PLAGIARIZED: Saffron (Crocus sativus L.) Strategies for Enhancing Productivity
(Case No. 31072013)
Professor Y.M. Agayev Agricultural Biotechnilogy Research Institute of Iran (ABRII) Mahdasht Road, P.O. Box 31535-1897, Karaj Iran, pointed out a plagiarism in a paper published in Research Journal of Medicinal Plant Volume 5 Number 6, 630-649, 2011.
On the receipt of Professor Y.M. Agayevs letter, the case forwarded to the
Ethics Committee of the Science Alert. As per the report of the Ethics Committee,
article entitled Saffron (Crocus sativus L.) Strategies for Enhancing
Productivity authored by Mushtaq Ahmad, Gul Zaffar, S.D. Mir, S.M. Razvi,
M.A. Rather and M.R. Mir from Division of Pant Breeding and Genetics, Sher-e-Kashmir
University of Agricultural Sciences and Technology of Kashmir, Shalimar Campus,
191 12-Srinagar, Kashmir published in Research Journal of Medicinal Plant Volume
5 Number 6, 630-649, 2011, contains substantial sections of text that have been
taken verbatim from earlier publication without clear and unambiguous attribution.
Science Alert considers misappropriation of intellectual property and duplication of text from other authors or publications without clear and unambiguous attribution totally unacceptable.
Plagiarism is a violation of copyright and a serious breach of scientific ethics. The Editors and Publisher have agreed to officially retract this article.
Science Alert is highly thankful to Professor Y.M. Agayev for pointing out this plagiarism.
Detail of article from which text has been copied by Mushtaq Ahmad:
Y.M. Agayev, A.M. Shakib, S. Soheilivand and M. Fathi, 2007. BREEDING OF SAFFRON
(CROCUS SATIVUS): POSSIBILITIES AND PROBLEMS, 203-207, 2007.
March 11, 2011; Accepted: April 30, 2011;
Published: June 25, 2011
Saffron, Crocus sativus L., is an important crop cultivated as
the source of its spice for at least 3,500 years. Dried stigmas of saffron flowers
compose the most expensive spice which has been valuable since ancient times
for its odoriferous, coloring and medicinal properties (Plessner
et al., 1989). Saffron introduction into new areas should be encouraged
as it is a unique crop in terms of its potential and is recognized as red gold.
It is the highest priced spice in the world at around $500 kg-1 of
saffron (Fernandez, 2007). The name saffron is commonly
used to refer both to the spice and the plant itself. Some archaeological and
historical studies indicate that domestication of saffron dates back to 2,000-1,500
years BC (Grilli Caiola, 2004). The origin of saffron
is obscure, but the plant is believed to have originated in the eastern Mediterranean,
probably in Asia Minor and Persia. The name saffron is derived from
Arabic Zafran which means be yellow (Winterhalter
and Straubinger, 2000). Owing to extremely high demand from the dye, perfumery
and flavoring industries, it is one of the most expensive spices on earth. The
components of the spice saffron are localized in the red stigmatic
lobes of C. sativus flower and these are responsible for its distinct
color, flavor and smell.
For color the principal pigment is crocin, for smell the main component is
safranal and for the special bitter flavor the main compound is the glycoside
picrocrocin (Basker and Negbi, 1999). These compounds
are derived from oxidative cleavage of the carotenoid zeaxanthin (Bouvier
et al., 2003; Moraga et al., 2004).
Besides these, a new class of defense chitinase namely Safchi A has recently
been isolated from saffron (Castillo et al., 2007).
There has been little success however in enhancing the levels of these bioactive
molecules of commercial importance. The information regarding flower initiation
can be exploited by plant breeders to apply conventional breeding procedures
suited to vegetatively propagated crops viz, mutation breeding or polyploidy
induction using physical/chemical mutagens can be tried at this stage to hit
the target site for generating variability with regard to floral traits or polyploidizing
agents like colchicines can be applied for inducing hexaploidy in an otherwise
sterile triploid (Zaffar et al., 2008).
A revolution in molecular biology, statistics and information technology has
stimulated the merger of some advanced technologies for understanding the complex
web of interactions linking individual components of a living cell to the integrated
behavior of an entire organism (Bruskiewich et al.,
2006). The marriage between these advanced technologies has given birth
to the discipline of bioinformatics. As large-scale expression profiling experiments
with saffron can generate huge amounts of data about the saffron transcriptome,
the discipline of bioinformatics can be used to extract information from the
data. Characterization of the transcriptome of saffron stigmas is vital for
throwing light on the molecular basis of flavor, color biogenesis, genomic organization
and the biology of the gynoecium of spices in general and saffron particularly.
The information derived can then be utilized to construct biological pathways
involved in the biosynthesis of the principal components of saffron i.e., crocin,
crocetin, safranal, picrocrocin and safchiA.
Saffron production in the world: Saffron is currently being cultivated
more or less intensely in Iran, India, Greece, Morocco, Spain, Italy, Turkey,
France, Switzerland, Israel, Pakistan, Azerbaijan, China, Egypt, United Arab
Emirates, Japan and recently in Australia (Tasmania). The worlds total
annual saffron production is estimated as 205 tons per year. Iran is said to
produce 80 percent of this total; i.e., 160 tons and Khorasan province alone
137 tons of the totals. Behdani et al. (2008)
reported that the age of saffron farms was the most important factor influencing
yield and five years aged farms had the longest flowering period, also there
is a positive linear relation between continuance of flowering and yield. The
Kashmir region in India produces between 8 to 10 tons mostly dedicated to Indias
self-consumption. Greek production is 4 to 6 tons per year. Morocco produces
between 0.8 and 1 ton. Saffron production has decreased rapidly in many traditionally
producing countries and is abandoned in others such as England and Germany.
Spain was used to be the traditional world leader and most reputed saffron producer
for centuries in areas of La Mancha and Teruel. Nowadays, the production is
only about 0.3-0.5 tons. Productions of Italy (Sardinia, Aquila, Cascia) 100
kg; Turkey (Safranbolu) 10 kg; France (Gâtinais, Quercy) 4-5 kg; and Switzerland
(Mund) 1 kg are nearly insignificant. All saffron producers in the European
Union, as well as in Turkey, suffer from increasing labour costs (Fernandez
et al., 2011). However, the demands of world industry for saffron
product are very high. Among countries cultivating saffron, Iran is at the first
place. More than 90% of the world production falls onto Iranian saffron which
has a great importance in the economy of the country. The area under saffron
cultivation in Iran reaches about 80,000 hectares with an annual production
of about 250 tons of the dry stigmas. In recent years this amount has been remarkably
increased which was achieved mainly by extension of the planting areas but not
due to the increased yield capacity in the area unit. Beside other factors new
high yielding cultivars of saffron are required to solve the problem.
Export and import of saffron: Export of saffron from India in small quantity has been a regular feature of international trade. However, among the saffron growing countries, Iran exports are the highest, with over 90% of its total produce being exported every year. Indian saffron is exported mainly to Spain, followed by France, USA, UK, UAE, Israel, Japan etc. However, the exports have been declining year after year, 9.77 MT in 1998-99 to 1.3 MT 2008-09, accounting for reduction of about 87%. This is primarily due to the decline in area and net productivity, lack of high yielding varieties. It is possible that the disillusionment of the farmers of the state with this crop could be also due to the declining trend in the domestic and international prices during the above period, thus contributing to the reduction in the net returns to the producers. Added to this is the major challenge thrown by the aggressive exports being made by Iran every year and the rampant adulteration that has plagued the saffron trade.
Saffron uses: The use of saffron goes back to the ancient times. It
is most commonly used in medicine, as well as dye and spice in food industry
(Basker and Negbi, 1999). Saffron has been also used
as a drug to treat various human health conditions such as coughs, stomach disorders,
colic, insomnia, chronic uterine haemorrhage, femine disorder, scarlet fever,
smallpox, colds, asthma and cardiovascular disorders (Giaccio,
1990; Winterhalter and Straubinger, 2000; Abdullaev,
2003). It has been shown that saffron is a protective agent against chromosomal
damage (Premkumar et al., 2001). Antinociceptive
and anti-inflammatory (Hosseinzadeh and Younesi, 2002),
antiseizure (Hosseinzadeh and Khosravan, 2002) and blood-pressure
reducing (Fatehi et al., 2003) effects in animals
were also reported. Saffron extract or its active constituents, crocetin and
crocin, could be useful as a treatment for neurodegenerative disorders accompanying
memory impairment (Zhang et al., 1994). Crocin
extracts have been used for the treatment of nervous, cardiovascular and respiratory
systems (Abe and Saito, 2000; Abdullaev,
2002) Crocin is also unique antioxidant that struggle with oxidative stress
in neurons (Ochiai et al., 2004). The antidepressant
effect of saffron petals and stigma in mice was also reported (Karimi
et al., 2001). The positive effect of saffron extracts has also been
described in patients suffering from allergic asthma (Haggag
et al., 2003). Hartwell (1982) reported that
saffron extracts were used against different kinds of tumors and cancers in
ancient times. Therefore, liver, spleen, kidney, stomach and uterus tumors have
been treated with pharmaceutical preparations of saffron. A number of studies
in animal model systems have showed an antitumor effect of saffron on different
malignant cells (Abdullaev, 2004). Khori
et al. (2006) for the first time has explained the role of saffron
on the protective mechanism of artrioventricular node against supraventricular
arrhythmia. The results also showed the non-specific effect of saffron on the
transitional cells of fast nodal pathway which was manifested as a rate-independent
increase of basic and functional (facilitation and fatigue) parameters of artrioventricular
Adulteration: Adulteration in Kashmir saffron is rampant and a most serious malpractice, making it a major constraint in reviving saffron cultivation in the state. It is a common sight to find imported Iranian saffron being mixed with Kashmir saffron and sold as Kashmir saffron to extract a higher price from the consumers/buyers. The adulterants detected were tetrazine-dyed starchy fibrous material (probably wheat flour and coconut fruit shell), dyed saffron stamens and dyed tender adventitious roots of Salix. Other adulterants reported are ray florets of marigold, safflower dyed with coal tar dyes viz, sunset yellow or matanil yellow; corn silk (stigma of maize), fibers of shredded meat coloured with saffron water, fibrous roots of various grasses, slender roots of willow and coloured nylon fiber. Fats, oils and glycerin are sometimes used to increase the weight of saffron.
Pests and disease: The major biotic stress being faced by saffron since
last several years is the corm rot fungal infection, a soil borne disease caused
by Fusarium monliform var intermedium and an unidentified mycelium
of Basidiomycetous fungus. Out of six fungicides Blitox (copper-oxychloride),
Difolatoan (captafol), Folpat (captafol), Bavistin (carbendazim) and Tecto (Thiobendazole)
evaluated by Sud et al. (1999), only Bavistin and
Tecto @ 0.2% as adip or drench gave a complete disease control. They further
concluded that use of healthy corms followed by application of Bavistin or Tecto
as a drench in the subsequent year appeared to be the best management strategy.
Saffron belts of Kashmir are also infested with plant parasitic nematodes, namely
Helicotylenchus sp. Tylenchorynchus sp. Pratylenchus sp.
Xiphinema sp. etc as well as free living mites and thrips. Rodents and
field rats also pose a serious problem.
WHY SAFFRON EXISTS JUST AS ONE CULTIVAR
In all over the world saffron is known as one cultivar, as descent of certain
triploid sterile plant arisen once spontaneously in nature which was caught
by sight of man and involved into cultivation (Mathew, 1977).
It has been propagated and still continues to be propagated vegetatively. There
is a supposition that saffron as a clone can be scarcely changed genetically
and its improvement is hardly possible through clonal selection (Dhar
et al., 1988; Piqueras et al., 1999).
Meanwhile, other suppositions exist as well. For example, Rzakuliyev (1959)
investigating in Apsheron (Baku) specimens of saffron obtained from 6 regions
(2 regions in Italy, France, Istanbul, Yalta and Mashtaða) during 3 vegetations
in 1934-1937 concluded that it is possible to create a new high yielding cultivars
of this plant on the basis of clonal selection. Kapinos (1965)
studied the morphogenesis and cytoembryology of Crocus sativus also under
climatic conditions of Apsheron and came to the conclusion that this plant represents
variegated blend of genetically heterogeneous forms clones and clonal
selection on it would be very promising. Apparently, the lack of new cultivars
of saffron at present may not be explained by the impossibilities of the improvement
of this plant through clonal selection. To solve this problem, it is necessary
for a researcher to be properly acquainted with the biology and genetics of
saffron and work up subtle puzzled methods of the clonal selection especially
for saffron, different from those of the other plants. Two main difficulties
of saffron breeding through clonal selection, in our opinion, are as follow:
(1) Difficulty in the recognition of the clones. In any plantation saffron is
represented by plants existing in highly different ages of individuals
connected with different sizes of corms underground. Above the ground, these
plants differ in the size and number of their flowers (at the stage of flowering),
also in their number and size of leaves. If some plants are sharply different
from the others in certain characters, interesting for the aim of breeding,
a researcher can not practically identify them. Naturally he does not see corms
underground and can not elucidate the cause of the mentioned differences: whether
these differences are due to the age (size) of corms, or because of the genotypes
of plants. So, genetically different (if exist) and similar plants will continue
to grow together and not be used as a subject for breeding. (2) Difficulty in
the multiplication of clones and bringing them to cultivars. Let us suppose
that farmer recognizes certain plant (s) which could be used as a clone with
good economic characters. Multiplication of such clone (s) would be turned as
insoluble problem. One saffron corm at planting with proper care produces on
an average 4 corms of middle sizes during vegetation (one year). At such intensity
of propagation it could be brought to about 100,0000 corms only after 17 years.
This amount could be enough for planting on the area of 2 ha. It is clear that
a farmer will accomplish never such an exhausting work of many years. Therefore
the farmers would not pursue the aim to make new cultivar of saffron even if
they have been lucky to find some clones with very highly expressed economically
valuable characters. Concerning to the researcher, in our opinion, he could
be able to do it in the favorable conditions, namely in the case of worthy appreciation
of his long term hard work on this way by existing law on breeding. That is
why in all over the world saffron has been remained as one cultivar despite
of existence in culture during hundreds and thousands of years (Agayev
et al., 2010). Alternatively, genetically changed superior plants
of saffron could be propagated rapidly via in vitro technique. Investigations
in this direction are very promising. Unfortunately, the experiments pursuing
rapid corm propagation of saffron have not been successful so far and a few
in vitro developed corms had been produced. Matured corms of in vitro
origin in mass production had not been produced.
Origin: Seven species comprising this aggregate are: C. sativus Linn, C. nivevs, C. cartwrightianus Herb., C. hadriaticus Herb., C. thomassi Ten., C. dispathacea Bowles and C. pallassii Ancestry of C. sativus and its phylogenetic kinship have to be sought within this aggregate by using conventional and molecular techniques of genome analysis. The DNA polymorphism based AFLP method has confirmed the close relationship between these species, C. sativus to be derived from C. cartwrightianus.
Breeding strategies: Crocus sativus L., is a sterile triploid and propagated only vegetatively so conventional breeding methodologies applicable only to vegetatively propagated crops viz clonal selection, mutation, polyploidy and tissue culture have been tried and are being discussed briefly.
CLONAL SELECTION OF SAFFRON
With the object of breeding new cultivars of saffron with economically valuable
traits, two principles can be applied: a) searching, identification and separation
of superior clones in existing plantations, b) creating new valuable forms experimentally.
Suggesting that in the existing plantations clonal selection of saffron is possible
and promising, we are guided by following considerations. Having an ancient
history of cultivation, saffron apparently should contain a lot of genetically
changed forms (clones) as the result of mutations in somatic cells. The task
is to find and study them individually, to separate the economically valuable
forms and to bring them to the new industrial cultivars. But the matter is how
to do it? In order to recognize the changed forms, all of the corms were separated
into groups according to their weights. Difference between adjacent groups was
1.0 g, or 0.5 g, or even less than 0.5 g. For the first time, we contented the
difference of 1.0 g. The groups were as 3.0-3.9 g, 4.0-4.9 g, 5.0-5.9 g, etc.
up to 16.0-16.9 g and more. Ignoring slight differences between weights of corms
within each group (not more than 1.0 g), we conditionally accepted that they
were of the same weight. The corms of each group were separately planted in
lines in soil. In each hole (cluster) only one corm of a given weight was planted.
So we were able to compare plants within each group separately and to investigate
the existence and kind of changed forms. In this way, we could recognize within
each group changed forms with valuable breeding characters such as multiflowering,
larger flowers, flowers with stronger color and more aroma stigmas, tall flowers,
early or late flowering, simultaneously flowering, lack of leaves at flowering
time, number of leaves, their vigor, etc. These investigations were reiterated
a few years for elucidation of the inheritance of established changes. At the
end of such work, new clones with economically valuable characters were finally
established. Next work will involve operations to propagate and use them in
the breeding programs (Agayev et al., 2010).
Mutation and polyploidy: Increasing variability in Saffron through mutagenesis
or changing ploidy level with chemical treatment has not received much attention.
In vegetatively propagated crops, mutagenesis is considered a useful method
for increasing the genetic variability to be exploited for improvement of different
traits through selection. Akhund-Zade and Muzaferova (1975)
irradiated Saffron corms with gamma rays which results in increase in corm production,
flower number and stigma weight for 3 consecutive years in the population treated
with 0.5 Kr. Mutagenesis in saffron should be done when the floral shoot has
come out from the corm because meristem differentiation and maximum mitotic
activity takes place during this stage. Formation of chimera will be low and
recovery of solid mutants likely to increase Slower growth rate could be because
of reduced rate of cell division, lower rate of growth hormones or lower metabolic
activities. Increase in size of stomata reduction in its number may be due to
higher gene doses as a result of chromosome number increase. Role of colchicines
in bringing about chromosome doubling is well known. The plants showing variability
for vegetatively floral traits have been tagged and will be observed during
ensuing season. Evaluation data of C3 generation along with chromosomal
(root tip/PMC) studies will further confirm role of mutation/polyploidy in inducing
variability in Saffron (Zaffar et al., 2004a).
Preliminary results of induced genetic variability through gamma irradiation
and induction of polyploidy through colchinization are not completely hopeful
and probably require further work.
Molecular and biotechnological approach: Clonal selection offers the
best chances of improvement in saffron provided a lot of genetic variability
is present in its natural population. However measures of molecular and morphological
genetic variations or often used to set conservation priorities and design management
strategies for plant texa (Zaffar et al., 2009a).
It seems that the genetic improvement of saffron and creation of new high yielding
cultivars in the past was impossible owing to the complexity of the problem.
Only just the traditional methods of breeding are not promising here. The literature
on saffron breeding is very limited. There are known old works from Azerbaijan
Republic more or less concerning with the breeding problems (Rzakuliyev,
1948, 1959; Kapinos, 1965; Muzaferova,
1970; Agayev et al., 1975). A lot
of work has been carried out using tissue culture (Homes
et al., 1987; Dhar et al., 1988; Munshi
and Zargar, 1991; Munshi, 1992; Piqueras
et al., 1999; Homes et al., 1987).
Ascertaining the specified activities at the same time it should be admitted
that for today on arena there is only one cultivar of saffron. Our attitude
to the given problem differs. The urgency of saffron breeding problems and the
necessity to solve them with the application of new extraordinary approaches
was stated before (Agayev, 1994a,b).
It is believed that saffron plant being cultivated in different countries under
different and constantly varying land and climatic conditions, has faced with
countless different stress situations during a rather long period of time (thousands
years) and undergone the various mutations. Although these mutations were not
redistributed between plants because of their sterility, but are accumulated
in populations and kept till now. With mutations, small and large, on our view,
should be encompassed all attributes, morphological and physiological, peculiar
to saffron plant. Clonal selection independently and in combination with the
experimental polyploidy and hybridization involving wild close relatives of
C. sativus is mostly promising. Methods of in vitro technique and molecular
genetics should be also applied if necessary. RAPD marker and SSR markers have
been used in saffron studies. Inter-SSR and inter-retroelement methods and gene
based markers (PCR and hybridization based) may also be used to add the precision
of molecular characterization studies.
TRANSCRIPTOMICS, TRANSGENOMICS AND GENE MINING
Gene profiles from DNA microarray technology provide a snapshot of life that
maps to a cross section of genetic activities controlled by thousands of genes
simultaneously. Transcriptome analysis of saffron plants, subjected to different
photoperiod and temperature regimes can throw light on to genes that get up
or down-regulated. Variable temperature during corm dormancy and subsequent
low temperatures appear to be effective factors in saffron flower initiation
(Koocheki et al., 2007). Comparison of environmental
and management practices for saffron in Iran (Khorasan) and India (Kashmir)
throw light on some basic climatic and topographic differences between the two
regions viz., humidity, altitude, rainfall, soil-type and irrigation. The main
similarities being in time of planting, harvesting and low temperatures (Kafi
and Showket, 2007). How these differences and similarities, translating
into gene expression, can be known using DNA microarray technology and bioinformatics
tools. This kind of huge data bases generated by physiological, agronomic and
gene expression studies can be analyzed under in silico to find agronomically
important candidate genes in saffron and to identify chemical agent (s) that
simulate the effect of these variable factors, so saffron can be grown under
controlled conditions. Furthermore, molecular biologists and biotechnologists
can utilize the knowledge generated to specifically tailor saffron plants for
new geographical areas such as the East Midlands of England (Yadollahi
et al., 2007). This will involve development of novel traits and
agriculturally relevant characteristics through changes in gene regulation.
Software tools can be employed for in silico analysis of the impact of such
molecular intervention i.e., introduction of a regulatory sequence or a transgene,
for enhanced adaptation to new geographical areas.
Bioinformatics tools and DNA microarray technology can be useful in locating
sources of resistance and agronomically interesting traits for transfer to saffron
by appropriate biotechnological tools. The removal of stamens and the hand separation
of stigmas from saffron flowers is labour intensive and leads to the high cost
of saffron stigmas (Tsaftaris et al., 2004). It
is desirable to have saffron flowers which do not form stamens, or even have
carpels in place of stamens, thus doubling saffron production in a single flower
while lowering the production cost. As C-class MADS-box gene function is essential
for both stamen and carpel formation (Tsaftaris et al.,
2005) recently characterized the expression of MADS-box genes in crocus
flowers using several molecular biology techniques, bioinformatics tools and
database resources. Such studies help in understanding and exploiting the molecular
mechanisms that control flower development in crocus and in realization of the
objective of producing flowers with carpels in place of stamens. Further, this
knowledge can even be used in molecular medicine. Recently T and B-cell epitopes
of Iranian Crocus sativus were mapped using bioinformatics tools and
the predicted peptides were found useful for vaccine development.
FUNCTIONAL GENOMICS OF SAFFRON
The primary motivation of functional genomics research in saffron is to narrow
the list of candidate genes implicated in the biological processes involved
in the production of flavoring compounds and stigma pigments, so that their
expression can be enhanced using a transgenic approach and hence improve quality
of the saffron stigma. Bioinformatics can play an enormous technical role in
sequence-level structural characterization of saffron genomic DNA. Earlier,
the only major information resource for modern genotyping and sequence characterization
available to saffron biologists was the Arabidopsis thaliana (L.) Heynh.
genome, published in 2000 (The Arabidopsis Genome Initiative,
2000). This information resource was further strengthened with the completion
of the rice (Oryza sativa L.) genome project and the availability of
the rice genome sequence. Even though several crop genome-sequencing projects
are rapidly constructing a rich and diverse repository of information about
plant DNA sequences, an important database for saffron has been designed recently
to manage and explore the Expressed Sequence Tags (ESTs) from saffron stigmas
(DAgostino et al., 2007). The database is
the first reference collection for the genomics of Iridaceae, for the molecular
biology of stigma biogenesis and for the metabolic pathways underlying saffron
GENES EXPRESSED IN CROCUS STIGMAS
DAgostino et al. (2007) produced 6,603
high quality ESTs from a saffron stigma cDNA library and grouped these into
1,893 clusters, each corresponding to a different expressed gene. The complete
set of raw EST sequences and their electopherograms are maintained in a database.
This allows users to investigate sequence qualities and EST structural features.
Putative transcripts determined to be associated with enzymes are organized
into classes and can be viewed in terms of enzyme assignments to metabolic pathways.
This represents a straight forward way to investigate the properties of the
stigma transcriptome which contains a series of interesting sequences (putative
sex determination genes, lipid and carotenoid metabolism enzymes, transcription
factors), whose function can now be tested using in vivo or in vitro
approaches. A contig (from contiguous) is a set of overlapping DNA segments
derived from a single genetic source and is used to deduce the original DNA
sequence of that source. Several such contigs have been characterized in saffron
genome and based on the presence of tentative consensus sequences categorized
into groups of putative function. The important ones include:
||Cl000944: 1 encoding non-heme-β-carotene-hydroxylase,
highly expressed in saffron stigmas (Castillo et al.,
||Cl000627: 1 encoding a putative glucosyltransferase, very similar
to UGTCs2 which is able to glycosylate crocetin in vitro (Moraga
et al., 2004)
||Cl001532: 1 and Cl001032:1 encoding putative isoprenoid GTases,
one of which could represent the still missing enzyme responsible for the
glycosylation of picrocrocin
||Cl000348: 1 encoding a Myb-like protein with high similarity to
LhMyb (from Lilium, GenBank accession BAB40790), Myb8 (from Gerbera) (Elomaa
et al., 2003) and Myb305 (from Antirrhinium) (Jackson
et al., 1991) and probably acting as a putative transcription
Further, a large number of Cytochrome P450 sequences are expressed in saffron
stigmas, some at very high levels (DAgostino et
Tissue culture studies of monocots: Saffron is a monocotyledon member
of the large family Iridaceae. Comparatively, bulbous and cormous monocotyledons
are regarded as difficult in vitro material. Contamination is a serious
problem during micropropagation of monocots especially if below ground organs,
such as corms, bulbs, rhizomes and tubers, are used as an explants source. The
size of geophyte, physical damage and dormancy are the other problems which
make tissue culture studies difficult. Tissue culture is presently mainly used
as a tool to facilitate a better understanding of the bio-chemical synthesis
of Saffron secondary products. Regeneration/proliferation ability of the corms
was dependent on genotype, type of explants, culture initiation time and composition
of the culture medium. The plants were able to form shoots or corms within 5-30
weeks of the start culture (Zaffar et al., 2004b).
Schenk and Hildebrandt (1972) reported the importance
of medium composition and techniques for induction and growth of monocotyledonous
and dicotyledonous plants in cell culture. They found that a high level of auxin-type
growth regulating substances generally favored cell cultures of monocotyledonous
plants, while low levels of cytokinin were essential for most dicotyledonous
cell cultures. Within the last few decades, an increasing number of bulbous
and cormous monocotyledons have been successfully cultured. Tissue culture technology
was greatly influenced by the demand of rapid multiplication and clonal propagation
of slow-growing monocots. Several economically important monocot species constituting
nutritional, medicinal or ornamental groups of plants were used for in vitro
clonal propagation (Sutter, 1986) and production
of secondary metabolites (Aslanyants et al., 1988).
Organogenesis and somatic embryogenesis from differentiated tissues of bulbous
and cormous monocots, such as Crocus sativus seeds. A corm survives for
only one season, producing up to ten "cormlets" that eventually give rise to
new plants. Therefore, reproduction is human dependent; the corms must be manually
dug up, broken apart and replanted. The natural propagation rate of most geophytes
including saffron is relatively low. Besides conventional methods of propagation,
in vitro cultural methods contribute importantly for the propagation of many
important and economic plants. Conventional propagation methods are very slow
and propagation by tissue culture represents an important potential to effectively
propagate it (Fernandez, 2004). Darvishi
et al. (2006) reported that treatment containing 2 mg L-1
NAA and BAP with highest Mean Rank had the best effect on induction of nonembryogenic
callus and treatment containing 1 mg L-1 2,4-D and BAP had the best
effect on induction of embryogenic callus.
Organogenesis of saffron: Organogenesis means the development of adventitious organs or primordia from an explant source. Direct and indirect are the two types of this method. In direct organogenesis, a cell or a group of cells differentiate to form organs. Indirect organogenesis is the development of adventitious organs originating from an intervening callus phase. Callus is an unorganized or undifferentiated mass of proliferative cells produced in culture and also in nature. It is made up of a mass of loosely arranged thin walled parenchyma cells arising from the proliferating cells of parent tissue. Many plant regeneration studies concerning saffron via direct and indirect organogenesis have been reported.
Organogenesis studies for in vitro propagation purposes: Plessner
et al. (1990) reported in vitro corm production in saffron.
Corms smaller than 1cm in diameter and isolated apical buds from larger corms
were used as explants. The nutrient medium consisted of MS (Murashige
and Skoog, 1962) minerals, supplemented with sucrose (3%), nicotinic acid
(5 mg L-1), pyrodoxine-HCL (1 mg L-1), thiamine-HCL (0.5
mg L-1), myo inositol (100 mg L-1), adenine sulphate (160
mg L-1) and casein hydrolysate (500 mg L-1). The medium
was solidified with 0.9% agar. 1 mg L-1 2,4-D (Dichlorophenoxyacetic
acid), 3-12 mg L-1 kinetin and 3 mg L-1 zeatin were used
as growth regulators. They reported that cytokinins, particularly zeatin and the
auxin 2,4-D were essential for regular bud development in vitro. They also
examined the effects of ethylene and ethapon on organogenesis. Ethylene and ethaphon
pretreatments inhibited leaf development but on the other hand, induced corm production
as well as dormancy. However, rooting could not be achieved. Direct adventitious
shoot regeneration from ovary explants of Crocus sativus L. was revealed
by Bhagyalakshmi (1999). Media components, incubation conditions
and age of the explants were the factors influencing the regeneration. Full strength
MS medium supplemented with NAA (naphthelene acetic acid) and BA (benzyladenine)
produced the best response towards caulogenesis (28%) with highest shoot numbers
per ovary. On the whole, the best response toward shoot growth, both in terms
of leaf length and number, was on the medium with 0.54 μM NAA and 2.22 μM
BA. Ovaries of different growth stages having stigmas of pale yellow, pale orange
and bright orange regenerated a maximum mean number of shoots per ovary. Further
development of ovary-derived shoots was influenced by the composition of basal
salts in the culture medium where full strength MS salts gave the best response
of those tested. Regenerated shoots produced normal photosynthetic leaves and
corms. Plant regeneration studies of saffron via indirect organogenesis from callus
cultures have also been reported. Ilahi et al. (1987)
described the morphogenesis in saffron tissue culture. Corms of saffron were cultured
on half strength MS medium supplemented with different combinations of growth
regulators; i.e., auxin and cytokinins and coconut milk. Callus was induced in
a medium containing 0.5 mg L-1 each of 2,4-D and BAP and 2% coconut
milk. The same culture was used for differentiation of callus into buds. They
reported that an increase in 2,4-D also enhanced callus formation but suppressed
shoot-bud formation. These shoots were induced to root when inoculated on a medium
containing 2 mg L-1 NAA for 24 h. However, further growth of these
roots was slow when re-inoculated on half strength MS containing 0.1 mg L-1
each of 2,4-D and BAP. In another set of experiments when a piece of callus, growing
in similar conditions, was transferred to MS medium containing 0.5 mg L-1
NAA, 0.1 of either BAP or kinetin and 2% coconut milk, the nodules gave rise to
roots after 4 weeks of culture with subsequent suppression of the shoot development.
The micro-corms obtained with the help of tissue culture using Murashige and Skoogs
medium supplemented with 6-12% sucrose, 0.1-1.0 mg L-1 it-benzylamino-purine
(BA) and Paclobutrozol (PAC) 1-10 mg L-1 it have a high survival rate
upon transferring directly in soil (Zaffar et al., 2009b).
Chaloushi et al. (2007) reported that the treatment
of NAA and BAP (each 1 mg L-1) as the best hormonal treatment for the
plantlet regeneration from the domestic saffron calli.
Somatic embryogenesis of saffron: Somatic embryogenesis is the process
of a single cell or a group of cells initiating the developmental pathways that
lead to reproductive regeneration of non-zygotic embryos capable of germinating
to form complete plants. There are few studies in the literature about the regeneration
of saffron via somatic embryogenesis. Ahuja et al.
(1994) indicated the somatic embryogenesis and regeneration of plantlets
in saffron. Somatic embryogenesis was initiated in Crocus sativus from
shoot meristems on LS medium containing 2x10-5M BA and 2x10-5M NAA. They observed
the various stages of somatic embryogenesis in the same medium and the development
was asynchronous. Maturated embryos could be germinated on half strength MS
medium containing 20 mg L-1 gibberellic acid (GA3). Complete
plantlets with well developed root system and corm formation were obtained on
transferring germinated embryos to half strength MS supplemented with 5x10-6M
BA, 5x10-6M NAA and 2% activated charcoal. Somatic embryogenesis in saffron
was described by also Blazquez et al. (2004).
They used MS culture medium supplemented with 0.5 mg L-1 BAP and
0.1 mg L-1 2,4-D for induction of somatic embryogenesis. Embryogenic
calli were subcultured in MS medium containing 1 mg L-1 BAP and 0.05
mg L-1 NAA for multiplication in solid medium. Temporary immersion
systems (TIS) were used for this purpose. A four-fold increase in the production
of embryogenic calli (fresh weight increase) was observed in tissue culture
when compared to solid medium. They obtained the best result when 1 mg L-1
of paclobutrazol was added. They also improved the development of somatic embryos
on solid medium supplemented with 0.5 mg L-1 jasmonic acid (JA) and
obtained plant regeneration via somatic embryogenesis after eight weeks of treatment
of JA in combination with sucrose According to Raja et
al. (2007). Organogenisis and somatic embryogenesis have shown that
auxin such as 2,4-D along with cytokinin BA is essential for somatic embryogenesis
from leaf explants of saffron (Crocus sativus L.); the auxin or auxin
in combination with cytokinins used in the medium can greatly influence the
frequency of induction and also on maturation of somatic embroys.
Karamian (2004) indicated the plantlet regeneration
via somatic embryogenesis in four species of Crocus. Shoot meristem culture
on LS medium containing 4 mg L-1 NAA and 4 mg L-1 BA or
1 mg L-1 2,4-D and 4 mg L-1 kinetin was used for somatic
embryogenesis in C. sativus, C. cancellatus, C. michelsonii and C.
caspius. Asynchronous somatic embryogenesis in all of the four species was
investigated and various stages of somatic embryo development were observed
when embryogenic calli with globular somatic embryos were transferred into half
strength MS medium containing 1 mg L-1 abscisic acid. According to
Karamian (2004), maturated embryos could be germinated
on half strength MS medium supplemented with 25 mg L-1 GA3.
Finally, complete plantlets were obtained by transferring germinated embryos
into half strength MS medium supplemented with 1 mg L-1 NAA and 1
mg L-1 BA at 20°C under 16/8 h (light/dark) cycle.
Organogenesis studies for secondary metabolite production purposes: In
vitro production of stigma-like structures of Crocus sativus for
the purpose of crocin, picrocrocin and safranal induction was also reported.
Stigma-like structures were reported to be induced from almost every part of
floral organs, including half ovaries (Himeno and Sano, 1987;
Loskutov et al., 1999), stigmas (Koyama
et al., 1988; Sarma et al., 1990),
petals (Lu et al., 1992), anthers (Fakhrai
and Evans, 1990) and stamens (Zhao et al., 2001).
Himeno and Sano (1987) described the synthesis of crocin,
picrocrocin and safranal by saffron stigma-like structures proliferated in
vitro. One young halfovary (0.3 mg, fresh weight) was placed on each medium
and incubated at 20°C, in the dark. Some of the stigma-like structures were
formed on Linsmaier-Skoog medium (LS) supplemented with NAA (10 ppm) and kinetin
(1 ppm) and the others were formed on Nitsch medium supplemented with NAA (1
ppm) and BA (1 ppm). The stigma-like structures were formed directly on the
explants after 10weeks. They found that the average of concentration of crocin
and picrocrocin in the stigma-like structures grown on Nitsch medium were about
3-fold higher than those grown on LS medium, while the total contents in each
structure were about the same. Loskutov et al. (1999)
studied the optimization of in vitro conditions for stigma like structure
production from half-ovary explants of Crocus sativus. The optimum proliferation
of stigma-like structure was observed on B5 basal medium (Gamborg B5 medium)
containing NAA (5.4 μM), BA (44.4 μM), MS organics, casein hydrolysate
(0.05%) and L-alanine (11.2 μM). They reported that the amounts of crocin,
crocetin, picrocrocin and safranal in stigma-like structure, as determined by
high performance liquid chromatography analysis, were similar to those found
in natural saffron. Sarma et al. (1990) also reported
in vitro production of stigma-like structures from stigma explants of
Crocus sativus L. MS medium supplemented with NAA (10 mg dm-3) and BA
(1 mg dm-3) induced the optimum response. NAA was found to be an important additive
to achieve a good response. Zeng et al. (2003)
recorded the increased crocin production and induction frequency of stigma-like
structures from floral organs of Crocus sativus by precursor feeding.
MS medium supplemented with 5 mg L-1 kinetin and 4 mg L-1
NAA was used as the basal medium. Almost all of the stigma-like structures formed
directly from explants, instead of from callus. Induction of crocin, crocetin,
picrocrocin and safranal synthesis in callus cultures of saffron was reported
by Visvanath et al. (1990). Callus cultures were
obtained from floral buds on MS medium supplemented with 2 mg L-1
2,4-D and 0.5 mg L-1 kinetin. The cultures could be induced to produce
red globular callus and red filamentous structures which produced crocin, crocetin,
picrocrocin and safranal. Crocin production using Crocus sativus callus
by two stage culture system was reported by Chen et al.
(2003). Saffron callus was grown in a two-stage culture on B5 medium supplemented
with casein hydrolysate (300 mg L-1) at 22°C in dark with 2 mg
L-1 NAA and 1 mg L-1 BA to give maximum biomass (16g dry
wt/L) and with 2 mg L-1 IAA (indole-3-acetic acid) and 0.5 mg L-1
BA for crocin formation. The maximum crocin production (0.43 g L-1)
was achieved by this two-stage culture method. Chen et
al. (2004) also examined the promotion of growth of Crocus sativus
cells and the production of crocin by rare earth elements. They reported that
La3+and Ce3+, either individually or as a mixture, promoted crocin production
of Crocus sativus callus but Nd3+(40 μM) had little effect and all
metal ions were toxic above 100 μM. La3+(60 μM) promoted growth of
callus significantly but increased crocin only slightly. Ce3+(40 μM) significantly
promoted crocin production but had little effect on cell growth. They showed
that La3+(60 μM) and Ce3+(20 μM) together gave the highest dry weight
biomass (20.4 g L-1), crocin content (4.4 mg g-1) and
crocin production (90 mg L-1). Although most of the studies about
in vitro production of saffron secondary metabolites via direct and indirect
organogenesis were reported in the literature, there were very few studies related
to the achievement of a whole plant. All these studies make clear that there
were no problems with the callus and shoot formation of saffron. However, corm
and especially root formation were very rare events in organogenesis of saffron.
Sheibani et al. (2007) reported that Thidiazuron
(TDZ) concentrations affected the induction of somatic embryogenesis significantly
while different types of corm explants showed no significant effect on this
process. Among TDZ concentrations used, 0.5 mg L-1 was the most effective
treatment for embryogenesis induction. Embryogenic calli (globular stage) proliferated
well when subcultured into MS medium supplemented with 0.25 mg L-1
TDZ before transferring to hormone-free MS medium containing 6% sucrose for
maturation (scutellar or horn-shape stage).
Ex vitro studies of saffron: The effect of gibberellin on functional
activity of dormant saffron corms were reported by Azizbekova
et al. (1982). At the time of transplantation, saffron corms were
immersed in a solution of gibberellin (100 mg L-1) for 4 h. Corms
immersed in water for 4 h served as the control. After these treatments, cormswere
planted in soil. They demonstrated that treating saffron corms with gibberellin
leads to subsequent acceleration of growth and development processes, increase
of leaf and root length and increase in the number of flowers. Unfortunately,
there were not many ex vitro studies about saffron in the literature.
However, ex vitro studies of Gladiolus were reported more than
saffron as explained in the following section. This species is also monocot
with corms and is very similar to saffron. Therefore, it was thought that these
studies could also be applicable for saffron.
The effectiveness of chemicals such as gibberellins (Arora
et al., 1992), BA (Goo et al., 1998),
ethephon (Suh, 1989) and methyl disulfide (Hosoki
and Kubara, 1989) in breaking the dormancy of Gladiolus corms and
cormels has been studied extensively. Ram et al. (2002)
indicated that plant growth regulators affect the development of both corms
and cormels in gladiolus. Three plant growth regulators, BA (25, 50 and 100
mg L-1), ethephon (100, 200 and 400 mg L-1) and GA3
(25, 50 and 100 mg L-1) were tested on fresh cormels. The cormels
were soaked in the solutions for 24 h and they planted in the field. In conclusion,
ethephon at 400 mg L-1 was most effective treatment for many of the
growth parameters including days to sprouting, percentage sprouting, corm diameter
and cormel production and weight.
Ex situ conservation and multiplication: Genetic materials are
preserved in the form of corms (saffron and wild relatives) and seeds (only
wild species). General conservation and multiplication strategies for the collections
are the standards for international genebanks outlined by Engels
and Visser (2003). In addition, specific strategies and bank design for
the ex situ conservation were established mainly based on information regarding
the source of the species, reproductive biology, mode of multiplication, sample
type and objectives of the collection. The design comprises three main collections.
||Reserve vegetative collection (saffron and allies):
Ten corms of each accession were sown in special flower-pots with substrate
of the collecting zone and specific mixture (soil enriched with organic
matter mixed with sand) and placed in a greenhouse with semi-controlled
conditions. Irrigation and weeding were done by hand when necessary (Khoury
et al., 2010)
||Exchange vegetative collection (saffron and allies): The accessions
(40 corms each) were sown in the experimental farm (field conditions) in
a 10-15 cm furrow, with 15 cm among plants and 50 cm among furrows. Conventional
laboring was used to prepare soil for seeding and previous fertilization
applying N (80 UF), P (100 UF) and K (100 UF) was carried out. Irrigation
schedule was applied depending on the climatic conditions
||Seed collection (wild crocus): The acquired seeds both from the
wild and from seed harvesting in BGV-CU were placed in hermetic jars including
silica gel inside and stored in a refrigerated chamber at 4°C and 30%
relative humidity (Agrawal et al., 2007)
Germplasm distribution: A basic criterion for supplying plant materials
including dates of request and sending has been established based on phenology
of the materials and facilities at the BGV-CU. At the short time the provision
of genetic materials (corms, leaves, styles, anthers and seeds) is addressed
to carry out the complete characterization/evaluation of the collection. From
the next 2 years users can consult the availability of materials (through the
CIS or by contacting the curator) in order to request accessions. Before the
user receives the materials a Material Transfer Agreement between donor and
recipient must be signed. Germplasm characterization descriptor definition A
descriptor list for whole characterization and evaluation of the genus has been
defined and improved during the last 3 years (unpublished data). It includes
characterization descriptors (95 traits), evaluation descriptors (47 traits)
and descriptors based on genetic markers technologies and cytological characters
(14 traits) and embraces a diverse set of data (morphological, phenological,
agronomical, phytochemical, molecular, etc.), with the aim of being a useful
tool for the description of the genetic variation in the genus Crocus. Characterization/evaluation
Germplasm characterization is an important operation for a genebank since the
value of the germplasm collection depends on the availability and quality of
the information relative to the preserved accessions. Therefore, one of the
main goals of CROCUSBANK action is to strategically characterize and evaluate
germplasm of saffron and allies at different levels. A partial characterization/evaluation
of the collection have been developed during last years taking into account
morphological, phenological, agronomical, resistance to salt stress, phytochemical
and molecular characters. Sixty-six saffron accessions have been characterised/evaluated
for morphological, phonological and agronomical traits and the existence of
variability has been observed, suggesting the existence of genetic differences
among the accessions related to the geographic origin of the materials. These
preliminary results are being confirmed by the data obtained in other approaches.
A different sensitiveness to saline stress has been recorded among some of the
above mentioned saffron accessions in relation to genotypes origin (unpublished
data). The phytochemical analysis using gas chromatography (GC-MS) and/or spectroscopic
methods (FT-IR, Raman) also indicates that potential variability occurs among
saffron accessions (unpublished data). In the same way genomic AFLP and SNPs
markers have been identified in a subset of accessions, showing clearly the
existence of genetic variation in saffron crop (unpublished data). In addition,
these genetic markers provide a specific genetic fingerprint that could be very
useful for the rationalisation of the bank. The significant genetic variability
found in saffron, evidenced with the on-going characterization/evaluation studies,
opens the door to unravel the peculiarities of >land varieties=
of this minor but highly appreciated Mediterranean crop. Accordingly, these
results scientifically support the importance of conserving the local and precious
cultivated germplasm worldwide. Similar studies have been programmed or are
being developed indeed for other Crocus species integrated in the WSCC, however,
the shortage of materials in most accessions is by the moment a limiting factor
to develop more extensive studies. Anyway, preliminary trials considering different
kind of traits (mainly morphological, phenological and molecular, but also salt
stress resistance and phytochemical in a lesser extent), have revealed both,
interspecific and intraspecific variability, in 34 accessions belonging to 21
species (unpublished data). That information may be of interest for different
purposes (commercial gardening, bank rationalization, taxonomic or evolution
studies, etc.), although much work remains to be done in the future with these
materials. Future actions and prospects on the WSCC (World saffron crocus
collection). The WSCC has already a wide representation of the Crocus germplasm
of plausible utility in saffron breeding which has never been achieved before.
Additionally, for the first time worldwide it has been created a unique collection
which contains a large part of the variability of the saffron crop and wild
relatives at global scale for common use. Therefore, priority actions to make
useful the genetic resources to potential users are needed (Pastor
et al., 2010).
Evaluation and genetic enhancement: A coordinated and integrated effort
to evaluate the WSCC(World saffron crocus collection) is needed to identify
useful genotypes (e.g.) sources of biotic and abiotic tolerance, genotypes with
optimal production of secondary genotypes interesting for improving saffron
yield, etc.) Rationalization of the collection (Fernandez,
2007). The reduction of the collection size, without loss in the genetic
variability is a very desirable approach in order to preserve the collection
and to promote its utilization (e.g. creation of a core collection) Utilization
and dissemination. A new more dynamic portal exclusively for the WSCC (World
saffron crocus collection) utilization and disseminations is being designed.
Its based on Joomla CMS (www.joomla.org)
and MySQL database with allows large numbers of people to contribute and share
stored data and to improve communication between users through blogs, forums,
Future strategies: Conventional breeding methodologies have not led
to any improvement in saffron, necessitating exploring alternative procedures
like biotechnological, molecular biological interventions. In order to achive
short, medium and long term goals for bringing about enhancement in productivity
per unit area which can lead to increase in net returns to farmers and encourage
them to continue growing saffron, the following strategies are recommended.
||Exploring possibilities of induction of site specific mutations
through in vitro culture for enhancing stigma length, size and its
||Induction of polyploidy (hexaploidy) through in vitro culture in
cell cultures for breaking sterility barriers and open vistas for over coming
sterility barrier and application of breeding procedures in enhancing yield,
tolerance to biotic and abiotic stresses
||Identification of putative parents (through molecular biological interventions)
and their collection/conservation for future allele mining
||High priority should be given on collection/Characterization of Crocus
taxa, wild relatives and local varieties
||Detailed genetic maps to be developed would provide valuable tool for
the identification of important genes
||The establishment of markers for important genes should enable the selection
of superior genotypes and the pyramiding of genes from several genetic background
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