hybridization was analyzed using hybrids Nicotiana tabacum + Atropa belladonna. Blothybridization of parental species DNAs to (32)P-rDNA specific probes revealed two classes of ribosomal repeats in both tobacco and nightshade; their length was 11.2 kb, 10.4 kb (tobacco) and 9.4 kb, 10.2 kb (night-shade). For analysis of hybrids, labelled (32)P rDNA specific probes were hybridized to DNA of parental species and somatic hybrids digested with restriction endonucleases EcoR1, EcoRV and BamH1. A new class of ribosomal DNA repeat, absent in parental species, was found in hybrid line NtAb-1. Possible mechanisms of appearence of a new rDNA class in the process of somatic cell fusion are discussed.
Chapter
Flowering and fruiting are key processes in the biology of higher plants, ensuring the transfer of genetic material from one generation to the next. In addition, as almost all of the world's agricultural and horticultural industries depend on the production of flowers, fruits and seeds, the study of the reproductive biology of cultivated plants is of fundamental importance to humankind. Surprisingly, therefore, this topic has received relatively little attention from environmental physiologists compared with studies on the growth and development of vegetative structures. This book, based on a meeting held by the Environmental Physiology Group of the Society of Experimental Biology, sets out to correct this deficiency. The topic is given a broad and comprehensive treatment, with chapters covering the onset of flowering through to the development and growth of fruits and seeds, and finally to ecological and evolutionary aspects of fruiting. This volume will therefore serve as a useful introduction to the various aspects of flowering and fruiting and will also provide a thorough general overview of the subject for students and researchers alike.
Article
Floral organogenesis and development of two Solanaceae species, Anisodus tanguticus and Atropa belladonna, were studied by using scanning electron microscopy (SEM) as part of a project on systematics and evolution in the tribe Hyoscyameae. These two species share the following common characters of floral organ initiation and development: (1) initiation of the floral organs in the two species follows Hofmeister's rule; (2) the mode of corolla tube development belongs to the "late sympetaly" type, namely, petals are initiated separately and later become joined by fusion of their basal meristem, then rise together and form a corolla tube; (3) primordia of the floral appendages are initiated in a pentamerous pattern and acropetal order: sepals are initiated first, followed by the petals and stamens, and finally the carpels. The whorl of five stamen primordia forms almost simultaneously and originates opposite the sepal primordia, but initiation of the sepal primordia shows different modes in the two species. The sepal primordia of Anisodus tanguticus have simultaneously whorled initiation, while those of Atropa belladonna have helical initiation. The systematic significance of the present results in the genera Anisodus and Atropa is discussed in this paper.
Article
Asymmetric nuclear hybrids have been obtained by fusion of cells from a nitrate-reductase deficient mutant of Nicotiana plumbaginifolia (cnx20) and gamma irradiated protoplasts of Atropa belladonna (irradiation doses tested were 10, 30, 50 and 100 krad). The hybrid formation frequency following selection for genotypic complementation in the NR function was in the range of 0.7%-3.7%. Cytogenetic studies demonstrated that all hybrid plants tested possessed multiple (generally tetra- or hexaploid) sets of N. plumbaginifolia (n = 10) chromosomes along with 6-29 Atropa chromosomes (n = 36), some of which were greatly deleted. Besides the cnxA gene (the selection marker), additional material of the irradiated partner was expressed in some of the lines, as shown by analyses of multiple molecular forms of enzymes. Surprisingly, rDNA genes of both parental species were present and amplified in the majority of the hybrids. Whenever studied, the chloroplast DNA in the hybrids was derived from the Nicotiana parent. Regenerants from some lines flowered and were partially fertile. It is concluded that irradiation of cells of the donor parent before fusion can be used to produce highly asymmetric nuclear hybrid plants, although within the dose range tested, the treatment determined the direction of the elimination but not the degree of elimination of the irradiated genome.
Article
Hairy root cultures from diploid and haploid Atropa belladonna plants were established by co-culture method, using the Ri plasmid of Agrobacterium rhizogenes (strain A4) and its insertion and deletion mutants. The hairy roots integrated β-glucuonidase and neomycin phosphotransferase genes were also established by binary vector method. Diploid hairy roots grew well in phytohormone-free Murashige and Skoog (HF MS) liquid medium, especially those induced from the bacterium with the insertion mutation at rol c. Haploid hairy roots could be maintained in HF MS liquid medium though their growth was inferior to that of diploids. Both, diploid and haploid hairy roots, contained hyoscyamine (ca 0.2-0.6% dry weight) as the main alkaloid. A higher yield of hyoscyamine was obtained in the diploid hairy roots (400-1300 μg/100 ml flask for diploids against 200-400 μg/100 ml flask for haploids). Transgenic plantlets were regenerated on MS solid medium with or without phytohormones at 25°C under 16 h day light. Except for the rol b and rol c insertion mutants, the diploid plantlets showed the typical hairy root syndrome, while the haploids exhibited much more dwarf features. The co-existence of the two rol loci b and c determined the hairy root syndrome expression. Alkaloid concentrations in roots and leaves of normal and transgenic plantlets were much lower than those of the corresponding hairy roots (less than 0.09% dry weight hyoscyamine), and no relationship in alkaloid concentration was found between the regenerated plantlets and the original hairy roots.
Article
Callus cultures have been initiated from excised cultured roots of Atropa belladonna and A. belladonna cultivar lutea and used to establish suspension cultures in a synthetic culture medium (referred to as SSM) containing 2.0 mg/1 α-naphthaleneacetic acid (NAA). Visible cellular aggregates develop in these suspension cultures and when such aggregates are cultured in an NAA-omitted medium, large numbers of roots arise superficially on the aggregates. Root development is enhanced by incorporating into the auxin-free medium either tropic acid or I-naphthoxyacetic acid and both these substances can promote root initiation in the presence of levels of NAA which alone suppress organogenesis. The aggregates also give rise under appropriate conditions of culture to shoots and to embryo-like structures. The nature and frequency of such morphogenesis in the aggregates is dependent not only upon the composition of the culture medium but upon the number and the length of previous culture passages through which the callus and suspension cultures have been propagated in the SSM. The ultimate loss of morphogenetic potential in serially propagated callus is discussed. The structure of the aggregates in SSM and in the media promoting root initiation is described. Examination of the aggregates for their content of alkaloids indicates that the major belladonna alkaloids can only be detected in cultures forming roots.
Article
Growth and nitrate reductase activity (NRA) of Atropa belladonna cells were studied in medium supplemented with NaNO3, NH4NO3, and amino acid precursors to tropane alkaloids. Growth and NRA were stimulated by NH4+ and by proline, by proline plus ornithine, but not by glutamate, in NO3--containing medium. Tested amino acids inhibited neither utilization of inorganic nitrogen nor growth.