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The advent of genomics in mulberry and perspectives for productivity

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DOI: 10.1007/s00299-011-1059-1 · Source: PubMed

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Plant Cell Rep (2011) 30:825–838
DOI 10.1007/s00299-011-1059-1

REVIEW

The advent of genomics in mulberry and perspectives

for productivity enhancement
Paramjit Khurana • Vibha G. Checker

Received: 31 January 2011 / Accepted: 10 March 2011 / Published online: 23 March 2011
Ó Springer-Verlag 2011

Abstract Sericulture in India is a highly remunerative Keywords Amelioration  Functional genomics  Genetic
industry, especially for the rural population. Mulberry is an transformation  Mulberry  Stress
extremely versatile plant, having multifaceted applications,
the most important being the sole feed for the monophagus
silkworm, Bombyx mori. Profitability of the sericulture Introduction
industry is directly correlated with production of high-
quality mulberry leaves. However, mulberry productivity is Silk, known as ‘‘Queen of textiles’’, originated in ancient
severely impacted by abiotic as well as biotic stresses. China where its use was reserved for the royalty and has
Therefore, to develop stress-tolerant mulberry with desired continued to lure people through antiquity. Silk is associ-
characteristics, a comprehensive understanding and utility ated with luxury, elegance, class, and comfort. Sericulture,
of biotechnological resources is essential. Research efforts both an art and a science of raising silkworms for silk
on mulberry encompass broad range of fields in plant production, has better prospects in developing countries as
biology from breeding, molecular markers, transcripto- silk production is largely a cottage industry. India has the
mics, proteomics, and metabolomics. Additionally, a large distinct advantage of practicing sericulture throughout the
number of mulberry germplasm accessions have been year, yielding about 4–6 crops/year as a result of its trop-
maintained and evaluated in several countries. Identifica- ical climate (Gangopadhyay 2008). Trends in international
tion of superior cultivars under stressed regimes is extre- silk production indicate that India is the second largest
mely important, and therefore, physiological traits have producer of raw silk, accounting for more than 18% of
often been used as proxy genetic markers for assessing global raw silk production. India has also the unique dis-
stress tolerance index. Mulberry genomic resources have tinction of being the only country producing all the four
provided a limited but an important list of novel candidate kinds of silk—mulberry, eri, muga, and tasar, but the major
genes, thus enhancing the scope for future investigations quantity of silk is produced by cocoons of Bombyx mori,
for improvement of its productivity. The present review the silkworm. The insect being monophagus feeds only on
article gives a bird’s eye view of current initiatives of the leaves of mulberry plants; therefore, mulberry culti-
genomics advancements in mulberry research and enu- vation plays a cardinal role in the sericulture industry.
merates the prospects for enhancing its productivity. Mulberry is an important component of combined pastoral
system (Talamucci et al. 2000) as its leaves are used as a
forage supplement for animal husbandry (Benavides 2000).
Communicated by R. Reski.
Mulberry contains unique medicinal compounds of great
A contribution to the Special Issue: Plant Biotechnology in Support of pharmaceutical worth in its leaf, fruit, stem, seed, and roots
the Millennium Development Goals. having anti-microbial, anti-hyperglycemic, anti-hyperlipi-
demic, antidiabetic, chemopreventive, neuroprotective, and
P. Khurana (&)  V. G. Checker
anti-oxidative potential (Andallu and Varadacharyulu
Department of Plant Molecular Biology, University of Delhi,
South Campus, Dhaula Kuan, New Delhi 110021, India 2003; Chen et al. 2006; Chen and Li 2007; El-Beshbishy
e-mail: param@genomeindia.org et al. 2006; Kang et al. 2006; Konno et al. 2006; Singab

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826 Plant Cell Rep (2011) 30:825–838

et al. 2005). Mulberry is also used for landscaping in Asia, established by Linneaus in 1753, was mainly based on
Europe, and America (Tipton 1994). Global climatic morphological characters, but major contributions to the
changes and scarcity of land and water in near future make systematics of Morus were made by Koidzumi (1917,
it mandatory to develop varieties suitable for different 1923). Mulberry species were grouped into two sections,
agro-climatic conditions for sustainable development of the namely the Dolychostylae and Macromorus, based on the
sericulture industry. This review illustrates in a broad length of the styles in the female flowers; each section was
sense, how recent biotechnological advances in mulberry subdivided by the nature of stigmatic hairs, and a total of
will generate advanced technologies for mulberry cultiva- 24 species were recognized. Four mulberry species (Morus
tion leading to economic improvement in sericulture, indica, Morus alba, Morus laevigata, and Morus serrata)
impacting quality of life of those involved in sericulture have been reported from India, and more than 68 species
practices. Figure 1 broadly depicts the information flow in are widely recognized in the world (Datta 2000). Conser-
diverse aspects of genomics research in mulberry. Refer- vation of genetic variability in mulberry is of utmost
ence list has been kept minimal in this article for the want importance for present and future use of mulberry breeders.
of space. Mulberry genetic resources are conserved mainly through
Mulberry (Moraceae) is a fast-growing perennial tree clones as mulberry is a clonally propagated plant. Mulberry
maintained as a shrub, occurring in wide range of areas germplasm conservation involves several activities such as
around the world, i.e. from the tropical, sub-tropical, to acquisition and characterization of germplasm, conserva-
temperate climates. Mulberry is believed to have originated tion procedures, and evaluation of genetic and agronomic
in the lower slopes of the Himalayan range, bordering traits (Tikader et al. 2009). Central Sericultural Germplasm
China and India. Currently, it is grown in warm and moist Resources Center is actively involved in conservation and
climates between latitudes 50°N and 10°S which include strengthening of mulberry germplasm in India as 1,109
southeastern region of Asia, Europe, North and South diverse accessions including 845 indigenous and 264 exotic
America. Mulberry is a dioecious plant with male and accessions have been conserved. Cryopreservation proto-
female plants occurring separately, and this fact deserves cols have been standardized for conservation of diverse
special attention. The breeding work in this plant is ham- mulberry germplasm accessions ensuring sustainable uti-
pered due to the extremely asynchronous flowering, espe- lization of these diverse resources. Storage potential of bud
cially in the tropical varieties. In vitro modification of sex grafts of different Morus species maintained at a temper-
expression in mulberry has been reported by Thomas ature of -1.5°C proved to be the best where the survival of
(2004). In general, mulberry is diploid (2n = 2x = 28), but bud grafts was observed up to the fourth month of storage
the ploidy level varies from 2n = 28 to 308 (Dandin and with the survival ranging from 63.33 to 86.67%. Genetic
Basavaiah 1995; Katsumata and Ishiguro 1980; Yile and stability studies based on DNA markers generated with
Oshigane 1998). The classification of genus Morus, firstly ISSR primers indicated no difference between mother plant

Fig. 1 Information flow in

DNA Markers EST Libraries
existing areas of research
(dashes) and under-represented
areas (boxes) of genomic
approaches in mulberry

Genetic linkage maps Germplasm evaluation Genome annotation

Breeding Genetic Engineering

Metabolomics/ Proteomics

Mulberry Improvement

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Plant Cell Rep (2011) 30:825–838 827

and in vivo regenerated cryosamples (Atmakuri et al. germplasm for stress tolerance is listed in Table 1. Being
2009). Low survival of the wild species necessitates stan- an important crop, trait-based investigations have been
dardization of specific protocols. carried out extensively providing a benchmark for deci-
phering stress tolerance index. To exploit genetic diversity
in mulberry, a number of methods like in vitro (Hossain
Screening for genotypic stress tolerance et al. 1991; Lal et al. 2006; Rao et al. 1997; Tewary et al.
2000; Vijayan et al. 2003, 2004a), pot cultures (Ahmad and
Indigenous and exotic mulberry germplasm accessions Sharma 2010; Chaitanya et al. 2001, 2009; Lal and Khur-
exist in several countries. Genotypic and phenotypic vari- ana 2009; Vijayan et al. 2008), and leaf disk assays (Das
ations exist in mulberry germplasm as different physio- et al. 2011a) have been adopted for screening of tolerant
logical, biochemical, and morpho-anatomical responses are cultivars. In vitro screening is labor-inexpensive and has
evoked in a plant under different environmental conditions. several advantages over pot culture method as large num-
In order to utilize these genetic resources efficiently, ber of genotypes can be rapidly screened. Stress combating
comprehensive evaluation of specific traits is necessary for strategies that allow higher plants to tolerate prolonged
breeding programs. Mulberry plantation is adversely periods of stress can involve numerous attributes; there-
affected by a wide range of stresses, drought and salinity fore, integration of different parameters for identification of
being the most devastating as nearly 50% of mulberry superior cultivars under stressed regimes is extremely
acreage falls under arid and semi-arid conditions. More- important for an agronomical viewpoint. Candidate traits
over, high yielding mulberry cultivars have high water conferring yield stability under multiple stresses should be
demands due to faster growth rates, large cumulative leaf identified, and for the optimal exploitation of wide genetic
area, and canopy size; therefore, water deprivation can variability occurring in mulberry, field evaluations must be
arrest the growth and yield performance of mulberry conducted to ensure multiple stress tolerance attributes
genotypes (Guha et al. 2010a). A summary of the important (Guha et al. 2010c). Initial attempts were made (Hossain
reports available on characterization of mulberry et al. 1991; Vijayan et al. 2003) for screening salt-tolerant

Table 1 Summary of the

Stressor No. of Parameters studied References
important reports available on
varieties
characterization of mulberry
germplasm for stress tolerance PEG 9 Morpho Rao et al. (1997)
Coastal saline soils 12 Phy, Biochem Agastian and Vivekanandan
(1997)
NaCl 2 Phy, Biochem Giridara Kumar et al. (2000)
NaCl 5 Morpho Tewary et al. (2000)
High temperature 1 Biochem Chaitanya et al. (2001)
Water withholding 1 Biochem Barathi et al. (2001)
NaCl 63 Morpho Vijayan et al. (2003)
Water withholding 5 Phy, Biochem Ramachandra Reddy et al. (2004)
NaCl 4 Phy, Biochem Lal et al. (2006)
NaCl 5 Morpho, Biochem, Anat Vijayan et al. (2008)
NaCl 5 Phy, Biochem Lal and Khurana (2009)
Drought 4 Phy, Biochem Ren (2009)
Drought 5 Biochem Chaitanya et al. (2009)
NaCl 11 Morpho, Biochem, Phy Vijayan et al. (2010)
Drought 15 Morpho, Biochem, Phy Guha et al. (2010a)
Drought 4 Morpho, Biochem, Phy, Guha et al. (2010b)
Anat
Drought 4 Morpho, Biochem, Phy Guha et al. (2010c)
NaHCO3 2 Biochem, Phy Ahmad and Sharma (2010)
Morpho morphological, Phy Control 9 Phy, Mol Mishra and Dandin (2010)
physiological, Biochem Aerial drying and 10 Biochem, Phy, Mol Das et al. (2011a)
biochemical, Anat anatomical, NaCl
Mol molecular

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828 Plant Cell Rep (2011) 30:825–838

lines by axillary bud culture under saline conditions. the effect of nutrient deprivation, a controlling factor for
Salinity tolerance was also assessed by scoring for seed biomass production and yield efforts were initiated to study
germination in different mulberry genotypes under in vitro morphology and oxidative physiology of magnesium-,
conditions (Vijayan et al. 2004a). Effect of salt stress has boron-, phosphorous-, and sulfur-deficient mulberry plants.
also been investigated on growth of popular Indian culti- Nutrient deficiencies aggravate oxidative stress via
vars by considering physiological and biochemical enhanced generation of reactive oxygen species in mul-
parameters like free proline accumulation, photosynthetic berry plants (Subbarayappa et al. 2009; Tewari et al. 2006,
yield, nitrate reductase activity, cellular membrane stabil- 2009, 2010).
ity, relative water content, and carbon isotope discrimina- As for biotic stresses, bacterial and fungal leaf spot,
tion (Lal et al. 2006; Lal and Khurana 2009). These powdery mildew, leaf rust, and root knot are the major
analyses were supplemented by expression analysis of diseases which not only reduce mulberry leaf productivity
cellular transporter, like MiNHX in contrasting genotypes (10–20%) but also affect leaf quality abysmally. Therefore,
(Lal and Khurana 2009). In mulberry, variability occurs selection of promising mulberry germplasm with stable
among closely related species in response to salinity stress resistance integrated into breeding programs is of utmost
which may be attributed to a wide range of modifications at importance. However, single or multiple genes for resis-
different levels of organization in the plant. Various tance in the identified sources need further qualitative and
approaches for enhancing salt tolerance in mulberry have quantitative genetic analysis. Efforts to evaluate resistance
been elaborated by Vijayan (2009). Attempts to screen have been made to explore mulberry germplasm for
mulberry genotypes for drought tolerance started almost resistance against bacterial (Banerjee et al. 2009) and
two decades back. Nodal explant culture under drought fungal pathogens (Biswas et al. 1996; Chattopadhyay et al.
conditions served as an effective evaluation technique for 2010; Gangwar and Thangavelu 1998; Maji et al. 2009)
screening mulberry genotypes under simulated drought with cultivated varieties and/or breeding lines, but with
conditions (Rao et al. 1997; Susheelamma and Datta 1990). limited success. Therefore, innovative and modern
Presently, drought stress tolerance in the existing gene pool approaches to develop highly effective resistant varieties
is correlated with high yield, relative water content (RWC), for integration into breeding program are needed to evolve
activity of antioxidant enzymes, proline accumulation, multiple disease resistance in mulberry.
glycine betaine content, and increased ABA content (Bar-
athi et al. 2001; Chaitanya et al. 2009; Guha et al. 2010a, b,
c; Ramachandra Reddy et al. 2004; Ren 2009). In a detailed Deciphering genotypic diversity through molecular
investigation by Das et al. (2011a), ten genotypes of mul- markers
berry were screened against both salinity and drought
stresses, and expression profiles of stress-responsive genes The genetic characterization of promising varieties of
like halotolerance 3a, dehydrin, NHX1 and ABC trans- mulberry is important for their utilization in the sericulture
porter were compared in these genotypes. Mulberry industry. Traditionally, genetic diversity in mulberry has
genotypes were also analyzed for genetic polymorphism been determined by examining morphological and physi-
based on their photosynthetic efficiency to identify effec- ological traits (Biasiolo et al. 2004; Dandin and Jolly 1986;
tive parental combinations for the development of mapping Huo 2002; Machii et al. 1997), but characterization through
populations (Mishra and Dandin 2010). Singhal et al. morphological features fail to serve as unambiguous
(2010) studied leaf surface scanning electron microscopy markers as most of the phenotypic traits are under the
of 16 mulberry genotypes (Morus sp.) with respect to their control of developmental stages and environmental influ-
feeding value in silkworm rearing to study which varieties ences. With the advent of molecular biology, DNA markers
can be exploited for mulberry improvement programs are universally accepted as they are highly polymorphic,
considering favorable leaf surface characteristics in Indian multiallelic, universally reproducible, dispersed throughout
subtropics. Complementing morpho-anatomical studies the genome, and independent of tissue and environmental
with physiological characteristics, variability in moisture influence. A summary of work on molecular markers used
retention capacity (MRC) was examined by screening 290 to detect genetic diversity in mulberry is given in Table 2.
diverse mulberry accessions. Leaf MRC varied signifi- ISSRs have been the most common marker system used in
cantly among accessions and correlated strongly with leaf mulberry. They were also combined with RAPD (Awasthi
wax amount. This study indicated that mulberry accessions et al. 2004; Naik and Dandin 2005; Srivastava et al. 2004)
having elevated leaf surface wax amount and crystal size and SSRs (Zhao et al. 2007b) to reveal genetic variation
and density exhibit reduced leaf post-harvest water loss and among mulberry varieties. Single nucleotide polymor-
could provide the foundation for selective breeding of phisms (SNPs) have recently emerged as a powerful tool in
improved cultivars (Mamrutha et al. 2010). To investigate marker technology as they are the most abundant

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Plant Cell Rep (2011) 30:825–838 829

Table 2 Summary of work on molecular markers in mulberry

Marker Description References

RAPD (randomly Single short oligonucleotide primer which binds to many Xiang et al.( 1995), Feng et al. (1996), Lou et al. (1998),
amplified different loci, is used to amplify random sequences from a Bhattacharya and Ranade (2001), Naik et al. (2002),
polymorphic DNA) complex DNA template such as a plant genome Zhao and Pan (2004)
AFLP (amplified AFLP analysis is a technique through which selected Sharma et al.( 2000), Wang and Yu (2001), Kafkas et al.
fragment length fragments from digestion of total plant DNA are amplified (2008)
polymorphism) by polymerase chain reaction.
SSR (simple SSR also known as microsatellites, are tandem repeat motifs Aggarwal et al. (2004), Zhao et al. (2005)
sequence repeats) composed of one to six nucleotides, which are ubiquitous,
abundant and highly polymorphic in all eukaryotic genomes
ISSR (inter-simple Inter-simple sequence repeat (ISSR) is a novel PCR technique Vijayan and Chatterjee (2003), Zhao et al. (2006, 2007a),
sequence repeats) that uses repeat-anchored or non-anchored primers to Vijayan et al. (2004b, c, 2006), Kar et al. (2008)
amplify DNA sequences between two inverted SSR
SRAP (sequence- SRAP is based on two primer amplification that preferentially Zhao et al. (2009)
related amplified amplifies open reading frames. The forward primers
polymorphism) preferentially amplify exonic regions and the reverse
primers preferentially amplify intronic regions with
promoters.

polymorphism present in the genome of any organism test cross mapping strategy to provide reference informa-
(Collins et al. 1997). SNPs are single base variations tion for future molecular breeding work on Morus and its
widely distributed throughout the genomes with the fre- relatives. They selected 100 RAPD, 42 ISSR, and 9 SSR
quency of[1% in a population. However, the usefulness of primers that amplified 517 markers. Two separate male and
SNPs depends on sequence information available, and female maps were constructed using 94 each of female-
despite a reasonable number of ESTs in public databases, and male-specific test cross markers, containing 12 female
no SNPs have been reported from mulberry as of today. linkage groups and 14 male linkage groups. However, the
This necessitates the importance of expressed sequence female map covered an average distance of 15.75 cM and
tags (ESTs) to be generated in public databases for SNPs maximum map distance of 37.9 cM between two loci,
mining and developing other DNA-based markers. SNP while average distance of male map was 18.78 cM, max-
detection in mulberry can open new perspectives for imum distance between two loci being 34.7 cM. Though
genetic studies and will also be useful for association the markers were randomly distributed between the linkage
analysis of candidate genes with phenotypic variation, groups, the information generated has limited utility. A
quantitative trait loci (QTL) mapping, assessment of detailed linkage map integrating information from multiple
genetic diversity, and for synteny-based comparisons from mapping populations with sufficient markers is necessary
model crop species. Further, accumulation of available for QTL identification and marker-assisted selection. These
molecular markers will contribute to identification of QTLs maps need to be constructed with the aim of determining
for traits related to abiotic and biotic stresses. the relative position of transferable markers, increasing the
number of available DNA markers, obtaining saturated
maps, and comparing the locations of quantitative trait loci
Linkage map: the elusive hunt (QTL) and candidate genes of interest across germplasm.
The construction of a consensus linkage map for mulberry
A genetic map constructed from a population segregating should enable us to determine the stability of locus posi-
for a trait of interest is required for QTL identification tions across mulberry germplasm, as well as increase the
(Hong et al. 2010); therefore, genetic maps are an impor- number of loci in the linkage map.
tant component of mulberry research, underpinning the
improvement programs. Nevertheless, mulberry research
still lacks adequate tools and resources for genetic, geno- Mulberry breeding
mic research, and breeding, and therefore, expansion of the
basic knowledge of the genetic control of complex traits is Palatability of mulberry leaves to silkworms and produc-
required. First, genetic linkage map of mulberry was gen- tion of appreciable number of good quality cocoons are the
erated by Venkateswarlu et al. (2006) with 50 F1 full-sib main targets in mulberry breeding which depend on several
progeny using RAPD, ISSR, and SSR markers and pseudo- quantitative traits such as leaf retention capacity, leaf size

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830 Plant Cell Rep (2011) 30:825–838

and weight, total biomass, resistance to pest and diseases, species like M. laevigata and M. serrata in the existing
tolerance to abiotic stresses like drought, salinity, and cold breeding programs can provide sufficient genetic diversity
stress. The horizontal expansion of sericulture has suc- as they may harbor novel genes regulating important traits
ceeded as traditional methods of plant breeding have made like abiotic and biotic responses (Tikader and Dandin
significant contributions to mulberry improvement by 2007). Mulberry being a perennial woody plant has long
developing several varieties with desirable agronomic traits juvenile periods, and it takes approximately 15–20 years to
which have been released for commercial exploitation. The develop a new variety. Therefore, efforts need to be
importance of evolving a new mulberry variety is to bring directed towards understanding genetic control of major
all desirable characteristics in a single genotype to feed the agronomic traits of the domesticated species. Parallely,
silkworm (Bombyx mori) to obtain high-quality cocoons. intervention of advances in genomics and molecular biol-
Although considerable efforts have been directed for ogy techniques have the potential to overcome problems
mulberry breeding for salinity resistance (Vijayan et al. associated with conventional breeding and open new vistas
2009), it is necessary to develop mulberry varieties specific for amelioration of mulberry productivity under adverse
to different agro-climatic zones for vertical development of environments to revolutionize the sericulture practices
sericulture, and at present suitable mulberry varieties are (Vijayan 2010). Integration of various disciplines of bio-
not available for alkaline, saline, acidic soil conditions as technology for mulberry improvement is depicted in Fig. 2.
well as for inundated conditions (Tikader and Kamble
2007). Traditional breeding approaches have been slow due
to obstacles like inherent genetic limitations, heterozygous Tissue culture and transgenic revolution
and perennial nature of the plant system, non-availability
of information about inheritance of various planta traits The efficient regeneration of plants from tissue culture is of
and genetic markers. Moreover, abiotic stresses like salt great value as it can be applied in direct cultivar
and drought which are inimical to mulberry’s productivity improvement and is a prerequisite for genetic manipulation
are complex quantitative traits and thus difficult to and transformation studies. Over the past 40 years
manipulate through conventional breeding and phenotypic numerous reports on reliable and efficient regeneration
selection. Another factor which fends off genetic protocols of commercially important genotypes of mul-
improvement of mulberry is the lack of sufficient vari- berry have been published. Axillary and apical buds are
ability, a pre-requisite to initiate the breeding programs. In most commonly used explants for in vitro propagation of
spite of sufficient genetic variability in mulberry germ- mulberry although successful regeneration has also been
plasm, the cultivated forms of mulberry belong to M. alba obtained from leaf, cotyledon, and hypocotyls explants
and M. indica and are used in existing breeding programs (Bhatnagar et al. 2000; Kapur et al. 2001; Raghunath et al.
(Tikader and Kamble 2008). Integration of wild/exotic 2008; Tewari et al. 1999). Improvement of mulberry by

Fig. 2 Integration of various

fields of biotechnology for
mulberry improvement

Abiotic Stress
(Hva1, osmotin, bch1)
Lal et al. 2008,
Das 2009,
Das et al. 2010b

Regeneration Transgenics

Genes of
Interest

Germplasm
Characterization Irradiance Stress
BioticStress
(bch1)
(osmotin)
Das 2009
Das et al. 2011b

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Plant Cell Rep (2011) 30:825–838 831

somatic hybridization via protoplast fusion has been dem- biochemical, and molecular evidence indicated increased
onstrated in temperate and tropical mulberry, but practical performance of transgenic mulberry plants when sub-
realization of this technology is yet to be achieved (Umate jected to simulated salinity and drought conditions.
et al. 2005; Wei et al. 1994). Another area of production of Transgenic and non transgenic lines behave differentially
haploid plants in mulberry has also been an arduous pro- under stress conditions and show better cell membrane
cess. Despite initial progress on mulberry androgenesis stability, photosynthetic yield, less photo oxidative dam-
(Jain et al. 1996; Katagiri and Modala 1993; Sethi et al. age, and high relative water content under salinity and
1992; Shoukang et al. 1987; Tewari et al. 1994), significant water stress than non-transgenic lines. Hva1 thus confers
success has not yet been achieved. Similarly, although broad spectrum of tolerance against various abiotic con-
gynogenic plants of mulberry were reported by Lakshmi ditions in transgenic plants. Preliminary studies regarding
Sita and Ravindran (1991) and Thomas et al. (1999), these suitability of these transgenic plants for silkworm rearing
have not been translated into practical ventures. Tissue was also performed. However, transgenic mulberry plants
culture studies in mulberry have been reviewed in detail with barley hva1 gene under the control of CaMV35S
earlier (Khurana et al. 2003; Thomas 2002; Wakhlu and promoter displayed growth retardation under normal
Bhau 2000). conditions. Therefore, stress-inducible rd29A promoter
Although mulberry is a hard to deal woody recalcitrant was used instead of constitutive CaMV35S promoter for
tree species, several protocols have been optimized to the overexpresssion of Hva1 to minimize the negative
transform Morus indica using particle bombardment effects on plant growth. Results indicate that a combina-
(Bhatnagar et al. 2002) and via Agrobacterium tumefaciens tion of rd29a and hva1 is useful for tolerance against
(Bhatnagar and Khurana 2003). These protocols are par- diverse stresses and minimizing negative effects on plant
ticularly efficient on cultivar K-2 but can be extended to growth (unpublished data). Subsequently, efforts have
other cultivars. Although seedling hypocotyls and cotyle- been initiated for field trials of these transgenic mulberry
don explants are regenerative (Bhatnagar et al. 2002, 2003; lines for assessing their growth under field conditions.
Lal et al. 2008), mature leaf-based transformation via Similar success was achieved in genetic transformation of
Agrobacterium tumefaciens proved to be a rapid and effi- mulberry with tobacco osmotin under the control of a
cient transformation system in this heterozygous plant constitutive (CaMV 35S) as well as a stress-inducible
allowing the transformation efficiency of 90% (Das et al. promoter rd29A. Osmotin and osmotin-like proteins are
2011b). Absence of significant achievements in traditional stress proteins belonging to plant PR-5 group of proteins
plant breeding for tolerance to abiotic stresses and the in several plant species in response to various types of
economic losses incurred due to abiotic stresses in recent biotic and abiotic stresses. Physiological analysis of
years have paved the way for molecular tailoring of crops transgenic plants under simulated salinity and drought
through transgenic approaches. Genomic technologies stress as well as fungal challenge was undertaken to test
generating valuable information on molecular basis of the effect of the integrated gene. Transgenic plants with
stress tolerance is the starting point for targeted gene-based stress-inducible promoter were able to tolerate salt and
transgenic approaches for introgression of beneficial genes, drought stress efficiently than those with constitutive
conferring stress tolerance in mulberry. There are reports of promoter, but in case of fungal tolerance 35S: osmotin
multiple stresses being simultaneously regulated by using transgenic plants performed better. Therefore, transfor-
single gene encoding stress-inducible transcription factor mation of mulberry with the osmotin gene would confer
(Kasuga et al. 1999). Putative transgenic plant should be tolerance against drought, salinity, and fungal pathogens
tested in range of relevant environments to study the effect (Das et al. 2011b). These transgenic plants were also well
of inserted gene at whole plant level if impact of powerful accepted by the silkworms—the ultimate users of mul-
transgenic approach has to be achieved. Further, to have berry leaves. Genetic transformation of Morus indica cv
enduring effect by these contentious transgenic strategies K-2 has also been achieved with bch1 (b-carotene
in this cross pollinated crop, proper discretion is definitely hydroxylase-1), and screening of the transgenic mulberry
required. revealed better tolerance of transgenic mulberry for high
Functional evidence indicate that expression of Hva1 temperatures, high light, and UV radiation stress. These
has shown landmark achievements in combating stress transgenic plants accumulate higher levels of xanthophylls
response (Chauhan and Khurana 2011; Fu et al. 2007; under stress conditions than non-transgenic plants as
Maqbool et al. 2002; Sivamani et al. 2000). Transgenic revealed by ultra performance liquid chromatography
mulberry plants were therefore generated by over (UPLC). This is the first attempt of manipulating the
expression of barley Hva1 using Agrobacterium mediated carotenoid biosynthesis pathway in mulberry by over-
transformation (Lal et al. 2008). Detailed physiological, expressing b-carotene hydroxylase-1 gene (Das 2009).

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832 Plant Cell Rep (2011) 30:825–838

Mulberry plastomics novel genes and utilizing them for genetic engineering.
Sequencing the genomes of diverse set of crops has pro-
Chloroplast genome sequencing helps in rapid, accurate, vided insights into the large set of gene repertoires, evo-
and automated identification of species providing plant lutionary complexities and unravels the consequences of
systematists/taxonomists with an easy-to-use species-spe- genome duplications (Paterson 2006). The complete
cific DNA barcode catalog (Ravi et al. 2008). Conservation sequencing for mulberry is not yet justifiable due to large
in gene order, content and lack of recombination make it size of genome and lack of suitable genetic and physical
ideal for phylogenetic studies. One of the pioneering areas maps. Large-scale EST sequencing is the first relatively
of mulberry research include determination of the complete inexpensive step in functional genomics of mulberry and
sequence of chloroplast genome of mulberry using a permits the direct identification of a large repertoire of
combination of long PCR, and shotgun approach represents genes. Additionally, ESTs are crucial for accurate genome
the first case study from this subclass Hamamelidae and annotation and provide information about gene structure,
even the first complete genome from India. The 158,484 bp alternative splicing, expression pattern, and transcript
circular double-stranded DNA comprises two identical abundance. As sequence and annotation data continue to
inverted repeats of 25,678 bp each, separating a large and a accumulate, genomic analysis will become increasingly
small single-copy region of 87, 386 bp and 19, 742 bp, valuable to the plant science community. Until 2009, there
respectively. Genes from sequenced chloroplast genomes was paucity of information available for functional
were used to assign homology to a total of 83 protein- genomics tools in mulberry, but as of today several EST
coding genes including five genes duplicated in the inver- projects have been completed, generating wealth of infor-
ted repeat regions, eight ribosomal RNA genes and 37 mation for functional genomics studies (Gulyani and
tRNA genes (30 gene species) representing 20 amino acids. Khurana 2011; Lal et al. 2009). Overview of the sequence
The mulberry plastome lacks the genes infA, sprA, and assembly results of different mulberry libraries constructed
rpl21 and contains two pseudogenes ycf15 and ycf68 (Ravi is listed in Table 3. Sequencing and analysis of ESTs from
et al. 2006). Based on sequence similarity, Morus appears mature mulberry leaf set the platform for transcriptomics in
closer to Cucumis and Lotus. Comparative analysis at mulberry. Based on sequence homology with available
genome level indicates a similarity to Eucalyptus, followed databases, ESTs were assigned into functional categories,
by Cucumis apparently due to inclusion of non-coding but the largest group among the total ESTs did not show
regions. This indicates that genic and intergenic regions any significant homology with known proteins. Of partic-
undergo differential selection pressure during the course of ular interest would be the identification of novel genes as
evolution. Exploring the genetic repository of this orga- they are not previously characterized and represent chal-
nelle is vital as cpDNA sequence information has been lenging genes for further investigation. The information
instrumental in phylogenetic and molecular taxonomy of gained from the analysis of genes related to various cate-
plants, and accordingly, mulberry plastome sequences have gories like abiotic and biotic stresses related (LEA, RD22,
been used to resolve the relationship between the orders dehydrins and Hal3), membrane transporters (vacuolar
solving rosid puzzle and placing Rosales closer to Fabales Na?/H? antiporter and aquaporins) under different stress
phylogenetically based on 32 taxa details (Ravi et al. conditions may act as starting point to adopt different
2007). The importance of sequencing this plastome lies in strategies for genetic improvement of mulberry. The
the fact that several components of the photosystems and identification of genes encoding enzymes involved in var-
proteins involved in biosynthetic pathways are also enco- ious secondary metabolites also opens up new avenues for
ded by the chloroplast genome, and therefore, cpDNA metabolomics research in mulberry (Lal et al. 2009).
sequences have promising ability for transgenic expression Realizing the enormous potential of transcriptome
for mulberry improvement as these cpDNA genes have approach, identification of ESTs from root tissue of Morus
negligible chances of dispersal in this cross-pollinated indica cv K-2 was also undertaken. Comparative analysis
crop. of ESTs generated from leaf and roots provides additional
information about snapshot of tissue-specific transcrip-
tional activities. A total of 2,599 unigenes from mulberry
Mulberry transcriptomics leaf and root ESTs were compiled in the present study and
were analyzed for the identification of EST-SSRs as SSRs
Mulberry breeding is a time-consuming process; therefore, are a cost-effective tool of choice and will contribute to
it is desirable to incorporate genetic and genomic methods new ideas for marker-assisted breeding programs (unpub-
that could speed up the detection of functionally important lished data). Other utilized genomic approaches to tran-
regions of the genome. Mulberry genomics toolbox can scriptional analysis in mulberry include suppression
serve as an important asset for studying the functions of subtractive hybridization (SSH). SSH followed by

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Table 3 Overview of the

Descriptive category Leaf (normal) Leaf (subtractive) Root
sequence assembly results of
different mulberry libraries Total cDNA sequenced 1,500 1,920 2,400
constructed
No. of cDNA after quality trimming 958 1,514 1,878
Unassembled singletons 517 151 1,420
Clustered ESTs 647 1,182 458
Total number of contigs 147 208 148
Unique sequences (%) 80 87.8 79.8
Reference Lal et al. (2009) Gulyani and Khurana (2011) Unpublished data

validation studies is a powerful technique that enables enzyme involved in the zeaxanthin biosynthetic pathway
obtaining cDNA libraries representing differentially catalyzing the conversion of b-carotene to zeaxanthin. A
expressed transcripts which are uniquely expressed. In the partial clone of bch1 was isolated from mulberry leaf
quest to understand drought tolerance in mulberry, a PCR- cDNA library (Lal et al. 2009), and full length clone was
based subtractive cDNA library containing water stress- developed by 50 RACE using BD SMARTTM RACE.
induced transcripts was constructed. The sensitive and Expression analysis of bch1 in mulberry revealed that it is
tolerant genotypes were identified based on physiological ABA, salicylic acid, high temperature and UV inducible,
evaluation by determination of proline content, electrolyte but its expression increases markedly with high tempera-
leakage, and measurement of relative water content. The ture and UV stress (Das 2009). Pan and Lou (2008) have
ESTs generated from this cDNA library comprises a broad isolated 1-aminocyclopropane-1-carboxylate oxidase gene
repertoire of stress-responsive genes, which contribute to involved in a key step in ethylene biosynthesis from mul-
the process of drought tolerance in mulberry. Additionally, berry and investigated its stress-responsive expression
23% of the cDNA library is represented by transcripts of pattern. Lectins are multivalent carbohydrate-binding pro-
unknown function. Characterization of these stress- teins and are used as biochemical tools in many fields of
responsive genes by cDNA macroarray and northern research. Lectins have attracted much interest of mulberry
analysis indicated that many of the genes isolated are researchers, and work on mulberry lectins was initiated
involved in drought stress response. In support of the before the advent of transcriptomics. Purification of
drought response of mulberry, orthologs of a set of genes N-glycolylneuraminic acid-binding lectins from mulberry
from this stress cDNA collection were confirmed to be leaves was reported in 1998 by Ratanapo et al., and later
induced in Arabidopsis under drought stress by in silico these lectins were shown to have antibacterial activity
analysis. This resource has provided a short list of potential against P. syringae pv mori, a specific bacterium of mul-
candidate drought-responsive genes, expressed in leaves, berry (Ratanapo et al. 2001). Bark of black mulberry
which are involved in both initial and subsequent stages of (M. nigra) tree accumulates large quantities of galactose-
stress and elucidate the transient regulatory proteins that and mannose-specific jacalin-related lectins (Van Damme
regulate adaption to drought in this important crop (Guly- et al. 2002), and structural studies have revealed that their
ani and Khurana 2011). exclusive specificity towards glucose or mannose relies on
Some novel genes have been functionally characterized reduced size of carbohydrate-binding site (Rouge et al.
from mulberry ESTs. Remorins represent a very large, 2003). Binding properties of these mannose-binding lectins
plant-specific multigene family. Since the isolation of first was studied in further detail, demonstrating its unique
remorin gene from potato (Reymond et al. 1996), cDNA specificity for various ligands. This study help in under-
clones encoding remorin genes have been identified from standing of cell surface carbohydrate ligand–lectin recog-
variety of plant species. It is estimated that there are 16 and nition and also highlights the utility of lectins in
19 remorin genes in Arabidopsis and Oryza genomes, biotechnological and clinical studies (Wu et al. 2004).
respectively (Raffaele et al. 2007). Therefore, in order to
define its function, characterization of its expression pat-
tern was undertaken for understanding the signaling net- Mulberry proteomics
works in which remorin genes are involved. It is
hypothesized that some of these proteins might have key Proteomics has been defined as the systematic analysis of
functions during biotic and abiotic stimuli and might pos- the proteome in a tissue, cell, or sub-cellular compartment
sibly be involved in hormone-mediated responses and of an organism and provides the opportunity for direct
signal transduction (unpublished data). b-carotene investigation of changes in protein expression patterns.
hydroxylases present in the thylakoid membrane is the Changes in protein component of a genome vary by cell

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and environmental influence and can give important clues activities were found in all the constituents of alkaloids,
about important gene products relevant to biological flavones, and amyloses, the alkaloid constituent being the
function. Mulberry proteomics is in its infancy as at the strongest (Yuan et al. 2006). These alkaloidal sugar-mimic
present time there is lack of data of mulberry proteomics to glycosidase inhibitors present in higher concentrations in
be exploited for functional genomics. Further, the most mulberry latex (1,4-dideoxy-1,4-imino-D-arabinitol, 1-de-
daunting aspect of application of proteomics in mulberry is oxynojirimycin, and 1,4-dideoxy-1,4-imino-D-ribitol), are
the lack of large-scale sequence resource to match the toxic to caterpillars but not to the silkworm Bombyx mori
sequences of peptides generated in tryptic digests to their implying the coevolution of the silkworms and the mul-
proteins of origin. Nonetheless, the accomplishment of the berry tree’s defense. Furthermore, these results indicate
B. mori genome sequence has catalyzed the applications of that latex ingredients could be good sources to discover
proteomics to B. mori (Zhou et al. 2008), but in mulberry, novel compounds (Konno et al. 2006). Realizing the
proteomic approaches have been used by very few importance of anthocyanin pigments in mulberry, antho-
researchers. Kumari et al. (2007) studied the effect of cyanin composition, content and further comparison of the
salinity on proteomic changes in two contrasting cultivars antioxidant capacity of five major mulberry cultivars were
of mulberry as identification of genes responsible for investigated by Bae and Suh (2007). Song et al. (2009)
salinity tolerance can give important cues for improvement examined mulberry fruits of 38 cultivars and mulberry
of salt tolerance in mulberry using genetic manipulation leaves of 33 cultivars for their 1-deoxynojirimycin (DNJ),
techniques. Three-month-old plants were subjected to dif- resveratrol, oxyresveratrol, anthocyanin, and flavonoid
ferent concentrations of salt for 7 days, and soluble pro- content. Significant differences in concentrations of these
teins were analyzed by two-dimensional electrophoresis. compounds in different species and cultivars were
Salt stress induced both qualitative and quantitative chan- observed. Phytochemical investigation of the stem bark of
ges in the polypeptide patterns in both the cultivars. These Morus cathayana led to the isolation and identification of
differentially regulated proteins provided a broad view of six new compounds, cathayanons F–J and cathayanin A,
biochemical and molecular mechanisms involved in and two known compounds, cathayanins B and C. Some of
acquiring salt stress tolerance. Similarly, proteomic anal- these compounds exhibited weak activities against human
ysis has provided new insights into mulberry dwarf cancer cell lines (Ni et al. 2010). Bioactive secondary
responses caused by phytoplasma in mulberry. Mulberry metabolites were induced by UV-B irradiation in vitro, and
dwarf is one the serious infectious disease caused by the induced products were extracted and isolated by HPLC.
phytoplasma. To understand the pathogen stress response Five significantly changed chromatographic peaks were
of mulberry (M. alba), two-dimensional electrophoresis observed in HPLC–DAD fingerprint, two of which were
protein profiles of infected and healthy leaves were com- identified as chalcomoracin and moracin N. This study
pared, and quantitatively changed spots were identified by further hypothesized that harvest season impacts the
mass spectrophotometry to reveal complex plethora of quantity for different active components present (Gu et al.
proteins involved in response to infection (Ji et al. 2009). 2010). The expression pattern of MiSMT (Morus indica,
These proteomic investigations are of great significance as Sterol Methyl Transferase) and MiVR (Morus indica,
they identify novel protein components responsible for Vestitone reductase) was investigated in ten genotypes of
acquiring stress tolerance. mulberry under control and salt stress conditions by
northern blot analysis. Significant genotypic differences
were observed for both of these genes in different geno-
Mulberry metabolomics types. The analysis of biochemical pathways and response
towards environmental stresses of these important bioac-
The potential applications of metabolomics, a new and tive components suggested their involvement for health
evolving science, in plant biotechnology are enormous as it benefits opening several new avenues for research in var-
has the power to decode the biochemical functions of plant ious medicinal and natural products (Tetorya 2010).
genes and regulatory networks in plant metabolism Therefore, metabolic profiling of mulberry on a genomic
(Trethewey et al. 1999). Recent investigations have scale will provide definitive insights to study gene func-
revealed that the fruits and leaves of mulberry plants tions and its metabolic status.
contain many bioactive components of great interest
(alkaloids, flavonoids, steroids, and anthocyanins) because
of their influence on human health as they have antioxi- Concluding remarks and future prospects
dant, antimicrobial, and anti-inflammatory properties.
Screening of glucosidase inhibitors from various fractions Mulberry is one of the most economically important plants
of mulberry leaves revealed that glucosidase inhibitory with significant contributions to the Indian economy.

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Abiotic stresses are major factor affecting the productivity Ahmad P, Sharma S (2010) Physio-biochemical attributes in two
of mulberry worldwide. Comprehensive evaluations of cultivars of mulberry (Morus alba L.) under NaHCO3 stress. Int
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genotypic variations suggested new perspectives for effi- Andallu B, Varadacjaryulu NC (2003) Antioxidant role of mulberry
cient use of its genetic resources. Genetic markers have (Morus indica L. cv. Anantha) leaves in streptozotocin-diabetic
been employed in mulberry to study its unique genotypic rats. Chin Chim Acta 338:3–10
variation. However, only limited progress has been made in Atmakuri AR, Chaudhury R, Malik SK, Kumar S, Ramachandran R,
Qadri SMH (2009) Mulberry biodiversity conservation through
development of linkage maps in mulberry, and therefore, cryopreservation. In Vitro Cell Dev Biol Plant 45:639–649
efforts are needed for development of mulberry genomic Awasthi AK, Naik NGM, Sriramana GV, Thangavelu K, Jav-
toolbox which would provide valuable basic information aregowda N (2004) Genetic diversity and relationships in
for functional genomics accelerating the creation of mulberry (Genus Morus) as revealed by RAPD and ISSR marker
assays. BMC Genet 5:1–4
improved mulberry varieties. Biotechnology has energized Bae SH, Suh HJ (2007) Antioxidant activities of five different
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DNA markers, for mapping expressed genes to a linkage phased screening strategy. Plant Cell Rep 21:669–675
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and marker-assisted selection. The potential of proteomics ation in commercially valuable Indian mulberry, Morus indica
and metabolomics has yet to be exploited; however, an cultivars K2 and DD. Plant Biotechnol 18:61–65
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Biochemical responses to drought stress in mulberry (Morus
which renders the data to be universally useful. alba L.): evaluation of proline, glycine betaine, and abscisic acid
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Acknowledgments This work was financially supported by Chattopadhyay S, Ali KA, Doss SG, Das NK, Aggarwal RK,
Department of Biotechnology, Government of India. VGC acknowl- Bandopadhyay TK, Sarkar A, Bajpai AK (2010) Evaluation of
edges University Grants Commission for Junior and Senior research mulberry germplasm for resistance to powdery mildew in the
fellowship. field and greenhouse. J Gen Plant Pathol 76:87–93
Chauhan H, Khurana P (2011) Use of doubled haploid technology for
development of stable drought tolerant bread wheat (Triticum
aestivum L.) transgenics. Plant Biotechnol J 9(3):408–417
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