Abstract
Traditional sericulture represents rearing of silkworm for production of silk that provides livelihood opportunity to millions of people in the country besides earning foreign exchange. The time has come to diversify the whole sericulture process for meaningful realization of its output under the present day scenario. The advent of modern biotechnology and its application have opened a new arena of the synthesized science for silk production. The vast potential of silk industry can effectively be exploited by the application of modern day biotechnological approaches like, marker assisted selection and expression of foreign protein through transgenic approaches. On the other hand, the silk quality has been enhanced using probiotics and providing artificial feed to the silkworm. The potential of silk has been further exploited for biomedical applications. In this communication the comprehensive account of biotechnological applications in sericulture and its byproducts for the development of sericulture industry are compiled while emphasizing the need of applying modern biotechnology for meaningful growth and development of sericulture and silk industry.
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Introduction
Sericulture, the synthesized science of silk production provides employment opportunities to around 6 million people of the country in its entire value chain extending from soil to silk. Since independence, India has increased its silk production from 11,000 MT in 1989–1990 to 31,906 MT in 2017–2018 [203] and has become the second largest producer in the globe. The major thrust areas for sericulture are the production of superior mulberry and silkworm varieties for the tropical conditions of the country, identification of new silkworm rearing technologies, suitable methods for containment of pests and diseases in sericulture etc. [195]. Owing to changing climate and complex farming conditions of the sericlulture farmers, focus needs to be shifted from the traditional interventions to application of advanced scientific knowledge. Applicability and potentiality of biotechnology in increasing the productivity of sericulture have been acknowledged unequivocally in the recent past in many scientific discussions. It has been proved to be a powerful tool for sustainable growth and development of sericulture and silk industry. Silk is one of the most precious natural fibers and the unit price of raw silk is roughly estimated to be twenty percent more than raw cotton [132]. Increased demand of natural fibre, application of silk as biomaterial as well as silkworm as a model organism for the production of foreign protein encouraged the scientific community to explore biotechnology among many other techniques to improve production and application of silk for both textile and non textile industrial purposes.
In the recent past, research contribution of various organizations enabled Indian sericulture industry to make a good stride by way of introducing new and improved mulberry and silkworm varieties, improved silkworm rearing technologies for tropical conditions and management strategies of diseases and pests in sericulture [226]. Even though conventional breeding approaches in sericulture have successfully introduced many productive breeds and hybrids, but their full potential could not be utilized in the field due to adverse agro climatic conditions and infections by pathogens while practicing repeated rearing. Situation worsens further because of limited ground water, soil differentiation with poor native fertility and improper cultivation practices [213]. The use of modern biotechnological tool for improvement of silkworm, host plant, development of artificial feed, seri waste utilization, production of silk-protein based biomaterials etc. holds great potential not only to meet production demand of silk but also to improve the industry in a comprehensive manner. On the other hand, Morus alba which is rich in folic acid, carotene, vitamins, flavonoids, tannins, saponins, ascorbic acid and antioxidents with phytoconstituents such as anthocyanins, anthroquinones, glycosides and oleanolic acid has been explored using biotechnological applications for its pharmacological significance. Recent advances have reported use of Morus alba as antidiabetic, anticancerous, immunomodulatory and antimutagenic agents. The possible areas of biotechnological interventions in sericulture are presented in Fig. 1. The plethora of information provided by the several genome projects, advances in functional genomics and transgenic technologies have added new insight into the field of seri-biotechnology. The potential application of these new approaches into sericulture may bring us closer to achieving the growing demands of silk in the country. Therefore, present review is an attempt to focus on the applicability of different modern biotechnological approaches like molecular marker system, transgenic technologies, development of artificial feed, immunodiagnosis techniques, expression of foreign protein in silkworm, development of silk-based biomaterials and control of diseases, pests etc. in sericulture.
Schematic representation on the possibilities of biotechnological application in sericulture
Molecular markers for mulberry improvement
The principal application of mulberry plants in sericulture is its leaves which are used for silkworm rearing to produce silk. According to an estimate on the economic return of sericulture point of view, mulberry cultivation cost alone constitutes more than 60% of the total expenditure for the entire cocoon production [56]. This further necessitates development of mulberry varieties with high leaf yielding potentials. Traditional mulberry breeding strategies have evolved several mulberry high yielding varieties viz. S-1, K-2, Kokuso-13, Shin-ichinose, V-1, S-13, S-34, S-799, S-30, S-36, S-54, Tr-4, G-9, RC-1, RC-2 [192, 210, 223]. But mulberry breeders encountered several problems in the breeding programmes, like limited information on genetics and inheritance pattern of mulberry, absence of pure lines, lack of definite parents, dioecious nature, inbreeding depression, perennial nature with long juvenile period, lack of genetic markers and efficient screening strategies [236]. Most of the yield and associated traits like leaf qyality and yield, resistance to abiotic (water, alkalinity, frost, etc.) and biotic stress (root rot, root knot nematode, whitefly, tukra etc.), efficiency in nutrient uptake capacity. Leaf quality of mulberry is polygenic in nature and influenced highly by genotype and environment interaction while manifesting its characteristics [239]. Therefore, situation demands application of modern biotechnological tools like use of molecular markers, transgenesis for introduction and over expression of desirable genes or silencing of undesirable genes through RNA interference technology [250].
Molecular marker (DNA/RNA) is a specific physical unit on a chromosome whose inheritance pattern can be observed and studied well. Exploration of mulberry genome has created Morus database which is a boon in identifying the vital regions of mulberry genome for exploiting in breeding process. Morus DB—Mulberry Genome Database (https://morus.swu.edu.cn/) is one of the sites where all the details of genetic makeup and molecular markers such as SNPs, ISSRs, RFLPs can be accessed.
Molecular markers enable fast and accurate identification of breeding lines, hybrids, cultivars and species, facilitate analysis of genetic diversity and also allow establishment of phylogenetic relationship with more precision than that was previously possible with morphological and biochemical techniques. The DNA markers are extensively used for screening genotypes, identification of gene of interest, characterization of germplasm collections and other gene related studies in mulberry [73, 122]. There are different DNA markers such as restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), inter simple sequence repeat (ISSR), simple sequence repeats (SSR) and single nucleotide polymorphism (SNP) [125]. Marker assisted selection (MAS) has got many advantages over conventional breeding methods as it enables the breeder to select desirable hybrids at the seedling stages, free from environmental interference, time saving, accurate and precise. MAS provide the potential for improving selection efficiency by allowing early selection, reduced number of generations and population size [208]. Genetic diversity, molecular characterization of germplasm and varieties, development of linkage and quantitative trait locus (QTL) map, association mapping, parental selection schemes and marker-assisted selection (MAS) are some of the important areas where molecular markers can play a great role for improvement of mulberry plants [105, 251].
There are plenty of reports on application of molecular markers for improvement of mulberry. Application of molecular markers namely RAPD [10, 42, 93, 141, 155,156,157, 165, 167, 225, 262, 274] AFLP [85, 92, 198] ISSR [98, 117, 240, 243, 245, 247, 256, 261, 275, 276, 277] RAPD with ISSR [19, 27, 46, 47, 51, 86, 94, 189, 200, 206, 238, 242, 244, 248, 263] RAPD, ISSR and SSR [12, 78] has been reported by many authors. Markers like genomic-SSR [109, 159, 166, 170, 257, 258], sequence-related amplified polymorphism (SRAP) [278], internal transcribed spacer (ITS) [260, 266], start codon targeted polymorphism (SCoT) [185], Cleaved Amplified Polymorphic Sequences (CAPS) [17], direct amplification of minisatellite DNA (DAMD) [33] were also used not only to ascertain molecular diversity in cultivated and wild mulberry species but also to identify its route of introduction and proliferation in India. Studies on molecular profile for genotypes and estimation of the genetic diversity among different germplasm collections of mulberries also resulted identification of SSR markers and SNPs from different mulberry species [148, 188]. The details of molecular markers used in mulberry improvement during last two decades have been listed in supplementary table (Table A).
Introduction of marker assisted breeding in mulberry further got a new momentum when whole-genome shotgun sequencing of haploid mulberry species (M. notabilis) was reported, the draft genome of 357 Mb included 128 Mb repetitive sequences with 27,085 high confidence protein coding loci in tandem with complete gene structure [82]. Attempts were made to develop QTL (Quantitative Trait Locus) maps that are specific to important agronomic traits such as water use efficiency (WUE), root traits and yield attributing characters in mulberry, using dominant markers such as RAPD and ISSR. These resulted few markers linkages in the QTL map governing the character of interest [139, 158]. But identification of marker intricately linked to the particular trait is still lacking in mulberry. Table 1 depicts some of the significant attempts on the development of molecular maps in mulberry improvement.
Improved mulberry characters using molecular markers in India
Molecular markers assisted breeding has benefited the Indian sericulture in many ways. The researchers in India have indentified many RAPD and ISSR markers associated with genetic diversity leading to development of desirable characters targeting specific gene/proteins encoding such as high leaf yields, abiotic stress resistance and, drought resistance using over expression of certain genes through molecular approach. The Morus notabilis genome database (https:/morus.swu.edu.cn) published in 2017 will help to extrapolate new primer design and their application in generatinge linked markers with quality traits.
Conventional breeding and molecualar breeding
Breeding is an important method to maintain silkworm races based on their characters and phenotypes. It also provides us the base material for development of new breeds specific to a particular region or climate, Conventional breeding does not establish a particular character improvement in organism. On the other hand, molecular breeding helps us establish specific characters in a species using molecular markers associated with desired phenotype of the organism in which one is interested. Outcome of this molecular breeding in turn will help in increasing the productivity of that specific phenotypic character item in the sericulture industry.
Tissue culture in mulberry genetic improvement
The main applications of plant biotechnology are to promote conservation, diversification, and sustainable use of plant genetic resources for food and agriculture besides improving food security and fostering socio-economic development. The role of plant biotechnology and its use of advanced methods in sericulture is to improve the productivity trait and their survivability under stress conditions. However, perennial nature of the plant coupled with prolonged juvenile period slow down the process of mulberry improvement [103]. Further, cross-pollination is the rule rather than an exception, enormous heterozygosity occurs in the plant [57]. The diploid (2n) chromosome number of the species is 28, but highly polyploid numbers, up to 308, can be found. Among the polyploids, the triploids have many desirable traits, including quality, and resistance to cold and disease. However, the production and multiplication of triploids are time consuming [57].
Mulberry can be vegetatively propagated through stem cuttings, grafting or budding. However, success of these methods depends on several factors such as genetic makeup of the plant, age and physiological conditions of the parental cutting, climatic conditions and others. Additionally, newly developed mulberry varieties cannot immediately be propagated through stem cuttings as at least 6–7 month maturity is required to make the cuttings from the parental plant [96]. Moreover, the dioecious nature of mulberry and the occurrence of genetic linkage with both desirable and weak traits limit improvements that can be attained through conventional hybridization.
The emerging application of tissue culture technology to clonal propagation provides an alternative to the routine vegetative propagation of woody species with such desirable traits [36, 222]. In fact, the in vitro culture of cells, tissues, and organs offers unparalleled opportunities for tree improvement [101]. Regeneration of plants via tissue culture is based on the principle of totipotency originally proposed by Haberlandt in 1902 [234]. Plant cell and tissue cultures play an important role in the manipulation of plants for improved crop varieties. Plant regeneration systems are an essential part of micropropagation and molecular approaches leading to plant improvement in mulberry. The possibility for qualitative and quantitative improvements in mulberry varieties is vital to the advancement of the sericulture industry.
As we know tissue-culture techniques have been used extensively for mass propagation of forest and cultivated trees, including plantation crops [24]. Plant tissue culture has found its applications not only in basic and applied research but also in commerce and various trades. In vitro regeneration of whole mulberry can be achieved using diverse types of explants such as meristems/buds, protoplast-dervived somatic hybrid, embryo, ovule, anther, and cells. Over the past two decades many reports have surfaced concerning plant regeneration from different explants of mulberry which have been detailed in Table 2. Tissue culture techniques such as, micropropagation, callus culture, organogenesis, somatic embryogenesis, and somatic hybridization were employed to improve the qualities of mulberry plant. Additionally, in vitro screening of genotypes for stress tolerance and yield, production of haploids and triploids, and improvement of secondary metabolites through transgenic approach are recomended.
Molecular markers in silkworm improvement
The art of silkworm rearing for silk production in our country is an ancient culture and even today sericulture constitutes an essential component of the rural economy in India [60]. The country is bestowed with extraordinarily rich silkworm genetic base in the form of mulberry silkworms and wild silk producing insects. Despite having such a wide silkworm genetic base, information is hardly available either on the unique features of many of these genotypes, or to the extent of genetic diversity between or within the genotypes/races. Till date, genetic improvement of these precious insects is usually ascertained based on their morphological characteristics which, are highly biased and environmentally dependent, thus requiring an authentic technique like MAS for genotype characterization and evaluation. As far as mulberry silkworm, Bombyx mori L., is concerned, more than 400 visible mutations have been placed in the linkage maps [64] which represent 217 loci consisting of mostly morphological and a few isozyme markers. Research on genetics of silkworm across the world has helped in establishing silkworm genome database called silkbase (http://silkbase.ab.a.u-tokyo.ac.jp/cgi-bin/index.cgi). This has become an integral part in exploring the genome of silkworm for improvement of sericulture industry. Exploring genome of silkworm has revolutionized the sericulture industry by identifying the molecular markers using linkage mapping. This led in identifying key regions in the genome of silkworm for specific phenotypes that have changed the face of the silkworm breeding. Hence, a lot of work on DNA based genetic markers in silkworm started emanating in the 1990s and a preliminary linkage map of 169 loci using RFLPs [201] and RAPD [172] was constructed. Studies on PCR-based markers, RAPD markers, and DNA fingerprinting with minisatellite probes in respect to Indian silkworm have also been carried out [152]. Further, thirteen silkworm strains were used for genetic characterization using inter simple sequence repeats (ISSR). The ISSR-PCR produced 39 fragments of which 76.98 per cent were polymorphic and the diversity index was observed to be 0.957 percent [153]. Afterwards, for complete genome analysis of the silkworm, Bombyx mori L., expressed sequence tag (EST) database was constructed [142, 143] covering about 55% of all the genes of silkworm. To identify and mapping of sex-linked traits in the silkworm genetic mapping of Z chromosome and identification of W chromosome- specific markers in the silkworm were carried out [151]. Fang et al. [72] while studying genomic analysis of cocoon and associated traits in silkworm through restriction-site-associated DNA sequencing (RAD-Seq) reported identification of a total of 11 cocoon yield-related QTLs on 7 chromosomes using the composite interval mapping (CIM) algorithm. The tasar silkworm studies also reveal that molecular markers are always better in identifying the genetic makeup, determining population structure and interrelationship among different ecoraces of Antheraea mylitta. Rao et al. [181] while studying molecular characterisation of Daba and Andhra ecoraces of tasar reported occurrence of certain common bands in both the ecoraces indicating possible genetic relationship between these two ecoraces. Chatterjee et al. [41] reported considerable genetic variation through ISSR markers in six different wild tasar ecoraces. Considerable intra and inter population diversities through ISSR markers within and among semi domesticated Daba bivoltine, Daba-trivoltine and nature grown Daba were also reported by Kar et al. [97]. Their report further suggested that semi domesticated population of Daba ecorace was at the threshold level of discriminating themselves. Genetic diversity between and within population of Raily, Daba and Modal was also studied by using 12 ISSR and 10 RAPD primers [246]. Phylogenetic relationships of nine tasar silkworm ecoraces were reported by Mahendran et al. [129] by RFLP method. Population structures of eight different ecoraces using ten amysat microsatellite loci were studied by Chakraborty et al. [38].
Exploration of the silkworm genome has helped to identify specific markers for thermotolerance, no glue eggs, silkworm resistance to viruses, regulation of moulting, regulation of body shape etc. Silkworm research in India by Central Silk Board has made advances by successfully identifying microsatellite markers linked to thermotolerance by Bulk Segregation Analysis [39]. Thermotolerant silkworm is under development in India for the tropical environment. Based on the available data we have summarized the important development of molecular map and use of markers in improvement of silk work in Tables 3 and 4.
Transgenics research in sericulture
Advancement of modern biotechnology enables us to identify a gene of interest that can be separated, cut off, inserted, transformed, and expressed into a foreign system. Such genetic manipulation is referred as genetic engineering. Knowledge on modern biotechnology has made it possible to improve our understanding on the living organisms and to apply the knowledge to the life and activities of mankind which include food, agricultural production, forestry, fish production, animal rearing and horticulture etc. [70, 164]. Modern biotechnology, especially genetic engineering enables us to obtain organisms carrying a gene or genes of interest from an unrelated organism. Scientists are taking advantage of recombinant DNA technology for the development of transgenics due to the limitations of conventional breeding in introducing desired characters in both mulberry and silkworm. Transgenic research has introduced desirable characters in silk and its associated products. Spider silk is one such example that has changed the silk industries intensely.
Transgenic mulberry
Mulberry is a woody tree species. Several protocols have been standardized so far for a time phased screening strategy to transform Morus indica L. using particle bombardment [32] and via Agrobacterium tumefaciens [30], yet there is a lot of difficulties in handling the same. Mulberry is very much susceptible to stresses like salinity and drought and leaf moisture content drops drastically while countering these stresses [111]. It has been observed that mulberry transgenics developed with the osmotin gene under a drought inducible promoter which showed abiotic stress tolerance and tolerance against biotic fungal pathogens [58]. Development of transgenic mulberry with Hva1 gene from barley for drought, salinity and cold tolerance has been reported [44, 111]. Some of the significant attempts of mulberry improvement through genetic engineering have been enlisted in Table 5.
Transgenic silkworm
In silkworm also, germline transformation of Bombyx mori L. using microinjection of PiggyBac (PB) derived vectors and its optimization was elaborated extensively Tamura and his associates [217] have developed a transgenic silkworm using the DNA transposon PiggyBac as a vector for inserting the target gene into the silkworm chromosome. Use of transgenic silkworms to produce different recombinant protein as well as for produce high quality silks employing silk-gland as bioreactor have also become a reality now a days [108, 227, 279]. The ongoing development of transgenic silkworm for monoclonal antibody drugs for cancer [119, 214], orphan disease drugs and animal drugs has also been happening in recent times. Therefore, attempts towards developing genetically modified silkworms may lead to many innovations in sericulture and sericology [150]. Production of recombinant human serum albumin (HSA) in transgenic cocoon, production of glycoproteins with reduced antigenicity in transgenic silkworms are some of the recent advances paving new avenues of transgenic research in sericulture [135, 173].
Limitation of transgenesis
Although there have been many advantages of transgenesis in improving silkworm characters and mulberry plants characters, it comes with certain risks. The disadvantages are the possible risks of such transgenes escaping in the nature and blend through gene flow with naturally occurring population, which can lead to undesirable character in the organisms.
Artificial diet in sericulture
Among the several factors which contribute successful cocoon crop, good quality mulberry leaves alone contribute around 38.2 percent of the total share [144]. Moreover, quality of leaves also varies greatly in different growth stages of silkworm like young stage worms prefer leaves with high moisture content whereas insects at later stage require slightly coarser leaves. Apart from these there are some practical difficulties in feeding the silkworm particularly during rainy seasons because of wet leaves. Feeding of wet leaves may cause to build up extra humidity in the rearing bed which ultimately causing outbreak of several diseases. Moreover, shrinking of farmland for mulberry cultivation and labour shortage are some of the other reasons which compelled the scientists to work on the development of artificial diet. Therefore, the first ever attempt to rear silkworm on artificial diet was made way back in 1960 [74, 87] where entire rearing of the silkworm, Bombyx mori L., had been achieved on artificial diets which could not be so successful if imbalance of natural food nutrition for rearing silkworm due to crop failure would not have happended. There after several researchers have come up with different kinds of artificial diets [83] and advocated two different kinds of low-cost artificial diets for silkworm. One is these “Pellet diet”, produced by using a twin-spindle extruder and can be fed to the silkworms by soaking with a suitable quantity of water just before use and another one is “Yuneri diet” that can be administered to the silkworms by mixing with hot water at around 80 °C without steaming. Trivedy et al. [228] developed a semi synthetic artificial diet’Seri Nutrid’ upon evaluation on the performance of five multivoltine and six bivoltine strains, cocoons and associated parameters were found at par with that of mulberry leaf reared worms. Nair et al. [160] reported potentiality of chawki rearing for multivoltine silkworm exclusively on artificial diet to create breeding resource material for prospective hybrids. Further rearing of PM × CSR2 a popular silkworm cross breed on ‘Seri Nutrid’ recorded having significantly higher average cocoon weight, shell weight, and shell ratio as compared to sole feeding of mulberry [145]. In addition to mulberry leaves, feed supplements or fortification are also tried in silkworm but recent trend in use of probiotics has opened a new area for improvement of silkworm and cocoon production. Effect of probiotic and neutraceutical agent on growth, development, and commercial characteristics of B. mori, has recently been reported by many authors [23, 133, 202, 212, 252].
Non mulberry or wild silk culture is dominated by tasar silkworm A. mylitta which feeds primarily on Terminalia arjuna and T. tomentosa with many numbers of secondary and tertiary food plants. Unlike mulberry, rearing of tasar is totally outdoor and exposed to the natural vagaries causing a frequent crop loss. Therefore, efforts were also put forth for the development of artificial feed for tasar silkworm. Continuous research efforts finally resulted in an effective formulation of artificial feed named ‘Tasar Amrit’, especially for rearing of juvenile silkworm. Field studies with ‘Tasar Amrit’ as feed for tasar silkworm have shown a great promise with higher survival rate and effective frequency of rearing compared to complete outdoor rearing [190]. The performance of ‘Tasar Amrit’ was further substantiated by Kumar et al. [110] where they achieved higher survival of young age worms (88.82%) and effective rate of rearing (46.95%) in comparison to feeding fresh leaves in indoor rearing and complete outdoor rearing of tasar silkworm.
Application of biotechnology in silkworm disease management
Diseases of silkworm have always been a matter of great concern for sericulture and silk production. Silkworms are known to be affected by number of diseases namely grasserie, flacherie, muscardine and pebrine caused by virus, bacteria, fungi and microsporidia, resulting frequent crop failure in practical situations [20, 22, 89]. Around 15 to 20 kg of cocoons per 40,000 silkworm larvae are estimated to be lost due to silkworm diseases in India [196]. The most common methodologies used for containment of silkworm diseases include preventive measures, microscopy, in vivo assays like enzyme-linked immunosorbent assay (ELISA) [232], DNA hybridization [18], colloidal textile dye-based dipstick immunoassay [161], protein-A linked monoclonal antibody latex agglutination test (PALMAL) [197] and viral DNA transfection [131] are some of the techniques that were developed with respect to silkworm diseases management. But early and rapid detection of pathogens such as nuclear polyhedrosis virus, densonucleosis virus, infectious flatcherie virus, Nosema bombycis in silkworm will help in the rejection of crop and replacement with new one to prevent further spread of diseases. Therefore, use of modern biotechnological tools for rapid detection and management of silkworm diseases are gaining momentum now a days. A series of different PCR based rapid diagnostic method particularly with reference to rapid detection of microsporidian and viral diseases of silkworm have been reported in recent times [1, 21, 90, 106, 186, 219, 254]. Use of LAMP (Loop Mediated Isothermal Amplification) for rapid detection of pebrine and engineering silkworm for baculovirus resistance through RNA Interference (RNAi) technology are some of the emerging areas of biotechnological innervations in management of silkworm diseases [53, 69, 95, 209, 233].
Silkworm as bioreactor for expression of foreign protein
Isolation, cloning, characterisation, and expression of a gene of interest have become possible because of the improvement in the modern techniques of genetic engineering. The expression of pharmaceutically important proteins, namely cell/viral surface proteins, membrane proteins, and guanine nucleotide-binding protein (G protein) coupled receptors in silkworm larvae or cocoons has opened a new area in the silkworm biotechnology. Further, use of silkworm larvae rather than cultured cell lines as recipient for expression of foreign protein in Bombyx mori nucleo polyhedrovirus (BmNPV) has become an effective alternative for the large-scale synthesis of commercially important biomolecules. Maeda et al. [128] first reported the production of human α-interferon (IFN-α) in the hemolymph of silkworm larvae using BmNPV expression system. Likewise, in the recent past, a lot of reports were surfaced on the production of different proteins or important biomolecules of commercial importance using silkworm as a model. Production of recombinant erythropoietin (rEpo) in silkworm larvae using Bm NPV expression system has been attempted successfully in recent times [100]. To overcome the problem of degradation of foreign protein in BmNPV system, cysteine protease depleted BmNPV was also tried for isolation of bovine interferon-τ from silkworm larvae [154]. While Muneta et al. [149] purified 500 μg of bovine interleukin-21 (IL-21) from 30 ml of hemolymph using a hybrid baculovirus. Construction of a recombinant baculovirus, containing a gene of interest requires transfection of cell culture with the baculovirus and the transfer vector which is time consuming and cumbersome one. Therefore, development of a recombinant baculovirus vector (Bacmid) that can replicate in E. coli as a large plasmid is known as Bac-to-Bac baculovirus expression system has come into picture [124]. Bac-to-Bac system has addressed many issues related to expression of foreign protein in BmNPV expression system. Motohashi et al. [147] while constructing a BmNPV bacmid established the Bac-to-Bac system using BmNPV. GFPuv was expressed only by the injection of BmNPV bacmid DNA into silkworm larvae and pupae. Production of capsid proteins for foot-and-mouth disease virus (FMDV) in the hemolymph of silkworm larvae and vaccination of 30 fold diluted hemolymph for protection of cattle against FMDV [115] is another significant attempt in this regard. Expression and purification of human (pro) renin receptor (hPRR) and its complex with human prorenin were also tried in silkworm larvae, with the same expression level as some secretory proteins [65, 66]. In the country like India, where art of rearing of silkworm is as old as our heritage, holds a lot of promise and opportunities to utilize it as biofactory for production of important proteins, vaccines, and biomaterials.
Silk based biomaterials
Fabrication and use of silk based biomaterials like sature, silkbased hydrogels, cosmetics, scaffolding matrix, 2D films, nanofibrous mat have drawn considerable attention to the scientific fraternity in recent times. India is bestowed with all four kinds of silk which provide immense opportunity to explore use of silk for non textile purposes, particularly in production and application of silk protein based biomaterials. Padamwar et al. [168] while studying in vivo effect of sericin on human skin reported prevention of transepidermal water loss, responsible for skin dryness confirming the moisturizing effect of sericin. The study indicated action of sericin in increasing the level of hydroxyproline and hydration of the epidermal cells. Recombinant silk protein/ spider silk has also been used in many avenues such as commercial, military and clinical research. Spider silk has been used in prepration of bullet proof jacket and utilized for its wound healing property, especially in surgery as its biodegradable. Role of silk protein in stimulating the migration, proliferation, and production of collagen provided evidence on the healing properties of sericin, [13,14,15]. Aramwit et al. [14] also highlighted the importance of amino acid methionine of sericin in collagen synthesis, essential in the healing process. In vivo skin wound healing properties of fibroin films [210] and fibroin-alginate sponges [183] have also been found to be better compared to other clinically used materials. Mixing of silk fibroin with human hair derived Keratin has also been examined for wound healing application in skin tissue engineering [126]. Another encouraging attempt of present times is the fabrication of tasar silk fibroin based corneal films [81]. Application of tasar silk fibroin as cardiac patch because of its fibronectine like properties has also been documented [169]. Recent report suggests that silk and silk fibroin films with suitable mechanical properties may be useful as artificial blood vessels as well as for vascular graft [28, 115, 117, 127, 194]. Further, manufacturing of 2D matrices from silk gland fibroins of muga silk and tasar silk along with cocoon fibroin of mulberry silk have been studied recently [99]. In another attempt fabrication of 3D scaffolds under single and double-seeded conditions using tasar silk fibroin extracted from silk gland showed improvement of oestrogenic potentials of the scaffolds [130]. Muga silk fibroin also has got tremendous potentiality to act as an appropriate material for fabrication of functional scaffolds to support cartilage tissue engineering [29]. Orthopaedic and dental surgeries are very much prone to loosening, inflammation, bacterial infections. Tasar silk fibroin nanoparticles coupled with gentamycin supports better in osteoblast adhesion when compared to bare Ti surface [199]. Therefore, use of silk protein for synthesising novel biomaterials may open a new area in sericulture.
Waste utilization
Apart from silk, several other by-products of economic importance are being generated by silk industry including waste mulberry shoots, silkworm moth, unused silkworm pupae, tasar peduncle, cut cocoons. Among the several kinds of waste generated by the silk industry unused silkworm pupae known to contribute maximum share. The pupae, which are obtained after reeling of silk threads from cocoons, are generally thrown away though they are very rich in amino acids, oil, carbohydrate and minerals. According to an estimate approximately 40,000 MT of silkworm pupae is produced through sericulture, per annum in our country. Traditional practice of drying and disposal of silkworm pupae cause environmental pollution besides loosing important nutrients in them. But this huge waste of silk industry holds a great promise to convert them into meaningful wealth. Consuming 100 gm of (dry weight) of silkworm pupae may substitute ingestion of approximately 56 gm of protein, which satisfies the recommended dietary allowance of protein for an adult [136]. Silkworm pupae protein is considered to be a newly available source that contains all the amino acids needed by the human body. Recently, it has been documented that silkworm pupae have excellent antioxidant potential to scavenge free radicals with good antityrosinase activity [61, 62]. Proximate analysis of pupa showed that it contains 55–60% protein, 25–30% lipid, 4.96% fiber, and other substances like, hormones, trace elements and vitamins, thus indicating the pupa as a good protein source for various purposes [120, 121, 180, 269]. The silkworm pupal oil is highly enriched with α-linolenic acid which has got many uses in oleo chemical and food processing industries. Chitin and chitosan, the two important products can be obtained from silkworm skin and pupae and both these materials are known to possess antimicrobial properties with capacity of biodegradability and biocompatibility. In India, the indigenous population of north east India uses different insects as food and among them eri and muga silkworms are two important insects which are most popular as traditional food. The consumption of pupa is maximum for eri (87%) followed by muga (57.4%) and mulberry (24.6%) [140]. Additionally, silkworm pupae are known to be an important food for poultry and fish industries along with its application as base materials for production of highly valued mushrooms [37].
Waste silkworm pupae hold a great promise in many advanced fields including cosmetics, animal nutrition and pharmaceuticals with commercial importance. However, no comprehensive work has been carried out on the utilization of high potential secondary wastes of silk industry which can further substantiate the profit for the silk industry. This excellent source of good quality protein offers huge potential for nutritional and economic benefits to marginal sericulture farmers if proper utilization is made from these by products of the silk industry.
Conclusion
Sericulture implies rearing of silkworm for production of silk and ultimately its usage for textile and garment. Time has come to diversify it to make sericulture more sustainable, lucrative, and remunerative one. Therefore, in the present day, focus has started shifting on the use of silk for non textile purposes. Inclusive development of sericulture is only possible if improvement in silk productivity and application of silk for non textile purposes grows together side by side. Modern biotechnological advancement has assumed greater importance in the development of sericulture and its diversification. The science of biotechnology and its application particularly in the area of identifying new genes, molecular markers, development of diseases resistant breed and feed, synthesis of biologically important molecules, formulation of artificial diet and conversion of seri waste into wealth may take sericulture into a new height. Further application of silk protein biomedical science through modern techniques has proved to be one of the new and promising area of applying sericulture other than non textile purposes. The increased approaches of biotechnological applications can certainly revolutionise the entire seri industry besides playing its role in maintaining ecological balance. The present paper, therefore, is a comprehensive document where an attempt to accumulate different reports and research findings on thrust areas of sericulture like molecular markers, transgenesis, embryogenesis, synthesis of biomolecules, chromosome engineering, use of artificial feed and waste management issues have been discussed which will prove to be useful for further development of the silk industry in coming years.
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The authors are thankful to Raiganj University and University of Kalyani, West Bengal, India for providing instrumentation and library facilities.
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KA provided the conception and design of the study, drafting the article; RVS: supplied the acquisition of data, drafting of manuscript; TC: supplied the acquisition of data, drafting of manuscript; AB: supplied the acquisition of data; PDG: supplied the acquisition of data; SS: provided the critically revised article with important intellectual content and gave final approval of this version to be submitted.
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Alam, K., Raviraj, V.S., Chowdhury, T. et al. Application of biotechnology in sericulture: Progress, scope and prospect. Nucleus 65, 129–150 (2022). https://doi.org/10.1007/s13237-021-00355-2
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DOI: https://doi.org/10.1007/s13237-021-00355-2