Realizing the importance of impending global climatic changes and threatened sustainability of biodiversity wealth in India at faster rate, systematic survey and exploration for collection of mulberry biodiversity have gained greater momentum in the recent past. Consequently, CSGRC at Hosur has been aptly established in the year 1990 by Central Silk Board (CSB), Ministry of Textiles, and Government of India under prestigious National Sericulture Project. It is the nodal agency for mulberry germplasm management in India and recognised as National Active Germplasm Site (NAGS) for mulberry by National Bureau of Plant Genetic Resources (NBPGR), New Delhi, India under Indian National Plant Genetic resources System (IN PGRS). CSGRC, Hosur so far conducted 46 survey and exploration trips covering more than 50 districts from Himalayan belt to Andaman and Nicobar Islands covering forest areas, biosphere reserves, national parks, back yards, kitchen gardens, agricultural lands and farmhouses etc. and collected 516 diverse mulberry germplasm resources which mainly belong to 4 Indian species (Table- 3 and Fig-1). Out of 4 species of genus Morus reported in India, M.laevigata is distributed throughout India both under natural and managed habitats and M. serrata is confined to northwest India in wild condition. M. indica and M. alba are mostly available in cultivated forms (Ravindran et al. 1977). The sericulture research institutes like Central Sericultural Research and Training Institute (CSRTI) at Berhampore (West Bengal), Mysore (Karnataka) and Pampore (Jammu and Kashmir) have started collection of Morus species in Central Himalayas, North-Eastern India, North-Western Himalayas, Kumaon and North Eastern India and Western ghats and Kerala. All these germplasm resources are collected and introduced in the field gene bank.
India being signatory to both CBD and World Trade Organization (WTO) needs to develop the efficient conservation strategies of its vast seribiodiversity best suited to India’s national interest. Exploration for collection of mulberry biodiversity and development of efficient conservation strategies has gained momentum in the recent past in India. The conservation of mulberry genetic resources includes their augmentation, safe holding for medium and long-term preservation; protection in natural habitats and it is interlinked to the sustainable utilization of germplasm to justify long-term investments on managements and maintenance of the germplasm. Studying the geographical spread, distribution and genetic architecture, arborescent nature and physiological storage behaviour of mulberry, two basic conservation strategies i.e. Ex situ and In situ methods composed of various techniques covering entire gamut of genetic diversity have been worked. The flowchart of the conservation methods presently followed at the centre and the proposed future plans of in situ conservation is presented in Figure-2.
Field gene bank
Mulberry being perennial outbreeding tree exhibits high degree of heterozygosity. Hence, for conservation of mulberry outside its natural habitat, ex situ field gene bank (clonal repository) has been developed by planting vegetative clones of mulberry accessions for maintaining the genetic integrity of the conserved material. Rooted cuttings (saplings) after six-month establishment in the nursery are transplanted in the field gene bank. Saplings of some exotic accessions and wild Morus species with poor rooting rate, are developed through bud grafting to the local scions and then established in the base collection. The plants (four plants/accessions) are maintained as a dwarf tree with spacing of 2.4 ´ 2.4 m between plants with the crown height of 1.5 m following recommended cultural practices and plant protection measures. Pruning is being followed once in a year (June-July) to renew the germplasm with new sprouts. However, the species, which cannot sustain repeated pruning are left unpruned. The entire collections in field gene bank are fenced and protected. Presently, the ex situ field gene bank of CSGRC, Hosur holds 908 mulberry accessions (Indigenous-647 and Exotic-261) from diverse genetic and geographical origin representing 13 Morus species collected from 26 countries (Table-4). The list of Morus species in the ex situ field gene bank is given in Table-5. The classification of the entire germplasm holdings is presented in Table-6. CSGRC, Hosur being the nodal agency for mulberry germplasm in India maintain the entire mulberry germplasm available in the country. However, the Sericulture Research Institutes, Universities, State Sericulture Departments are also maintain the mulberry germplasm for research purpose, which also serves as backup conservation centres (Table-7). Each accession contains permanent label with unique identification number. National Accession Numbers (Indigenous collections: IC. No: 313662- 314262 and Exotic collections: EC. No: 493758-493928) have been provided for all the mulberry accessions conserved in the ex situ field gene bank by NBPGR, New Delhi for protection of mulberry genetic resources at global level.
On-farm participatory conservation
The advent of high yielding varieties of mulberry like V-1 and S-1635 and their large scale spread in the traditional sericultural zones under monocropping pattern replacing the local landraces and more particularly in the irrigated system which leads to reduced genetic base and increased the genetic vulnerability of the crop. The seribiodiversity otherwise greatly threatened because of unlawful habitat destruction, natural calamities, fragmentation of forests and social disruption and this large scale genetic wipeout disturb the coexistence of sericigenous flora and fauna. Under these circumstances on-farm conservation linked with Farmers Participatory Breeding (FPB) to be given due emphasis for achieving twin goals of sustainable conservation of biodiversity and in turn utilization on-farm biodiversity (Eyzaguirre and Iwanaga, 1996). In India, rich Morus diversity exists under managed habitats i.e. in the backyards, kitchen gardens, farmhouses (Fig-3), horticultural gardens, agricultural lands and roadside plantations. These are the firsthand selections of the farmers and tribals for varied utilizations hence; conservation of potentially interesting alleles and development of diversity is promoted. In mulberry the wild species like Morus laevigata and Morus serrata and other wild species, which do not get attention in the formal sector for cultivation for sericulture purposes. So, these associated valuable species or otherwise utilized for non-sericultural purposes (horticulture and agroforestry) flourish well in the on-farm conservation procedures promoting farmers/tribals livelihood development while conserving Morus biodiversity. This particular sector of Morus biodiversity lies mainly in the public domain, which needs to be taken care and bring them under definite set of legal framework of Indian Forest Act (1972).
Botanical gardens and national herbarium
In the context of PGR, the herbarium provides the basic material for detailed monographic and phyto-geographical information of the species. In India there are many herbarium centers and some of the main herbarium centers like: Botanical Survey of India with its regional centers, Presidency College, Chennai; Baltter Herbarium, St. Zavier College, Mumbai; St. Josephs College, Tiruchirapalli and National Herbarium, NBPGR, New Delhi maintain Morus species herbarium. Botanical Survey of India, Pune preserves the oldest herbarium of genus Morus dates back to 1886. Seribiodiversity museum of CSGRC, Hosur maintains large number of herbaria and it serves as National Herbarium Centre for Morus species in India. In India there are about 33 botanical gardens and the some of them like: Indian Botanical garden, Howrah; Lloyd Botanical garden, Darjeeling; National Botanical Garden, Lucknow maintain mulberry arboreta.
Conservation using biotechnology In vitro and cryopreservation
Conservation of mulberry germplasm in the field gene bank is simple and technically less demanding. However, maintaining a large collection is costly, requiring huge resources. Besides, it is risky since it is exposed to different biotic and abiotic stresses. Cryopreservation technology forms an alternative to ex situ conservation strategy for crop like mulberry, which is vegetatively propagated utilizing liquid nitrogen (LN) at ultra low temperature of -150 °C (vapour phase). Cryopreservation method ensures the genetic stability of the germplasm and provides an alternate cost effective non-dependence on electricity long-term strategy for conservation of mulberry germplasm. Keeping this in view, establishment of in vitro and cryopreservation laboratory facilities at CSGRC, Hosur is planed in collaboration with NBPGR, New Delhi. Efforts have been made to standardize the efficient techniques on cryopreservation of winter buds, embryonic axes, pollen of mulberry accessions (Niino et al. 1992 a, b, c, 1993 and 1995) comprising different species, landraces, local cultivars, wild and polyploid accessions in liquid nitrogen (-196°C). In vitro conservation techniques involve conservation of active collections under active growth stage and base collection germplasm under suspended growth stage using growth retardants.
Even though lot of work on mulberry regeneration with or without callus formation in in vitro has been reported. Very little work has been carried out in in vitro conservation of mulberry genetic resources. Single shoots of M. nigra L stored on multiplication medium at 4 °C for 16-hour photoperiod survived only for six months. Survival was enhanced to 42% at nine months by storing them at 25 °C with activated charcoal as supplement. High viability (80%) for six months was observed in 15 genotypes of M. alba stored at 4 °C dark in shoot proliferation medium (Sharma and Thorpe, 1990). Rooting was observed in all the shoots and shoots retained their multiplication potential.
Cryopreservation procedures have been standardised for about 100 different plant species cultured in various ways including cell suspension, callus, apices and zygotic and somatic embryos. Cryopreservation, possibility for the first time was demonstrated using mulberry twig by Sakai (1960). Since then, considerable work on cryopreservation of mulberry has been undertaken especially in Japan. Shoot tips of pre-frozen winter buds of M. bombycis Koidz. was able to withstand storage in LN. However, grafts and cuttings did not survive in LN. With modification of this method it was able to regenerate plants of M.multicaulis P. through tip culture of frozen winter buds. Shoot segments were pre-frozen at - 3 °C for 10 days, - 5 °C for three days, - 10 °C for 1 day and - 20 °C for one day before immersion in LN. Buds were cultured on MS medium after thawing in air at 0 to 20 °C. Survival rate was 55 to 90%. Excised shoot tips from winter buds of M. bombycis Koidz. pre-frozen at 10 °C per day. Prior to pre-freezing at -20 °C partial dehydration to 38.5% improved the recovery rates. The survival rates of the winter buds stored in LN from the month to 3-5 years did not change. Direct dehydration with silicon gel at 25 °C of excised shoot tips (2 mm long) from winter bud could be done before immersion in LN. With decreasing water content shoot formation increased and at about 19% of water content, a maximum of 80% survival rate was observed. Encapsulation by alginate coating of winter hardened shoot tips of many Morus species had 81% of shoot formation with 22-25% water content. In vitro grown shoot tips of thirteen cultivars of mulberry were tested for cryopreservation. Slow freezing (0.5°C/min. to - 42°C), vitrification (PVS2, 90 min) and air- drying (24% water content) or encapsulation dehydration (33% water content) was tested for survival, which ranged from 40-81.3%. It was also reported the long-term storage of mulberry winter buds by cryopreservation. Winter buds from M. bombycis with about 10 mm vascular tissue were kept at 0°C for 1 day before freezing. Buds were cooled to 10°C steps at daily intervals from 0 to 30 °C prior to immersion in LN or before transferring to -135 °C. After storage, buds were rapidly thawed at 37 °C in a water bath and then cultured on MS medium supplemented with 2% fructose and 1 mg/l with 6-BAP. Rate of shoot formation did not vary much in buds stored in LN or deep freezed at -135°C after a storage period of 3-5 years.
In India, the application of in vitro technique for mulberry conservation has been recently attempted. In vitro technique has been attempted in mulberry mainly for propagation of popular mulberry varieties and poor rooting materials, evolution of some clonal variants and elite materials and screening of genotypes for tolerance to salt and somatic stresses. (Tewary et al. 1996 and 2000). Bapat et al. (1987) developed method for propagation of Morus indica L. (Mulberry) by culturing encapsulated shoot buds. Conservation of mulberry germplasm accessions through cryopreservation technique has been recently attempted in India.
In situ conservation promotes the conservation of eco-system and natural habitats and the maintenance and recovery of viable population of species, which can survive, and best perpetuate in their natural microclimate. It also simultaneously permits continued evolutionary development under natural selection pressures, thereby promoting the fitness of the species. In situ conservation demands the establishment of nature or biosphere reserve and national parks to protect the endangered species. The National Committee on Environmental Planning and Coordination (NCEPL) and Man and Biosphere (UNESCO) already identified 14 Biosphere reserves in India and among them Uttarkhand, Nandadevi, Namdapha, Kaziranga, Manas, Nokrek, North Andaman and Great Nicobar are the potential reserves for in situ conservation of mulberry. Keeping this in view, efforts have been made to collect information on the location of availability of mulberry germplasm with details on “declared protected area network of India” including biosphere reserves, national parks, wild life sanctuaries etc. Mulberry is not fully protected under Indian Forest Act (1972) in many parts of India except in some states of North-East India. Survey map, exact location, landowner with postal address, survey number and its jurisdiction are not available with CSB units or State department. Under the circumstances, a suggestive and advisory role can be contemplated with greater thrust on repeated survey and exploration.
Since ages, mulberry is being worshiped by tribals of Uttaranchal of Himalayan region and conserved in the name of “Sacred grooves”. The sacred mulberry tree (M. serrata) at Joshimath (altitude of 2 000 above MSL) being worshipped by the pilgrims of Badrinath, is said to be the oldest (1200 years) and biggest mulberry tree (21.6 m of circumference) in the world. The great Indian sage Adiguru Shri Shankarcharya is said to have meditated under this tree. Because of this reason all M. serrata trees are found unaxed and worshiped with taboo at several places in Garhwal and Kumaon regions of Himalayas. Similarly, trees of M.laevigata are conserved in Punjab. Attempts are being made to locate the concentrated areas of wild/natural mulberry trees of Morus laevigata at Island of Andaman & Nicobar biosphere, Arunachal Pradesh and areas of Himalayan region. Large numbers of naturally grown M.laevigata trees are also available in Eastern Himalayas of south and west Sikkim, West Bengal, Assam, Meghalaya and Manipur. Besides, developed gene pool of M.alba, M.indica and M.laevigata are also available in western ghats covering Kerala, Tamilnadu and Karnataka, Madhya Pradesh, Maharastra, Rajasthan (Fig-4), Yarcaud hills of Tamilnadu and Simlipal reserve forest of Orissa etc. which are adapted to local environment for many years are also required to be protected under in situ conservation.
Characterization of mulberry genetic resource determines the structural and functional attributes of the accessions and is highly essential for their genetic identity under IPR regime. The comprehensive information on variability recorded helps breeders and users for effective crop improvement. For choosing the donors for hybridisation programmes, the background information is highly essential. The detailed information is required to describe an accession. The data need to be standardised in terms of terminology, measurement codes, encoding methods and data recording procedures and hence, characterization becomes mandatory and forms a bridge between conservation and utilisation.
Systematic characterization of mulberry germplasm resources was started after establishment of CSGRC, Hosur in year 1990. CSGRC, since its inception actively engaged in characterization works and during the first phase and second phase of the project (1991-2000) 628 mulberry accessions (Indigenous-455 and Exotic-173) have been systematically characterised for 22 morphological, 26 reproductive behaviour, 15 leaf histology, 14 growth and yield, 2 cytological characters and the data of 328 mulberry accessions are published in the form of catalogue (Thangavelu et al. 1997) and the data with query based retrieval is also available in the website (WWW.SILKGERMPLASM.COM). The second volume of catalogue with data of 300 mulberry accessions will be published shortly and the website will also be updated.
2.2.1.1 Morphological characterization
Morphological characterization is mainly based on visual observation and wherever it is necessary it is supported by quantitative measurements. These characters are strongly heritable in nature and manifested equally in all the environments and hence used in the genetic identify of the accession and this also enable an easy and quick discrimination between the phenotypes. Thus, the characterization and estimation of the morphological variability provides useful measure of genetic diversity prevalent in the gene pool. At CSGRC, Hosur morphological characterization has been completed for 628 mulberry accessions (Rao et al. 2002). The frequency distribution of some important morphological characters is presented in Fig-5.
2.2.1.2 Reproductive characterization
The knowledge of floral structure and reproductive behaviour of mulberry accessions are of utmost important to undertake any breeding programme for maximum utilization of genetic resources for future needs.
At CSGRC during the first phase 617 mulberry accessions (Indigenous-446 & Exotics- 171) have been characterised with 26 reproductive descriptors. The sex expression, variability of different male, female reproductive characters and the frequency distribution of mulberry accessions on sex expression in relation to leaf yield are presented in Table 8-9 and Fig-6. Three major groups viz. dioeciously male, female and monoecious with regard to sex expression and sexual composition of various accessions are observed. Out of 617 mulberry accessions studied 368 (59.64%) produced strictly pistillate (female) inflorescence, 75 (12.48%) staminate (male) inflorescence and 172 (27.88%) are monoecious types. Monoecious accessions with high leaf yield base may utilized for developing inbred lines. Increased degree of dioecy (femaleness) was observed in most of the locals and wild genotypes and this prevalence of unisexuality and preponderance of femaleness in mulberry irrespective of genetic and geographical origin might have resulted through evolutionary changes by suppression and progressive reduction of one sex in bisexual flower (Rao et al. 2000).
2.2.1.3 Leaf histological characterization
Leaf is the vital organ in a plant where the major physiological activities take place. Largely stomatal characters possibly influence stomatal resistance, transpiration rate, and photosynthesis rate. The assimilatory tissue such as number of chloroplast in the guard cells of the stomata is directly correlated to the ploidy status. Physio-anatomical features related to drought tolerance in mulberry were reported (Susheelamma and Jolly, 1986 and Dorcus and Viveknandan, 1997). Thus, the leaf anatomical characterization of mulberry is highly essential not only for the phylogenetic studies but also for the identification of stress tolerant varieties.
At CSGRC during the first and second project periods 628 mulberry accessions (Indigenous-455 and Exotics-173) have been systematically characterised with fourteen anatomical descriptors. The variability and frequency distribution of mulberry accessions on leaf histological characters are presented in Table-10 and Fig-7.
2.3.1.4 Cytological characterization
Mulberry is characterised by different ploidy levels ranged from x to 22x. Among the different ploidy levels triploids are considered to be superior in most of the economical characters and wider adaptability to different agro climatic conditions. Most of the genotypes which belong to M. alba L. and M. indica L. are diploids (2 n = 2x =28). The wild species of mulberry belong to M. laevigata Wall. and M. serrata Roxb. are available in different ploidy levels. Hexaploid mulberry varieties belong to M. serrata Roxb. (6x=2n=84) M. cathyana has been reported. Octaploidy in M. cathyana, docosoploidy in M. nigra and Polysomaty in M. multicaulis and M. alba was also reported. The karyotype morphology will help in understanding the phylogenetic relationship among the different species and its distribution has a great value in modern taxonomy. A detailed karyo-morphological study in some popular mulberry varieties was reported (Janaki Ammal, 1948 andDandin et al. 1987).
2.2.1.5 Molecular characterization
WTO requires its member countries to accord protection to plant varieties under TRIPS agreement. The protection intended to buoy innovations, which can be provided by patents, by an effective sui generis system or by any communication thereof. Molecular markers on the other hand exhibit high degree of non-tissue specific polymorphism and simple inheritance pattern with minimum influence of environmental and epistasis. In the last two decades, geneticist has started using biochemical and molecular techniques to add valuable information for characterization of germplasm. In addition to the fact this genetic information can be generated quickly, they are environmentally independent, accurate and reliable. Isozyme markers have been successfully used to characterise plant genetic resources. Even though, isozyme analysis is simple and relatively cost effective they are confine to small portion of genome and often they do not offer sufficient polymorphism to reveal the genetic difference. In the pursuit, molecular approaches like RFLP, RAPD, microsatellite markers, AFLP etc. effectively overcome this problem by offering high level of polymorphism. These techniques along with computer based software programmes are being routinely used in characterization and germplasm related studies like marker based gene tagging, gene cloning, diversity analysis and marker assisted selection of desirable genotypes all over the world.
Isozyme analyses
At CSGRC, Hosur the isozyme study was initiated in 17 representative Morus species using peroxidase enzyme system to assess the allelic variation at isozymic loci and to ascertain the genetic diversity. All the species showed one common band but variability was restricted to minor bands. Based on the number of bands and Rf values, these species were categorised into six groups. Fifty mulberry landraces and popular varieties were subjected to Isozyme analysis using three enzyme system namely anodal peroxidase, acid phosphotase and cathodal peroxidase to study the extent of genetic diversity. Peroxidase banding pattern showed greater variability. M. laevigata is distributed in different parts of the country both in wild and managed habitats. In order to assess the genetic diversity in 32 M. laevigata explored collection were subjected to isozyme analyses. Peroxidase, ß- esterase and Cathodal peroxidase enzyme systems showed wider genetic polymorphism. Acid phosphotase enzyme again did not show any genetic polymorphism among 32 M.laevigata accessions. Isozyme banding pattern did not show any correlation with geographical distribution of collections. However, M.laevigata collections from north-eastern India revealed maximum genetic diversity at isozymic loci indicating potentiality for further exploration and collection. Polymorphism at Isozyme loci is comparatively low in mulberry as is the case in most of the cultivated crops. Hence, there is a need to take up more number of enzyme systems for Isozyme analyses to obtain conclusive result (Anonymous, 1997-1998).
DNA Finger printing
Molecular characterization of mulberry germplasm was initiated in collaboration with Seri Biotech Research Laboratory (SBRL), Bangalore. Initially, protocol for mulberry DNA extraction, RAPD and Inter SSR- PCR analysis was standardised. Fifteen morphological distinct species were analysed using RAPD and Inter SSR-PCR assays. In RAPD assay, 19 oligonucleotide primers were utilised. These analyses also identified few species-specific amplification products. Inter SSR-PCR analyses were carried out in 15 mulberry species using four primers. However, the species-specific markers have to be studied over populations of individual species so as identify the unknown species. The RAPD analyses of 46 mulberry varieties using five oligonucleotide primers revealed polymorphism suggesting the genetic variation. However, clustering pattern did not show correlation with regard to regional/geographical distribution of these varieties (Chatterjee and Awasthi, 2000). National Botanical Research Institute, Luknow studied nine mulberry germplasm varieties (triploids, species and popular varieties) using 23 arbitrary sequence decamer primers for RAPD and three minisatellite core sequence primers for DAMD polymorphism. Nearly 85% RAPD and 91% DAMD bands were polymorphic across nine varieties differentiating ployploids and popular varieties (Bhattacharya and Ranade, 2001).
2.2.1.7 Taxonomical status of genus Morus L.
Ever since sex in plants first demonstrated in mulberry, the sexual polymorphism in genus Morus continues to be enigmatic and forms the study material for many classical taxonomists. Linnaeus (1753) for the first time established genus Morus with 7 species. Hooker (1885) placed genus Morus L. under Urticales and tribe Moreae. Takhatajan (1980) rightly placed genus Morus under Moraceae family of order Urticales which is considered to be advanced in the woody flowering plants. Different authors have classified genus Morus based on morphological and phenological characters (Koidzumi, 1917; Engler and Prantl, 1924; Bounocore, 1941; Ledebour, 1951; Hotta, 1954 and Katsumata, 1972). The taxonomical status of genus Morus L. has been critically reviewed by Sanjappa (1989), Tewary et al. (1989), Ramesh and Basavaiah (1989).So far more than 150 species (vide Index Kewensis) have been described under genus Morus, however, most of them have been now reduced as synonymous or varieties of the same species. Presently there are about 68 recognized species endemic to 13 nations (Sanjappa, 1989). The comprehensive classification of genus Morus with 25 species by Koidzumi (1917) mainly based on style length and stigma hairiness though holds good but there is no place for male unisexual accessions in his classification. Tikader et al., (1999) and Tikader and Rao, (2001) used morpho-reproductive characters of quantitative nature for divergence analysis of ten Morus species and found that Morus alba formed separate cluster for all the traits studied and reproductive characters have contributed maximum forming seven clusters. Dandin et al. (1987) studied the chromosome configuration and phylogenetic relations of different varieties of four mulberry species. Recently, using molecular techniques with four ISSR and 19 octamer random primers, the species-specific markers in 15 mulberry species were identified. (Chaterjee and Awasthi, 2000). However, these markers are to be linked and correlated to the qualitative and quantitative traits. Reviewing the taxonomical status of genus Morus based on literature available, it is clear that various authors have classified genus Morus with limited number of mulberry species of particular geographical origin. Nevertheless, an agreeable system with diagnostic keys of different species for correct classification of genus Morus L. is yet to emerge out. There is an urgent need to procure the wild relatives of Morus species from different geographical endemic regions through survey and explorations and study the numerical taxonomy using various descriptors including molecular techniques for the revision of genus Morus on world basis.
The assessment of genetic potentiality of mulberry germplasm is a vital for crop improvement programmes. The breeders mostly depend on the evaluated material for choosing parental lines. Evaluation of germplasm is mainly done for propagation, growth and yield, nutritional quality, biotic and abiotic stress tolerance and region and season specific performance.
2.2.2.1 Propagation parameters
Mulberry is chiefly propagated through vegetative stem cuttings to maintain the genetic potentiality of evolved varieties. Rooting behaviour of mulberry accessions is an important trait in the breeding programme. In the temperate belt, usually the introduced exotic varieties such as Goshoerami and Kosen propagated through grafts, on the stocks raised as seedlings of local varieties. Hence, scion and stock compatibility of different mulberry accessions are to be studied for utilization of temperate varieties.
Earlier attempts were made at CSR&TI, Mysore with 61 accessions taking root length and root-shoot ratio (Susheelamma and Jolly, 1986). Screening of rooting ability in cuttings of different ploidy levels of mulberry accessions have indicated that triploids are better rooters followed by diploids and then tetraploids. Correlation of rooting traits with growth parameters was worked out in 31 accessions (Bindro et al. 1996). Morus multicaulis is a good rooter with high yield potential for exploitation. Sujathamma and Dandin, (1998) studied sprouting and survival of 25 germplasm varieties under arid zone.
At CSGRC, Hosur, propagation studies related to survival and rooting behaviour were conducted in 530 accessions (indigenous-390 and exotic-140) during spring and rainy seasons of 1996-99 following RBD design (Goel et al. 1998). Exotic varieties mainly from temperate belt have showed poor rooting ability.
2.2.2.2 Growth and yield attributing parameters
Earlier studies at CSR&TI, Mysore and CSR&TI, Berhampore were restricted to the regional requirement with limited no. of genotypes and various mutants/hybrids. The S series such as S-30, S-36, S-41 and S-4 were yield tested at various levels before release for commercial cultivation. This was followed by screening of genotypes under rainfed conditions, which resulted in identification of RFS-135 and RFS-175 followed by S-13 and S-34 (Susheelamma et al. 1990). Similarly, evaluation studies conducted at CSR&TI, Berhampore have resulted in isolation of triploid varieties such as TR-4, TR-8, TR-10 followed by BC-2-59, C-776, S-799, and S-146 etc. Evaluation of few exotic varieties for tree plantations in temperate areas of Northern States of India such as Jammu and Kashmir have been carried out at CSR&TI, Pampore to recommend Goshoerami, Kosen and China White for commercial cultivation.
Field gene bank evaluation under high bush
The yield attributing parameters of mulberry accessions are first to be assessed in the field gene bank to group and shortlist mulberry accessions for further screening through primary and final yield trials. In mulberry, there is a paucity of information available on the evaluation of germplasm as dwarf tree for different growth parameters. At CSGRC, Hosur during the year 1993-1997, 628 mulberry accessions (Indigenous-455 and Exotic-173) were assessed for their yield attributing characters for 16 growth and yield parameters. The analyses of variance indicated that both the seasons and accessions are significant. Exotic varieties exhibited higher mean values for different leaf characters whereas indigenous accessions showed higher mean values for shoot characters. Range of variability of mulberry accessions on growth parameters under dwarf tree condition is presented in Table-11. The promising accessions on different growth parameters presented in Table-14 may be utilized by the breeders for crop improvement.
Evaluation under augmented RBD design
Evaluation of mulberry germplasm suited to farmers package of practices is an added advantage for selection of superior genotypes. Hence, an experiment was conducted at CSGRC, Hosur with 316 accessions (234 indigenous + 78 exotic + 4 checks) for preliminary evaluation for growth and yield parameters under ARBD. This would facilitate to shortlist promising genotypes for further screening at breeders level after evaluation for quality parameters through bioassay studies. A total of 45 mulberry accessions were finally selected for further evaluation. Bioassay (full rearing test) of these 45 accessions has been conducted to select promising accessions.
2.2.2.3 Evaluation for quality parameters
The contribution of mulberry leaf for successful harvest of cocoon crop is well recognised. Varietal differences in moisture, protein, mineral and sugar content in mulberry germplasm are reported by various authors (Cheluvachari and Bongale 1996 and Sujathamma and Dandin 2000). At CSGRC, Hosur, biochemical characterization was completed in 628 accessions using the leaf samples collected from base collection. Chlorophyll-a, Chlorophyll-b, carbohydrate and protein (water soluble) content were estimated as per standard procedures. These studies have indicated the potentiality of exotic varieties in relation to quality parameters. With limited number of commercial varieties of mulberry, quality assessment through moulting test have been undertaken at CSR&TI, Mysore and other Research Institutes. Such studies need to be extended to the germplasm accessions after preliminary screening and a full rearing test also need to be conducted for further utilization.
2.2.2.4 Evaluation for biotic stress
Mulberry is affected by large number of diseases caused by fungi, bacteria, viruses and nematodes and pests. The diseases besides causing heavy crop loss (10 to 30%) also reduce the leaf quality. One of the most promising and economic ways of evaluating the disease loss in mulberry is to evaluate germplasm collections in the hot spots, which would also help in identifying genotypes for breeding disease resistant varieties (Govindaiah, et al. 1989 and Philip et al. 1996 and Yadav et al. 1993). Ninety-two mulberry accessions were screened for various diseases at CSR&TI, Mysore of which 17 were found to be resistant to leaf spot and mildew diseases with 5% disease incidence. Only three accessions were moderately resistant to rust disease. The varieties Kaliakuthai and Bilidevalaya were found to be completely resistant to leaf spot/powdery mildew.
At CSGRC, Hosur preliminary screening of 328 mulberry accessions against three major diseases such as powdery mildew, leaf rust and leaf spot was carried out in the field gene bank to select disease tolerant varieties. (Thangavelu et al. 1997). However, these accessions are to be tested in the hot spots for their conformation.
2.2.2.4 Evaluation for abiotic stress
It is estimated that various abiotic stress conditions such as moisture (drought), salinity and alkalinity result in 50-60% yield loss in case of mulberry, thereby affecting the economics of sericulture.
Drought resistance
Susheelamma et al., (1990, 1992) have successfully demonstrated the need of wider genetic base and importance of mulberry germplasm characterization and preliminary evaluation with reference to leaf anatomical characters (high thickness of leaf, smaller stomata in lower frequency, thicker cuticle, and higher spongy palisade layer ratio), rooting parameters (length of root, dry weight of root, root shoot ratio by length and weight) and yield characteristics (number and length of branches, leaf weight and area, internodal distance, leaf shoot ratio, moisture content and retention capacity of leaves) for identification and evolution of drought resistant mulberry cultivars. These studies have resulted in releasing several drought tolerant varieties such as S-13 etc.
Field studies (Dorcus and Vivekandan, 1997) have indicated that chlorophyll stability index is an important drought resistance parameter since the increase in level of total chlorophyll (a & b) is found to be minimal under severe stress condition in potential drought resistant varieties of mulberry. Studies on drought induced cellular events in mulberry (Das et al. 1998) have indicated the increased activity of proline and glutathione. Such methods of quantification of bio-chemicals can be extended to preliminary screening of germplasm accessions for drought resistance. Earlier observations (Dorcus and Vivekanandan, 1997) in Mulberry though have indicated marginal to significant level increase of osmotins such as proline and glycine betaine, however without significant difference between drought susceptible and drought resistant genotypes. Hence, suggesting that level of such osmotins do not lead to any meaningful conclusion to be associated with stress resistance.
Mulberry germplasm accessions selected through different morpho-physiological, propagation and yield evaluation characters at CSGRC can be subjected to preliminary pot screening test under stress and non-stress conditions to assess drought tolerance based on wilting percent and wilting recovery and other growth characteristics of saplings. The tolerant genotypes are to be tested in the hot spots for selection of drought tolerant varieties.
Alkaline or acidic tolerance
Mulberry is cultivated often in soils, which are alkaline or acidic as in some parts of Karnataka (Mysore district) and Tamil Nadu (Salem district) of Southern plains and West Bengal and Bihar in Eastern plains. Mulberry can tolerate pH range of 5 to 8, however it grows best in the soils having pH around 6.5. Empirical testing in hot spots for saline or acidic soil is the best way to screen genotypes tolerant to such soils. CSGRC, Hosur in collaboration with its regional net working stations of CSB units has to take up the evaluation trials of the promising mulberry germplasm accessions to select alkaline tolerant varieties. The hot spots identified in Southern plains for alkaline soils are Kinakanahalli under RSRS, Chamarajanagar (pH 8.5-9.5) and RSRS, Salem in Tamil Nadu. Similarly for acidic soils (Kar, 94-95), Kishanganj (Bihar) is a hot spot with pH around 5.25. CSRTI, Mysore has conducted yield trials at Kinakanahalli under RSRS, Chamarajanagar resulted in identification of alkaline resistant varieties.
Saline tolerance
Salinity is one of the limiting factors of crop yield in nearly one third of irrigated land. Sericulture productivity is more dependent on irrigation or partial irrigation since mulberry responds well to irrigation; as evidenced by intensive cultivation in Southern and Eastern plains of India, where nearly 40% of mulberry cultivation is under irrigation. Salinity influence growth in mulberry through reduction in root growth at lowers levels and at higher level it has greater effect through reduction of shoot growth. Above 30 mM of salinity drastic reduction in growth of both shoot and root are reported. Salinity also strongly decreases the protein, starch, and sucrose levels in leaves. Protocol for screening of large number of germplasm accession of saline tolerant lines in mulberry, through in vitro screening, using lower concentrations of NaCl at 0.1% and 0.2% level to know the effect on rooting has been suggested (Chakraborti et al. 1997-98). Rooting parameters such as number of roots/plant, average root length and length of longest root are considered important in such screening.
Raising of saplings from mulberry cuttings in hydroponics culture solutions with different concentrations of NaCl have been suggested as a method for screening large number of mulberry accessions for saline tolerance (Shaik and Vivekanandan, 1994). The specific effects of salinity on rooting ability, early growth and level of chemical constituents are assessed in this procedure. Level of increase in total chlorophyll content in particular and total proline, glycine, betaine and peroxidase in saline conditions are known to be useful markers.
In vivo screening of 26 popular mulberry varieties on saline soils of West Bengal (EC below 9dsm-1 resulted in identifying saline tolerant mulberry variety C-776 with yield potential of 25.96 tonnes/ha/year. for yield in different seasons, followed by bio-assay to select varieties with the combination of higher assimilability and digestibility is the best way to select saline tolerant varieties for coastal saline soil under medium range of salinity less than 9 EC (Chakraborti et al. 2000).
2.2.2.6 All India Co-ordinated Experiment (AICE)
The research institutes of Central Silk Board like CSRTI, Mysore (Karnataka), Berhampore (West Bengal) and Pampore (Jammu and Kashmir) located in different agroclimatic regions and the sericultural state departments have evolved high yielding varieties of mulberry through controlled selection, hybridisation and triploid breeding. These varieties have high yield potential in different agroclimatic and cultural conditions. Realizing the wider adoptability of these varieties and to select region and season specific varieties, All India Co-ordinated Experiment (AICE) was conducted by CSB wherein 16 high yielding varieties were tested in 22 testing centers under four agroclimatic conditions to select region specific varieties.
2.2.2.7 All India Mulberry, Silkworm Germplasm Evaluation Programme (AIMSGEP)
For the first time in India, the germplasm multilocation evaluation programme at national level was initiated by CSGRC, Hosur involving eight regional backup conservation centers located in different agroclimatic zones during 2001. Fifty mulberry accessions (indigenous-24 and exotic-24), which stand promising in the field gene bank evaluation trials along with regional checks were planted under simple lattice design for multilocation evaluation. The data will be computed through Mulberry Germplasm Information System (MGIS) at CSGRC, Hosur so as to develop national database on mulberry.
Studies so far conducted have revealed large amount of genetic variability as could be seen from analysis of variance (Table 8-11). Based on the same top scoring accessions on important parameters related to economic traits have short listed and circulated among all the institutes of CSB for making use of the same as donor potential parents in their mulberry improvement programmes. Some of the genotypes scoring higher ranks with respect to majority of the important traits have to be short-listed and PYT/FYT of these lines has to be taken up. The information thus generated along with other left over parameters needs to be utilized to prepare the phylogenetic tree and establish the genetic divergence/closeness among species and accessions. Hence, the following future line of studies has been identified as priority area.
1. Cyto-morphological characterization of the mulberry genetic resources
2. Embryological investigation of all the species and important cultivars which hitherto lacking
3. Screening of short listed mulberry genetic resources for various biotic and abiotic stress in the hot spots
4. Identification of species-specific markers for taxonomical and phylogenetic studies and bimolecular trait specific molecular markers for drought/salinity stress condition.
5. Identification of resistance genes for the important diseases viz. leaf spot, powdery mildew, leaf rust, bacterial leaf blight, root rot, stem canker and other important disease
6. Identification of resistant sources for mealy bugs/leaf roller/stem borer and nematodes
7. Promising top scoring lines be put to yield and quality tests
8. Development of core collections (grouping on taxonomic characters, quantitative parameters, qualitative characters etc.) for effective utilization of mulberry genetic resources
Sustainable utilization of genetic resources for global food security has been greatly thought and discussed after the most important Forth International Technical Conference on Plant Genetic Resources for Food and Agriculture (PGRFA) of FAO held in Leipzig, Germany in 1996. The Global Action Plan was drawn representing the input of 198 countries, NGO, scientific experts and over 2 000 recommendations were passed and about 20 priority areas for utilization of PGR were identified. “Sustainable Use”, means, the use of components of biological diversity in such a manner and at a such a rate that does not lead to long-term decline of the biological diversity thereby maintaining its potential to meet the needs and aspirations of present and future (National Biological Diversity Act India, 2000.). The most effective way to conserve mulberry germplasm resources is to put them in continuous use meeting competing demands of the society. Hence, multipurpose uses of mulberry germplasm have to be greatly exploited for their sustainable conservation (Fig-8).
2.2.3.1 Utilization of mulberry genetic resources in breeding
Mulberry improvement programmes utilizing genetic resources in India has travelled a long way (Dandin et al. 1997). Mulberry genetic resources have been used in the breeding programmes mainly through controlled hybridisation, polyploidy breeding, mutation and selection. The Indian sericulture in early 1950s-1960s has witnessed the use of local landraces belong to species Morus indica like Kajli in West Bengal and Mysore local in Karnataka with highly dissected leaves. During 1960s introduction of broad entire leaved varieties like Gosheorami, Tsukasaguawa and Ichinose from Japan in the temperate region of Jammu and Kashmir and Matigara black in north eastern India under wet tropical climate, which have marginal, increase in the leaf yield. The mulberry improvement programmes utilizing germplasm resources were started in India during 1970s after selection of open pollinated hybrid Kanva-2 from Mysore local and the clonal selection Ber-S1 from Mandalaya (Introduction from Burma). Utilization of local landrace Berhampore local in West Bengal probably paved the way for developing high yielding varieties like S-30, S-36, S-41 and S-54 developed through mutation breeding techniques and V-1 through controlled hybridisation between S-30 (Parent Berhampore local) × C-776 in South India. This resulted in recognition of potentiality of diverse germplasm resources with wider genetic base for crop improvement programmes. In West Bengal, Mandalaya (Morus alba) introduction from Burma has been extensively used for polyploidy breeding using local Morus indica (HP) as a female parent because of its copious female flowering and a series of triploids and tetraploids have been evolved. The most promising triploids from West Bengal like Tr-4, Tr-8 and Tr-10 released for hilly regions and C-1730 and S-1635 for irrigated regions. The success of triploid varieties for commercial cultivation in west Bengal has attracted the breeders and prompted them to use high yielding diploid parents like S799, Chinese white, CRS for developing triploids. The promising triploids like S-1635, C-1730 are released in West Bengal during 1990s. The conceptual utilization of different mulberry (Morus species) in crop improvement resulted many high yielding varieties in many countries. Out of 68 recognised species of genus Morus, approximately 35 species so far identified, which possess genes for exploitation for quantity and quality improvement of leaf yield in mulberry (Anonymous, 1975). The potentiality of species Morus latifolia/Morus multicaulis in crop improvement programmes have been well recognised by many breeders in China and Japan.In India Morus multicaulis M.latifolia (var. Kosen) has been used along with Morus indica (var. Black cherry) for developing high yielding varieties like C-763, C-776and BC-2-59 through controlled hybridisation and back cross breeding in West Bengal during 1980s. Utilization of diverse germplasm resources for abiotic stress tolerance for wide range of environments has yielded varieties like S-13, RFS-135 and RFS-175 for semi-irrigated conditions. The mulberry varieties developed for commercial cultivation is presented in the Table-12. The year wise supply of mulberry germplasm is given in Table-13.
Realizing the increased use of mulberry germplasm resources for crop improvement programmes, CSGRC, Hosur being the nodal Institute for mulberry germplasm conservation and utilization in India taken up massive experiments like characterization and evaluation of the entire germplasm holdings at national field gene bank to group and short listing of trait specific promising accessions which will facilitate breeder/users. The list of promising accessions for growth and yield traits is given in Table- 14.
2.2.3.2 Utilization in agro forestry and horticulture
Mulberry is fast growing tree and produce large quantity of renewable biomass in the form of branches, shoots, leaves and fruits. One hectare of mulberry garden yields up to 60-70 tonnes/ha/year of green leaf and 12.1 tonnes of mulberry sticks (V1 mulberry variety). The energy generated per hectare is 27 940k calories @ 4 600 calories/kg of mulberry wood (Chinaswamy and Hariprasad, 1995). It promotes employment generation of 12 persons/ha/year. The green leaf with rich protein content (up to 28% on dry wt. basis) is used as fodder for cattle in many parts of India. The wood is used for making furniture, carving, toys making, sports goods, agriculture implements and papermaking. The fruits are eaten fresh and also used for jam, jelly and wine preparations. In the under-utilised fruit genetic resources category mulberry has a great potential for exploitation. It can be cultivated in diverse habitat both in temperate and tropical climate. Mulberry fruits are having 100% edible portion with 80% of moisture and rich in protein, iron, phosphorus, calcium, minerals and vitamins like carotenes, thiamine, riboflavin, niacin and vitamin C (Ashok Kumar and Gupta, 1996). An average single mulberry plant under dwarf tree condition can yield about 6 to10 quintals/acre/year of fresh mulberry fruit (estimated yield) and this may vary with species and cultural practices.
2.2.3.3 Utilization in medicinal purposes
It is estimated by World Health Organization (WHO) that 80% of world population still relies on traditional system for primary health care (Farnsworth et al. 1985). The various parts of mulberry plant are used in ayurvedic preparations The root bark, twigs and fruits which contain phenolic compounds, morasin and cyclomorasin are used as restorative, tonic, pectoral, diuretic and are prescribed to treat cough, asthma, phthisis and other chest complaints, dropsy and rheumatism. The decoction of the leaves possess blood purifying properties as a febrifuge, diuretic and galactugogue and stringent. The syrup made from fresh fruits is used as laxative, refrigerant in fevers and as an expectorant in cough and sore throats dyspepsia and Melancholia. The root extracts is having hypoglycaemic properties and used in treatment of diabetes. The root bark is used as a purgative and vermifuge. Mulberry root juice is administered to patients with high blood pressure. The shoot contains latex, which is used as plaster for sores and for preparation of skin ointments (Ashok Kumar and Gupta, 1996 and Ravindran et al. 1999).
The relation between trade and global exchange of germplasm became more intricate under Intellectual Property Rights (IPR) regime by TRIPS agreement under GATT. The Indian government preferred to use Sui generis option because of three major advantages i.e. flexibility, better protection of farmer rights and stronger researchers exemption.
Registration of germplasm is an important aspect for protection of breeders right under national and international agricultural/sericultural research systems. Outstanding varieties of mulberry are released for commercial exploitation after authorization. However, many promising germplasm do not get authorized due to one or other limitation in spite of their outstanding performance. Registration of such germplasm is required so as to give due recognition to the scientists who have developed the material. Lack of formal recognition of such useful germplasm materials discourages the scientist/breeders for supply of these materials to others for utilization. Consequently, such valuable materials remain under utilized or may even get lost. Moreover, there is urgent need to document such materials in wake of GATT and IPR regime at national and international level. Keeping these points in view, CSGRC, Hosur has developed a set of guidelines for registration of sericultural germplasm under National Sericultural Research System of CSB. For further details, the concerned scientists/Institutions responsible for development of the germplasm/genetic stock material may contact the Director, CSGRC, Central Silk Board, P.B.No-44, Thally Road, and Hosur- 635 109.
In India there is a single window system for germplasm exchange of small samples of PGR for research purposes and it is negotiated through NBPGR, New Delhi which is the nodal agency to plan, co-ordinate, execute and monitor PGR management in India under Indian Council of Agriculture Research (ICAR) in the Department of Agriculture Research and Development (DARE). In India, the Directorate of Plant Protection, Quarantine and Storage (DPPQS), Ministry of Agriculture, New Delhi monitor plant quarantine regulations with its regional network centers at airports, seaports and land frontiers. Under enactment of new policy on seed development (Regulation of import into India order (1989), any plant breeder intending to import planting material of mulberry including in vitro cultures has to get import permit addressing to Director NBPGR, New Delhi on prescribed format. The Phytosanitory certificate which is to be met by the supplier along with the sample ensuring that the material is not coated treated with chemical pesticide and the envelop to be addressed to the Director, NBPGR New Delhi who takes the delivery in full of the consignment and contain required for quarantine check. The material so introduced shall, after quarantine clearance and National registration (accessioning), will be made available to the indenter. Some of the introduced material may also be examined through post entry quarantine growing under controlled conditions for hidden pest/pathogen. The export of germplasm to any country should also be regulated through NBPGR, New Delhi. The material will be completely processed for quarantine check and issued a Phytosanitory certificate. Quarantine process includes examination of plant material for the presence of any pest/pathogen, unwanted weeds, through pest detection techniques like visual microscopic examination, X ray radiography, soaking seeds, sieving, staining, blotter method, growing out test, infectivity test and Enzyme Linked Immuno-sorbent Assay (ELISA). Under Article 19.3 of CBD, the potential risk posed by transboundry movement of Living Modified Organs (LMOs), which may have an adverse effect on biodiversity and its components will also be taken into account if necessary (Cartegene Protocol on Bio safety, January, 2000)
The International Treaty of Plant Genetic Resources established “ Multilateral System” for facilitated access to a specified list of PGR for food and Agriculture, benefit sharing in the areas of exchange of information, technology transfer and capacity building. At present 60 plant genera, which includes approximately 35 crops complexes, are listed in the Multilateral System. Paradoxically, mulberry is not included in the Multilateral System.
