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Black cumin (Nigella sativa L.) – a review

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Reveiw Article

BLACK CUMIN (NIGELLA SATIVA L.) – A REVIEW

Animesh K. Datta1*, Aditi Saha2, Arnab Bhattacharya1, Aninda Mandal1, Rita Paul3
and Sonali Sengupta4

1. Department of Botany, Cytogenetics and Plant Breeding Section, Kalyani University,

Kalyani 741235, West Bengal, India
2. Department of Botany, Narasinha Dutt College, Howrah 711101
3. Charuchandra College, Department of Botany, Kolkata 700029
4. P.G. Department of Botany, Hooghly Mohsin College, Hooghly 712101
*Corresponding author email: dattaanimesh@gmail.com

Abstract: Black cumin (Nigella sativa L., Family: Ranunculaceae) is an annual herb possessing wide range of medicinal
uses apart from its commercial significance as a spice yielding plant. Black cumin seeds are used in folk (herbal) medicine
all over the world for the treatment and prevention of a number of diseases. Prophet Mohammad (Peace be Upon Him) said:
"Use this Black Seed; it has a cure for every disease except death" (Sahih Bukhari). The plant species is also important
cytogenetically and may be used as a model plant for better understanding of gene and chromosome relationship. Despite the
major advancement of modern medicine in human health-care, it is still intangible and beyond reach to ailing humanity,
especially the destitute and therefore in recent years plant based system has been utilized for traditional medicine and
phytotherapy. 'Medicinal plants are gift of nature' and black cumin is one such plant with potential uses, which can be
explore for safe and effective herbal medicine for human benefit. Considering nearly all essential aspects of the species
(synonym(s), common names, origin of the name, distribution, varieties, plant description, floral biology, pollination
biology, scanning electron microscopy of seed surfaces, cultivation, economy, diseases, pest, microscopical and powdered
characteristics, biochemical constituents, extraction methods of essential oils, therapeutic uses, insecticidal activity, other
uses, clinical trials, biosafety, tissue culture and patents), a monograph is prepared on the laid formulation of WHO (World
Health Organization) as well as on other significant parameters (cytogenetics and molecular genetics) with the following
objectives: to provide an unabridged repository of references regarding the species for its effective and safe utilization as a
'Potential Medicinal Herb'; for creating awareness regarding the use of plant based medicine; understanding economic status,
biosafety and patents for regulating herbal medicinal market Nationally and Internationally and exploration of cytogenetical
and genetical aspects.

Keywords: Black cumin, Herbal medicine, Nigella sativa

INTRODUCTION oil) and its quality. Existing germplasms may not

substantiate the need for future, if not, at present.

N igella sativa L. (Family: Ranunculaceae;

commonly known as Black Cumin) is an annual
Therefore, it is of utmost essentiality to raise
desirable plant type(s) in N. sativa through induced
herb possessing a wide range of medicinal uses1,2 not genetic variations and efficient breeding endeavour.
withstanding its commercial significance as a spice Considering nearly all essential aspects of N. sativa,
yielding plant3. Black cumin seeds are most revered a monograph is conducted with the laid formulation
(Holy herb of the Middle East – Yarnell and of WHO as well as with other significant parameters
Abascal4; can heal every disease except death – which will provide unabridged repository of
Islamic prophet Mohammad; stimulates body’s references for present and future researchers who are
energy and helps recovery from fatigue and looking to eugenize the species as a ‘potential
dispiritedness – The Canon of Medicine, Avicenna; medicinal herb’ for human benefits.
included in the list of natural drugs of ‘Tibb-e-
Nabavi’; valuable remedy for number of diseases – Synonym(s)
Unani Tibb system of medicine) medicinally. WHO
(World Health Organization) is providing emphasis Nigella indica Roxb. ex Flem., Nigella truncata
on the exploration of medicinal plant species for Viv.5
benefit of human care system. Emphasis has been
laid mainly on scientific information, on the safety, Common names
efficacy, quality control / quality assurance, dosage,
toxicity description of the plant species, therapeutic English: fennel flower, nutmeg flower, Roman
uses, clinical trials, drug interactions amongst other coriander, blackseed or black caraway, black sesame;
but genetic resources and its induction must also be India: Assamese - kaljeera or kolajeera, Bengali -
taken into consideration for significant utilization of kalo jeeray, Kannada – Krishna Jeerige, Tamil -
a plant species under consideration. Effective karum jeerakam, Hindi/Urdu - kalaunji/mangrail;
utilization of N. sativa for therapeutic purposes as Russian: Chernushka; Hebrew: Ketzakh; Turkish:
well as for trade will vastly depend upon yield (raw çörek out; Arabic: habbat al-barakah; Persian: siyâh
plant product- seeds; bioactive compounds- essential dâne; Indonesian: jintan hitam; Bosnian: urekot6;

________________________________________________
Journal of Plant Development Sciences Vol.4 (1): 1-43. 2012
ANIMESH K. DATTA, ADITI SAHA, ARNAB BHATTACHARYA, ANINDA MANDAL, RITA PAUL AND SONALI
SENGUPTA.

French: nigelle de Crète, toute épice; Germany: indehiscent style and composed of variable number
Schwarzkümmel; Portuguese: cominho-negro; of multi ovule carpel, developing into a follicle after
Spanish: ajenuz, arañuel; Swedish: svartkummin7. pollination; fruit single partially connected to form a
capsule like structure (capsule 5 to 45; mean
Origin of the name 20.0±3.37; capsule fertility 94.5%) dehiscence
through suture; fruits (length – 0.4 to 1.7 cm, mean
Originally black cumin was the common name for 1.03 cm ± 0.13; seta per capsule 4 to 8, mean
Bunium persicum and later named as Carum 5.10±0.10) with numerous seeds (59.29±3.2; average
bulbocastanum, which is now near extinction and seed production/plant - 935±177.9; seed yield – 1.91
slowly Carum carvi graduated to the name and due gm; seed viability 80% to 90%); seeds ovate,
to inability of the species to all over India, later N. tetrangular, angles sharp, acute, more tapering at the
sativa was adopted from Portuguese or Turkish end (Fig. 4), color black (000021 – British Atlas of
merchants6. Colour, 2007); seed size 2.33 mm ± 0.1 × 1.14 mm ±
0.02.
Distribution The quantitative data of the species were provided
from plants grown in the Experimental garden of
The species is cultivated and distributed all over Department of Botany, University of Kalyani (West
India especially in Punjab, Himachal Pradesh, Bengal plains, Nadia, latitude 22°50' to 24°11' N,
Gangetic plains, Bihar, Bengal, Assam and longitude 88°09' to 88°48' E, elevation 48 feet above
Maharashtra. Apart from India, the species is also sea level, sandy loamy soil, organic carbon 0.76%,
grown in Syria, Lebanon, Israel and South Europe8 soil pH 6.85 – Mandal et al.13) during the months of
as well as in Bangladesh, Turkey, Middle-East and November (15th Nov – sowing; 40 cm between rows
the Mediterranean basin9. and 30 cm between plants) as rabi crop and harvested
in last week of March or in first week of April14.
Varieties
Floral biology
Following varieties of cultivated Kala-Zira reported
with seed yield (g/plant) from Zira and Saffron Andersson15 suggested that increased allocation to
Research Station, Sangla, district Kinnaur (Himachal perianths leads to reduced allocation to direct
Pradesh), India: Rarang (1.7), Pangi (1.4), Stang component of fitness. Plants both with and without
(2.1), Barang (1.3), Sanji (1.9), Rispa (2.0), Kanam perianths did not differ in fecundity of total flower
(2.4), Kilba (1.7), Ribba (1.8), Singla (2.3), Telangi number. Further, perianthless plants produced
(1.4), Thangi (1.9), Lobsang (2.1), Maiber (2.4), heavier seeds with earlier germination dates than the
Rogi (1.5), Kothi (1.8), Spillow (2.4), Morang (1.7), control plants. No detectable effect of perianth
Purbani (1.8), Sharboo (1.8) and Sunam (1.8)10. removal was noted on seed viability or the fecundity
Variety NRCSS AN 1 to different agrotechniques is of plants in the progeny generation. High seed mass
also reported11. Cheikh-Rouhou et al.12 also reported and germination speed had positive and independent
varieties namely, Tunisian and Iranian. effects on progeny fecundity. The author was of
opinion that it is necessary to determine whether
Plant description large conspicuous perianths enhance the amount of
cross pollination and in such case perianth is to be
The species is an erect annual herb (Fig. 1) attaining under stabilizing selection, the optimum phenotype
30.0 cm to 67.6 cm (mean: 52.18 cm ± 4.42) at being a compromise between pollinator-mediated
maturity. Number of primary branches per plant selection for larger floral displays and trade off with
ranges from 4 to 10 (mean: 7.0±0.71); leaf seed size and/or germination speed. The species are
arrangement alternate, leaf phylotaxy 1-2, pinnae of capable of setting seed without being cross
leaves broad, number of pinna per rachis 5-6; total pollinated, an advantageous feature in seed crop
branches per plant 22.5±4.1 (6-48); flower which should be under strong selection for increased
hermaphrodite with determinate flowing patterns, seed production. Finally, the author concluded that
main axis terminate with a solitary flower (Fig. 1), resource trade-offs with seed mass and time to
delicate; flower size 2.74 cm × 2.78 cm; color (Fig. germination may facilitate evolutionary reductions in
2) - french blue (43/3 – Horticultural Color Chart); flower size.
flowers without any involucre of bracts, pedunculate;
peduncle long, erect; petalloid sepals broad, ovate in Pollination biology
a single whorl, 4-6 mostly 5 and characterized by the
presence of nectaries; flower fertility 89.89%; Self pollinated; onset of the male stage stamen stand
stamens in 3 to 4 whorls (Fig. 3), numerous (32 to erect, curved outwards one by one, roughly in whorls
66; 49.6±2.7) and shed their pollen as the filament and strictly reflecting the order of initiation, pollen
bent outward during male phase; gynoecium 5, grains released when anthers reach a horizontal
completely united follicles, each with a long position; male phase initiated a few days before the
JOURNAL OF PLANT DEVELOPMENT SCIENCES Vol.4 (1) 3

stigmas became receptive and lasted for five days; of 2 cm. After 20 days of sowing thinning of the
anther receptivity occurred between 8.00 p.m. to plant to a distance of 20 cm is done.
13.00 p.m. for one day only, male and female stages Sowing by bulbs (previous year root stock) is
synchronized on the last day of the flowering; weight possible when soil moisture content of the field
of pollen 0.064 mg/flower whereas the volume of is favorable for deep ploughing i.e. neither too
nectar 0.13 µl16; empty anthers curved up; pollinated wet nor too dry10.
stigma erect and made an angle of 180º with the 6. Manure and Fertilizer: NPK (5:3:2) is
ovary; style and anther length nearly equal 1.73 cm; generally applied every year along the side of
pollinator honey bee, one bee per flower, visited in the planted bulbs10.
morning around 7.00 a.m.; high temperature effect 7. Weed Control: Frequent weeding reduce weed
fertilization success by affecting stigma receptivity competition and produce good environmental
and accelerating ovule degeneration17. condition for growth and development. About 3-
5 weeding at an interval of 20 to 25 days is
Scanning electron microscopy of seed surfaces recommended by hand hoe or khurpi10.
8. Irrigation: One or two irrigations at flowering
Datta and Saha14 studied seed surface ornamentation and seed formation stage are helpful to increase
and found that surfaces were with distinct grain size and oil content10.
reticulation marks; reticulation more prominently 9. Harvesting: Black cumin grown as rabi crop in
raised, pentagonal to polygonal, ovoid or irregular in West Bengal Plains are generally harvested late
outline; reticulate rows consisting of smaller March to first week of April. The crop harvested
tuberculate raised cells, cells either uni- or multi before shedding at little green stage gives high
seriate or in aggregation along corners or junction; aromatic oil contents providing good market.
cells of reticulate lines showed shrinkage structure; Black cumin retains seed viability longer when it
bound area with variable number of cells (2-5), each is full ripe. It is rather essential that harvesting is
cell comparatively larger, penta-, hexa-, polygonal or done before shedding (shattering of fruits is a
rounded in outline; lumen floor depressed or shallow major problem) and therefore 2 to 3 or more
glabrous (Figs. 5-10). pickings can be done to avoid loss of seeds due
to shattering of the capsules. The harvested crop
Cultivation is dried under sun and threshed by beating with
the stick10.
In India N. sativa is mostly grown once in a year as 10. Post Harvest Management: N. sativa requires
rabi crop during the months of October (late)– extensive labor in collection and harvest as the
November to March-April in plains; while, rarely in capsules (fruit) tend to shatter at maturity. Post
hills in May-June18. harvest management of the fruits usually
1. Area of Cultivation and Production: Area of involves their harvest, one by one, by hand and
cultivation and annual production (source – dry storage till natural dehiscence. The mature
Comparative Sales Report 2010, VDM Verlag fruits do not require much attention as they are
Dr. Muller AG & Co.) were reported to be – self-preserving and their essential oil is a great
India: 6234600 ha, 254000 t; Turkey: 8122010 deterrent to fungal attack, insect attack as well as
ha, 689350 t; USA: 16420 ha, 11200 t; UK: 500 rodent infestation19.
ha, 10 to 20 t respectively.
2. Climate: Grows well in cool-dry with light Shelf life
snowfall areas to warm-humid areas. Cool and
humid weather favors flowering and seed The seeds of N. sativa store well for one year as
setting10. planting material and as a spice, they are stored in
3. Soil: Sandy, loam rich in microbial activity is airtight conditions to prevent the loss of aroma. As a
the most suitable soil for cultivation. The sloppy spice, it is recommended to be stored away from
soils of heavy rainfall areas and leveled and well other species as the species has a overbearing flavour
drained soils of moderate rainfall areas are quite and aroma and disturb the flavour of other species10.
suitable for cultivation. Soil pH 7.0 to 7.5 is
favorable for cultivation10. Economy
4. Preparation of Land: One ploughing followed
by 2-3 harrowing and leveling will be suitable10. Reports
5. Method of Sowing: Seed sowing or by
replanting previous year root stocks. Seed 1. Rs. 275-300/kg in local market (Pakisthan-
sowing is done during October-November by Mingora, Din, Peshawar, Pindi, Lahore, Gilgit
broadcasting (1.5 kg/hectare) or seed drill and Astore), whereas in down country it cost Rs.
method or by line sowing keeping space 450-500. In Internatinal market it is sold for Rs.
between lines (30, 40 or 50 cm) and at the depth 850-1000/gm20.
ANIMESH K. DATTA, ADITI SAHA, ARNAB BHATTACHARYA, ANINDA MANDAL, RITA PAUL AND SONALI
SENGUPTA.

2. Germany: Black cumin oil 1000 ml – 23.90 globules; powdered characteristics brownish black,
EUR + shipping cost21. parenchymatous cells and oil globules27,28.
3. Black cumin MGS Heirloom Seeds; Product
code: NIG02:300 seeds – 1.90€22. Biochemical constituents
4. Black cumin USA. (i) Product No. 1130.F, 29.57
millilit. - $1.40. (ii) Product No 1130.G, 59.14 Constituents of N. sativa seeds are fixed oil – 32 to
millilit. - $1.99. (iii) Product No. 1130.H, 118.29 40% (saturated fatty acids- about 30%; palmitic acid,
millilit. - $2.46. (iv) Product No. 1130.I, 236.58 stearic and myristic acid; unsaturated fatty acids:
millilit. - $3.50. (v) Product No. 1130.J, 476.16 arachidonic, eicosadienoic – 3%, linoleic – 50 to
millilit. - $6.00. (vi) Product No 1130.K, 5lbs 60%; oleic acid – 20%; dihomolinoleic fatty acids –
minimum - $5.00. (vii) Product No. 1130.N, 10%), volatile oil- 0.4 to 0.45% (nigellone,
10lbs min. - $4.50. (viii) Product No. 1130.O, thymoquinone, thymohydroquinone, dithymo-
25lbs min. - $3.9523. quinone, thymol, carvacrol, and -pinene, d-
5. Black Seed 100 capsules - $9 limonene, d-citrlnellol, p-cymene), proteins 16-
Black Cumin Tea (Organic) 20 Bag - $624. 19.9% (arginine, glutamic acid, leucine, lysine,
6. In Indian market Rs. 250-300/kg. Since its methionine, tyrosine, proline, threonine), minerals
cultivation fetches high income per unit area, 1.79-3.74% (calcium, phosphorus, potassium,
therefore, it is highly suitable for cultivation by sodium, iron), carbohydrate 33.9%, fibre 5.50% and
marginal farmers10. water 6.0%29. Ramadan and Morsel30 reported that
apart from physical constants: 2% w/w, foreign
Diseases matter; 6% w/w, total ash; 0.2% w/w, acid insoluble
ash; 20% w/w, alcohol soluble extractive; 15% w/w,
Sinha and Singh25 reported Macrophomina water soluble extractive; 3.91% w/w organic matter;
phaseolina infection in roots causing its deformation. 4% w/w, loss on drying31. The seeds contain
Wilt (causal organism grows along the seedling and carotene, which is converted to vitamin A in liver32.
leaves and branches look light green in colour, leaves Acetylated triterpene saponin (penta hydroxyl
shed and plant dries up; control: spray Dithane M-45 pentocyclic triterpene) has been isolated from the
0.2% or Dithane Z-78 or Blitox 5 w.p. at 15 days species33.
interval) and rotting of bulbs (emit a special odour; Phytochemical Compounds: Categorically different
control: dipping bulbs in 0.3% bavistin for 30 mins. phytochemical compounds of seeds are nigellone34,
before planting, field kept free from stagnant water) nigellicine, nigellimine, nigellimine-N-oxide,
are diseases reported10. Early report by McRae and avenasterol-5-ene, avenasterol-7-ene, campesterol,
Shaw26 also suggested Fusarium wilt in the species. cholesterol, citrostadienol, cycloeucalenol, 24-ethyl-
Prolonged survival of F. udum for upto 8 years was lophenol, gramisterol, lophenol, 243-
reported in roots. methyllophenol, obtusifoliol, sitosterol, stigmastanol,
stigmasterol, stigmasterol-7-ene, beta-amyrin,
Pest butyrospermol, cycloartenol, 24-methylene-
cycloartanol, taraxerol, tirucallol, 3-O-[ -D-
1. Caterpillar – Makes holes in the bulbs and cut xylopyranosyl(1 3)- -L-rhamnopyranosyl(1 2)- -
down seedlings. L-arabinopyranosyl]-28-O-[ -L-rhamnopyranosyl
Control: Dust the soil at the sowing or hoeing (1 4)- -D-glucopyranosyl (1 6)- -D-
with 5% Aldrine, 10% BHC at the rate of 25 kg glucopyranosyl] hederagenin, volatile oil (0.5-1.6%),
per hectare; application of well-decomposed fatty oil (35.6-41.6%), oleic acid, esters of
farmyard manure10. unsaturated fatty acids with C15 and higher
2. Armyworm and semi-looper – Feed on the terpenoids, esters of dehydrostearic and linoleic acid,
flowers, seeds, and damage the crop. aliphatic alcohol31,35,36, nigellidine37, carvone, d-
Control: Spray with 0.05% methyl parathion – 1 limonene, cymene, , -unsaturated hydroxy ketone,
ml/l water or Thiodian or Endosal 35EC, 1 ml/l steroids, hederagenin glycoside, melanthin,
of water at 15 days interval10. melanthigenin, bitter principle, tannin, resin, protein,
reducing sugar, glycosidal saponin, 3-O-[ -D-
Microscopical and powdered characteristics xylopyranosyl-(1 2)- -L-rhamnopyranosyl-(1 2)-
-D-glucopyranosyl]-11-methoxy-16,23-dihydroxy-
Transverse section of seed show single layered 28-methylolean-12-enoate, stigma-5,22-dien-3- -D-
epidermis, thick walled cells, covered externally by a glucopyranoside, cycloart-23-methyl-7,20, 22-triene-
papillose cuticle and dark brown contents; 2-4 ,25-diol, nigellidine-4-O-sulfite38, nigellamines
layered, thick, tangentially elongated A3, A4, A5, C39, nigellamines A1, A2, B1, and B240.
parenchymatous cells followed by reddish brown Seed Oil: The seed oil contains cholesterol,
thick walled rectangular cells; endosperm thin campesterol, stigmasterol, -sitosterol, -spinasterol,
walled, cells rectangular to polygonal filled with oil (+)-citronellol, (+)-limonene, p-cymene, citronellyl
acetate, carvone41, nigellone, arachidic, linolenic,
JOURNAL OF PLANT DEVELOPMENT SCIENCES Vol.4 (1) 53

linoleic, myristic, oleic, palmitic, palmitoleic and process that involved apoptosis and cell cycle arrest
stearic acids. Fixed oil: linoleic acid (55.6%), oleic with little effect in non-cancerous cells –
acid (23.4%) and palmitic acid (12.5%). Volatile oil: Buyukozturk et al.63), anti-hypertensive64, antiviral
trans-anethole (38.3%), p-cymene (14.8%), limonene (Infections Laryngotrachietis virus – Zaher et al.65),
(4.3%), and carvone (4.0%)42, 2-(2-methoxypropyl)- anti-asthmatic (crude seed extracts exhibits
5-methyl-1, 4-benzenediol, thymol and carvacrol43. spasmolytic and bronchodilator activities mediated
Root and shoot are reported to contain vanillic acid44. possibly through calcium channel blockade – Kalus
et al.66), anti-allergic (oil is an important adjuvant for
Extraction methods of essential oil the treatment of allergic disease – Dahri et al.67),
anti-diabetic, antilipidemic, antiobesity9,
43,68 69
1. Conventional method – extraction by hexane in anticonvulsant , antitoxic properties apart from
Soxhlet45. having immunomodulatory (extract inhibit human
2. Enzymatic extraction46. neutrophil elastase activity which is mainly attributed
3. Ultrasound assisted extraction47. to carvacrol – Mansi70), hematological (oil play role
4. Microwaves assisted extraction48. in modulating the balance of fibrinolysis/thrombus
5. Supercritical solvent extraction49. formation by modulating the fibrinolytic potential of
6. Surfactant assisted method; based on the use of endothelial cells – Gilani et al.71, Zaoui et al.72),
aqueous solution polyethylene glycol sorbitan gastro-protective (thymoquinone protect gastric
monolaurate (Tween 20)50 amongst other mucosa against injurious effect of absolute alcohol
methods. and promote ulcer healing – Naz9),
73,74,75 76
Oil extracted were analyzed and characterized by nephroprotective , diuretic , cardiovascular
using classical analytical procedures, spectroscopic (active ingredient thymol has shown to lower blood
and chromatographic methods. pressure through blockade of calcium channels -
Gilani et al.71, Paarakh8) properties as well as the
Therapeutic uses species is protective against heavy metal77,78, effects
nitric acid production79, possesses analgesic activity
Traditional Uses: In traditional system of medicine (volatile oil – Ramadhan et al.80) amongst others.
black cumin seeds are effective against cough, Moreover, essential oil was found to be effective
bronchitis, asthma, chronic headache, migraine, against Cr(VI) hazard and may be a promising
dizziness, chest congestion, dysmenorrheal, obesity, candidate against different environmental
diabetes, paralysis, hemiplegia, back pain, infection, pollutants81,82 reported that the species is a good
inflammation, rheumatism, hypertension, and absorbent for the removal of cationic metals coming
gastrointestinal problems such as dyspepsia, from wastewater. Tasawar et al.82 reported that black
flatulence, dysentery, and diarrhea51. It has also been cumin (tested on 80 subjects, divided randomly into
used as a stimulant, diuretic, emmenagogue, 2 groups) is effective to change the lipid profile
lactagogue, anthelmintic and carminative52 as well as significantly in a way which is beneficial to heart.
it is applied to abscesses, nasal ulcers, orchitis, Black seed has also been used externally where it is
eczema and swollen joints51. Seed oil is considered to applied directly to abscesses, nasal ulcers, orchitis,
be local anesthetic53,54. eczema and swollen joints51. N. sativa is also a
Pharmacological Significance: The species potential source for antidermaphytic drugs. The ether
possesses antimicrobial (diethyl ether extract and extract of seeds and its active principle
methanol and chlorophyll extract and plant extract as thymoquinone are found to be effective after clinical
well as seed oil were found to inhibit Staphylococcus trials against many species of three important genera
aureus, Pseudomonas aeruginosa, Escherichia coli of dermatophytes: Trichophyton, Epidemophyton and
and a pathogenic yeast Candida albicans – Hanafy Microsporum83,84. The volatile oil inhibited the
and Hatem55, Hosseinzadeh et al.56, Chaieb et al.57, spontaneous movements of rat and guinea pig uterine
Khalid et al.58), anti-malarial59, antioxidant smooth muscle and also the contraction induced
(thymoquinine constituent of seed oil, enhance the oxytocin suggesting its anti-oxytocic potential69. Hot
oxidant scavenging system – Salem60), anti- water extract of NS as well as whole seeds in large
inflammatory (the oil and thymoquinone – Salem60; oral doses causes abortion in human pregnant
thymoquinone has the ability to attenuate allergic females85. The species is also used in long term
airway inflammation by inhibiting Th2 cytokines and treatment of opioid defense86. Thymoquinone has
eosinophil infiltration into the airways and been reported to exhibit effect on dopaminergic
exploratory effects – Isik et al.61), anticancerous neurons against Parkinson’s disease87.
(methanolic extract of plant exhibits potent inhibition
of cancerous cell growth against HL-60 and U-937 Insecticidal activity
cell lines with IC50 value 13.50 µg/ml and 28.31
µg/ml respectively – Raval et al.62), antitumerogenic Essential oil from dried fruits was isolated by
(active components – thymoquinone and hydrodistillation and tested for its repellent, toxic and
dithymoquinone; tymoquinone kill cancer cell by a developmental inhibitory activities against wheat
ANIMESH K. DATTA, ADITI SAHA, ARNAB BHATTACHARYA, ANINDA MANDAL, RITA PAUL AND SONALI
SENGUPTA.

flour pest Tribolium castaneum88. Results indicated peroxidation levels in hyperlipidemic rabbits, thereby
that the essential oil reduced the oviposition potential indicating that seeds may be considered as a useful
and increased the developmental period of T. therapy for hyperlipidemia.
castaneum in comparison to control group. Abbas et al.93 reported that N. sativa oil possesses
Fumigation of essential oil inhibited development of anti-inflammatory and bronchodilator activities.
larvae to pupae and the pupae to adults and also Clinical trial with mouse model suggested that N.
resulted in the deformities in the different sativa significantly reduced blood eosinophil count;
developmental stages of the insects. All the IgG1 and IgG2a levels, cytokine profiles and
responses were found concentration-dependent. inflammatory cells in lung tissue. These effects were
comparable to the effects of dexamethasone except
Other uses unchanged IFN-y level.
Abou-Gabal et al.94 studied the effect of the oral
N. sativa seed cakes in the feed of buffalo and lambs administration of aqueous suspension of N. sativa
improved their body weight and reproductivity as (50 mg/kg.b.wt) against chromosomal aberrations
well as seeds in the food of broiler chicks improved and ultrastructural changes of the bone marrow cells
their immunity and feed conversion efficacy89,90. in mice treated with carbon tetrachloride CCl4 (two
dose level: 1.9 ml/kg.b.wt and 3.8 ml/kg.b.wt).
Clinical trials Mitotic activity decreased in bone marrow cells of
animals treated with CCl4 as well as significant
Significance of the species has been documented increase in the number of bone marrow cells with
from some clinical trial experiments. Al-Ghamdi91 different types of chromosomal aberrations was
administered aqueous suspension of the seeds orally recorded. Ultrastructural changes were also dose-
at two dose levels (250 mg/kg and 500 mg/kg) for dependent including both nucleus and cytoplasm of
five days to assess carbon tetrachloride (CCl4)- erythroid and myeloid elements of the bone marrow
induced liver damage. CCl4 (250 microl/kg cells. Treatment of the animals with N. sativa
intraperitonelly/day in olive oil) was given to the improved both genotoxicity and ultrastructural
experimental group on days 4 and 5, while the changes induced by CCl4.
control group was only treated with the vehicles. Al-Kubaisy and Al-Noaemi95 reported protective role
Animals treated with CCl4 showed remarkable of seed oil against effect of CCl4 on the liver cells.
centrilobular fatty changes and moderate Samir Bashandy96 reported that administration of NS
inflammatory infiltrate in the form of neutrophil and oil to hyperlipidemic rats improved their
mononuclear cells when compared to the controls. reproductive efficiency (increase in seminal vesicle
This effect was significantly decreased in animals weight, testosterone level, sperm motility and sperm
pretreated with N. sativa. Histopathological or count and a decrease in sperm abnormalities) and
biochemical changes were not evident following produced additional protection against
administration of N. sativa alone. Serum levels of hyperlipidemia induced reduction in fertility.
aspartic transaminase (AST), L-alanine Najmi et al.97 performed clinical study (2 groups of
aminotransferase (ALT) were slightly decreased 30 patients each) to evaluate the adjuvant effect of
while lactate dehydrogenase (LDH) was significantly seed oil on various clinical and biochemical
increased in animals treated with CCl4 when parameters of the metabolic syndrome. Group I
compared to control group. LDH was restored to (standard group) patients were given Atorvastatin 10
normal but ALT and AST levels were increased in mg once a day and tablet Metformin 500 mg twice a
animals pretreated with N. sativa. In conclusion, it day along with N. sativa seed oil 2.5 ml twice a day
appeared that seeds are possible safe and protective for six weeks. Results indicated that Group III
against CCl4-induced hepatoxicity. patients showed significant improvement with
Ali and Blunden92 examined the hypolipidemic and reference to total cholesterol, low density lipoprotein
antioxidant effects of dietary black seed in and fasting blood glucose, thereby indicating that
hyperlipidemic rabbits (24 male rabbits were fed seed oil is effective as an add-on therapy in patients
with 0.5% cholesterol diet for 1 month, randomly with metabolic syndrome and also possessing
assigned to two groups – control group received the therapeutic activity in diabetic and dyslipidemic
hypercholesterolemic diet and the black seed group patients.
was fed 7.5 g/kg b.w/day crushed black seed + 0.5% Al-Sa'aidi et al.98 determine the effect of alcoholic
cholesterol diet, each for 2 months). Fasting blood extract of black seed N. sativa on fertility parameters
samples were obtained at baseline, after in white rat. A total of 60 mature males were divided
hyperlipidemia, 1 month and 2 months of treatment into 3 groups – the first group (control) intake
to determine serum lipid profile, malondialdehyde drinking water, while the other two groups (T1 and
(MDA) level, total antioxidant status (TAS), T2) intake the extract in two doses (0.5 and 1.5 g/kg
superoxide dismutase (SOD) and glutathione respectively) daily for 53 days. The results revealed
peroxidase (GPX). Results indicated that black seed that treatment with alcoholic extract of N. sativa led
can favorably decrease serum lipid profile and lipid to significant increase (P<0.01) in body weight gain
JOURNAL OF PLANT DEVELOPMENT SCIENCES Vol.4 (1) 37

(g), reproductive parameters (seminiferous tubules Attia et al.104 performed experiment on male rats and
thickness and diameters, account of spermatogonia, were of opinion that omega-3 polysaturated fatty acid
primary and secondary spermatocytes, spermatids, 3) and seed oil of N. sativa might prevent oxidative
free spermatozoa, account of sertoli and Leydig cells, stress and attenuate the changes in the biochemical
diameter of Leydig cells and the height of epithelial parameters (levels of urea, creatine, total bilirubin
cells entirely covered epididymal caudal), hormones and uric acid contents and aminotransferase,
(testosterone and follicle stimulating hormone) as phosphatases, and lactic dehydrogenase) induced by
well as protein concentration and significant decrease Lindane (r-HCH-r-hexachlorocyclohexane).
(P<0.01) in leutinizing hormone and cholesterol El-Gohary et al.105 studied the effect of carboplatin (a
concentration. synthetic antineoplastic agent used for cancer
Mohammad et al.99 from clinical trial experiments treatment) and N. sativa oil alone or in combination
with male albino rats suggested that the aqueous on human breast cancer cell (MCF-7) in vitro and
extracts of N. sativa have increased spermatogenesis Ehrlich as cites tumor bearing female mice (in vivo).
activity in seminiferous tubule. The in vitro experiment on MCF-7 cells illustrated
Al-Attar and Al-Taisan100 reported the preventive that IC50 of carboplatin was 11.8 µg/ml, IC50 of N.
effects of black cumin seeds (seed extract – 300 sativa oil was 39 µg/ml and IC50 of the combination
mg/kg/day) on Spragu Dawley Rats (clinical trial between carboplatin and black cumin oil was 3.78
performed with 50 male rats, divided into four and 40 µg/ml respectively. The in vivo experiment
groups) exposed to Diazinon. Results indicated that illustrated that carboplatin (10 mg/kg) increased the
seeds can be considered therapeutic agent against enzyme activity of aspartate amino transferase
hematotoxicity, immunotoxicity, hepatoxicity, (GOT) and aniline amino transferase (GPT) by
nephrotoxicity and cardiotoxicity induced by 56.52% and 51.14% respectively as compared to
diazinon and may be against other chemical both healthy control (non-tumor transplanted mice)
pollutants, environmental contaminants and and negative control. The activity of GOT and GPT
pathogenic factors. was increased by 14.75% and 19.84% respectively as
El-Naggar101 investigated the cytotoxicity of N. compared to healthy control under the effect of N.
sativa dry methanolic extract on cultured cortical sativa oil (12 ml/kg); while, the enzyme activities
neurons and its influence on neurotransmitter release, decreased in comparison to negative control. The
as well as the presence of excitatory (glutamate and combination of carboplatin and oil appeared to
aspartate) and inhibitory amino acids (gamma- increase the enzyme activity of GOT and GPT by
aminobutyric acid-GABA- and glycine). The 62.41% and 49.39% respectively compared to both
secretion of different amino acids was studied in healthy control and negative control. Agarose gel
primary cultured cortical neurons by HPLC using a electrophoresis revealed that carboplatin induced
derivation before injection with dansyl chloride. NS DNA damage of liver tissue but N. sativa oil showed
modulated amino acid release in cultured neurons; intact DNA without any damage.
GABA was significantly increased whereas secretion Parhizkar et al.106 studied the estrogenic activity of
of glutamate, aspartate, and glycine were decreased. N. sativa by vaginal cornification assay using an
Mohamed et al.102 investigated protective role of N. ovariectomized rat model (40 ovariectomized
sativa in DAB (dimethylaminoazobenzene) induced Sprague Dawley rats, weighing 250 to 350 g were
liver carcinogenesis. The study included 140 Albino used; NS powder given at 300, 600 and 1200 mg/kg
mice weighing 40-50 gm divided into 4 groups: for 21 consecutive days; compared with 0.2 mg/kg
Group I - normal control; Group II - N. sativa treated conjugated Equine estrogen as positive control). Data
control; Group III – treated with DAB; Group IV – obtained from vaginal smear suggested that NS
treated with N. sativa and DAB. Biochemical possesses estrogenic function which can be helpful in
investigations, flow cytometric analysis and managing menopausal symptoms as an alternative
histopathological examination of the liver tissue were for Hormone Replacement Therapy.
performed and the results showed significant change Rayan et al.107 studied the effect of black cumin oil
in the DNA content, histomorphology, and (BSO) against Toxoplasma gondii Me 49 strain in a
antioxidant enzymes in liver tissues of the DAB murine model of infection. After clinical diagnosis
treated group. These changes were restored to normal with mice (35 mice were studied in 3 groups) and
with N. sativa treatment. Further, it was noted that assessment of survival rate and brain cyst burden,
treatment with N. sativa only showed comparable brain histopathological lesions and
result with control untreated group. Thus, it was immunohistochemical expression of inducible nitric
inferred that N. sativa lonely induce no harmful oxide synthase (iNOS) it was noted that BSO in
effect on the liver rather it exerts hepatoprotective prophylactic or therapeutic regimens significantly
effect against liver carcinogens. enhanced protection of infected mice against death
Al-Naqeep et al.103 reported (experiment conducted (P=0.01) and reduced brain cyst burdens at 5, 7 and
on HC rabbit) that N. sativa seeds powder or oil 12 weeks post infection compared to the infected
showed hypocholesterolemic and antiatherogenic untreated control.
cardioprotective properties.
ANIMESH K. DATTA, ADITI SAHA, ARNAB BHATTACHARYA, ANINDA MANDAL, RITA PAUL AND SONALI
SENGUPTA.

Antitumor- Ait et al.108 suggested that essential oil lipid peroxidation products and antioxidant enzymes
(IC50=0.6% v/v) and ethyl acetate (IC50=0.75%) like catalase, superoxide dismutase, reduced
extracts were more cytotoxic against P8-15 cell line glutathione and glutathione peroxidase in liver and
than butanol extract (IC50=2%). The authors further kidney. Meral et al.118 suggested that NS might be
suggested that TQ induced apoptosis and inhibited used in diabetic patients to prevent lipid
proliferation in pancreatic ductal adenocarcinoma peroxidation, increase in anti-oxidant defense system
cells. TQ also increased P21WAF1 expression, activity and also to prevent liver damage. al-Awadi et
inhibited histone deacetylase activity and induced al.119 reported the significance of NS seeds for its use
histone hyperacetylation. TQ is reported that it acts in non-insulin dependent diabetic mellitus. An
as a novel inhibitor of pro-inflammatory pathways aqueous decoction of a plant mixture containing NS
which combines anti-inflammatory and proapoptotic was found to lower blood glucose level after oral
modes of action. Banerjee et al.109 performed in vitro administration120. Al-Hader et al.121 suggested that
studies on pancreatic cancer cells preexposed with intraperitoneal administration of volatile oil of seeds
thymoquinone (25 µmol/l) for 48 h followed by produced a significant hypoglycemic effect in normal
gemcitabine or oxaliplatin resulted in 60 to 80% and alloxan induced diabetic rabbit.
growth inhibition compared with 15 to 25% when Oral supplement of N. sativa seeds to normal rats
gemcitabine or thymoquinone was used alone which was investigated and the results showed intrinsic
suggested that the mechanism of thymoquinone cardiac properties without evidence of an increased
could potentiate the killing of pancreatic cancer cells cardiac work load or energy consumption in vivo
by down regulation of nuclear factor kappa B (NF- which makes the seeds an isotropic agent with
kappa B), Bcl-2 family, and NF-kappa B-dependent hemodynamic profile77,122,123. Shafei et al.124
antiapoptotic genes. Breyer et al.110 tested 4- examined the effects of aqueous and macerated
acylhydrazones and 6-alkyl derivatives of extracts from N. sativa on heart rate and contractility
thymoquinone for growth inhibition of human HL- of the isolated heart. Results showed a potent
60, leukemia, 518A2 melanoma, KB-VI/Vbl cervix inhibitory effect of both extracts on both heart rate
and MCF-7/Topo breast carcinoma cells. The 6- and contractility of guinea pig heart that was
hencosahexaenyl conjugate was most active in all comparable and even higher than that of diltazem
resistant tumor cells, with IC50 (72 h) values as low which may be due to calcium channel inhibitory or
as 30 Nm in MCF-7/Topo cells. Nagi and Almakki111 an opening effect for the plant on potassium channels
investigated the effect of thymoquinone (TQ) in vivo of the isolated heart. Dichloromethane extract of
and in vitro male albino rats on fibrosarcoma induced seeds (0.6 ml/kg/day), essential oil and
by 20-methylcholanthrene. It was found to inhibit unsaponifiable matter of oil, volatile oil and
tumor incidence and tumor burden significantly. thymoquinone found to be cardioprotective125,126,76,67.
Shafi et al.112 reported methanol (IC50-2.28 µg/ml), Gilani et al.127 reported that thymol has shown lower
n-hexane (IC50-2.20 µg/ml) and chloroform (IC50- blood pressure through blockade of calcium
0.41 µg/ml) extracts of the seeds effectively killed channels. The effect of oral treatment of Wister
HeLa cells by inducing apoptosis. albino rats with different doses of powdered seeds
Diabetic and Cardiovascular Activities- Meddah et (100, 200, 400 and 600 mg/kg/day) for four weeks on
al.113 observed improvement of glucose tolerance and the levels of serum lipid was investigated, and it was
body weight in rats after chronic oral administration found that it causes significant decrease in low
in vivo, which validate the traditional use of black density lipoprotein-cholesterol levels, triglyceride
cumin seeds against diabetes. Chandra et al.114 levels and increase in high density lipoprotein-
reported that HIV protease inhibitors, nelfinavir (5- cholesterol level128.
10 µM), saquinavir (5-10 µM) and atazanavir (5-20 Pulmonary Activity- Nigellone was found to inhibit
µM) with N. sativa seed extract decreases glucose effectively the histamine release from the mast cells
stimulated insulin secretion from rat pancreatic beta- suggesting its use in asthma129. Padmalatha et al.130
cells. Altan et al.115 were of opinion that combined studied the antinaphylactic effect of a polyherbal
treatment with NS and hPTH alone in improving formulation containing NS on mesenteric mast cells.
bone mass, connectivity, biomechanical behavior and The antinaphylactic activity was possibly due to the
strength in insulin-dependent diabetic rats. NS membrane stabilizing potential, suppression of
treatment alone or in combinations significantly antibody production and inhibition of antigen
increased the area of insulin immunoreactive beta- induced histamine release. Gilani et al.127 suggested
cells in diabetic rats suggesting that NS might be that bronchodilatory effect of NS seeds was mediated
useful in the treatment of diabetic osteopenia. Kanter possibly through calcium channel blockade.
et al.116 and Kaleem et al.117 suggested that oral Keyhanmanesh et al.131 studied the prophylactic
administration of ethanol extract of black cumin effect of TQ on tracheal responsiveness and WBC
seeds (300 mg/kg body weight/day) to streptozotocin (white blood cell) count in lung lavage of sensitized
induced diabetic rats for 30 days significantly guinea pigs. The results suggested the preventive
reduced the elevated levels of blood glucose, lipids, effect of TQ on tracheal responsiveness and
plasma insulin and improvement altered levels of inflammatory cells of lung lavage of sensitized
JOURNAL OF PLANT DEVELOPMENT SCIENCES Vol.4 (1) 93

guinea pigs. Suddek132 was of opinion that TQ- tolerability of powdered N. sativa seeds in capsules
induced relaxation of the precontracted pulmonary on serum lipid levels, blood sugar, blood pressure,
artery is probably by the activation of ATP-sensitive and body weight in adults (123 patients were
potassium channels and possibly by non-competitive recruited; 64 and 59 patients were randomized to the
blocking of serotonin, alpha-I and endothelin intervention and the control arms respectively; 39
receptors. patients in the intervention group and 34 in the
Immunomodulation- Islam et al.133 studied the control group completed the study). Favourable
effect of volatile oil of N. sativa seeds (NSVO) for its impact of powdered N. sativa seed in capsule was
immunomodulating and cytotoxic properties in rats noted on almost all variables; however, larger study
and it was found that there was a significant decrease with adequate sample size was recommended.
in splenocyte and neutrophil counts, but a rise in
peripheral lymphocytes and monocytes in rats. LC50 Biosafety
values for NSVO were 155.02±10.4, 185.77±2.9,
120.40±20.5, 384.53±12.1 and 286.83±23.3 micro 1. Seed powder did not produce any toxic effects at
g/ml respectively against the SCL, SCL-6, SCL-376, very high doses (28 gm/kg orally) in rabbits140.
NUGC-4 cancer lines and 3T6 fibroblast line. 2. Seed oil safe when given orally to rats (LD50 of
Results indicate NSVO as a potential 28.8 ml/kg)72.
immunosuppressive cytotoxic agent. Swamy and 3. Oral thymoquinone was found safe (LD50 of 2.4
Tan134 performed in vitro cytotoxicity of seed g/kg)141.
extracts (in ethyl acetate fraction) in different cancer 4. Oral thymoquinone (LD50 of around 1000mg/kg)
cell lines P388, Molt 4, Wehi 164, LL/2, HePG2, SW and intraperitoneal (LD50 of around 100 mg/kg)
620 and J82 as measured by 3-(4,5-dimethylthiazol- in mice/rat, safest142.
2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay
and the ethyl-acetate column chromatographic Cytological and cytogenetical studies
fraction (CC-5) showed selectivity against HePG2,
Molt 4 and LL/2. CC-5 was relatively non-toxic Karyomorphology: Gregory143 was pioneer to
against human umbilical cord endothelial cells at 50 enumerate the number of chromosomes (2n=12) in
µg/ml. Results therefore indicated that CC-5 somatic complement of N. sativa. Bhattacharyaya144
possesses a potent cytotoxic effect as well as a revealed five pairs of very long (L1) to long (L)
potentiating effect on the cellular immune response. chromosomes with median to sub median primary
Contraceptive Activity- Hexane extract of the seeds constrictions and a single pair of medium-sized (M)
prevented pregnancy in Sprague-Dawley rats treated chromosomes with sub-terminal primary
orally at 2 g/kg daily dose on day’s 1-10 post – constrictions in the species. Secondary constrictions
coitum. The active hexane extract exhibited only were located in two of the long pairs of
mild euterotrophic activity comparable to ethinyl- chromosomes and karyotype formula was suggested
estradiol, but was devoid of any estrogenicity in the as 2n=12=2L1+4LS+4L+2M.
immature rat bio-assay135. Agarwal et al.136 reported Saha and Datta145 reported four morphologically
that ethanolic extract of seeds possesses antifertility distinct chromosome types (A, B, C, D) in N. Sativa
effect in male rats which is probably due to inherent (2n=12) on the basis of chromosome length (very
esterogenic activity. long 15.0 to 20 µm; long 10.0 to 14.9 µm; median
Nephroprotective Activity- Ali137 investigated the 5.0 to 9.9 µm), nature of primary constriction and
effect of oil (oral treatment: 0.5, 1.0 or 2.0 ml/kg/day presence or absence of secondary constriction (Figs.
for 10 days) on gentamycin induced nephrotoxicity 11-12). The somatic complement possessed one pair
in rats. A dose-dependant amelioration of the AA (very long, 19.13 µm; F% 44.01), one pair BB
biochemical and histological indices of GM (very long, 16.70 µm; both primary- F% 44.88 and
nephrotoxicity that was statistically significant at the secondary constriction were present), three pairs CC
two higher doses. Treatments enhanced antioxidant (C1C1- very long, 15.31 µm; C2C2- long, 14.86 µm
status in plasma and also reduced glutathione and C3C3- long, 13.80 µm; F% 44.04 to 45.42) and
concentrations in renal cortex and enhanced growth. one pair DD (medium 6.64 µm, F% 7.23)
Badary et al.138 studied the effect of TQ on the chromosomes (TF% 41.38, haploid chromatin length
nephropathy and oxidative stress induced by 86.50 µm ± 3.3). The somatic chromosome types
doxorubicin (DOX) in rats (10 mg/kg/day – could easily be marked in meiotic plates (Figs. 13-
supplemented with drinking water for 5 days before 14).
DOX and daily thereafter) and found that TG, TC Ghosh and Datta146 karyotyped N. sativa through
and serum urea lowered significantly. TQ has been Image Analyzing System (Micro Image TM Lite
suggested to be protective agent for protienuria and Software, Version 4.0 for windows, 47N40155 2000
hyperlipidemia associated with nephritic syndrome. 0515 MAN VG MIX) and revealed four
Effectiveness- Qidwai et al.139 performed clinical (2n=12=4A+4B+2C+2D; karyotype formula:
trial experiment (study design was randomized, 2Lsc1sm+2L1m+2Lsm+2St) morphologically distinct
double-blind trial) to assess effectiveness, safety, and chromosome types (L1= very long 15.0 µm, L= long
ANIMESH K. DATTA, ADITI SAHA, ARNAB BHATTACHARYA, ANINDA MANDAL, RITA PAUL AND SONALI
SENGUPTA.

13.0 to <15.0 µm, M= medium 7.0 to <13.0 µm, S= between 1.67 µm and 8.30 µm) nucleoli (Figs. 20-
short <7.0 µm; m= metacentric, sm= sub- 25) in different mutant (1-2: Lax branching and
metacentric, t= telocentric and sc= satellites). The viridis; 1-5: bushy, chloroxantha, crinkle leaf,
somatic chromosome complements in the species feathery leaf, narrow leaf) lines of N. sativa.
formed graded karyotype which was symmetric in Nucleoli was either free or found in association to
nature (TF% 42.90). Total haploid chromatin length different bivalents but occasionally two nucleoli of
was noted to be 78.62 µm ± 2.87. different or same sizes were seen attached to a single
Meiotic Analysis and Pollen Fertility: Saha and bivalent. Multiple and variable sized nucleoli
Datta145 reported regular 6 bivalents formation at formation were presumed as an outcome of disturbed
diplotene and metaphase I (MI) in most PMCs (Figs. genetic state of the plant types caused by gene
15-17); while, the rest demonstrated 5II+2I mutation and the mutant genes possibly have induced
formations (175 meiocytes assessed). Frequency of changes in the regulatory system of the cell thereby
bivalent and univalent per cell varied from 5.88 to activating various latent loci capable of synthesizing
6.0 and 0.00 to 0.23 respectively. Frequency of tiny nucleoli. Hiko-Lchi and Chen-Hui Kao150
bivalent and univalent per cell was 5.95 and 0.10 attributed size variation of nucleolus on the basis of
respectively. The bivalents formed rings (range- difference in the intensity of nucleolar forming
2.85±0.51 to 3.58±0.24/cell) and rods (range- power.
2.41±0.24 to 3.15±0.51/cell). Average frequency of Mitotic and Meiotic Abnormalities Arising out of
ring and rod per cell over the plant was 3.18 and 2.77 Irradiations: Kumar and Nizam151 assessed the
respectively. Chiasma per nucleus range between effect of X-rays on dry and pre-soaked seeds of N.
8.87±0.21 and 9.62±0.32 (average: 9.34±0.28). sativa and noted that the frequency of mitotic and
Frequency of bivalents, ring and rod configurations meiotic aberrations in the pre-soaked seeds was
per cell and chiasma per nucleus showed random higher than that of the dry seeds. The aberrations
distribution over the plants (p>0.05) but univalents encountered were mostly related to spindle
per cell was non-random (p<0.01) as evidenced from organization and formation of dicentrics, rings,
2
test of heterogeneity. Mostly (99.49%- pooled micronuclei and acentric fragments. Mandal and
over the plants) anaphase I (AI) cells manifested Basu152 studied X-ray induced chromosomal
equal 6/6 separation (Fig. 18) of chromosomes, rare aberration from leaf meristems, pollen mother cells
often unequal separation (5/7), lagging chromosome and endosperm and reported that aberration
and bridges were also noted. Pollen fertility among percentage increased with an increase in doses and
black cumin plants varied from 95.2% to 100.0% decrease with time lapse from 2 to 24 hours after
(average: 98.06%). Saha and Datta145 were further of irradiations. Most resistant tissue was endosperm
opinion that the meiotic chromosomes could easily though it had the largest Interphase Chromosome
be identified and marked in meiotic plates. Volume (ICV).
Pachytene Chromosome Analysis: Datta147 Datta and Biswas153 (X-irradiations to dry seeds,
reported that the length of pachytene chromosomes doses- 6, 8, 10, 20, 30 kR, LD50- lie between 8 kR
(Fig. 19) in the species ranged from 51.86 µm to and 10 kR), Datta et al.154 (gamma irradiations- 5,
140.55 µm with mostly median primary constrictions 10, 20, 30, 40, 50 and 60 kR doses, seed moisture-
(F%: 41.60 to 47.56; arm ratio: 0.71 to 0.91). A 1.8%, LD50- lie between 20 kR and 30 kR, treatments
telocentric (F%: 12.36; arm ratio: 0.41) was also beyond 30 kR were lethal) and Mukherjee and
marked in the pachytene complement. Four Datta155 (gamma irradiations- 50, 100, 150 and 200
(chromosome type A- 140.55 µm; type B- 109.75 µm Grey, moisture content- 19.04%, LD50 lie between 50
and 97.89 µm; type C- 94.93 µm and 89.32 µm; type Gy and 100 Gy) reported physiological (germination
D- 51.86 µm) morphological types were suggested and seedling growth under petriplate conditions) and
with 2 bivalents (B type) documenting secondary chromosomal disturbances (mitotic and meiotic
constrictions. However, further studies on the including pollen fertility) in irradiated samples.
somatic complement has suggested that one pair of Frequency of total mitotic anomalies enhanced in
chromosome were with secondary constriction145,146. treatments but the percentage of dividing cells
Accessory Nucleoli: Rang and Datta148 revealed decreased with an increase in the radiation doses, and
consistent presence of single nucleolus (size 8.36 µm it was suggested that mitotic disturbances have
± 0.08) in PMCs (pollen mother cells) of N. sativa affected physiological processes like germination and
and it is in accordance to the number of chromosome seedling growth. Apart from normal chromosome
with secondary constriction in the configuration 2n=12 (Fig. 26), irradiations (X-
complement145,14,146; however, nucleolus is not irradiation as well as gamma irradiations) have
commensurable to the number of secondarily induced chromosomal aberrations like fragments,
constricted chromosomes and it has been proven that ring configuration of chromosome, pseudochiasma
those chromosomal regions which code for 18S and like configurations, diplochromosomes, cells with
24S RNA are nucleolar organizing in nature149. polyploid and aneuploid chromosome number and
Rang and Datta148 found 1 (48.39% to 65.57% deformed cellular configurations at metaphase (Figs.
PMCs; size: 8.30 µm ± 0.18) to 5 (size variation 27-31), and bridges (single, double, criss-cross, inter-
JOURNAL OF PLANT DEVELOPMENT SCIENCES Vol.4 (1) 11
3

locked and incomplete) with or without fragments (2 induced cytological aberrations viz., fragments,
to 4 identical sized and rare often with one fragment- laggards, micronuclei, grouping and stickiness of
Figs. 32-41), and multipolar organization of chromosomes and reduced mitotic index in
chromosomes at anaphase (Figs. 42-43). At resting prolonged treatments and in higher concentrations.
cells micronuclei (1-4 variable sizes; condensed as The authors were of opinion that the chemicals
well as uncondensed) and giant cells were also noted possibly affect nucleic acid synthesis in differential
(Figs. 44-47). Meiotic abnormalities studied manner which ultimately causes hazards in
following irradiations (apart from normal 6II replication thereby inducing chromosome breakage.
formation- Figs. 48-49) were univalents (2-8, Figs. Kumar and Nizam158 studied induced somatic pairing
50-52), fragments (paired identical sized- Fig. 53), of homologous chromosomes from root tip mitosis
multivalents (Figs. 54-57), stickiness (Fig. 58) and following treatment with mitomycin C. It was
cell fusion (Fig. 59) at metaphase I (MI); while, observed that the homologous chromosomes become
fragments, bridges with or without an accompanying juxtaposed to each other with remarkable regularity
fragment were observed in anaphase I and II cells in the prometaphase cells following treatment for 40
irrespective of normal segregation of chromosome at minutes, whereas the untreated cells showed no such
AI153,154,155 (Figs. 60-65). Mukherjee and Datta155 associations. It was presumed that these movements
noted enhanced frequency of quadrivalents (mostly may be due to kinetochore activity which normally
ring- 89.79%, rest were of chain configuration) was causes congregation of chromosomes towards the
noted in higher doses of treatments. Most of the ring equatorial plate of the spindle but which does not
quadrivalents were of adjacent orientation (63.64%); occur contemporaneously in all chromosomes. In
while, the rest were alternate (34.09%) and rare often view of the observation, the authors were inclined to
non co-oriented (2.27%). A PMC at 200 Gy was believe that kinetochores were responsible for
observed to posses 6II + two nearly identical sized placing homologues near each other and stickiness
(2.93 µm and 2.59 µm) fragments (1.21%) thereby has been attributed to be a factor for association of
suggesting localized breakage in chromosome due to homologous chromosomes.
irradiation. Paired identical sized fragments (5.38 Chand159 reported that pentachlorophenol (PCP)
µm) at AI was also studied in one of the two inhibited mitosis in shorter duration of treatments
telocentric chromosomes (one telocentric is marked and cytological abnormalities were formed.
intact at one pole). Pollen sterility and meiotic Incorporation studies revealed that PCP inhibited
anomalies studied have shown dose dependent DNA synthesis. The chemical was found to affect
increasing tendencies thereby indicating that former nuclear membrane cycle, chromosome division
is an outcome of the latter. cycle, spindle organization and chromosome
Rang and Datta156 exposed dry, pre-soaked (12 hours movement, condensation and spiralization of
in distilled water), totally dehydrated and stored (one chromosomes and DNA and protein synthesis.
year six months stored under desiccation; one season
stored seed) seed samples (moisture content: 7.5%) Induced mutagenesis
of N. sativa to gamma irradiations (5, 10 and 20 kR
doses) and also that some amount of the dry Variants in M1 Generation: Datta and Biswas160
irradiated materials were treated with ethyl methane reported that as compared to the erect nature of the
sulfonate (EMS) and hydrogen peroxide (H2O2) for stem in untreated control plants, stem anomalies
six hours at 0.25 percent to evaluate the cytogenetic including bifurcation (Fig. 66), trifurcation (Fig. 67-
changes that might occur due to gamma-irradiation 68), twisting (Fig. 69), unbranched (Fig. 70) and
influenced by the physical and chemical factors. twining nature of stem (Fig. 67) were observed at 4,
Assessment of radio-sensitivity has been made from 10 and 30 kR of X-ray doses and 2 and 4 hours
attributes like seed germination, rate of seedling treatment with 0.75% and 0.50% EMS respectively.
growth, mitotic index, frequency and spectrum of Interesting floral anomalies were found to occur in
chromosomal aberrations in root tip cells and pollen all treated doses of EMS and only 20 kR X-
and seed sterilities of M1 plants as well as M2 irradiation. In relation to control flower (Fig. 71)
mutation (macromutants) frequency. Results interesting floral variations like adnation of sepals,
indicated that the factors (physical and combined elongated and strap shaped petals, two gynoecium in
treatments) have influenced gamma radiation the same flower and presence of bract like structures
sensitivity in inducing cytogenetical and genetical (incompletely forked) similar to that of the petalloid
changes along with M2 mutation frequency. sepals were observed (Figs. 72-78). The
Mitotic Abnormalities Induced by Chemical abnormalities studied at M1 have not recurred in M2
Treatments: Biswas and Bhattacharyaya157 studied generation and these were non-inheritable changes
the effect of some mutagenic chemicals like mateic (chimeric in nature) possible arising out of somatic
hydrazide (MH), acridine orange (AO), ethyl mutation.
urethane and ethylene-diamine-tetracetic acid Macromutants and Their Inheritance Pattern:
(EDTA) at variable concentrations and durations on Kumar and Nizam161 induced (X-rays and gamma
the root tip mitosis of the species. The chemicals rays) few viable mutants such as multicolor capsular
ANIMESH K. DATTA, ADITI SAHA, ARNAB BHATTACHARYA, ANINDA MANDAL, RITA PAUL AND SONALI
SENGUPTA.

fruits and color fruit coat with ornamentations crumpled leaf mutants showed that the mutant traits
including mutation affecting branching pattern and were controlled by two pairs of recessive genes.
fertility at M2. Datta and Biswas160 induced (X-ray Datta and Rang166 spotted a viable chloroxantha
and EMS) several chlorophyll (albina > xantha > mutant in EMS treated population at M2. The
chlorotica > chloroxantha > albescens > albino- seedlings of chloroxantha (Fig. 86) were pale
terminals > xantha-terminals > lutea > viridis = greenish yellow in color (2012 – “Dictionary of
marginata = coeruleovirens) and morphological (13 Colour” by Maerz and Paul 1950) and the mutant
different types; 9 viable – lax branching - Fig. 80, could be easily marked at the very seedling stage.
feathery leaf, bushy, male sterile, crumpled leaf, The mutant plants showed delayed flowering (17 to
dwarf, early flowering, prostrate - Fig. 81, and 29 days from control plants) and maturity, which
brown seed coat; 4 non-viable types– cup - Fig. 85, indicates that the mutant being deficient in
needle leaf, crinckle leaf and cotyledonary leaf) chlorophyll content might have utilized their
mutants in relation to normal trait (Fig. 79). buffering capacity to maintain the photosynthetic
Threshold doses were effective and efficient and efficiency by increasing the number of branches
0.5% EMS, 2 hours treatment was the best among all (consequently pinnae of the leaves increased in the
the treated doses. Chlorophyll mutatuions occurred mutant) and duration of the crop to complete their
predominantly than other types and among them life cycle successfully. The inheritance of the mutant
viridis and chloroxantha were the viable types and trait was recessive and was under the control of two
were found to be controlled by two pairs of recessive gene loci. The mutant was compared with control at
genes; while, the mutant trait(s) of bushy, dwarf, M4 and results indicated that chloroxantha possessed
feathery leaf, lax branching and early flowering higher number of primary branches and capsules per
mutants were controlled by a single pair of recessive plant and had smaller seed (length) than normal;
gene. Datta and Biswas162 assessed different mutants although, other traits were more or less comparable
(lax branching, feathery leaf, bushy, early flowering, to normal plants. The authors presumed that the color
prostrate, dwarf, brown seed coat and viridis) for of chloroxantha may be exploited as genetic marker
different quantitative traits at M2, M3 and M4 for efficient breeding.
generations (ANOVA performed in mutant lines with Rang and Datta167 spotted five dark reddish brown
control at M4) and were of opinion that the mutants (color code - 3/2), one yellowish brown (5/4) and one
have exhibited superiority over the control plants in peach (512/1) color (colors were confirmed from
some of the characters only but not in all the Horticultural Color Chart 1968 and Munsell Soil
parameters. This observation was significant as it Color Chart 1975) seeded plants at M2 following
offered scope of improvement through hybridization different treatments of gamma irradiations and EMS.
and selection. Mutation frequency of dark reddish brown color
Mitra and Bhowmick163 induced ten different types (Fig. 89), yellowish brown color (Fig. 88) and
of chlorophyll mutation in two cultivars of N. sativa bicolor (peach color was associated with blackish
following treatments with gamma irradiation and tinge at the base and the apical region – therefore
EMS. Higher doses of gamma-rays and lower designated as ‘bicolor’ - Fig. 90) was estimated to be
concentration and duration of EMS were reported to 1.92, 0.055 and 0.54 percent respectively (7956
be most efficient. Mitra and Bhoumick164 studied the plants scored). Dark reddish brown and yellowish
mutagenic effects (gamma irradiation and EMS) of brown seed-coat color traits were monogenic
some biological parameters in M1 generation and recessive to black seeds (Figs. 87-90); while, the
suggested that gamma irradiations were more inheritance of bicolor trait of seeds was under the
effective than EMS and the cultivar KS-1 was more control of two pairs of recessive genes (mutant ×
sensitive to mutagens under the tested doses and normal – reciprocal crosses were performed, F1 –
concentrations. black and F2 segregation analyzed following 2 – test
Datta and Rang165 screened seven viable analysis). Crossing experiments suggested that black
morphological mutants (lax branching, feathery leaf, coloration of seeds is dominant over other seed
bushy I - Fig. 82, bushy II - Fig. 83, lax pinnae - Fig. colors and gene symbols assigned were B for black,
84, needle leaf and crumpled leaf) from 7956 treated bdr for dark reddish brown and By for yellowish
plants at M2 following mutagen treatments (gamma- brown colors and p for peach color of seeds, and the
rays, EMS and H2O2 and their combined treatments) dominant form (P) of this gene has no effect on B or
to dry seeds (moisture content: 7.5%). F2 segregation on any allelic forms of B (bdr/by) and the mutation
(control × mutant, F1 normal) revealed that lax involving both the dominant genes (B-P) results to
branching, feathery leaf, bushy I (associated traits: bicolor seeds. Following genotypes were proposed
synchronous flowering, compact habit, thick dark for the seed-coat colors – BBPP, bdr bdr PP, bybyPP
green pinnae of leaves), bushy II (thick dark green and bbpp for black, dark reddish brown, yellowish
pinnae of leaves) and lax pinnae (pinnae elongated) brown and bicolor seeds respectively. The true
mutant traits were controlled by a single pair of breeding mutant plants were evaluated at M4 in
recessive genes; while, selfed lines of needle leaf and comparison to control for several agronomic traits
and it was noted that dark reddish brown seed coat
JOURNAL OF PLANT DEVELOPMENT SCIENCES Vol.4 (1) 133

mutant was as productive as normal; while the Datta and Biswas172 screened four extremely dwarf
bicolor and the yellowish brown seed coat mutants plants at M3 having identical leaf phenotype as their
were sort sized and small seeded plants. progenitor from the selfed M2 feathery leaf mutant
Polygenic Mutation: Datta and Biswas168 analyzed (0.50%, 2 hour EMS treatment). Meiotic studies
variations for quantitative characteristics (plant revealed the characteristic presence of paired
height, number of primary and total branches per fragments in the parent (M2) and multivalents in the
plant, total capsules, capsule chamber/fruit, capsule dwarf mutant plants (M3 as well as M4). The dwarf
length and seed per capsule) from 10 randomly plants were designated as telescopic mutants as the
selected plants of each of the M2 (X-irradiated and leaves were found to be clustered around the stem
EMS treated) lines and computed mean and forming a crown-like appearance. Out of four
coefficient of variations and also determined student telescopic mutant (Fig. 91), one of which showed
t-test between control and treatment. The magnitude prevalence of ring quadrivalent. The cytogenetically
of variability released (as evidenced from C.V.) marked telescopic mutant was semisterile and the
through induction of mutation was both positive as possible origin of the mutant lines has been ascribed
well as in negative direction, thereby suggesting due to deficiency of genes as an outcome of
random nature of mutation. chromosomal deletion in the parent.
Biochemical Studies on Induced Mutants: Saha and Datta145 induced 5 translocation
Electrophoretic characterization and evaluation of heterozygotes (P-14 and P-26 from 5 kR and P-32, P-
seed protein in control and EMS induced mutant line 36 from 10 kR) following gamma irradiations (5, 10
of the species were performed from seed samples169 and 20 kR) to dry seeds (moisture content 7.5%). P-
and the qualitative as well as quantitative variations 14 (possessing long drooping floral shoot), P-32 (lax
in banding pattern among the plants were noted. The branching) and P-36 (semi-dwarf with thick and non-
authors were of opinion that electrophoretic shattering capsules) were viable translocations;
characterization of the mutant lines may be used as while, P-26 and P-37 yielded only abortive seeds at
an additional parameter to supplement cytogenetic R1 following selfing and on open or controlled
data in understanding genetic variations. Das et al.170 pollination. The translocation heterozygotes
extracted protease from germinating seeds of wild exhibited the formation of either a ring or a chain of
type and seven EMS induced mutant lines of N. 4 chromosomes in 38.7% to 77.7% meiocytes apart
sativa and the activity was assayed with casein as from 6II formation (Figs. 92-101). Predominance of
substrate in the pH range 3.5-8.0. Results indicated rings occurred in all translocation heterozygotes
that most protease types showed pH 3.6-7.0 and excepting P-26 where rings and chains were nearly
more than one protease enzyme in the plant types equal. P-14 and P-26 had more adjacent orientation
tested. Amylase activity and variation of amylase of quadrivalents than alternate; while, P-32, P-36 and
isozyme pattern were also studied and it was reported P-37 demonstrated random orientations. The
that gene(s) controlling enzyme production/activity quadrivalent behavior was found to be persistent in
have been affected differentially in different mutants. all generations (R1, R2 and R3) of P-14, P-32 and P-
36. The rings showed preponderance of adjacent
Cytogenetical consequences of induced orientation and the chains demonstrated frequent
mutagenesis alternate orientation. Though normal 6/6 separation
of chromosomes at AI was observed in 85.8, 83.3,
Translocation Heterozygosity: Datta and Biswas171 69.4, 82.3 and 86.4% cells of P-14, P-26, P-32, P-36
isolated a cytologically marked plant (phenotypically and P-37 respectively (rest showed unequal
indistinguishable) from the R1 population of gamma separation of chromosomes and bridge formation
irradiations, which showed ring or a chain with a lagging fragment - Figs. 102-103), pollen
quadrivalent in 49.38% meiocytes at MI (241 cells fertility was reduced in the heterozygotes (8.2 to
scored). Although normal 6II formation was noted 37.5%). F1’s raised from intercrossing of P-14, P-32
predominantly (50.72%) at MI, the most common and P-36 were meiotically assessed and the results
type of configuration studied in the marked plant was indicated that same 2 non-homologus chromosomes
4II+1IV (34.25%); while in the remaining meiocytes were involved in translocation and the 2 longest pairs
the quadrivalent appeared in association with 2 were suggested to be associated.
univalents. PMCs with ring of four chromosomes Desynapsis (Synaptic Mutants): Datta and
(41.08%) occurred more frequently than those with Biswas173 noted desynaptic behavior of
chain quadrivalent (8.3%). Among the meiocytes chromosomes in a bushy mutant (M2 generation,
showing interchanged configurations, 65.55% were 0.5% EMS, 2 hour treatment) and the mutant trait
alternate and 34.45% were with adjacent was reported to be controlled by a single pair of
orientations. Anaphase I separation was mostly recessive genes. The bushy mutant plant could
(82.0%) balanced (6/6) although pollen sterility was always be characterized by their delayed
high (55.8%) with extremely poor seed setting germination, flowering and maturity, high frequency
(12.2±5.7) per capsule as compared to normal (pollen of sterile pollen grain formation, poor seed setting
sterility – 2.2 to 3.6%; seed setting 65.6±4.2/capsule) and univalent formation in the meiocytes. Desynapsis
plants.
ANIMESH K. DATTA, ADITI SAHA, ARNAB BHATTACHARYA, ANINDA MANDAL, RITA PAUL AND SONALI
SENGUPTA.

studied in the mutant was partial or weak because of developmental disturbances might be responsible for
high frequency of bivalents per cell (4.70 to 5.24) arrestation of pollen formation.
than univalent per cell (1.53 to 2.59). Compared to Rang and Datta176 reported a male sterile plant which
controls (5.24 ± 0.41 chiasma/cell), frequency of exhibit broad elongated lax pinnae along with
chiasma has been found to be decreased in M2 bushy yellowish green pinnae in the shoot apex of the
mutant (4.57 ± 0.49 /cell). Univalents formed in the primary axis at the onset of floral bud initiation (Fig.
mutant line were found to be distributed randomly in 113), and non-dehiscent and pollenless anthers at
most of the cases, which were not affected by the anthesis. The male sterile plant showed desynaptic
number of bivalents per cell. Less frequently, behavior of chromosomes and the chromosomal
however, occurrence of univalent in close proximity association studied at diplotene, diakinesis and MI
to each other could be marked, which may be an (168 PMCs scored) were 6II (4.76%) - Fig. 117,
indication of their belonging to same pair and their 5II+2I (9.52%), 4II+4I (4.76%) and 12I (80.95%) -
very recent separation. Anaphase I separation was Fig. 118-119. Mean frequency of univalents and
irregular (34.44% to 44.23%) in the mutant line bivalents per cell was estimated to be 10.10 and 0.90
leading to the formation of laggards and unequal respectively, chiasma frequency per nucleus
separation of chromosomes. Lagging chromosomes observed in the male sterile plant was 0.31±1.2 as
at anaphase II and unequal size of microspores in tri- compared to 9.9±0.74 in normal plants. Unequal
and polysporous condition were also noted. separation of chromosomes was studied in AI
Saha and Datta174 reported two synaptic mutants (71.43%) and AII (42.62%) from 82 and 122 cells
(DS-1, 5 kR gamma irradiations; DS-2, 10 kR) at M2 respectively. The male sterile plant produced tetrads
(screened from 6582 M1 plant progenies) possessing mostly with unequal spory (Fig. 120) followed by
distinctive phenotypic marker trait (lax branching). near complete degeneration of microspores (Fig.
The synaptic mutants (medium strong type) 121) compared to normal oval shaped fertile pollen
demonstrated fuzzy appearance of chromosomes at grains in control plants (Fig. 122).
early prophase I (Figs. 104-105) along with univalent Datta and Saha177 categorized male sterile mutant
frequency ranging from 0 to 12 (enhanced frequency plants into five (I to V) types on the basis of sterility
- Figs. 106-112) per cell (control: 0.10, DS-1: 2.47, and morphology. The mutants were type I: mutant
DS-2: 3.50), reduced number of chiasma and bivalent dwarf, pollen grains 100.0% sterile and showed sign
per nucleus (control: 5.95 II/cell, chiasma 9.34 ± 0.3; of degeneration, pollen grains were round and small
DS-1: 4.77 II/cell, chiasma 7.40 ± 0.3; DS-2: 4.25 sized 27.2 µm × 24.8 µm (Figs. 128-129) as
II/cell, chiasma 5.59 ± 0.4), few meiocytes with compared to oval shaped pollen grains (Fig. 130),
unequal separation (5/7, 5-1-6 and 4/8) at AI 39.0 µm × 38.08 µm; type II: mutant yellowish green
(control: 0.5%, DS-1: 15.6%, DS-2: 22.5%), color, 100.0% sterile pollen grains, small roundish
cytologically balanced AII cells and high pollen with thick wall; type III: mutant with crumpled and
fertility (control: 98.06%; DS-1: 96.57%; DS-2: deformed pinnae of leaves, anther small sized 3.74
93.95%). mm ± 0.05, brownish, shrunken and indehiscent and
Male Sterility: Male sterile mutants with distinctive were completely pollenless at maturity. Meiotic
phenotypic marker traits (bushy- EMS treatment; analysis revealed no clear bivalent formation, rather
Datta and Biswas175; chlorophyll deficiency- 6 hours the chromatin agglutinated into unequal masses
0.25% EMS - Fig. 113; Rang and Datta176; dwarf- (Figs. 123-125). Agglutination of microspores was
chlorophyll deficiency - Fig. 116, crumpled pinnae - also evident which consequently degenerated (Figs.
Fig. 115, bushy - Fig. 114 and lax pinnae, gamma 126-127); type IV: bushy, normal cytological
irradiation- 5, 10 and 20 kR and EMS- 0.25, 0.50 and behavior, 100.0% sterile pollen grains; type V: the
1.00%, 3 hours; Datta and Saha177) were isolated mutant plants were with long elongated and dissected
from M2 mutagenized population. Concomitant pinnae of leaves and the pinnae were lax in nature.
association of phenotypic marker trait(s) with male The mutant plants demonstrated normal meiotic
sterility was unique as it not only give selective chromosomal behavior and formed tetrads but the
advantage but will also be of immense value in the pollen grains were completely sterile. The male
breeding behavior of the crop. sterile mutants showed monogenic recessive (IV to
Datta and Biswas175 isolated a male sterile mutant V) as well as digenic recessive (II) mode of
which was indistinguishable at earlier stages of inheritance pattern. Type I and III were both male
growth, but the mature plant could be recognized by and female sterile. The mutants arising out of gene
its characteristics dark green, thick and leather like mutation and the mutant genes have favoured the
pinnae of the leaves and synchronous flowering. continuation of meiosis and thereafter they have
Although the mutant demonstrated normal behavior acted on microspores and on pollen grains. The
of meiotic chromosome with 6 bivalents in MI cells mutants were non-structural nuclear type as per
and usual formation of tetrads, none of the pollen classification proposed by Gottschalk and Kaul178
grains could be scored in the mutant which is an and Johns et al.179.
indication of complete inhibition of pollen grain Trisomic: Datta and Biswas180 isolated a trisomic
development leading to male sterility. Post tetrad (detected after male meiotic studies) plant from the
JOURNAL OF PLANT DEVELOPMENT SCIENCES Vol.4 (1) 153

selfed progenies of M2 lax branching mutant at M3. 2.6%, 5II+1I – 7.9%, 4II+3I – 7.9%, 6II – 26.3%,
Morphologically the trisomic plant was weak with 1IV+ 4II+2I – 5.3%, 7II – 5.3%, 12 II – 36.8%, 78
slender stem and drooping lamina at the seedling PMCs could only be analyzed) with remarkably
stage. At maturity the plant attained a height of 19.7 higher pollen sterility (78.5%). Both stained and
cm. Flowering in the trisomic was delayed by 10-11 unstained pollen grains were considerably smaller
days as compared to normal plants. Only four flower sized (10.05 µm ± 0.2; normal – 39.8 µm ± 0.6) than
buds of the trisomic bloomed, while the rest dried up. control pollen grains (Figs. 144-145). Moreover,
Flowers were smaller in size and at maturity the functional instability of the stained (fertile) pollen
stamens turned brownish in contrast to yellowish has been evidenced by the formation of only
green color in the control and ultimately rudimentary rudimentary seeds in the marker plant. The aberrant
capsules with abortive seeds were formed. The has been ascribed as the outcome of cytomixis noted
trisomic showed 6II+1 (87.8%) and 5II+ 1III (12.2%) in lax branching M2 mutant.
chromosomal associations in 72 and 10 PMCs
respectively (Figs. 131-133). At AI, either the extra Induced polyploidy
chromosome appeared as laggard or has been
incorporated in any of the two poles (Fig. 134). The Biswas and Chatterjee182 induced tetraploid plants
aneuploid plant appeared to be a primary trisomic, following seed and seedling treatments with various
with 58.17% pollen sterility and it was completely concentrations of colchicine and the plants were with
seed sterile. increased number of branches, enhanced size and
Cytomixis: Datta and Biswas180 studied transfer of frequency of stomata, increase in the number of
nuclear materials from one PMC to another at flowers, variation in pollen size, fruit setting and the
prophase I and MI while performing male meiotic rate of germination of seeds, increase in the number
analysis in M2 mutants (observed in lax branching of septa per fruit and seeds per septum and delayed
mutant). Chromatin transfer between adjacent flowering. Biswas and Datta183 performed meiotic
meiocytes occurred through cytoplasmic links and analysis in colchicine induced (seedling treatments)
the migration was at random within a group of PMCs autotetraploid plants and found prevalence of
(Figs. 135-137). The phenomenon of cytomixis was chromosome irregularities producing varying number
restricted between/among few clusters of meiocytes of quadrivalents (0-4), trivalents (0-2) and univalent
of a single microsporophyll squash preparation. (0-10). The tetraploids were seed sterile.
Cytomixis resulted in hypo- and hyperploid variation Saha and Datta184 induced one autotetraploid (C0-1; 5
in chromosome numbers (19.87%) in meiocytes, hour treatment with 0.5% aqueous solution of
thereby producing aneuploid and polyploid PMCs. colchicine for 3 consecutive days) following
The nucleolus of the meiocytes, undergoing treatment with colchicine at the apical meristematic
chromatin transfer, in most cases remained in the tips of young seedlings bearing only two
donor cell; rarely it passed to the cytoplasm of the cotyledonary leaves. The autotetraploid at maturity
recipient cell along with the chromatin materials. yielded 37 healthy seeds, 30 seeds were sown in C1
Clumping and sticky nature of the nuclear materials generation and 11 plants were obtained of which 4
were also noted in certain PMCs. were cytologically confirmed to be autotetraploids.
Meiotic Instability: Datta and Biswas181 identified a The seeds of C1 tetraploids were bulked and 25
phenotypically aberrant and sterile plant at M3 in the randomly selected healthy seeds were sown in C2
selfed progeny of EMS-induced M2 mutant (lax generation from which 5 plants were obtained and all
branching), which showed aneuploid variation in were meiotically confirmed to be tetraploids. The
chromosome numbers. Phenotypically, the aberrant most prominent morphological changes of C0-1
plant exhibited lax branching nature (Fig. 138) tetraploid and its progenies at C1 and C2 were
attaining a relatively shorter height (32.7 cm) at increase in flower and capsule sterility and reduction
maturity as compared to rather erect (43.65 cm ± in seed number per capsule and seed fertility
1.72) and compact habit of the normal plants. During (expressed as per cent of control). Seed set in the
the initial growth period of the plant the pinnae of tetraploid plants varied from 0.64 to 12.62% of
lamina were represented by linear, thicker appendage control, thereby demonstrating negative selection
like structures and at the latter stages few normal value of induced autotetraploids. However, one
leaves developed. Most of the flower buds autotetraploid (C2-2) possessed some useful traits
terminated in rudimentary flowers excepting a few compared to the diploid and other tetraploids (Figs.
which bloomed after 121 – 137 days after sowing 146-147). The C2-2 plant yielded 118 good seeds
instead of 70 – 98 days in control plants. The flowers (12.62% of control) and the flowers (significantly
had only 1–2 normal looking stamens, while rest of larger than those of diploids) of the plant
the microsporophylls were represented as leafy (synchronous flowering) remained in blooming stage
projections. These flowers produced only for a considerably long period (25 to 32 days) than
rudimentary capsules with abortive seeds. Meiotic the flowers of diploids (4 to 5 days) and the other
analysis revealed distinct chromosomal instability - tetraploids (8 to 12 days) studied over two
Figs. 139-143 (2II+5I – 2.6%, 5II – 5.3%, 11I – generations. Compared to normal (Fig. 148) diploids
(2n=12) the induced tetraploids (2n=4x=24) formed
ANIMESH K. DATTA, ADITI SAHA, ARNAB BHATTACHARYA, ANINDA MANDAL, RITA PAUL AND SONALI
SENGUPTA.

quadrivalents (0-4), bivalents (1-12) and univalent and between Mg and Ca and Mg and Cu. Based on
(0-14) in varying proportion at MI (Figs. 149-151). principle component analysis (PCA) six clusters
Trivalents (0-2) were only observed in C0-1 plant. were observed and it was suggested that the
The induced tetraploids formed 0.80 to 2.08 genotypes may be utilized in various combinations
quadrivalents per cell and the coefficient of for genetic improvement of the species. Iqbal et al.186
quadrivalent realization was low. Chi-square test of recorded genetic variation for plant height, days to
heterogeneity revealed that the frequency of first flower, days to 50% flowers, days to maturity,
bivalents and quadrivalents per cell among the biomass, capsule weight, yield, seed weight and
tetraploids was random (p>0.05) but number of harvest index while studing 31 genotypes under field
univalent per cell was non-random (p<0.001). The conditions with 3 replications. Three accession
mean chromosomal association in C2-2 was (MP00023, MP00111 and MP00120) were found
1.37IV+9.00II+0.50I (32 PMCs scored). The better for more than one character and are expected
univalent frequency among tetraploids demonstrated to be a potential for improvement of N. sativa.
significant positive correlation with abnormal AI
(laggards, bridges, groupings and unequal separation Tissue culture
- Figs. 152-158) cells (r=0.81; p<0.05). Anaphase II Callus Induction: Banerjee and Gupta187 raised
cells showed unequal and multisporic conditions calluses from leaf tissues and were of opinion that
(Figs. 159-160). induction of calluses depended on the balance
The abnormal AI cells showed significant negative between auxin and kinetin in the medium and
correlation with pollen fertility (r=-0.99, p<0.001). coconut milk factor as a source of kinetin. Chand and
However, the correlation between frequency of Roy188 used different concentrations of 2,4-D, NAA
abnormal AI cells and seed set and between pollen and IAA to explore maximum callusing in the
fertility and seed yield and between pollen fertility species. The concentration of kinetin has been kept
and seed fertility were non-significant. Cytological constant throughout the experiments. The calluses
examination of induced autotetraploids leads to the grown in medium containing NAA, have been found
conclusion that reduction in pollen fertility was the to be friable, soft and green in color than in media
result of chromosomal disturbances arising from containing IAA and 2,4-D. It was suggested that
pairing irregularities. Seed sterility seems to have a NAA was most favorable for producing callus tissue.
genetical rather than cytological basis. Ghosh and Gadgil189 initiated callus culture from
excised hypocotyl segment when cultured in MS agar
Genetic variability
medium supplemented with IAA, NAA, IBA and
Datta147 studied relationship between yield and its 2,4-D. Chand and Roy190 observed that in presence of
attributes (plant height, number of primary GA in the media the seeds as explant grew into
branches/plant, total capsules/plant and plantlet and there was no callus formation; while in
seeds/capsules) and found significant positive the presence of NAA in the media seeds first
correlation in all cases excepting for seed/capsule. produced calli from which plantlet developed. In the
Plant height was positively associated with primary presence of IAA seeds grew into plants but at the
branches/plant (r=0.71, p<0.01) and total base callus formation took place. In all cases amount
capsules/plant (r=0.69, p<0.01); while, number of of kinetin and coconut milk were kept constant. It
primary branches/plant was significantly associated was also pointed out that in presence of 2,4-D,
with capsules/plant (r=0.80, p<0.01). However, kinetin and coconut milk seed proliferated into callus
capsules/plant showed insignificant relationship with tissue without formation of plantlets. Datta et al.191
seeds/capsule (r=0.04, p>0.05). Path coefficient reported calli formation from hypocotyl segment in
analysis revealed that the direct contribution of total MS medium supplemented with 2,4-D (2 mg/l) and
number of capsules/plant (P35=0.7460) was very high kinetin (1 mg/l), and they were creamy white,
and the trait indirectly contributed in high amount compact ones. Youssef et al.192 reported that 0.05 per
through plant height and number of primary cent casein hydrolysate promotes callus growth;
branches. Direct contribution of plant height however, growth was reduced by increasing salinity.
(P15=0.2886) and seeds/capsule (P45=0.1296) to yield On the contrary, it was also suggested that
was relatively low. Primary branches/plant (P25=- accumulation of primary products in callus cultures
0.3374) showed negative contribution to yield. is enhanced by salt stress. Al-Ani193 cultured roots,
Results indicated that capsules/plant is the most hypocotyls and leaves in MS medium supplemented
important trait for selection and crop improvement. with 2,4-D (0.0, 1.0, 2.0, 3.0, 4.0 mg/l) and kinetin
Iqbal et al.185 studied 34 accessions with 2 check (0.0, 1.0, 1.5, 2.0, 2.5, 3.0, 5.0 mg/l) and best
genotypes of black cumin for assessment of mineral callusing was obtained from leaf explants with 1
nutrients. High variation was recorded for Fe, Ca, mg/l 2,4-D and 1.5 mg/l Kin. Such callus yielded
Cu, Mg, Pb, Zn, Co, Mn, Na, P, B, K and N amongst higher thymol concentrations after 75 days by HPLC.
genotypes suggesting sample selection based on the Suspension Culture: Banerjee and Gupta194 reported
composition of mineral nutrients. Correlation studies that in suspension culture 91% free cells of N. sativa
revealed significant association between Cu and Ca, was obtained in WHITE’s medium supplemented
JOURNAL OF PLANT DEVELOPMENT SCIENCES Vol.4 (1) 173

with casein hydrolysate, inositol and adenine. Ploidy anomalies like binucleate cell, micronuclei,
distribution pattern was similar in cell clumps of diplochromosomes, multipolarity, sticky bridges and
different sizes and free cells. Chromosomal ring chromosomes formation was also observed.
irregularities were more in free cells. A number of Anomalies might be due to endoduplication and
globular embryoid were formed when casein various mitotic disturbances.
hydrolysate, inositol and adenine were added in the
medium after subsequent omission of auxin and Molecular genetics
coconut milk. Al-Huqail and Al-Saad199 performed DNA
Embryogenesis: Banerjee and Gupta195 noted fingerprinting in 4 accessions from Saudi Arabia,
embryogenesis in leaf callus (MS media Ethiopia, Egypt and Syria with an objective of
supplemented with casein hydrolysate; coconut milk genotypic characterization between/among black
replaced). Casein hydrolysate suppressed the cumin taxa. Inter Simple Sequence Repeat (ISSR)
differentiating capacity at a concentration of 100mg/l method was employed in the PCR technique to detect
after fifth subculture. It was reported that 2,4-D and genetic polymorphism. The scored bands of the DNA
kinetin have inhibiting effect on morphogenesis. On fingerprints (17 primers representing 3 types of
the histological examination of differentiated tissue, intermicrosatelites – di, tri and tetra of short tandem
it was observed that roots, shoots, buds and leaves repeats) were 108 in Saudi Arabia, 106 in Ethiopia,
have originated from group of meristematic cells 100 in Egypt and 81 in Syria and the percentage of
whereas embryoids have initiated by the repeated dissimilarity was computed to be 21.5-36.3%.
division of single cell. Twenty four genes representing 24 different enzymes
Elhag et al.196 with an objective of inducing and and isozymes were selected and scanned via PCR
isolating somatic embryos for biosynthetic studies technique using suitable SSR primers and the
callus cultures were initiated from leaf, stem and root obtained results showed some changes in the genetic
explants of axenic seedlings on MSB5 basal medium structure of some of these genes. Iqbal et al.200
supplemented with kinetin (0.46 µM) and 2,4-D (4.5 carried out investigation to explore genotype specific
or 13.5 µM) or NAA (5.4 or 16.2 µM) in the dark. fingerprinting of 32 germplasms based on randomly
Cultures initiated and subcultured on medium amplified polymorphic DNA markers. From 58
containing NAA produced friable callus with random primers used, 15 primers generated 249
numerous roots regardless of explant type. These reproducible and scorable amplification products
cultures differentiated into somatic embryos on across all the genotypes, out of which 164 (66.0%)
medium containing NAA. The embryos developed fragments were polymorphic revealing a high level
into leafy structures on basal medium devoid of of polymorphism among the genotypes. The
growth regulators. When the embryogenic callus was proportion of common bands was low (34.0%). In 13
transferred to liquid medium containing NAA, genotypes, 27 bands of different masses (kilobases)
numerous embryos and clusters of embryos were were recorded and were considered specific. The
released into the liquid medium but, in contrast to specific/amplified PCR products were reported to be
solid medium, development remained arrested at the used as molecular markers for identification of
early embryonic stages. germplasms and resource protection. The result of
Chromosomal Instability: Chand and Roy188 genetic polymorphism was validated from UPGMA
reported very high number of chromosomes in media and PCA.
containing 2,4-D and kinetin; while NAA resulted
very minor chromosomal variations. Ghosh and Genes
Gadgil189 found shift in ploidy level from diploid to
higher polyploids in presence of 2,4-D and when 1. APETALA 3 – like protein (AP3-3) mRNA,
kinetin was mixed with 2,4-D or 2,4-D mixed with 746bp, linear, partial cds, accession –
coconut milk factor. Bansal and Sen197 reported that HQ694794201.
polyploidy has been a common feature of occurrence 2. APETALA 3 – like protein (AP3-2) mRNA,
in calluses induced from root, shoot and leaf tissues 865bp, linear, partial cds, accession –
and their appearance did not show marked difference HQ694795201.
in the tissues. Datta et al.191 studied numerical 3. PISTILLATA – like protein (PI-2) mRNA,
variations in chromosome number including 809bp, linear, partial cds, accession –
polyploidy, aneuploidy and haploidy as well as HQ694796201.
structural anomalies (Figs. 161-166) from callus 4. PISTILLATA – like protein (PI-1) mRNA,
tissues raised from hypocotyl segment. Frequent 840bp, linear, partial cds, accession –
chromosome elimination in different cell lines was HQ694797201.
noted; however, the marker chromosomes 5. microsatellite NIG_HSP 70 sequence, DNA,
(telocentric) were found constantly at different 345bp, linear, accession – HM803244.1202.
ploidy level. Kumar and Roy198 were of opinion that 6. Nigella sativa voucher A. Guener, M. Vural and
apart from occurrence of high frequency of aneuploid H. Sagban 9189 internal transcribed spacer 1,
and polyploid cells in callus tissues, structural 5.8S ribosomal RNA gene, and internal
ANIMESH K. DATTA, ADITI SAHA, ARNAB BHATTACHARYA, ANINDA MANDAL, RITA PAUL AND SONALI
SENGUPTA.

transcribed spacer 2, complete sequence, DNA, Patents

621 bp, linear, EU699463203.
7. Nigella sativa internal transcribed spacer 1, 5.8S Nigella sativa currently has five FDA (Food and
ribosomal RNA gene, and internal transcribed Drug Administration) separate patents in the U.S.A.
spacer 2, complete sequence, DNA, 621 bp, for the treatment of:
linear, EU699464203. 1. Inhibition of cancer cell growth, Patent no.- US
8. Nigella sativa beta-amyrin synthase (basl) 5,653,981, Inventor- R. D. Medenica.
mRNA, complete cds, mRNA, 2430 bp, linear, 2. Diabetes, No.-US 6,042,834, Inventor – Wasif
FJ013228204. Baraka.
9. Nigella sativa beta-amyrin synthase (basl) gene, 3. Improvement of the Immune System, No.- US
complete cds, DNA, 4444 bp, FJ013229204. 5,482,711, Inventor – R. D. Medenica.
10. Nigella sativa squalene epoxidase 1 (seq 1) 4. Viral Infections, No.- US 6,841,174, Inventor – S.
mRNA, complete cds, mRNA, 1566 bp, linear, I. A. Shalaby and E. M. A. H. Allah.
FJ232947205. 5. Psoriasis, No.- US 6,531,164, Inventor – H. H. R.
Credé.

Figs. 1-4. 1) Normal N. sativa plant. 2) Flower before pollination. 3) Flower after pollination. 4) Seeds of black
cumin.
JOURNAL OF PLANT DEVELOPMENT SCIENCES Vol.4 (1) 193

Figs. 5-10. Scanning Electron Microscopy of seed surfaces of black cumin. [Source: Cytologia 68, 2003]

Figs. 11-14. Chromosomes (2n=12) in Nigella sativa. 11) Mitotic chromosome. 12) Photoplate ideogram
showing 4 (AA, BB, CC, DD) chromosome types. 13-14) Diplotene plates where the bivalents are marked.
Bar=15 µm. [Source: Cytologia 67(4), 2002]
ANIMESH K. DATTA, ADITI SAHA, ARNAB BHATTACHARYA, ANINDA MANDAL, RITA PAUL AND SONALI
SENGUPTA.

Figs. 15-18. Meiotic configurations (2n=12). 15-16) 6II at diplotene. 17) MI showing 6II. 18)6-6 separation of
chromosomes at AI. Bar=15 µm.

Fig. 19. Pachytene chromosome configurations.

JOURNAL OF PLANT DEVELOPMENT SCIENCES Vol.4 (1) 3
21

Figs. 20-25. PMCs at prophase I showing variation in number and size of nucleoli in control and in mutant lines
of N. sativa. 20) One nucleolus. 21-22) Two unequal sized nucleoli, unattached to bivalents. 23) Three nucleoli.
24) Four nucleoli of which two are attached to a bivalent (a) and three unequal sized nucleoli (b). 25) Five
nucleoli. Bar=15 µm. [Source: J. Phytol. Res. 12(1-2), 1999]

Figs. 26-31. Mitotic consequences following irradiations at metaphase. 26) 2n=12 – normal configuration. 27)
Pseudochiasma like configuration. 28) Ring chromosome. 29) Diplochromatic nature of chromosomes in a
polyploid cell. 30) Abnormal shaped cell with chromosome bending. 31) Aneuploid cell with fragments and
unequal chromosome length. Bar=15 µm. [Source: Cytologia 48, 1983; Cytologia 51, 1986; J. Plant Dev. Sci.
3(1), 2011]
ANIMESH K. DATTA, ADITI SAHA, ARNAB BHATTACHARYA, ANINDA MANDAL, RITA PAUL AND SONALI
SENGUPTA.

Figs. 32-37. Anaphase bridge formation in irradiated samples. 32) Single bridge with a round globular fragment.
33) Double bridge. 34) Double bridge with equal sized round and rod fragments. 35-36) Criss-cross bridge. 37)
Interlocked bridge. Bar=15 µm. [Source: Cytologia 48, 1983; Cytologia 51, 1986; J. Plant Dev. Sci. 3(1), 2011]

38 39

40 41
Figs. 38-41. Anaphasic events following irradiations. 38-39) Incomplete bridge with two identical sized
fragments. 40) Paired fragments. 41) Four fragments. Bar=15 µm. [Source: Cytologia 48, 1983; Cytologia 51,
1986; J. Plant Dev. Sci. 3(1), 2011]
JOURNAL OF PLANT DEVELOPMENT SCIENCES Vol.4 (1) 233

Figs. 42-47. Mitotic events following irradiations. 42) Polyploid cell at anaphase showing multipolar
organization. 43) Multipolarity at anaphase. 44) Two condensed nearly identical sized micronuclei in resting
cell. 45) Condensed and uncondensed micronuclei. 46) Four unequal sized micronuclei. 47) Giant cell. Bar=15
µm. [Source: Cytologia 48, 1983; Cytologia 51, 1986; J. Plant Dev. Sci. 3(1), 2011]

Figs. 48-53. Meiotic consequences of irradiations at metaphase I. 48-49) 6II. 50-51) 3II+6I. 52) 2II+8I. 53)
6II+2 identical sized fragments. Bar=15 µm. [Source: J. Plant Dev. Sci. 3(1), 2011]
ANIMESH K. DATTA, ADITI SAHA, ARNAB BHATTACHARYA, ANINDA MANDAL, RITA PAUL AND SONALI
SENGUPTA.

Figs. 54-59. Meiotic events at MI. 54)1IV (adjacent orientation) + 4II. 55) 1IV (alternate) + 3II+2I. 56) 1IV
(adjacent) + 4II. 57) 1IV (non-co oriented) + 4II. 58) Sticky configuration of chromosomes. 59) Fusion of two
PMCs. Bar=15 µm. [Source: Cytologia 48, 1983; Cytologia 51, 1986]

Figs. 60-65. Meiotic configurations in irradiated samples at AI and AII. 60) 6-6 separation at AI. 61) Two
fragments at AI. 62) Dicentric chromatid bridge with an acentric fragment. 63) Double bridge formation at AI.
64)Two lagging chromosomes at AII. 65) A bridge with a fragment at AII. Bar=15 µm. [Figs. 26-65. Ref.:
Cytologia 48, 1983; Cytologia 51, 1986; J. Plant Developmet Sci. 3(1-2), 2011]
JOURNAL OF PLANT DEVELOPMENT SCIENCES Vol.4 (1) 3
25

66
Figs. 66-67. Stem anomalies. 66) Bifurcation. 67) Twining nature. [Source: Cytologia 50, 1985]

Figs. 68-70. Stem abnormalities. 68) Trifurcation. 69) Twining. 70) Unbranched. [Source: Cytologia 50, 1985]
ANIMESH K. DATTA, ADITI SAHA, ARNAB BHATTACHARYA, ANINDA MANDAL, RITA PAUL AND SONALI
SENGUPTA.

Figs. 71-78. 71) A normal flower. 72-78) Floral abnormalities. 72) Unequally dissected petaloid sepal. 73)
Shield shaped sepal. 74) Triforked sepal. 75) Elongated and strap shaped petal. 76) Presence of two gynoecium
in a same flower. 77) Small sized sepal in addition to the normal complement. 78) Incompletely forked bract
like structure. [Source: Cytologia 50, 1985]
JOURNAL OF PLANT DEVELOPMENT SCIENCES Vol.4 (1) 273

Figs. 79-84. Control and mutants of N. sativa. 79) Normal plant. 80) Lax branching. 81) Prostrate. 82) Bushy I.
83) Bushy II. 84) Lax pinnae. [Source: Cytologia 50, 1985]

85
Fig. 85.Cup leaf mutant.
ANIMESH K. DATTA, ADITI SAHA, ARNAB BHATTACHARYA, ANINDA MANDAL, RITA PAUL AND SONALI
SENGUPTA.

86

Fig. 86. Chloroxantha with normal plants in field condition. [Source: Ind. J. Genet. Pl. Breed. 61, 2001]

Figs. 87-90. Seed-coat color in N. sativa. 87) Black in normal. 88) Yellowish brown. 89) Dark reddish brown.
90) Bicolor.
JOURNAL OF PLANT DEVELOPMENT SCIENCES Vol.4 (1) 293

91

Fig. 91. Telescopic mutant in N. sativa. [Source: Cytologia 51, 1986]

Figs. 92-97. Meiotic configurations at MI (92, 94-97) and diplotene (93) in translocation heterozygotes. 92) 6II.
93)1IV+4II. 94) 1IV (chain, alternate) + 4II. 95) 1IV (chain, adjacent) + 4II. 96) 1IV (chain, alternate) + 4II. 97)
1IV (ring, alternate) + 4II. Bar=15 µm. [Source: Cytologia 67, 2002]
ANIMESH K. DATTA, ADITI SAHA, ARNAB BHATTACHARYA, ANINDA MANDAL, RITA PAUL AND SONALI
SENGUPTA.

Figs. 98-103. Meiosis in translocate heterozygotes. 98 and 101) 1IV(ring, alternate) + 4II at MI. 99-100) 1IV
(ring, adjacent) + 4II at MI. 102) 5-7 separation of chromosomes at AI. 103) Dicentric chromatid bridge with an
acentric fragment at AI. Bar=15 µm. [Source: Cytologia 51, 1986]

111 112

Figs. 104-112. Meiosis in synaptic mutants. 104-105) Early prophase I cells showing fuzzy chromosomes and
lack of pairing. 106-112) MI chromosome associations. 106) 6II. 107) 5II+2I. 108-109) 4II+4I. 110) 2II+8I.
111) 1II+10I. 112) 12I. Bar=15 µm. [Source: Plant Archives 2, 2002]
JOURNAL OF PLANT DEVELOPMENT SCIENCES Vol.4 (1) 313

Figs. 113-116. Phenotype of male sterile mutants. 113) Mutants showing chlorophyll deficiency in pinnae of the
apical part. 114) Bushy. 115) Crumpled pinnae. 116) Chlorophyll deficiency. [Source: Plant Archives 1, 2001]

Figs. 117-122. Meiosis in a male sterile mutant (117-121). 117) 6II at MI. 118-119) 12I at MI. 120) AII with
unequal spory (near complete degeneration of one pole). 121) Degenaration of microspores. 122) Fully stained
round to oval shaped pollen grains in normal plants. Bar=15 µm. [Source: Plant Archives 2, 2002]
ANIMESH K. DATTA, ADITI SAHA, ARNAB BHATTACHARYA, ANINDA MANDAL, RITA PAUL AND SONALI
SENGUPTA.

Figs. 123-130. Meiosis in male sterile mutants. 123-125) Agglutination of chromatin into unequal masses. 126
and 128) degenerative pollen grains. 127) Agglutinated pollen grains. 129) Small sized round unstained pollen
grains. 130) Fertile pollen grains in normal plants. Bar=15 µm. [Source: Plant Archives 2, 2002]

Figs. 131-134. Meiosis in a trisomic plant (2n= 13). 131-132) 6II+1I at MI. 133) 5II+ 1III at MI. 134) 9-7
separation of chromosomes at AI. Bar=15 µm. [Source: Cytologia 49, 1984]
JOURNAL OF PLANT DEVELOPMENT SCIENCES Vol.4 (1) 333

Figs. 135-137. Chromatin bridge (Fig. 136) and fusion of meiocytes (Figs. 135 and 137) in chromosome/
chromatin transfer. Bar=15 µm. [Source: Cytologia 49, 1984]

Figs. 138-145. 138) Aberrant plant showing lax branching nature and leaf deformity. 139-143. Meiosis in the
aberrant plant. 139) 2II+5I (2n=9) at MI. 140) 6II (2n=12) at MI. 141) 4II+3I (2n=11) at MI. 142) 1IV+4II+1I
(2n=13) at MI. 143) MI showing 12II (2n=24). 144-145. Pollen grains. 144) Stained and unstained small sized
pollen grains in the aberrant plant. 145) Normal stained pollen grains in control. Bar=15 µm. [Source: Cytologia
50, 1985]

Figs. 146-147. 146) Normal diploid. 147) Autotetraploid showing synchronous flowering. [Source: Indian J.
Genet. Plant Breed. 62, 2002]
ANIMESH K. DATTA, ADITI SAHA, ARNAB BHATTACHARYA, ANINDA MANDAL, RITA PAUL AND SONALI
SENGUPTA.

Figs. 148-151. Meiotic configuration at MI and AI (151). 148) 6II in diploid. 149-151. Meiosis in
autotetraploid. 149) 1IV+9II+2I. 150) 2IV+4II+8I. 151) 8-16 separation of chromosomes. Bar=15 µm. [Source:
Indian J. Genet. Plant Breed. 62, 2002]

Figs.152-155. AI configurations in the autotetraploid. 152) 11-13 separation. 153) 11-13 separation associated
with a fragment. 154) Tripolarity along with a lagging chromosomes. 155) Multiple bridges with fragments.
Bar=15 µm.
JOURNAL OF PLANT DEVELOPMENT SCIENCES Vol.4 (1) 353

Figs. 156-160. AI (156-158) and AII (159-160) configurations in autotetraploids. 156-157) Unequal (11-13)
separation of chromosomes. 158) Tripolarity with laggards. 159) Unequal spory. 160) Multiple spory. Bar=15
µm.

Figs. 161-166. Chromosome variations and abnormalities in callus tissue. 161) 2n=6. 162) 2n=24. 164-165)
Enhanced ploidy level. 166) Bridge formation with higher ploidy. 167) Five extremely variable chromatin
masses. Bar=15 µm.
 ANIMESH K. DATTA, ADITI SAHA, ARNAB BHATTACHARYA, ANINDA MANDAL, RITA PAUL AND SONALI
SENGUPTA.

CONCLUSION

Despite the major advancement of modern medicine in human health-care, it is still intangible and beyond reach
to ailing humanity, especially the destitutes. In recent years plant based systems has been utilized for traditional
medicine and phytotherapy. Medicinal plants are ‘Gift of Nature’ and N. sativa is one such plant with potential
uses which can be explored for safe and effective herbal medicine for human benefit. Cytogenetical studies also
revealed that the species can also be used as a model plant for better understanding of gene and chromosome
relationship.

ACKNOWLEDGEMENT

This monograph is dedicated to those individuals who believe in herbal medicine and also to researchers
working in the field of Cytogenetics.

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