Biotechnology of mulberry (Morus L.) - A review

Abstract

Mulberry is one of the economically important trees grown in Asian countries. It is cultivated to provide leaves for feeding the caterpillars of the silk producing insect. In addition, it adds value through production of edible fruits, timber and several pharmaceutically important chemicals. Improvement of mulberry through conventional breeding is limited due to high heterozygosity and long generation period. Attempts have recently been made to complement conventional breeding with modern biotechnological tools such as plant tissue culture, recombinant DNA technology and molecular markers to facilitate mulberry genetic improvement. The techniques of tissue culture have grown considerably in mulberry and encompassed areas including micropropagation, plant regeneration from leaf discs, and screening for stress tolerance. Recently, genetic engineering was adopted to enhance drought and salt tolerance in mulberry using HVA1 and Osmotin genes. Molecular markers such as Random amplified polymorphic DNA, inter simple sequence repeats and simple sequence repeats have been used for molecular characterization of mulberry germplasm, biodiversity analysis, genetic mapping and identification of molecular markers for growth and yield.

Headnote

Abstract

Mulberry (Morus sp.) is one of the economically important trees grown in Asian countries. It is cultivated to provide leaves for feeding the caterpillars of the silk producing insect (Bombyx mori L.). In addition, it adds value through production of edible fruits, timber and several pharmaceutically important chemicals. Improvement of mulberry through conventional breeding is limited due to high heterozygosity and long generation period. Attempts have recently been made to complement conventional breeding with modern biotechnological tools such as plant tissue culture, recombinant DNA technology and molecular markers to facilitate mulberry genetic improvement. The techniques of tissue culture have grown considerably in mulberry and encompassed areas including micropropagation, plant regeneration from leaf discs, and screening for stress tolerance. Recently, genetic engineering was adopted to enhance drought and salt tolerance in mulberry using HVA1 and Osmotin genes. Molecular markers such as Random amplified polymorphic DNA (RAPD), inter simple sequence repeats (ISSR) and simple sequence repeats (SSR) have been used for molecular characterization of mulberry germplasm, biodiversity analysis, genetic mapping and identification of molecular markers for growth and yield . However, still a number of issues such as resistance to fungal and bacterial diseases, combating infestation of pests and insects, and tolerance to drought and salinity are to be sorted out to achieve sustainable mulberry cultivation to meet the increasing demand of the silk industry. This review describes the developments of tissue culture, transgenesis and molecular markers in mulberry and highlights the current constraints and future prospects.

Key words: Cryopreservation, Gene transfer, Haploids, Molecular markers, Tissue culture, Triploids

Introduction

Mulberry (Morus) of the family Moraceae is an economically important tree grown commercially in India, China and several other Asian countries to feed the caterpillars of the silk producing Lepidopteran insect Bombyx mori L. (Vijayan et al., 2011a, 2012). Its leaf is also used for feeding cattle, goat and other animals as it is highly nutritious and palatable to herbivorous animals (Benavides et al., 1994), besides having several medicinal properties including antioxidant (Yen et al., 1996) and hypoglycaemial compounds (Kelkar et al., 1996). It is also grown for fruit, which is used for human consumption, production of jam, jelly, marmalade, frozen desserts, pulp, juice, paste, ice cream and wine (Koyuncu, 2004). Mulberry fruit is a traditional medicine for dysentery, constipation, hypoglycaemia and avulsed teeth (Lee et al., 2011) and it is a rich source of phenolic acids and flavonoids (Arfan et al., 2012). Further, mulberry trees have become an integral part of the landscaping in a number of countries (Tipton, 1994). Traditionally, mulberry (Morus) was placed in the tribe Moreae of the family Moraceae under the order Urticales (Takhatajan, 1980); however, based on molecular evidence, the angiosperm phylogenetic group (APG II, 2003) placed Moraceae in the order Rosales. More than 68 species of Morus have been widely recognized (Datta, 2000), of which M. alba, M. latifolia, M. mutlicaulis are grown for leaves while M. nigra is grown for fruit and M. serrata for timber (Vijayan et al., 2011a). Different cytomorphs such as diploids (Morus alba; 2x, 2n=28), triploids (M. alba, M. indica; 3x, 3n=42), tetraploids (M. laevigata, M. cathayana, and M. boninensis; 4x, 4n=56), hexaploids (M. serrata and M. tiliaefolia; 6x, 6n=84), octaploids (M. cathayana; 8x, 8n=112), and docosaploids (M. nigra; 22x, 22n=308) are available in mulberry, though diploids and triploids dominate mostly (Vijayan et al., 2012). It is believed that mulberry originated in the northern hemisphere, particularly in the Himalayan foothills and spread to the tropics of southern hemisphere (Benavides et al., 1994). At the present, mulberry is growing in various regions located between 50oN Lat. and 10oS Lat. including Asia, Europe, North and South America, and Africa at a wide altitude ranging from sea level to as high as 4000 m (Tikader and Dandin, 2005).

Need of biotechnological tools in mulberry

The main focus of mulberry breeding is to improve leaf productivity as it alone contributes more than 38.2% to the sericulture productivity (Banerjee, 1998). However, improving leaf productivity is difficult being a multifactorial trait and determined by a number of associated characters such as plant height, number of branches, leaf retention capacity, nodal length, leaf size and weight, and total biomass (Doss et al., 2011). High heterozygosity and inbreeding depression hinder the development of inbreds; hence, directional breeding failed to make much progress. Therefore, the heterozygous parents are used to generate F1 progenies, which are then subjected to different evaluation and selection procedures to identify the best one. This type of breeding system bars the possibility of introgressing genes of desirable traits from wild relatives/species due to genetic drag and subsequent difficulty in eliminating the undesirable traits that come along. Under such circumstances, the feasible means of improving specific traits without disturbing the current trait combinations is adoption of biotechnological tools like transgenesis, which enable introduction/over expression of desirable genes (Vijayan et al., 2011a), or knocking out undesirable genes via RNA interference technology (Vijayan et al., 2011b). Mulberry, being a tree with high heterozygozity, poses difficulties on improving traits of economic importance through conventional breeding and selection. Environmentally less influenced and developmentally stable molecular markers provide reliable tools for the breeders to characterize the germplasm and select parents and offsprings through marker-assisted selection. Thus, it would be prudent to use biotechnological tools to harness the vast benefit mulberry offers to mankind.

Tissue Culture in Mulberry

Tissue culture technique in mulberry has developed and ramified into different areas such as micropropagation, callus culture, organogenesis, and screening of genotypes for stress tolerance, induction of polyploids, cryopreservation, and transgenesis.

Micropropagation

Mulberry can be vegetatively propagated through stem cuttings, grafting or budding. However, success of these methods depends on a number of factors such as genetic makeup of the plant, age and physiological conditions of the parental cutting, climatic conditions and cultural practices. Additionally, newly developed mulberry varieties cannot be immediately propagated through stem cuttings as at least 6-7 months of maturity is required before cuttings can be isolated from the parental plant (Kapur et al., 2001). Micropropagation (Figure 1), on the other hand, allows multiplication of the plant in a short period under the controlled conditions. Further, in conventional method of propagation through stem cuttings, each stem cutting produces only one plant, whereas in micropropagation thousands of plants can be produced from a single plant piece (explant). Moreover micropropagation can provide plantlets throughout the year irrespective of seasonal variations. Thus, micropropagation is an efficient and cost effective method for rapid multiplication of mulberry in a relatively short time and limited space. Micropropagation also facilitates production of virus- free plants from the apical meristematic tissues. Ohyama (1970) initiated mulberry micropropagation by regenerating whole plants from axillary buds of M. alba. Later, a number of investigators used different media and explants as evident from Table 1. Shoot tips and dormant axillary buds were found suitable for mulberry micropropagation.

Composition of media is one of the factors that significantly affect micropropagation in mulberry. Among the different media compositions and hormones tested, MS (Murashige and Skoog, 1962) medium containing 2 mg L-1 6-benzylamino purine (BAP) is the best for shoot multiplication (Lalitha et al., 2013). However, it is also important to note that concentrations of 6-benzyladenine (BA) above 2 mg L-1 is inhibitory for shoot initiation and multiplication (Bhau and Wakhlu, 2003). Among the different sugarssuch as sucrose, glucose and fructose tested, sucrose is the best (Bhau and Wakhlu, 2003). Vijayan et al. (2000) found that 3% glucose is better for shoot formation from leaf explants. Among different pH levels tested, 5.6-5.8 is optimum for shoot multiplication (Enomoto, 1987). Agar in the culture medium also plays an important role in the success of micropropagation. Among various concentrations tested, 0.8% agar is the best whereas above 1% reduced shoot growth (Pattnaik and Chand, 1997; Thomas, 2003).

Rooting in mulberry is achieved mostly in half- strength MS medium as it is found better than full strength (Table 1). Among the plant hormones, 0.5 mg L-1 NAA is the best for M. alba, M. indica, M. multicaulis and M. latifolia resulting in 95% rooting (Vijayan et al., 2003), though indole-3- butyric acid (IBA) is the best auxin for M. nigra (Rao and Bapat, 1993). Vijayan et al. (1998) observed that higher concentrations of most of the auxins (>1.0 mg L-1) were inhibitory for root formation in mulberry and there was a strong interactive relationships among genotype, hormone type and concentration. Generally, rooting initiates is obtained within 12-18 days after transferring shoots to the rooting media (Hossain et al., 1992; Pattnaik and Chand, 1997). In vitro plantlets need to be hardened in the greenhouse before transfer to the field. Since the newly developed plantlets are grown in a pathogen free atmosphere in the laboratory, sudden exposure to outside conditions may put them into the risk of infestation by pests and diseases. In order to avoid/reduce such risks, hardening in autoclaved vermi-compost is considered as one of the most suitable substratum for the initial stages 2-3 weeks of plantlets establishment ex vitro (Yadav et al., 1990). After the initial hardening, the plants are transferred to earthenware pots of 12 cm doameter containing garden soil composed of 50% soil and 50% compost. The pots are kept under shade for another 2-3 weeks, before transferring to the field (Chattopadhya et al., 2011; Vijayan et al., 2011b). Biological hardening is an alternative method for hardening micropropagated plants before transferring to the field and in this technique plants are challenged with biotic stress caused by soil microbes. Many fungi like vasicular arbuscular mycorrhiza (VAM), Trichoderma, Piriformospora indica, and Azotobactor chroococcum are used for biological hardening (Kashyap and Sharma, 2006).

Organogenesis in mulberry

Organogenesis is a complex phenomenon involving de novo formation of organs. Successful organogenesis depends on a number of factors including appropriate selection of explants, age of the explants, media compositions, specific growth regulators, genotype, sources of carbohydrate, gelling agent, and other physical factors including light, temperature, humidity and other factors. Depending on these factors, plant regeneration may occur either directly or indirectly (Jain and Datta, 1992). In direct organogenesis, plants develop directly from the explants without formation of intermediate callus while in indirect organogenesis plant develops via callus formation. Again, callus induction depends on a number of factors such as nature of explants, genotype, medium and its composition. A variety of explants has been tested to initiate callus formation in mulberry (Table 1). Cambial regions (Narasimhan et al. 1970), hypocotyls segments (Shajahan et al., 1997), cotyledons (Thomas, 2003), stem segments (Vijayan et al., 1998), and young leaves (Chitra and Padmaja, 2005) have been tested and it is found that internodal segment from young shoot is the best explant for callus initiation in mulberry (Vijayan et al., 1998) and MS medium (Murashige and Skoog 1962) is most commonly used medium for callus induction in mulberry (Vijayan et al., 2011b). Similarly, 2,4-D is the hormone best suited for callus induction. Addition of Kn, IAA and NAA facilitates better proliferation and friability of callus. Supplementation of coconut water (150 ml L-1) and casein acid hydrolysate (100 mg L-1) enhances callus induction from foliar explants (Susheelamma et al., 1996). Multiplication of callus through repeated subculturing makes the callus more friable and responsive to shoot formation (Vijayan et al., 1998). Subculturing at an interval of 20-25 days favors better callus growth and lessening of phenol oxidation. Once the friable callus is transferred onto a medium supplemented with higher concentration of cytokinin and less auxin, shoot buds start develop. BAP is the most preferred cytokinin for shoot regeneration (Narayan et al., 1989). Indirect organogenesis often creates variations among the regenerated plants, a phenomenon called somaclonal variation, which often facilitates isolation of useful plants. Narayan et al. (1989) isolated such a variant (SV1) from plantlets developed from a productive variety S1. SV1 was found have better leaf yield (34,938 kg/ha) than S1 (28,048 kg/ha). Direct organogenesis from explants especially from cotyledons and leaf segments has great potential for transgenesis. In mulberry, direct plant regeneration from leaf explants was first reported by Kim et al. (1985) and later by Yamanouchi et al. (1999). Vijayan et al. (2000) obtained shoots from leaves on MS medium supplemented with BAP 2 mg L-1 and glucose as the sugar source (Figure 2). Subsequently, Bhatnagar et al. (2001) achieved 50% regeneration from hypocotyl and 70% from cotyledon using thidiazuron (TDZ) (0.5 mg L-1). The effectiveness of TDZ on direct shoot induction from leaf explants was further confirmed by Chitra and Padmaja (2005) and Raghunath et al. (2013). Currently, it is possible to regenerate plantlets directly from most of the (Table 2).

Somatic embryogenesis

Somatic embryogenesis provides a valuable tool to enhance the pace of genetic improvement of commercial crop species (Stasolla and Yeung, 2003). Several investigating groups attempted induction of somatic embryos in mulberry but the rate of success is less. Shajahan et al. (1995) obtained heart shaped embryos from M. alba hypocotyl segments cultured on MS medium supplemented with 2,4 D (0.45-4.52 µM) and BAP (2.2 µM). Agarwal (2002) and Agarwal et al. (2004) could obtain primary and secondary somatic embryoids by culturing zygotic embryos on MS medium containing 0.05 mg L-1 2,4-D + 0.1 mg L-1 BAP and 6% sucrose. However, due to the difficulty in hormonally controlling the formation of adventitious shoots and roots in mulberry, somatic embryogenesis has not been developed as it is in many other crop plants. Thus, concerted efforts are needed to make somatic embryogenesis successful in mulberry.

Haploid production

Haploid plants being gametophytic in origin possess only half of the normal chromosome number of the parent. They can be used to produce homozygous lines, which are invaluable for any breeding programmes especially for tree crops with longer generation cycle and high heterozygosity. Since the first successful report on regeneration of haploid plants from pollen grains of the cultured anthers of datura (Guha and Maheshwari, 1964), this technique has been extensively used in most of the agriculturally important plant species. However, only limited success could be obtained on tree species. In mulberry, though anther culture was first attempted by Shoukang et al. (1987) and later by Katagiri (1989), Sethi et al. (1992) and Chakraborti et al. (1999), till date no plants could be regenerated. Nonetheless, Thomas et al. (1999) regenerated gynogenic haploids by culturing immature female catkins on MS medium supplemented with BA (2.0 mg L-1) and 2,4-D (1.0 mg L-1) for the first three weeks, and the remaining period with 2,4-D (1.0 mg L-1), glycine (88.8 mg L- 1) and proline (15 mg L-1). However, no further report on haploidy is available in mulberry, though doubled haploidy is of much use in mulberry breeding.

Protoplast isolation, culture and regeneration of plantlets

Somatic hybridization through protoplast fusion has opened a new avenue for developing new characteristics, which are not possible through conventional breeding (Johnson and Veilleuz, 2010). There are only a few reports dealing with plant regeneration from protoplasts in mulberry (Tewary and Sita, 1992; Umate et al., 2005). A combination of 2% cellulase, 1% macerozyme and 0.5% macerase is found optimal for better isolation of viable protoplast. Protoplast fusion in mulberry was successfully achieved using chemical fusogen (Ohinshiand Kiyama, 1987) and electro-fusion (Ohinshi and Tanabe, 1989). Although protoplast isolation and regeneration was achieved, development of somatic hybrids in mulberry could not be achieved. Hence, efforts in this end need to be continued.

Other applications of tissue culture in mulberry Screening for stress tolerance

Since salt tolerance in plants is a complex phenomenon involving morphological, physiological and biochemical processes, screening of genotypes for salt tolerance need to be done in such conditions where the influence of external factors is minimal (Vijayan et al., 2011c). Maintenance of uniformity of salinity across the field and seasons is difficult, therefore, screening of the plants under in vitro is considered as an ideal option, where most of the environmental conditions can be controlled. Vijayan et al. (2003) using axillary buds of 63 mulberry germplasm accessions maintained at the Central Sericultural Research and Training Institute, Berhampore, West Bengal, India isolated salt tolerant genotypes by surface sterilizing the nodal explants and culturing on MS medium (Murashige and Skoog, 1962) supplemented with 2 mg L-1 BAP, 30 mg L-1 sucrose and 0.0% to 1.0% NaCl (Figure 3). Genotypes showing early sprouting and better growth rate in 1.0 % NaCl were selected as salt tolerant. Out of the 63 accessions tested, 16 sprouted in 1.0% NaCl, and 13 of them survived till 30 days and these genotypes were considered as salt tolerant. For rooting assessment, in vitro grown shoot apices (1-2 cm in length) were cultured on MS medium containing 2.6 mM NAA, 30 g l-1 sucrose and 0.1, 0.2, 0.3 and 0.4% NaCl. Out of 63 genotypes tested, only five genotypes could develop roots at 0.2% NaCl. Five genotypes, which showed better root and shoot growth in both experiments, were further tested in pot culture under different salt levels to confirm the efficacy of the in vitro screening. The study while confirming the higher salt tolerance of the selected genotypes also demonstrated the efficacy of in vitro screening to isolate salt tolerant genotypes in mulberry (Vijayan et al., 2003). Later, Ahamad et al. (2007) used this technique to investigate the effect of NaHCO3 on various characters of mulberry. This method is more economical, efficient and less time consuming for screening large number of mulberry accessions for salt tolerance.

Induction of tetraploidy

In general the mulberry is propagated through vegetative means. Hence, sterile high yielding varieties/cultivars do not pose any problems for their true to type multiplication. Triploidy in mulberry is considered as the optimum level of ploidy because triploids show several advantages over plants of other ploids. Triploids are superior in leaf yield, stress resistance and chemical components of the leaf (Yang and Yang, 1989). Considering these advantages, tetraploids are developed from diploids by colchicine treatment of the growing shoots. In this method, small cotton pads soaked with 1.0-2.0% colchicine solution is applied over the growing buds for 2-3 consecutive days. Though this method is easier to apply, it suffers from quick drying of the cotton pad, excessive loss of colchicines and difficulty in maintaining the uniform concentration of the colchicines solution. Application of colchicine in vitro solves most of these problems and also makes the system more economic. Chakraborti et al. (1998) cultured apical buds of field grown mulberry on MS medium supplemented with BAP (2 mg L-1) and four concentrations of colchicine (0.0, 0.05, 0.1 and 0.2%). It was observed that the optimal level of colchicine was 0.1% as it produced 39.4 ± 4.8% tetraploids. Higher concentrations of colchicine reduced the survivability of the buds and low concentration reduced the percentage of tetraploid formation. It is further observed that in vitro application of colchicine was 80.8% more efficient than the cotton pad method. Additionally, in vitro application of colchicine is more cost effective as the same medium can be used for at least 4 repeated treatments without reducing the efficiency of the conchicine to induce tetraploidy.

Another method of getting triploidy in mulberry is to culture the endosperm because in angiosperm, endosperm is a triploid tissue formed via double fertilization (Bhojwani and Razdan, 1996). In mulberry, Thomas et al. (2000), for the first time, successfully developed triploids from endosperm of the variety S36. Endosperm tissue isolated from young fruits was cultured on MS medium supplemented with 2,4-D (5 µM) and different concentration of BA, Kn, TDZ, IBA, NAA, gibberellic acid (GA3), along with tomato juice (TJ), yeast extract (YE), casein hydrolysate (CH) and coconut milk (CM). Shoot buds developed when the callus was subcultured on a medium containing a cytokinin or a combination of cytokinin and NAA. The best result for callus induction (70-72%) was obtained on MS medium with BAP (5 mM), NAA (1 µM) and CM (15%) or YE (1000 mg/l). The maximum number of shoot formation was on the medium containing TDZ (1 µM), or BA (5 µM) and NAA (1 µM). On cytological investigation, it was found that all the ten plants examined were triploids with 42 chromosomes. This clearly indicated the efficiency of the protocol and it could be used for developing more triploids in mulberry.

Synthetic seeds

Synthetic seeds are the encapsulated somatic embryos, which functionally mimic zygotic seeds and can develop into seedlings under sterile conditions. In a broader sense, it would also refer to encapsulated buds or any other form of meristems, which can develop into plants. In mulberry, synthetic seeds are produced mostly by encapsulating the apical/axillary buds or somatic embryos with 3-5% sodium alginate and 100mM calcium chloride solution as complexing agent (Kamareddi et al., 2013). Sodium alginate solution is mixed with culture medium containing all necessary ingredients essential for proper growth. Patnaik et al. (1995) successfully developed this technology for artificial seed sysnthesis in mulberry. However, adoption of this technology for mulberry propagation was limited to a few species of M. indica (Bapat and Rao, 1990; Patnaik et al., 1995). Researchers have explored the possibility of using in vitro derived vegetative propagules for synthetic seed production since it was found difficult to develop somatic embryos in mulberry (Pattnaik and Chand, 2000; Kavyashree et al., 2004). Shoot cultures established from axillary buds on Linsmaier and Skoog's basal medium (LSBM) supplemented with BAP (4 mg L-1) and TIBA (1 mg L-1) used for encapsulation. Sodium alginate and carboxy methylcellulose were added in the range of 2, 4, 6 and 8% (w/v) to liquid initiation medium separately. For complexation, 25 x 103µM, 5 x 104 µM, 75 x 103 µM and 10 x 104 µM calcium chloride solutions were prepared using distilled water. Gel complexation was done by mixing the axillary buds with hydrogels, dropping these into different concentrations of calcium chloride solution and incubating in orbital shaker for different time intervals (20, 30, 40, 50 and 60 min) to obtain uniform beads. The sodium alginate and carboxy methylcellulose embedded axillary buds were collected using a sterilized tea strainer and rinsed 2-3 times in sterile water to remove traces of calcium chloride. The synthetic seeds were tested for their conversion potential under in vitro and ex vitro conditions. Although different concentrations of sodium alginate were used, the best results were obtained at 4% alginate (Kamareddi et al., 2013). Although somatic embryos could not be developed easily in mulberry, dormant axillary buds proved to be an ideal material for the synthesis of synthetic seed, it can be used for cryopreservation of germplasm accessions.

Cryopreservation of germplasm

The high heterozygosity hinders conservation of mulberry germplasm through seeds as the progenies from such seeds are heterogenous in nature and getting true to the parental type is difficult. Thus, mulberry germplasm is conserved as ex situ germplasms, which is laborious, needs huge resources, and is in a risk of destruction by natural calamities, pests and diseases (Vijayan et al., 2011d). Thus, safe alternative methods with economically viability need to be explored. Cryopreservation is one such alternative wherein plant materials are stored at ultra-low temperatures (-196°C) in liquid nitrogen. At this temperature all the metabolic activities of the cell including divisions remain arrested; hence, the material remains unaltered for long period. Two different cryopreservation techniques in vogue are the classical one and the modern vitrification (Engelmann, 2000). In classical cryopreservation technique, the plant material is cool down slowly at a controlled rate of 0.1-4°C/ min to about -40°C and rapidly immersed in liquid nitrogen. In vitrification, plant material is physically or osmotically dehydrated and is subsequently subjected to ultra- rapid freezing resulting in vitrification of intracellular solutes, i.e. formation of an amorphous glassy structure without occurrence of ice crystals. Although different plant materials are used for cryopreservation, the most appropriate material for cryopreservation of mulberry is winter buds (Niino, 1995). Genetic constitution of the plant material also influences significantly the success and duration of the preservation (Rao et al., 2007). Cryopreservation techniques have been standardized for M. indica, M. alba, M. latfolia, M. cathayana, M. laevigata, M. nigra, M. australis, M. bombycis, M. sinensis, M multicaulis and M. rotundiloba species (Rao et al., 2007, 2009). The general procedure for cryopreservation of shoot tips is that the shoot segments are pre-frozen at -3°C for 10 days, -5°C for three days, -10°C for one day and -20°C for one day and immersed into liquid nitrogen. The cryopreserved material is brought back to the ambient temperature via thawing. During this process, the material is warmed either slowly at 0°C in air for more than 3 h or rapidly in agitated water at 37°C for 5 min. After thawing, surface-sterilize the bud with 70% ethanol for 1 min and sodium hypochlorite solution (0.5% effective chlorine concentration) for 20-30 minute and rinse it thoroughly with sterile distilled water for three to four times before culturing on MS medium containing all necessary supplements. Niino et al. (2006) obtained 65% survival of the buds cryopreserved at -135°C for 5 years. The Surviving buds resume growth within 5 days and develop shoots without intermediary callus formation. It is also found that partial dehydration of the bud up to 38.5% before pre-freezing at -20°C improves the recovery rate. Encapsulation of winter-hardened shoot tips with calcium alginate coating was also tested successfully (Padro et al., 2012). It is concluded from different experiments that dormant buds of mulberry can be cryopreserved for 11 years without reducing the viability of the buds (Fukui et al., 2011).

Problems associated in mulberry tissue culture Problems in bud culture

High rate of contamination is one of the major problems that limit the use of mature field grown stem cutting for in vitro studies, especially for flowering and in vitro fertilization (Vijayan et al., 2011b). Since the field grown plants are often heavily loaded with various epiphytic and endophytic microbes, it is very difficult to remove them, especially from the lenticels. Further, it is also noted that occurrence of microbial contamination depends to a great extent on genotype, maturity of the explants and seasons. During rainy season, heavy bacterial infection was observed. Patnaik and Chand (1997) reported that an initial thorough washing of field grown nodal explants with running water for 1-2 h followed by treating it with 5% (v/v) liquid detergent and 7% sodium hyphochlorite for 8-10 minutes are essential before doing the normal surface sterilization with 0.1% aqueous (w/v) mercuric chloride to contain contaminations. Narayan et al. (1989) washed the field grown nodal explants under running tap water, then immersed in 0.1% (w/v) carbendazim for 20 min and washed thoroughly with distilled water before treating with 0.2% (w/v) cetavlon for 10 min, and with 0.1% (w/v) aqueous mercuric chloride solution for 5 min for effective surface sterilization. Chitra and Padmaja (2002) washed the nodal explants first in running tap water for 30 min and surface sterilized with 70% alcohol for 1 min followed by 0.1% mercuric chloride (HgCl2) for 15 min under sterile conditions. Vijayan et al. (2011b) found that when mature nodal plants are used for micropropagation, fungal growth takes place after 20-30 days from the lenticels and scale leaves. In order to avoid this contamination the young shoots should be subcultured as soon as they emerge from the buds of the explants collected from the field.

Problems in callus culture

Oraganogenesis in mulberry is also not devoid of problems as often excessive blackening of the callus occurs due to exudation and oxidation of the phenol contents. The oxidative products of phenolic compounds lower the pH of the culture medium, thereby affecting the callus growth. This problem can be limited to an extent by the addition of activated charcoal (Mhatre et al., 1985) and or silver nitrate (Chakraborti et al., 1999) in the culture medium. Regular subculturing at an interval of 20-25 days is another way of controlling this problem (Narayan et al., 1989; Vijayan et al., 1998). Another major problem is the formation of roots from the callus before the shoot formation. Once the root formation starts shoot formation becomes difficult from the same callus. This serious problem should be controlled by hormonal manipulation such that the effect of cytokinin should not be less than that of auxin in the initial stages of organogenesis.

Problems associated with acclimatization and field transfer

During in vitro culture, plantlets grow under control conditions such as humidity, irradiance, temperature and other factors. The plantlets cultured in vitro in a water-saturated atmosphere generally wilt rapidly when transferred to normal greenhouse or field conditions and also are highly prone to microbial attacks, if adequate care is not taken. One of the major reasons for wilting of the transferred plant is the rapid loss of water from the leaves due to poorly developed stomata and epicuticular wax (Santamaria et al., 1993). Poor contact of roots with the soil also limits water up take (Fila et al., 1998). Although it is quite common that leaves with normal stomatal structure replaces the older ones, it is very common that plantlets die during the acclimatization process (Huylenbroeck et al., 1996). To minimise such casualties, it is essential to provide with a regulated temperature, humidity, irradiance, CO2 concentration and air flow rate during acclimatization (Bolar et al., 1998). Initially the in vitro developed plants can be kept inside the culture room under sterile conditions. Then, gradually it can be shifted to green house where humidity and temperature are under control. After a couple of months the grown plants can be shifted to shady places in the field, thereafter it can be transplanted to field.

Genetic engineering in mulberry

Although conventional plant breeding has contributed significantly by developing several high yielding mulberry varieties (Table 3), high heterozygozity and long generation cycles and inbreeding depression prevent introgression of traits from wild species and relatives. Transgenesis is one such technique that enables direct transfer of genes of interest. Out of the two popular gene transfer techniques viz., particle bombardment and Agrobacterium tumefaciens-mediated transformation, the latter received much attention in mulberry due to the easiness and efficacy. Bhatnagar and Khurana (2003) generated transgenic mulberry expressing GUS genes. In the process, it is found that A. tumefaciens strain LBA4404 was more infective than the other strains. Nearly 90-100% transformation was obtained with LBA4404, 70- 75% with GV2260 and 25-35% with A281. Among the plasmids, pBI121 and pBI101:Act1 were most efficient (100%). Subsequently, several transgenic mulberry plants overexpressing desired genes were developed (Table 4), including glycine gene AlaBlb, the oryza cystatin gene OC and the barley HVA1 gene (Wang et al., 2003; Lal et al., 2008; Checker et al., 2012). The transgenic mulberry over-expressing HVA1 from barley showed comparatively better growth under abiotic stresses (Lal et al., 2008; Checker, 2012). The transgenic plants with Osmotin gene under the constitutive expression of the CaMV 35S promoter and a stress- inducible promoter rd29A also expressed high salt, drought and cold stress tolerance (Das et al., 2011). Thus, transgenesis in mulberry seems to have come of the age. In view of the above developments, attention should be directed to some of the burning issues of mulberry cultivation. Major diseases like powdery mildew (Phyllactinia corylea) and bacterial blight (Xanthomona campestris cv. Mori) are causing a crop loss of 20-30% in the field (Philip et al., 1994). Therefore, sincere and serious efforts should be made to develop disease resistant transgenic plants against these diseases.

Molecular markers

From the research during the last couple of decades (Table 5) clearly demonstrates that four major types of molecular markers have been used in mulberry for various purposes which includes elucidation of genetic diversity among germplasm accessions, relationship among populations, cultivars, land races and species, identification of markers associated with economically important traits, and developing fingerprints of germplasm accessions as detailed hereunder.

Random amplified polymorphic DNA (RAPD)

Random amplified polymorphic DNA markers uses arbitrary short oligomers (usually 10 bases) to PCR amplify the genomic DNA (Williams et al., 1990) and the variation in the band pattern is due to base pair substitutions modifying the primer binding sites (Moeller and Schaal, 1999). In mulberry, RAPD was the first molecular marker used for genetic diversity analysis. RAPD has been used by many investigators to work out genetic diversity among the cultivars (Awasthi et al., 2004; Ozrenk et al., 2010; Chikkaswamy and Prasad, 2012; Chikkaswamy et al., 2012; Ipek et al., 2012; Naik, et al. 2013), to identify molecular markers associated with sex characteristics (Figure 4) in mulberry (Vijayan et al., 2009), to identify mutants (Anilkumar et al., 2012) and to develop linkage maps in mulberry (Venkateswarlu et al., 2006). The advantages of RAPD are requirement of small amount of template DNA, low cost of development, easiness in use and the major disadvantages are the poor reproducibility (Rafalski, 1997), underestimation of genetic distances between distantly related individuals (Powell et al., 1996) and are unable to distinguish homozygous from heterozygous ones.

Inter simple sequence repeats (ISSR)

Inter simple sequence repeat (ISSR) markers amplify DNA segments between two identical microsatellite repeat regions oriented in opposite direction by primers of 16-25 bp long designed from the microsatellite core regions bordering them. The primers either anchore at 3' or 5' end with 1 to 4 degenerate bases extended into the flanking sequences (Zietkiewicz et al., 1994) or to remain unanchored (Gupta et al., 1994). Usually di- nucleotide repeats anchored either at 3' or 5' end reveal high polymorphism (Joshi et al., 2000). Polymorphism occurs whenever one genome misses the sequence repeat or has a deletion or insertion or translocation between the repeats. ISSR markers are generally dominant markers following Mendelian inheritance (Ratnaparkhe et al., 1998), however, incidence of segregation as co-dominant markers also been reported (Sankar and Moore, 2001). ISSR markers have higher reproducibility than RAPD markers (Moreno et al., 1998). Since the development of ISSR markers does not need prior knowledge of the genome to be analysed, it has been used initially to assess the genetic diversity of mulberry. Vijayan and Chatterjee (2003) used 13 ISSR primers from University of British Columbia, Canada (set # 9) to estimate diversity among 11 indigenous cultivars. The study showed that AG, TG and AC repeat primers generated excellent band profiles but primers of AT repeats failed to amplify even at a low annealing temperature. The important tri and tetra nucleotide repeat primers generated excellent amplification are those from ACC, ATG, AGC, GAA, GATA and CCCT. The penta-nucleotide repeats GGAGA and GGGGT also amplified well. Awasthi et al. (2004) used ISSR in combination with RAPD to characterize 15 germplasm accessions and proved the efficiency of ISSR once again. Later, Vijayan (2004a) worked out the genetic relationships between Japanese and Indian species (Figure 5). ISSR has also been used to estimate the biodiversity of wild populations of mulberry (Vijayan et al., 2004a). Populations of M. serrata present in Uttaranchal (29o22'- 30o45' N latitude and 75o52'- 80o12' E longitude) and Himachal Pradesh (30o30' - 30o54' N latitude and 77o06' - 77o40' E longitude) were assessed and conservation strategies were formulated. Likewise, the phylogenetic relationship among nineteen genotypes belonging to five mulberry species viz., M. latifolia, M. bombycis, M. alba, M. laevigata and M. indica were also worked out using both RAPD and ISSR markers (Vijayan et al., 2004b). The study revealed that M. laevigata can be considered as a separate species while the other four species may be grouped together and treated as sub-species. Subsequently, Vijayan et al. (2005) demonstrated the admixturing of the mulberry genetic pool of the eastern India and the southern India using 34 mulberry cultivars collected from different regions of India. In China, Zhao et al. (2007) used ISSR along with RAPD to estimate the genetic diversity of 27 mulberry accessions. Similarly, genetic diversity of 73 local mulberry varieties from Shanxi Province were assessed with 15 primers and with the help of stepwise clustering and random methods and the modified heuristic algorithm, 21 core collections were constructed and the ratio of core collection was 28.77% (Lin et al., 2011). Recently, Chikkaswamy et al. (2012) used ISSR along with RAPD to estimate the genetic diversity of 20 mulberry varieties. Along with RAPD, it has also been used to develop linkage map of mulberry (Venkateswarlu et al., 2006).

Amplified fragment length polymorphism (AFLP)

Amplified fragment length polymorphism (AFLP) is a combination of RFLP and polymerase chain reaction (PCR) techniques wherein the speed of PCR combines with the precision of RFLP (Vos et al., 1995) though it requires only a small amount of DNA, it can be readily automatable. AFLP is more robust, reliable and reproducible than RAPD and ISSR (Jones et al., 1997). The process of AFLP is initiated by extraction of DNA and subsequent digestion with rare and frequent cutter restriction enzymes like Eco-R1 and Mse-I. The digested fragments are then ligated to double-stranded oligonucleotide adapters and PCR amplified with primers that bind to the adapter sequence, restriction site sequence and adjacent selective base(s). The products of the PCR amplification are subsequently run on a polyacrylamide gel to resolve it. Because of the use of two restriction enzymes with different properties, a large number of DNA fragments suitable for PCR amplification are generated. Since poly acrylamide gel electrophoresis can resolve fragments differing in length even by a single base pair (Miyashita et al., 1999), a large number of fragments are resolved in the gel. Like RAPD, AFLP also not required any prior information on the target genome (Vos et al., 1995) and the markers are dominant in nature (Powell et al., 1996). AFLP was first used in mulberry to study the genetic diversity of 45 mulberry accessions from different eco-geographic regions of Japan and other parts of the world. Five primer combinations were used and an average of 110 AFLP markers was generated by each primer pair. The size of the bands varied from 35bp to 500 bp. The polymorphism ranged from 69.7 to 82.3% across all the genotypes. The UPGMA-dendrogram grouped the accessions into four major clusters agreeing with the genetic relatedness established among them by conventional methods (Sharma et al., 2000). Further, it is concluded that mulberry cultivars are naturalized because they have been established, adapted and persisted in areas far away from their origin, making their classification very difficult and unreliable when based solely on morpho-phenological traits (Sharma et al., 2000). Later, it was used to estimate the genetic variability within as well as among different mulberry species. Botton et al. (2005) assessed 48 accessions belong to three species collected from Japan, Brazil, South Caucasus, Middle East, Philipines, and Italy but were maintained in Italy. From the study it is concluded that since spontaneous and artificial hybridization is possible, and due to continuous variation of most phenotypic characteristics, the taxonomy of the genus Morus, especially for M. alba, M. latifolia and M. bombycis species, is not well defined. Later, Kafkas et al. (2008) characterized 43 mulberry accessions from different regions of Turkey using fluorescent dye amplified fragment length polymorphism (AFLP) markers and capillary electrophoresis. Unweighted pair- group method of arithmetic mean (UPGMA) clustering grouped the accessions according to the species they belong. Furthermore, the study also clearly brought out the ability of AFLP markers to identify the accessions of M. nigra, M. rubra, and M. alba without any ambiguity. These studies demonstrated unequivocally, the resolving power of AFLP, which can be used for identifying genotypes for conserving genetic resources, eliminating duplicate accessions from germplasm collections and monitoring erosion of genetic diversity within the populations.

Simple sequence repeats (SSR)

Simple sequence repeats (SSR) or microsatellites or short tandem repeats (STR) or simple sequence length polymorphism (SSLP) are tandem repeats of short (2-6 base pair) DNA fragments present throughout the genome (Litt and Luty, 1989). Variations at SSR loci are generated through (a) replication slippage (b) unequal crossing-over and (c) genetic recombination. Among them, replication slippage is considered to be a major factor affecting the repeat number for short tandem repeat sequences, whereas unequal crossing-over is thought to result in a very large number of alleles for long tandem repeat arrays (Huang et al., 2002). SSR markers are co-dominant, stable, robust and are highly reproducible. However, the major disadvantage of SSR is the need for prior information on the target genome to develop suitable primer sets. With the introduction of next generation sequencing techniques the cost of sequencing has come down considerably. This facilitates development of SSR primers much cheaper than earlier. Further, it is interesting to note that due to, synteny of genomic regions; primers developed in a closely related species can be used to amplify microsatellite loci of other species (Hormaza, 2002). In mulberry, the first attempt to isolate microsatellite markers was made by Aggarwal et al. (2004), wherein six primer sets were developed from the genomic DNA of M. indica (Table 6). The markers developed by these primer sets produced high polymorphism when tested on a set of 43 elite genotypes including 13 related Morus species. The markers could easily differentiate the species. Later, Zhao et al. (2005a) developed another 10 primer sets (Table 6) and validated their suitability by testing in 27 mulberry accessions. Using these SSR markers, Wangari et al. (2013) and Wani et al. (2013) studied the genetic diversity among mulberry genotypes present in Kenya and India respectively. Likewise, these SSR markers have also been used in constructing a linkage map along with RAPD and ISSR markers (Venkateswarlu et al., 2006). Similarly, Anuradha et al. (2013) used SSR primers along with RAPD and ISSR to test the quality of mulberry genomic DNA extracted with a new protocol. Although SSR markers have several advantages over other dominant marker systems, they have not yet been exploited widely in mulberry. Hence, attempts should be made to develop more number of SSR markers so as to utilize them in identification of QTLs for enabling marker assisted selection breeding in mulberry.

Conclusions and Prospective

Biotechnology of mulberry has advanced far and wide in areas like tissue culture and molecular biology and contributed to micropropagation of hard to root genotypes, isolation of somaclonal variants, screening of germplasm for tolerance to abiotic stresses, induction of polyploids, production of synthetic seeds, and cryopreservation of genetic resources, development of transgenic plants, characterization of germplasm accessions and identification of markers associated with economically important traits. However, there is much more to do than what has been accomplished. Inbred lines are urgently required for elucidating the genetic basis of most of the economically important characters in mulberry. Considering the difficulty to develop inbreds through conventional breeding, developing the same through doubled haploidy should be attempted. The development of a reproducible system for the production of doubled haploids, either using anther cultures, microspore cultures and/or cultivation of ovary segments containing unfertilized ovules, need to be developed. Deeper insight into each particular step in the process of haploid plant production can help to develop more sophisticated and more successful protocols for rapid application of the gametic embryogenesis. Although a few transgenic plants harbouring some of the desired genes have been developed, regeneration of plants from leaf disc of most of the high yielding varieties is still remain as the major bottle neck. Thus, efforts need to be made to develop easy protocol for these mulberry varieties so that genes of special interest can be inserted into their genome easily. Regarding the molecular marker systems, only a few SSR primers are still available for use. These few primers are not enough to make saturated linkage maps to identify QTLs tightly linked to economically important traits. Thus, it is important to develop large numbers of SSR and SNP (Single Nucleotide Polymorphism) markers for wider use of these marker systems. In this context it is heartening to note that the first draft sequence of mulberry genome has just been published (He et al., 2013), which will facilitate development of more information on mulberry genome to enable fast improvement of this very important crop plants of Asia.

Acknowledgement

The authors express their deep gratitude to Dr. S. Gandhi Doss, CSR & TI, Mysore for providing photographs of micropropagation, to Dr. S. Mohan Jain, Department of Agricultural Science, University of Helsinki, Finland and Dr. A. J. Cheruth, United Arab Emirates University, UAE for language correction.

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