eChapters

Bone being a dynamic tissue undergoing constant biological changes, organic and mineral phases of fracture healing need an optimum general body metabolism and even a slight deviation from normal condition of the body is likely to cause considerable alterations in the healing process. Facture healing can be modified by variety of endogenous or exogenous factors that affect the metabolic function of cells. These factors include transforming growth factor beta, bone morphogenetic proteins, hormones, vitamins and prostaglandins. Anabolic steroids have the property of preventing the destruction and elimination of nitrogenous substances and minerals from the body. They help, thus, to have a positive nitrogen and mineral balance which is essential for healing and regeneration of any kind of tissue in a living organism. This chapter is undertaken to observe the effect of anabolic steroid (Durabolin) on fracture healing.

This study is conducted on ten clinically healthy adult New Zealand White rabbits of either sex divided into two equal groups (X and Z) consisting five animals each. All the animals are kept in standard diet and management throughout the experiment. In all animals, midshaft segmental defect (1cm) are created in both the ulnae under thiopental (2.5%) anaesthesia. In test group (Gr X), 10mg of Nandrolone phenyl propionate (durabolin) is injected intramuscularly in 5 days interval at 7 occasions. Lateral and anterio-posterior radiographs are taken on days 0, 7, 14, 21, 28 and 35 postoperative days. The radiographs are observed for progress of fracture healing and complications, if any, at different intervals.

Inflammation at the fracture site is increased upto 4^th postoperative days in all the animals of both groups, and then the limb is normal in appearance by day 7. Surgical wound healing is by first intention in all the animals. Earliest return of weight bearing is seen in animals of test group followed by control. Body weight and food intake are increased two-fold in the test group of animals.

India is bestowed with an enormous wealth of medicinal plants and has a rich heritage of treating human and animal ailments by using herbal medicines. However, there is need for a systemic, scientific approach to validate the authenticity of claims on plant-based medicaments. This approach has paid rich dividends in developing cheaper, safer, effective and eco-friendly remedies, which recently has gained worldwide attention. Cissus quadrangularis, Linn (Syn. Vitis quadrangularis, Wall; Heliotropium indicum, Linn) is a tender climber with fleshy quadrangular stems and grows in the hotter parts of Indian subcontinent, Sri Lanka and East Africa. In Hindi it is known as hardjore; in English as adamant creeper, bonesetter; in Ceylon as hiressa; in China as di xanh voung; in Burma as shazam-lese and in Bangladesh as harbhanga. Considering the advantage of herbal medicines, it is planned in this chapter to investigate the efficacy of Cissus quadrangularis, Linn, in one of the most commonly encountered problem of fractures in small animals.

Dried ethanolic extract of Cissus quadrangularis (CQ) is obtained from 500g of dried finely powered stems using soxhlet apparatus. It is dissolved in a vanishing base to make a semi-solid paste for local application. For parental use, this extract is reconstituted with distilled water (100 mg/ml) and filtered.

Twelve clinically healthy mongrel adult dogs aged 2-3 years and weighing 10-15 kg is randomly divided into two equal groups (A and B). In all animals operative site is prepared for aseptic surgery. Premedication is done with atropine sulphate @ 0.06 mg/ kg body weight subcutaneously and anaesthesia is achieved with a combination of Xylazine hydrochloride @ 1 mg/kg body wt. and Ketamine hydrochloride @ 10 mg/kg body wt. intravenously. Unilateral comminuted diaphyseal femoral osteotomy is created under general anaesthesia and the bone defect is immobilized with neutralization bone plating (AO/ASIF technique). All the animals are administered 5 mg/kg b wt Enrofloxacin intramuscularly for 5 d and 1 mg/ kg b wt Diclofenac sodium injection intramuscularly for 5 d postoperatively. Skin sutures are removed after 10 days. In the animals of group B, in addition to the postoperative treatment, the ethanolic extract of the herb, Cissus quadrangularis is applied on the skin twice daily along with subcutaneous injection (50 mg/ kg body wt.) on every alternate day for 20 d postoperatively.

The action of the gonadial hormone is not confined to the growth and development of the sexual organs alone, but the normal body growth as well as the growth of the skeleton are to a great extent influenced by these hormones. Of the female gonadial hormones, the action of estrogen and progesterone on skeletal growth has not been studied in detail. Some reports implicate estrogen deficiency in post menopausal osteoporosis and shows that estrogen replacement therapy can help to prevent that post menopausal stage of accelerated rate of bone loss. It’s reported that the progesterone treatment play a significant role in regulating cortical bone cell activity and as such, suggest that progesterone may play a part in the prevention and slowing down of cortical bone loss in females following menopause or oophorectomy. The effect of gonadotropic hormone on bone healing has not yet been studied in detail. The chapter is thus, undertaken to investigate the effects of gonadotropic hormone (Gn-rh)) on cortical bone healing in female rabbits.

Clinically healthy adult female New Zealand white rabbits (10) are divided equally into groups A and B each consisting of 5 animals. All animals are kept on standard diet and management throughout the experiment. In all animals midshafts simple transverse fracture is created in both the ulnae under thiopental (2.5%) anesthesia. In test group (group A) 0.1 ml of Gn-rh analogue is injected intramuscularly at 3 days interval up to 30 days post operatively.

Lateral and anterio-posterior radiographs are taken on days 0, 10, 20. 30. and 40-post fracture. These radiographs are observed for progress of fracture healing and complication, if any, at different intervals. The specimens for histopathological study are collected from the healed site of test bone immediately after euthanizing the animals on day 40. Every specimen consists of better healing site and adjacent host bone. The bone sections are preserved in 10% formalin for 9 to 10 days and then are decalcified using Goodling and Stewart’s solution (15% formic acid, 5% formalin and 80% distilled water). Completely decalcified bone sections are then processed routinely for H & E staining. These sections are observed for status of healing at fracture site.

Bone healing is an extraordinary process involving not only an inflammatory reaction and formation of callus but also reconstruction of the defect to regain its original biomechanical strength. Bone healing can be modified by variety of endogenous or exogenous factors that affect the metabolic function of bone cells. Prostaglandins (PG), a family of cycled derivation of unsaturated long chain fatty acids, are intimately associated with bone metabolism, and are important as locally active agents with significant effects on bone and bone cells in vitro. In some experimental studies, it is suggested that the local increase of prostaglandin in response to trauma may act to stimulate differentiation and proliferation of osteoprogenitor cells during fracture healing, whereas, specific prostaglandin antagonists, indomethacin, inhibited fracture healing. The objective of this chapter is to investigate the effects of locally (defect-site) administered prostaglandin (PGE₂) on cortical bone healing in rabbit model.

Clinically healthy adult New Zealand White rabbits (10) of either sex are divided equally into groups A and B, consisting of 5 animals each. They are kept on standard diet and management throughout the experiment. In all the animals, mid-shaft single transverse fracture is created in both the ulnae under thiopental (2.5%) anaesthesia.

In test group A, 0.1 ml (25 µg) prostaglandin (PGE₂) is injected at fracture site on alternate day for 30 post operative days, whereas, group B is served as control. In both the groups lateral and antero-posterior radiographs are taken on days 0, 10, 20, 30 and 40 postoperatively to observe progress of fracture healing and complication, if any at different intervals.

During orthopaedic surgery, it is very difficult and sometimes impossible to stop the flow of blood from fractured bone. For this reason, there has been a longstanding interest in haemostatic agent for osseous tissue surgery. Bone –wax, oxidized cellulose, sanguistat, bone dust, marrow paste and combination using polyethylene glycols have been tried for osseous hemostasis, however, bone wax appears to be the most promising bone haemostatic agent and is widely used today. But, very littlie work has been done to define the local effect of bone wax on healing and osteogenesis and its eventual fate in animals.

The objective of this study is thus, undertaken in this chapter to investigate the effects of bone wax on cortical bone healing.

Clinically healthy adult New Zealand white rabbits (10) of either sex are divided equally in to two groups A and B, consisting of 5 animals each. All animals are kept in standard diet and management throughout the experiment. In all animals’ mid-shaft simple transverse fracture are created in both the ulnae under Xylazine-Ketamine anaesthesia. In animals of group A, bone wax packed in between two fracture ends, whereas, in animals of group B, fracture site kept as it is.

Lateral and anterio-posterior radiographs are taken on days 0, 10, 20, 30 and 40 post-fracture. The radiographs are observed for progress of fracture healing and complication, if any at different interval. The specimens for histopathological study are collected from the healed site of test bone immediately after euthanizing the animals on day 40. Every specimen is consisted of both healing site and adjacent host bone. The bone sections are preserved in 10% formalin for 9-10 days and then are decalcified using Goodling and Stewart’s solution (15% formic acid, 5% formalin and 80% distilled water). Completely decalcified bone sections are then processed routinely for H&E staining. These sections are observed for the status of healing at the fracture site at cellular level.

Fracture healing is a specialized type of wound healing response in which the regeneration of bone leads to restoration of skeletal integrity. Though the process of fracture healing is usually considered to be biologically optimum; the healing of 5-10% is delayed or impaired. For management of these types of fractures, use of biological methods for the enhancement of fracture healing is essential. Furthermore, fracture-healing can be modified by almost any endogenous or exogenous factor that has an influence on the metabolic function of cells. These factors include transforming growth factor-beta, bone morphogenetic proteins, hormones, vitamins, prostaglandin and calcium containing feed supplement medicine.

Keeping in view the above, in this chapter, efficacy of herbal product-Magacal in promoting osteogenesis during fracture healing in experimental animals is discussed. Magacal optimizes absorption, bioavailability and utilization of dietary calcium, phosphorus and magnesium. It is claimed to have advanced chelating agent to form organic complex chelate with dietary Ca, P and Mg, besides containing natural Vitamin D metabolites in highly bioavailability organic form, naturally protected and stable biotin and modern delivery system for optimum translocation of Ca, P and Mg ions to the sites of requirements.

Clinically healthy, adult New Zealand White rabbits of either sex (10) are divided equally into two groups A and B each consisting of 5 animals. All animals are kept on standard diet and management throughout the experiment. In all animals, midshaft simple transverse fracture is created in both the ulnae under thiopental (2.5%) anaesthesia. In test group (Group A), Magacal is given @5ml/animal/ day, orally from day 5-20 post fracture, whereas, in group B, no drug is given which serve as control. Antibiotic dressing of the skin wound is done daily for one week with povidone iodine and himax ointment. All the animals are administered streptopenicillin @ 0.5 gm/animal/day for 5 days postoperatively. Skin sutures are removed on the10th postoperative day.

Clinically all the animals are observed for inflammation, pain and edema at the fracture site, surgical wound healing, appetite, general health, muscular atrophy of the limb, if any, at different intervals postoperatively.

Bone is the only tissue in the body able to heal without microscopical scarring. On the other hand fracture healing is a slow process causing long immobilization periods with consequently high costs for fracture healing giving rise to delayed healing, fibrous healing or non-healing with subsequent problems for both, the patient and the orthopedic surgeon. Fracture healing is complex phenomenon which involves numerous cells, regulators of cell function and biochemical interactions in the repair process. The healing potential of bone, in a fracture model is influenced by a variety of biochemical, biomechanical, cellular, hormonal and pathological mechanisms. A continuous occurring state of bone deposition, resorption and remodeling facilitates the healing process.

Biodegradable and bioinert ceramic materials such as hydroxyapatite (HA), tri-calcium phosphate (TCP), aluminum-calcium-phosphate (ALCAP) ceramics provides scaffold to support the attachment and migration of newly formed bone cells into the osseous defect and also help in formation of a vascular network through the newly formed bone. Bioceramic also act as a carrier to deliver stem cells for osteogenesis.

Specific bone-inductive proteins/growth factors can induce bone formation and healing in vivo. These morphogens are therefore, to be an ideal alternative to autogenous bone grafts. These findings initiating intensive research into bone regeneration orchestrated by putative soluble signals and lead to the discovery and identification of an entirely novel family of protein initiators collectively called the bone morphogenic protein (BMPs), which belong to transforming growth factors-ß (TGF- ß) super family. These growth factors might possess a biological activity that is site and tissue specific in the induction of bone formation. Different growth factors have been demonstrated to possess osteoinductive and fracture healing properties.

Stem cells have therapeutic potential in the realm of orthopaedic surgery because of their capacity to self-renew and differentiate into various types of mature cells and tissues, including bone. Embryonic stem cells (ESCs) are pluripotent cells derived from the inner cell mass of pre-implantation embryos and represent embryonic precursor cells that give rise to any cell type in the embryo. Specifically, it has been shown that ES cells can differentiate into osteogenic cells under selective culture conditions. These osteogenic cells are capable of in vitro producing bone nodules in three dimensional scaffolds.

Despite the benefits that minimally invasive surgery and osteosynthesis have brought to fracture management and bone healing, there are still many circumstances where bone healing remains challenging. Large bone defects are serious complications that are most commonly caused by extensive trauma, tumour, infection, or congenital musculoskeletal disorders. In nonunion cases repairing of bone defects with composite biomaterials as defect filler can promote bone regeneration. Currently, the gold standard for bone regeneration is use of autogenous bone graft. In order to avoid morbidity at the donor site or if large amount of autogenous bone is needed, bone substitution materials can be used. Bone substitution materials can be combined with cells such as mesenchymal stem cells (MSCs) to increase bone formation. Bone-marrow derived mesenchymal stem cells (BMSCs) represent an attractive cell population for tissue regeneration. Bone marrow stem cells are pluripotent cells that have been used to facilitate bone repair because of their capability of differentiating into osteoblasts. Several studies on the regeneration of bone have shown that cultured BMSCs, seeded on different bioabsorbable implants are able to induce bone formation in vivo and lead to improved healing of critical-size defects.

Calcium phosphate bone substitutes such as hydroxyapatite (HA) and tricalcium phosphate (TCP) are currently used for bone substitution in many different clinical applications such as repair of bone defects after trauma or tumour and bone augmentation in spinal arthrodesis. Although these bone substitutes are osteoconductive, they often lack the osteogenecity needed to support bone healing in large defects and are slowly degraded in the body. Studies have shown that coating of hydroxyapatite with a calcium silicate layer could encourage cell proliferation and osteogenic differentiation of human bone marrow-derived stromal cells. Silica-calcium phosphate composite in comparison to calcium phosphate-rich biomaterials has a faster resorption rate owing to greater dissolution of Si ions. The use of several growth factors have been studied in bone repair and these factors are known to play a role in differentiation of mesenchymal progenitor cells specific lineages like endothelial cells or osteoblast. Bone morphogenetic protein-2 (BMP-2) has been shown to accelerate bone healing in humans and animal models. BMP-2 acts by osteoinduction and is involved in the differentiation of mesenchymal progenitor cells into osteoblasts. The addition of recombinant human BMP (rhBMP-2) to a self-cross linkable cellulose hydrogels/biphasic calcium phosphate granules construct promotes bone regeneration in a critical-size segmental defect model of non-union in dogs. It is also reported that protein, insulin-like growth factor (IGF-1) stimulates direct migration of human mesenchymal progenitor cells (MPC) and contributes to the recruitment of MPC in bone formation and bone healing.

The process of fracture healing is unique, in which damaged bone restores its original architecture but it does not form a poorly organized replacement matrix, otherwise known as scar tissue, rather it regenerates the original matrix and retains its mechanical properties. In order to repair bone defects, a combination of cells with osteogenic activity and appropriate scaffolding material can stimulate bone regeneration and its repair. A potential substitute for autologous transplantation should possess 3 elements: osteoprogenitor cells, osteoinductive factors, and an osteoconductive scaffold. Stem cells from adult tissues are attractive materials for cell therapy and tissue engineering. These cells generally have restricted lineage potential when compared to embryonic stem cells, and this may be advantageous in certain therapeutic applications. Bone-marrow-derived mesenchymal stem cells (BMSCs) have proven to be beneficial in bone regeneration. Bone marrow stem cells are pluripotent cells with the capability of differentiating osteoblasts so that they have been used to facilitate bone repair. Several studies have shown that seeding of cultured BMSCs on bioabsorbable implants can induce bone formation in vivo and lead to improved healing of critical-size bone defects.

Mesenchymal stem cells (MSCs) because of their self replication and osteogenic differentiation capabilities are regarded as an excellent source of cells for bone tissue engineering. Autologous stem cells are not always preferable since the quality and quantity of such cells will be affected by metabolic diseases, old age, and osteoporosis. Allogenic BMSCs might be preferable to xenogenic, but they are always not readily available. In addition, allogenic MSCs have the potential for carrying some diseases. The use of non-autologous stem cells isolated from healthy donors offers a major advantage since these stem cells can be thoroughly tested and formulated into off-the-shelf medicine in advance. Major attractive advantage of BMSCs as a source of cell transplantation is their low immunogenecity. It is now well established that BMSCs are immune-privileged cells that do not elicit immune responses due to an absence of their immunologically relevant cell surface markers.

The knowledge and principles from interconnected disciplines of bioengineering, material science and life sciences are combined in tissue engineering with the aim of developing a construct, that can wholly or partly restore/maintain/augment the function of a damaged tissue or organ. Tissue engineering construct generally comprises cells on a suitable scaffold. The ultimate functionality of a tissue engineering construct is mainly dependent on the cell behavior on scaffolds. The proliferation and cell viability on scaffolds varies from one cell type to another and from one species to another. So it is important to investigate individual cell-scaffold combinations. The similarity in structure and composition to bone mineral, osteoconducive properties, ability to integrate with the bony tissue and absence of immune response makes hydroxyapatite [Ca₁₀ (PO₄)₆(OH)₂] the choice of calcium phosphate biomaterial with only disadvantage of slow rate of resorption. On contrary, silica–calcium phosphate composite in comparison to calcium phosphate is rich biomaterials having a faster resorption rate. Studies have shown that coating of hydroxyapatite with a calcium silicate containing layer encourages cell proliferation and osteogenic differentiation of human bone marrow-derived stromal cells. So in this chapter, a triphasic composite scaffold (calcium silicate, hydroxyapatite and tricalcium phosphate), namely HASi with elements in the following percentages: 66.36 % - calcium, 25.35 % - phosphorus and silicon - 8.29 % and porosity of 50 – 500 µm is reported to evaluate the rBMSC proliferation and differentiation properties in vitro.

The present chapter is therefore, included with the aim of assessing cytocompatibility of HASi and rBMSC in terms of cell attachment, cell morphology, cell proliferation and osteogenic differentiation.

Bone differs from other tissues not only in physio-chemical structure, but also in its extraordinary capacity for growth, continuous internal remodelling and regeneration throughout post fetal life. Bone formation is a complex process regulated by diet, vitamins, hormones and growth factors. Growth factors are polypeptides that increase cell replication and have important effects on differentiated cell function. Growth factors are initially considered as systemic agents, but current evidence indicates that they act primarily as local regulators of cell growth. The transforming growth factor-β family of proteins, which comprises polypeptide growth factors that have diverse effects on the growth, differentiation, and function of cells, is receiving considerable attention for potential clinical applications and is likely of physiological and orthopedically significance.

Transforming growth factors

It is one of the most important growth-promoting osteo-inductive substances, which have been identified at the site of fractures and other places. Transforming growth factors (TGFs) have been categorized into TGF-β (alpha) and TGF-β (beta). TGF-α has not been isolated from bone tissue and cannot be considered a local regulator of bone remodelling, although it is mitogenic for bone cells and stimulates bone resorption. TGF-α shared amino acid sequence, receptor-binding and functional activity with epidermal growth factors but had only weak transforming growth factors activity. Furthermore, TGF-β by itself is completely ineffective, but together with TGF-α, it potently induced growth of colonies of cell-line.

In 1965, Urist demonstrates that the implantation of demineralized bone at extra skeletal sites induced de novo formation of cartilage and bone and proposed that the decalcified bone matrix possess high osteoinductive property and the inductor is a unique chemical that differentiates mesenchymal type cells (pericytes) into new bone. Urist and Starates (1971) reported that the inductor is a protein and termed it bone morphogenetic protein (BMP) . BMP is the bone induction principle.

Sources: Bone morphogenetic protein isolate from bovine as well as human bone and designated as bBMP and hBMP respectively. The molecular cloning of the bone morphogenetic proteins and their subsequent expression in recombinant system, resulted recombinant bone morphogenetic protein designated as rhBMP. BMP has been extracted from dentin and bone matrix, as well as from bone tumours, by enzymatic and nonenzymatic methods. The sources of BMP, thus far explored are rabbit dentin, rat, rabbit and bovine bone, human bone, mouse or human osteosarcoma and human placenta.

BMP circulates in the blood and it can be detected by BMP-radioimmunoassay. BMP levels higher in growing children (20-72 ng/ml) and patients with Paget’s disease (over 259 ng//ml) than in normal adults (14-18ng/ml). Crude and partially purified BMP has been extracted with the aid of collagenase, ethylene glycol, calcium chloride urea and Guanidine hydrochloride (GuHCL) solution. Wang et al (1988) purified BMP factor that induces bone formation> 300,000 fold from Guanidine chloride extracts of demineralized bone. As littlie as 50ng of this factor can induce the formation of cartilage and bone in an in vivo assay, though only 40µg could be obtained from 400kg of bovine bone powder.

Fracture in animals occur frequently and is a concern of economic importance to the owner. The practice of keeping them as pets in households rather than in sheds or in confinements have increased the chance of encountering accidents like fall from coat, terrace, limbs being trapped in drains, being hit by vehicles, dog bites etc. leading to fracture. Fracture management in small animals/small ruminants can be successfully achieved owing to their light weight; they tolerate external coaptation relatively well and can ambulate in three limbs well in order to spare the injured limb. Fracture can be repaired successfully with regional anaesthetic techniques which they can tolerate very well. The constraint of cost effectiveness can be balanced by carefully selecting the appropriate technique according to the weight and limb involved. Many economical skeletal fixation technique have been evolved which can well fit into the budget of the owner. Knowledge of simple techniques can enhance healing in cases of bone loss and management of infection.

Fracture site stabilization

Fracture repair is usually elective, hence initial support and care of the fracture is necessary to prevent further trauma and complications like communication of fracture site to the exterior through wound or sinus and chances of infection. Initial support to splint the bone not only limits the movements but also supports the soft tissues from untoward consequences. Limb splinting reduces edema and pain, preserves neurovascular supply to the limb till the day of operation and helps to transport the animal safely. Fracture to limbs caudal to mid radius ulna/ tibia can safely be stabilized by splints or full limb casts. Wood, pliable bamboo or split poly vinyl chloride pipes positioned on the caudal and lateral aspects of the limbs and supported firmly with tapes or bandages can serve the purpose. Fracture proximal to mid radius ulna or tibia should not be stabilized using splits since the fulcrum effect created due to this can augment soft tissue injury. Careful assessment of the viability of the structures caudal to fracture site should be assessed prior to a decision regarding surgery, devitalized tissue hamper healing and is a subject to be considered for amputation.

Repairing fractures in small animal’s presence challenges to the veterinarians in their day to day service. These challenges include properly repairing fractures, each of which is unique, choosing the right fixation device, keeping an extensive inventory of costly instruments, and providing high standards of care for fracture repair. But fracture repair also gives veterinarians the opportunities to do something a little unusual in daily practice and to experience the immediate gratification of easing pain and promoting activity in injured animals.

Successful management of fractures mainly depends upon early diagnosis, type of fracture, timing of surgery and selection of proper method of fixation. The fixation of fractures can be achieved either by external or internal fixation methods. An ideal method of fracture fixation is one that can maintain early anatomical reduction, minimal soft tissue trauma, rigid stability and early motion of bone and joint. Restoring a fractured bone’s normal shape is important for return to function because it contributes to normal alignment of adjacent joint surfaces. Whereas, rigid stability of fracture fixation eliminates fragment movement and reduces pain. Soft tissue trauma adjacent to fractured bone should be minimum, which play a vital role in early vascularisation and prevents infection. Finally, early return to function maintains muscular tone of the limb allows normal weight mobility and joint nutrition and uses normal weight bearing forces to maintain bone density. These can be achieved with use of internal fixation devices.

1. Bone grafts

Bone grafting represents one of the earliest devised reconstructive approaches to the musculoskeletal system and remains among the most commonly used orthopedic procedures. Bone is the second most frequently transplanted tissue. The first recorded attempt to use bone graft is by the Dutch surgeon Job Van Meek’ren in 1668.

Definition

A graft is a living tissue that is transplanted to another site either within the same individual or into another individual for repair of a defect. An implant is a non viable material placed in a living system. Implants are composed of bone cement, metal or ceramic material. Grafts are structurally important for mechanical stability, but their use evolved to include the stimulation and enhancement of bone in response to injury.

Indications

Bone grafts are used, in general as framework, both to provide stability and to augment healing. It is used in the management of non union, delayed union, treatment of pseudoarthrosis, filling of osseous defects or cavities, arthrodesis, stabilization of spinal segments and addition of bone stock in total joint displacement.

Fracture healing is a specialized type of a wound healing response in which the regeneration of bone leads to a restoration of skeletal integrity. Because healing of a fracture in a living organism is accomplished by cells, it can be modified by almost any endogenous or exogenous factors like nutrients, vitamins, hormones, growth factor etc. that has an influence on the metabolic function of bone cells. Fracture healing may be induced by malnutrition that involves certain nutrients, notably calcium (Ca), phosphorus (P) and vitamin D, but also iodine (I), copper (Cu), zinc (Zn), manganese (Mn), fluorine (F), silicon, vitamin A and perhaps vitamin C as well as different endocrines. The number of these nutrients and their interaction is so large that dietary intervention in fracture management is likely to be the most effective if directed at consumption of a good diet that is completely balanced for growth, maturity, old age or stress. Substitution of a good diet for a bad one is also likely to be more convenient and safer than most attempts at partial supplementation of a faulty diet in order to bring it into balance. Underfeeding a good diet decreases size-for-age of bones during growth and delayed fracture healing. Underfeeding may engender osteoporosis in older animals. Underfeeding of Ca, P and vitamin D, causes delayed fracture healing. Over feeding a balanced diet induces rapid growth of newly formed bone and sometimes-accelerated fracture healing but it also causes obesity in adults.

Three major component of bone are osteogenic cells, organic matrix and inorganic matrix (mineral). The osteogenic cells include osteoblasts, osteocytes and osteoclasts, while the matrix consists predominantly of collagen and proteoglycans and constitutes approximately one-third of bone mass (on dry basis). The inorganic/mineral component that makes up approximately two-third of bone and it performs two essential functions : it serves as an ion reservoir and it gives bone most of its stiffness and strength. Bone is a reservoir for 99% of body calcium, 85% of body phosphorus and 40-60% of body sodium and magnesium. The main mineral composition of a long bone is: 85% calcium phosphate as hydroxyapatite, 12% calcium carbonate and 3% magnesium phosphate, calcium fluoride and magnesium carbonate and trace amount of chloride, potassium and sodium. For collagen synthesis, hydroxylase enzymes requires ferrous ion and vitamin C. Impaired collagen synthesis also occurs during deficiencies of vitamin D and E and zinc. The organic intercellular bone matrix is rich in mucoplysaccharide. Their synthesis involves enzymes that require manganese, zinc and vitamin A.

A. Definition

A fracture is a break in the continuity of hard tissues like bone, cartilage, etc.

B. Etiology

(a) Extrinsic causes

Direct violence

Indirect violence

Bone is an integral part of musculo-skeletal system of body. In the living animal it is bluish pink, whereas, fresh dead bone is yellowish-white in colour. It is hard and resistant to pressure but also have certain amount of elasticity. Its tensile strength is equal to the cast iron, though bone three times lighter and ten times flexible to cast iron.

Number of bones: The number of bones of the skeleton varies with age and species. Number of bones in cattle-210; Horse-208; Dog-271; Fowl-166 and human-208 (207-212)

Composition of bone: Bone contains different bone cells, organic and inorganic matrix and water. Organic matrix is about one-third and Inorganic matrix is two-third weight by volume. The organic material consists of 90% collagen [type-I (maximum), type V and type XII] and 10% non-collagenous glycoprotein and bone specific proteoglycans (osteocalcin, osteonectin, sialoprotein, bone phosphoproteins and small proteoglycans) and bone growth factors [TGF-beta family; Insulin-like growth factor-1, 2; bone morphogenetic protein (BMP); platelet derived growth factor (PDGF); Interleukin -1 (IL-1); Interleukin-6 (IL-6); colony stimulating factor]. Organic matrix of bone resembles the matrix of dense fibrous tissues such as tendons, ligament and joint capsule. It is not certain whether all of these proteins are synthesized by bone cells or whether they are synthesized by cells outside of the bone and then incorporated into the bone matrix. However, their presence within bone and their potential to affect the activity of bone cells suggests they have important role in controlling bone cell functions. Inorganic matrix of bone or mineral component of bone serves as an ion reservoir and gives bone of its stiffness and strength. It reserves 99% of body calcium, 85% body phosphorus and 40-60% body sodium and magnesium. Inorganic matrix of bone is composed of 85% calcium phosphate, 12% calcium carbonate, 3% of magnesium phosphate, calcium fluoride and magnesium carbonate. It also contains traces of chlorine, sodium, potassium. These minerals remain as hydroxyapatite crystal form within bone. However, bone mineral crystal is not pure hydroxyapatite, instead they contain both carbonate and acid phosphate groups (HPO₄) and bone crystal does not contain OH groups. Bone matrix has such great durability and stability that it can remain essentially unchanged and retain much of its strength for centuries after death. Removal of organic matters renders the bone fragile, brittle and shattered, whereas, decalcification renders bone soft and pliable and resistant to fracture.

11.1 Performance of Farm Irrigation Systems

The performance of farm irrigation systems is determined by the efficiency with which water is delivered to the field and utilized to the fullest amount possible with minimum losses due to seepage and percolation, runoff, evaporation and other operational losses. The efficiency of the irrigation system will increase if the conveyance losses, application losses and other losses are reduced to a great extent.

11.1.1 Overall Efficiency

The overall efficiency of a farm irrigation system is defined as the percent of water applied to the farm that is used for irrigation on the farm. Irrigation efficiency otherwise called as overall system efficiency is defined.

10.1 Application Efficiency and Irrigation Uniformity

All irrigation systems have some non-uniformity. This non-uniformity influences yields and irrigation efficiency especially application efficiency. If the net amount of water applied during irrigation is the same as that required to fill the soil water deficit, then because the distribution of water is never perfect, half of the field will receive too much water while the other half will receive too less water. In such a context, a farmer will try to apply more irrigation water to his field so that a larger fraction of the field has an adequate amount. This will tend to have higher percolation and runoff and thereby declining the application efficiency. An alternative to adding extra water is to improve the uniformity of water application. A major advantage when the uniformity is improved is that the amount of deficit in the area under irrigation is reduced and the potential for water logging and salinization is also reduced.

9.1 Introduction

After knowing when to irrigate and how much to irrigate, next important thing is how to irrigate the crop field. Methods of irrigation plays a very crucial role in deciding how to irrigate the crop fields since all the crops do not require same methods of irrigation. Depending on the sources of water available for irrigation, type of crop to be irrigated, quantity and quality of water to be applied and other economic considerations, methods of irrigation will vary. Irrigation water may be applied to the crops by flooding it on the field surfaces as surface irrigation, by applying it beneath the soil surface as sub irrigation, by spraying it under pressure as sprinkler or overhead irrigation or by applying it in drops around the root zone of the crop as trickle irrigation. The commonly used methods of irrigation are described in schematic diagram as follows

8.1 Introduction

For maximization of yield of crops, it is essential to provide irrigation to crops. When either the natural sources like rainfall, dew, snowfall and other forms of precipitation or when the capillary rise of groundwater is not sufficient to meet the crop water demand, then there is need to supply irrigation. This irrigation water is a precious resource and need to be used cautiously. There are three important things need to be understood for optimum utilization of irrigation water with basic purpose to maximize the yield and returns. 

7.1 Introduction

The entire amount of water that is diverted at the canal head regulator does not reach at the field for use of irrigation. Major amount of it is lost on way during its movement in the canal systems. During the passage of water from main canal to outlet at the head of the water course, water may be lost either by evaporation from the surface or by seepage through the peripheries of the channel. These losses are sometimes very high, and may go up to 50 % of the total water diverted into the main canal. Evaporation losses are low and may go to a maximum of about 7 percent of total losses. The major loss of water in the conveyance system is the seepage loss. Seepage loss depends on the channel geometry and evaporation loss is proportional to the area of free surface. In determining the designed channel capacity, a provision of these water losses must be made. The provision for the water lost in the watercourses and in the field however, already made in the outlet discharge factor, and hence no extra provision is made on that account. However, excluding the above two losses, there is operational loss which when added to the evaporation and seepage losses, the total losses goes up the maximum 50 percent level. The operational loss includes spillage, over-toping, leakage through rodent holes, dead storage, initial infiltration and evapotranspiration from the vegetation along the watercourse. Evaporation and seepage losses of the canals are discussed below.

6.1 Introduction

In order to increase the production and productivity of crops, provision of irrigation system is highly essential. A project that makes use of weir or a barrage for providing irrigation to fields is called as direct irrigation system. On the other hand, a storage irrigation system makes use of storage reservoir by construction of dams. In order to provide irrigation by both the schemes, it is necessary to have a network of canal systems. A canal is an artificial channel, generally trapezoidal in shape constructed on the ground to carry water to the fields. The canal systems that provide water to the fields include main canals, branch canals, distributaries, minors and watercourses. It is imperative to design the canal systems properly for a certain realistic value of peak discharge that must pass through them so as to provide sufficient irrigation to the command areas. The flow irrigation that provides irrigation water to the fields may be of two types i.e. Inundation irrigation and Perennial irrigation.

5.1 Soil

Soil and water are the two most important natural resources required for crop production. Without soil, no crop can be produced even if there is availability of plenty of water. It is vital not only for agriculture but also very vital for sustenance of life. No building can stand on any place without soil. Soil gives foundation support for any building. The term ‘soil ’is defined in various ways depending on the general professional field in which it is used. However to an agriculturist, soil is the substance existing on the earth surface, which grows and develops plant life. It contains all vital elements including its inherent fertility contents that support the growth of plants. For an engineer, soil is the unaggregated deposits of mineral and organic particles covering major part on the earth crust. It includes widely different materials like boulders, sand, gravel, clays and silts. Soil is produced by the weathering of solid rocks. The formation of soil is due to geologic cycle continuously taking place on surface of the earth. The geologic cycle comprises weathering or denudation, transportation, deposition and upheaval, again followed by the above mentioned processes. Weathering may be natural or artificial. In natural weathering, physical processes like temperature changes, flow of water, ice, wind and rain play the role. In artificial weathering animals especially human beings interference causes soil erosion and formation. It is obvious that it takes hundreds of years to form one inch of soil.

4.1 Introduction

A reservoir that collects and harvests in-situ rainfall as well as runoff from the catchment area can be used for many purposes: the most important of which is the supply of water to crops as supplemental irrigation. The water of the reservoir can be used for domestic and industrial uses. It is also a good source for recreational use. Total water demand of the reservoir including irrigation to different crops in different seasons, domestic and other non-agricultural uses are to be assessed. While estimating the crop water requirement and irrigation requirement, various crops to be grown in the service area of the reservoir, their water requirements, crop duration, extent of coverage and water management practices followed are to be taken into consideration. Multiplying the irrigation requirement with crop coverage area, volumetric irrigation water requirement of crop can be found out. Summation of irrigation requirement of all crops grown in service area of the reservoir gives total irrigation demand of the reservoir.

4.2 Water and Irrigation Demand of Crops

Of the various demand of water from the reservoir, irrigation demand of crops grown in the command area of the reservoir is very important. Most of the crop water demand is met from rainfall. However, when the rainfall is deficient to meet the crop water demand, then the deficit is met from the stored water of the reservoir in the form of irrigation, more commonly called as supplemental irrigation. The irrigation to the crops is required mostly to meet the evapotranspiration requirement of the crop. Before discussing the water and irrigation demand/requirement of crops, it is essential to know some commonly used terms frequently used.

3.1 Introduction

Irrigation water that is supplied to crop fields is defined by a term commonly called as discharge which is also called as rate of flow. Discharge is defined as the volume of water flowing through a cross section in a unit time. It can be expressed as cubic metre per second (cumec), cubic feet per second (cusec), litre per sec (lps) etc. one cumec is equivalent to 35.5 cusecs or 1000 lps. The measurement of discharge is very important in everyday life starting from deciding the amount of irrigation to be supplied to a crop at a particular growth stage, or amount of water supply for domestic and industrial uses, for flood control and power generation etc. It plays a vital role in water resources planning.

3.2 Methods of Discharge Measurement

The various methods of discharge measurement can be broadly classified into the following types: (i) volume method of discharge measurement (ii) flow measuring structures such as orifices, mouthpieces, flumes and weirs.

2.1 Introduction

Rain and snow are the two chief sources of water of the earth. In-situ conservation of rainwater helps the plants to meet some of their consumptive uses directly. Not all the amount of rainwater that falls at a point is conserved by the soil reservoir to support the plant growth. Some part of it flows over the soil surface as surface runoff and some seeps down as infiltration and finally adds to the underground reservoir. Therefore, rain or snow, which is not used directly by the plants, becomes a potential source of either surface or ground water for irrigation. In areas with extreme low temperature, snow contributes major source of water supply wherever, in humid and sub humid regions, rain is the principal source. Precipitation, rain or snow, is the first stage of the continual cycle of water in its round from atmosphere to the earth and return by evaporation from water bodies, snow surfaces and land surfaces. The continual cycle of distribution of water is governed by the process called as hydrologic cycle. Thus, precipitation or atmospheric water is the first form of water above the earth’s surface, the second form being the surface water on earth and the third form is the subsurface water or underground water. The study of occurrence, distribution and circulation of precipitation through the unending hydrologic cycle forms the basic part of study of hydrology. As mentioned above, the part of precipitation that is not used by the plants in-situ flows over the soil surface as runoff and contributes to either surface or ground water supply.

Engineering hydrology is the science that deals with the estimation of runoff and its transmission from one place to the other, besides dealing with the properties of water in nature. For, an engineer, surface and groundwater are the two most important components to deal with. But due to lack of data of stream flow, it is usually necessary to study precipitation, evaporation and percolation to indirectly determine the availability of surface and groundwater. For planning and management of water resources systems, an engineer has to estimate (i) water resources potential of a basin, (ii) dependable yield for irrigation and hydel power generation and industrial water demand (iii) probability of occurrence of floods with their magnitude, (iv) sediment yield and transportation (v) hydrometeorology (vi) new sources of water for irrigation (vii) stream flow forecasting by use of precipitation data (viii) water control, navigation, control of soil erosion and control of pollution of water bodies and (ix) operation and maintenance of river valley projects. For estimation of the above mentioned parameters, it is important to understand the hydrologic cycle properly.

1.1 Importance of Water for Agriculture

All animals and plants require water for their survival. Animals cannot prepare their own food. They depend on plants for their food. Plants need water for preparation food by the process of photosynthesis. Majority of this water requirement of the crops is met from different forms of precipitation. However, when the water requirement is not fully met from precipitation, then growth of the crops is affected. Water requirement of crops depend on many factors. It depends on the type of crops, irrigation schedules practiced, soil texture, position of groundwater table below effective root zone of the crop and management practice followed in crop cultivation. The dry land crops need less water compared to rice which is a water loving crop. The perennial crops like sugarcane, banana etc. need more water than other crops like cereals, pulses, oilseeds etc. Water requirement of crops is partly met by the upward flux of groundwater. The capillary rise of shallow water table in the form of upward flux helps in meeting the water requirement of crops to some extent. The upward flux depends on the type of the soil, effective root zone depth of the crop, depth of water table below effective root zone depth of the crop and potential gradient of soil moisture suction. If the soil is heavy like clay, effective root zone depth of the crop is more (deep rooted crop) and position of ground water table is near to ground level (shallow water table), then the upward flux is more.

Due to rainfall or application of irrigation water, some amount of water is recharged to soil reservoir. This recharged water is stored in the soil as soil moisture content. The stored moisture or called as water in the soil reservoir helps in supporting water requirement of crops also. The heavy soil like clay has more moisture retention capacity than the light soils like sandy soil. The residual soil moisture held in clay soil is released slowly and supports more soil moisture to the crops for a longer period than the sandy soil which has less soil moisture retention capacity. After meeting the water requirement of crops from all sources including different forms of precipitation, ground water contribution by capillary rise and residual soil moisture, if there is any shortage of water, then this shortage should be met from other sources which are called as irrigation. It is to be noted that irrigation is not the natural phenomenon of supply of water; rather it is the artificial means of supply of water to the crops. Irrigation may, therefore, be defined as the science of artificial application of water to the crop in accordance with the crop requirement for better nourishment of the crops.

Introduction

Plants are considered as immobile organisms with poor sensitivity and limited ability to respond. Within the plant kingdom, Mimosa pudica (also called the sensitive plant), Drosera (sundews), Dionea muscipula (flytraps) and tendrils of climbing plants which showed rapid and purposeful movements were considered as exceptions. These sensitive plants attracted attention of outstanding pioneer researchers like Pfeffer (1845-1920), Burdon-Sanderson (1828-1905), Darwin (1809-1882), Haberlandt (1854-1945) and Bose (1858-1937). Stahlberg, 2006 reported the presence of various mechanoreceptors in such species which could trigger action potentials (APs) which activated these sudden movements.

The first extracellular recording of a plant action potential (AP) development was initiated by Charles Darwin; later, the animal physiologist Burdon-Sanderson in 1873 performed similar studies on leaves of the Venus flytrap (Dionea muscipula Ellis). Haberlandt (1884) demonstrated that phloem strands were the actual paths for excitation by destroying the external, non-woody part of vascular bundles. This notion was confirmed in Mimosa and other plant species by a number of recent studies. Houwink (1935) carried out experiments with two species of Vitis (grape) and obtained electrical fluctuations which was similar and comparable with the action potentials he obtained with Mimosa. In 1907, Bose, used a D’arsonval galvanometer to demonstrate propagating electrical effects in different genera of plants such as Ficus, Artocarpus, Cucurbita, Corchorus, “fern” etc. APs have their largest amplitude in phloem sieve cells and cells adjacent to this area (Sibaoka, 1969). Other studies found that AP-like signals are propagated with equal rate and amplitude throughout the cells of vascular bundles.

Introduction

Plant architecture is defined as a three-dimensional organization of plant body in terms of its size, shape position of leaves, flower organs and branching pattern. Earlier this was the only criterion for systematic and taxonomic classification of plant species and the best means of identifying them (Stecconi, 2006). Study of plant architecture emerged as a new scientific discipline some 30 years ago. The subject derives its base from earlier works on plant morphology (Halle and Oldeman, 1970) but currently it is integrated with several disciplines of plant sciences ecology and engineering. Modification in plant architecture causes alteration in primary and secondary growth. This occurs due to differences in phyllotactic arrangement, branching pattern and floral differentiation. Plant architecture can be modulated by various parameters like climate, agronomic practices and human interventions. Manipulation of plant architecture is a major area in plant science which is currently adopted for productivity improvement by breeders, agronomists, horticulturists and landscape managers. However, the basis for such manipulation lies in the knowledge of the basic architecture of plants and its significance.

Introduction

Clean and renewable energy is a basic requirement for developmental activities. Global energy crisis is caused by increasing world population, escalating demand due to urbanisation, and continued dependence on fossil-based fuels. Non-renewable energy sources like coal, natural gas oil contribute 79 per cent of world electricity generation; only 21 per cent comes from renewable energy sources. Promoting the use of existing renewable energy sources and identifying new environment-friendly technology is gaining importance. Switching to renewable energy sources like wind energy, ocean energy and solar energy is gaining a lot of importance. These green technologies emit less greenhouse gases into the atmosphere and hence are pollution free and immensely available. Recently, investigators have identified plants as weak energy sources. Plants are versatile organisms capable of communicating with each other with a number of signaling mechanisms. It was observed that plants generate electronic signals inside their system. The concept of utilising electronic signals for studying various physiological plant processes was first put forth by Sir Acharya J. C. Bose, an eminent biophysicist, botanist, archaeologist and polymath from India (Fromm and Lautner, 2007). His ideologies and concepts of movements of plants laid the foundation for modern electrophysiology-a branch of physiology that deals with electrical phenomena associated with metabolic functions. Attempts have been made to harvest electricity from free electrons available in plant leaves produced during photosynthesis and from rhizosphere of plants formed as a result of bacterial activity. Currently, a lot of attention is being given to improvise and develop this technology. To proceed forward in this direction, it is very critical to understand the mechanism of electrical signaling in plants.

Introduction

Earth’s magnetic field (MF) is a natural component of environment and is an inescapable factor for all living organisms including plants. Earth has an inner core made of molten iron which rotates at a fairly high speed creating a magnetic field just like a dynamo. This Geo Magnetic Field (GMF) extends from the earth’s interior into outer space. It interacts with the solar wind, which is actually a stream of charged particles emanating from the sun. As earth is engulfed in a GMF, all living organisms, both plants, animals and all biological processes are under its influence. However, local differences in strength and direction of the geomagnetic field have been detected. The vertical component of GMF is maximum at the magnetic poles, which is around 67 μT, and the horizontal component is zero there. Conversely, at the magnetic equator the vertical component is zero and the horizontal component is maximum, about 33 μT (Kobayashi et al., 2004).

Turbulent flows of the fluid metallic inner core of earth mainly contribute to GMF and the effect of external MFs located in the ionosphere and the magnetosphere is very meagre (Qamili et al., 2013). As GMF extends beyond the earth’s surface it is this force along with magnetosphere that protects the earth and its biosphere, from the solar wind by deflecting most of its charged particles (Occhipinti et al., 2014).

Introduction

The ultimate aim of agricultural research or any other related activity is to increase yield. Yield improvement could be obtained mainly through the enhancement of photosynthetic efficiency. Photosynthesis is a process in which plants convert light energy to chemical energy. Sun is the ultimate source of energy for photosynthesis. However only half of the total solar radiation (51%) reaches the earth’s surface of these only 1.2% reaches the plant canopy. Solar spectrum contains an array of other radiations which can be harmful for plant and human life. With increasing global climatic change amount of harmful radiation reaching the earth’s surface has increased and this is found to influence plant productivity. Under this scenario, protected cultivation is gaining popularity in many countries as it helps to reduce harmful effects of undesirable radiation, to allow cultivation of crops irrespective of seasons and also expand cultivation to non-traditional areas by artificially manipulating crop environment. In this context, spectral manipulation for crop production has emerged as an important area in crop production system

Introduction

Plants possess complex, systematic and specific mechanisms to cope up with abiotic as well as biotic stresses in the existing environment. ‘Do plants respond to sound ? How can plants perceive sound without a hearing organ?’- are interesting queries among the scientific community. Responses of plants to different factors such as temperature, moisture, light, wind, pests and microbes are well understood. However, information on the effects of audible sound frequencies on plants are still lacking. Recent evidences suggest that audible sound stimulation can contribute to better plant growth and improved quality of plant produce. It also represents a new trigger for plant protection. Sound is an acoustic energy in the form of a mechanical wave which transmits through gases, liquids and solids. Acoustic spectrum consists of three regions, infra sound (less than 20 Hz), audible region (20-20,000 Hz) and ultrasound (greater than 20,000 Hz). Infra sound and ultrasound are used in clinical diagnosis and therapeutics.

Research on plant responses towards different stimuli started during 1880s by Charles Darwin. In 1848, Dr. Gustav Theodor Fechner, a German experimental psychologist, suggested that plants are capable of feeling emotions and that one could promote healthy growth of plants with talk, attention, attitude and affection. Later, J. C. Bose (1926), famous Indian botanist and physicist, made several studies on the effect of sound vibrations on plants. According to him, a plant treated with care and affection gives out a different vibration than a plant subjected to torture. In addition, Bose found that plants grew more quickly in pleasant music and more slowly in loud noise or harsh sounds. He also noticed that plants can “feel pain and understand affection” based on variation of cell membrane potential under different sound frequencies. Retallack (1973) also described several experiments involving plants and music.

Introduction

Plasticity is a unique attribute by which the otherwise sessile plant species are capable of adjusting to environmental vagaries by changing their phenotypic and morphological characters. Any plant population will be able to survive only if it can respond to an extremely variable environment by becoming more plastic and more genetically variable. To achieve this, plants should be able to suitably change their morphology anatomy and physiology or any one of these parameters based on environmental changes so as to improve their adaptability and survival in the altered environment. When such changes are genetically heritable, then they lead to species evolution.

Plasticity helps plants to adapt to new environmental conditions, after migration to new geographical areas, by genetic assimilation of traits that can help to improve their performance under a new set of conditions. Understanding plasticity is important for predicting and managing the effect of climate change on native species as well as crop plants. Response of plants to modified environmental conditions is critical for their persistence.

Phenotypic plasticity has been defined as the ability of an individual genotype to produce different phenotypes when exposed to different environmental conditions (Pigliucci et al., 2006). This includes the ability of a plant to modify its development in response to environmental cues and also its ability to bring about changes in its metabolism which increases the chances of survival of an organism under a specific condition.

The biofertilizer technology has paved way for the manufacturers and entrepreuners to establish a biofertilizer production unit due to an increasing demand and awareness among farmers. Biofertilizer production units have already been started with most of them in the southern states of India. The biofertilizers can be produced using different kinds of carrier materials (peat, coal, lignite, clays, inorganic soil), organic materials (composts, soybean meal, wheat bran, sawdust, etc.), or inert materials (e.g., vermiculite, perlite, kaolin, bentonite, silicates). Natural carriers like peat, plant waste materials, composts, sawdust, sludge lignite, clays, coal, vermiculite, perlite and bentonite and mixture of organic and inorganic materials may also be used.

The Government of India is also encouraging a low cost technology by providing a subsidy to establish a biofertilizer production unit . The major equipment’s and other materials required for a biofertilizer production unit with the capacity of 150 metric tonnes/annum are given below

There are many manufacturers of biofertilizers in both government and private sectors. Only some of them have been listed here for the information

The soil fertility is depleting in the current scenario of gap between nutrient removal from soil and supplies. Moreover, increase in the fertilizer prices has become unaffordable to small and marginal farmers. There is also a growing concern about the environmental hazards and its threat to sustainable crop production. Indiscriminate use of synthetic fertilizers for intensive crop production has resulted in large quantity of nutrient (particularly P) accumulation in soils which has resulted in generating a dead soil. In this context, the long term use of biofertilizers are considered to be economical, eco-friendly, more efficient, productive and accessible to marginal and small farmers over chemical fertilizers (Subba Rao, 2001). Biofertilizers mainly consists of specific living cells which supplies nutrients to plants through their root system. Microbes in these fertilizers provide nutrients to the plants by nitrogen fixation, phosphate solubilization, phosphate mobilization, and promotion of beneficial rhizobacteria (Bhat et al., 2010). Biofertilizer application improves the supply of nutrients, organic carbon, accumulation of soil enzymes, productivity, soil fertility index, economy of farmers, and maintenance of sustainability in natural soil ecosystem and horticultural crops. The production of efficient and sustainable biofertilizers for crop plants needs to be undertaken to make the biofertilizers popular and efficient. The important and specific research in future may be focused on the following aspects.

Microbes present in the biofertilizer formulations help in microbial processes in the soil which augment the availability of nutrients to be taken up by plants. They are of different types based on their nature and function such as nitrogen fixing, phosphate solubilizing, phosphate mobilizing or biofertilizers for micronutrients. Plants use different strategies for growth promotion at different stages of plant growth and development. Biofertilizers are mass multiplied cultures of specific beneficial microbial strains which improves the fertility of soil and productivity. The nutrients are added through natural processes of nitrogen fixation, solubilization of phosphorus and synthesis of growth promoting substances to stimulate the plant growth. The main sources of biofertilizers are bacteria, fungi and cyanobacteria.

11.1. Bio-enrichment of chemical fertilizers- A new approach

Yield response of different host plant depends on the mineral fertilizer and its fertilizer use efficiency. Generally, such fertilizers are low in efficiency due to environmental and soil factors. A large portion of synthetic fertilizers are lost due to fixation, leaching, run-off, erosion, volatilization, denitrification and precipitation without reaching plants. Hence, there is an economic loss and water and environmental pollution.

Microbial inoculants applied to the soil enhances the uptake of nutrient by plants and increase the efficiency of synthetic or organic fertilizers. In this context, it is possible to impregnate mineral fertilizers with specific microbial strain. A new approach is to incorporate the microbes or microbial compositions into fertilizer compositions.

It has been well established that the biofertilizers are beneficial to plants and are cost-effective inputs for sustainable production system. However, there are some constraints with respect to its production, which are presented below:

10.1 Production constraints

10.1.1 Raw material

Eventhough there are large number of biofertilizer formulations, only few are considered as good carrier materials for biofertilizers such as peat and lignite. Moreover, its availability in India is limited and the quality is lacking for these carrier materials. Hence, a cost effective and locally available material with increased shelf-life is the need of the hour.

10.1.2 Specificity and availability of strains

As the microbial strains used as biofertilizers are specific to soil and agro-ecological conditions, availability of specific strains for the specific host and soil edaphic factors are inadequate.

Quality Control is very important in biofertilizer production, which determines the efficiency of microbial strains. Generally, the quality control based on BIS are voluntary in nature. However, some of the biofertilizers are brought under the ambit of Fertilizer Control Order 1985 (FCO) during 2006 (Yadav and Chandra, 2014). At present, biofertilizers are under the FCO with specific quality standards. Biofertilizer production and its sales can be regulated and is a mandatory requirement for registration of manufacturing unit with the State Fertilizer Controller (who is generally the Commissioner or Director of Agriculture Department) under the statutory provision of FCO. Moreover, some officers of the Agriculture Department have been declared as Fertilizer Inspectors at the district level, who are authorized to inspect production and storage facilities and draw samples for quality analysis. National Centre of Organic Farming (NCOF), Ghaziabad under the Govt. of India and its six Regional Centre’s located at Bhubaneswar, Bangalore, Jabalpur, Nagpur, Hissar and Imphal have been declared as testing laboratories. Under the provisions of the FCO act, State Governments can also develop their own quality control laboratories and notify them under the FCO (1985). Peat, lignite, peat soil, humus, wood charcoal or similar material favouring the growth of microbial strain could also be used. The FCO specifications for different biofertilizers are presented below:

Biofertilizer is already proven to be beneficial for agricultural and horticultural crops. The most important pre-requisite for the success of biofertilizer application is the presence of high population of viable cells which will show the desired effect on plants. Biotic interactions along with competition from native microflora is a major challenge to an applied strains of biofertilizers. The formulation is an important parameter for biofertilizer production containing efficient microbial strain with suitable carrier material and additives. The microbial strain needs suitable protective environment to increase their efficiency at the target site and also make it user-friendly. Performance of biofertilizers under field condition is the major requirement for inoculants. Inadequate formulations and poor inoculant quality are the major hurdles for the successful use of biofertilizers.

Formulation of biofertilizers has to be consistant during production, distribution, storage and transport to the farmer/end users. The shelf-life of inoculated microbial fertilizer depends on the formulation with suitable carrier material. If local production and distribution of an efficient native biofertilizer formulations are developed, the present problems in the field performance of biofertilizers could be significantly solved. Specific formulations of native strains for use in saline soils, acid soils, drought, high temperature and eroded soils requires specific formulations. Therefore,use of native strains of biofertilizer formulations which are tolerant to the abiotic stress is an important approach. The formulations of biofertilizers are challenging and limited success has been observed in the commercialization of new biofertilizers. Hence, the search for new formulations has been a continuous approach. However, the choice of the formulation depends on the application method, available equipment, farmer’s convenience, plant inherent characteristics and developmental stage, cost, mode of action and colonization pathway of the inoculated strain. Conventionally, solid based formulations are used, which have problems of low viability during storage and application.

Biofertilizers can directly or indirectly influence the growth of plant. The direct influence can be through production of phytohormones or making the nutrients available to plants. The indirect effect can be through management of plant pathogens through biological control. Therefore, the purposeful introduction of biofertilizer inoculants to plant microbiome represents an environmentally sound option that holds a prominent position in order to reduce the dependency on agrochemicals (Adesemoye and Kloepper, 2009; Abhilash et al., 2016; Aloo, et al., 2019).

Microbes in soil can rarely exists as single cells and survive near the plant root canopy or in bulk soils or endophytes or even on plant surfaces (Lorito, 2006). The soil microflora occurs as complex members of microbiota (Hacquard, 2015). Microbial consortia (MC) are unique mixture of organisms which respond to the environment stress and adapt to stresses due to internal beneficial interactions among the microbial community. Ecosystem’s physiological functions by microflora is carried out by more than one microbial species with the functional diversity and succession among different microbial components. The microbial balance among the different microflora of a MC will consist of a continuous shift between actively growing cells and non-dividing cells of the microbial consortia. It has been reported that different microbial populations “talk” to each other by “quorum sensing” mechanism in a given environment by exchanging specific chemical signals (Thompson, 2015). Natural microbial consortia holds many specific properties like stability, functional diversity and perform complex functions. The natural consortia have generated an interest in manpulating synthetic consortia for the benefit of application (Minty, et al., 2013).

Fruits, vegetables, ornamentals, plantation crops and spices, medicinal and aromatic plants, tubers, are the major horticultural crops contributing to the economic growth in India. Even though the horticultural sector is growing fast, with India being the second largest producer of fruits and vegetables in the world, the productivity and quality have to be improved in order to meet the demand of increasing population. Under these circumstances, ecofriendly techniques needs to be adopted which could ensure food security and sustainable production. Indiscriminate application of agrochemicals and chemical fertilizers in horticultural crops and their residues could harm the human health, soil and environment. Horticultural crops need an ecofriendly technique, as most of the horticultural products are directly consumed. Hence, there should not be any residues of agrochemicals in food products. In the current context, use of ecofriendly, cost effective and organic-based inputs like biofertilizers can maintain health of the soil and good quality in horticultural crops. The application of biofertilizers offers an attractive and sustainable means of improving the quality of food products. Biofertilizers like Azotobacter, Azospirillum and AMF and PGPRs not only supplies the required nutrients to plants, but improves the fruit quality and quantity (Pathak et al., 2017).

The inoculum dosage and method of application determines the efficacy of biofertilizers. The quantity of biofertilizers to be applied depends on the type of crop, seed rate and soil edaphic factors. There are 3 methods for inoculation of biofertilizers

Seed treatment: One packet containing inoculant (200 g) is mixed with 200 ml of rice gruel or 40 % gum arabic solution to make a slurry (Fig. 23). Then the seeds required for an acre are mixed in the slurry in such a manner that they have a uniform coating of the inoculant over the seeds. Then, the inoculated seeds are shade dried for 30 minutes. The shade dried seeds should be sown within 24 hours. One packet of the inoculant (200 g) is normally sufficient to treat 10 kg of seeds.

Benefit of biofertilizers have been extensively studied for various agricultural and horticultural crops. However, the effectiveness of inoculated biofertilizers are influenced by several factors. If the use of biofertilizers have to be popularized in horticultural crops, the major factors affecting their performance particularly in relation to soil, plant, quality of the product and application methods have to be addressed. Factors affecting the performance of biofertilizers are:

4.1 Quality of the product

4.1.1 Production process

The high quality of biofertilizers are determined by the production process (Bashan et al., 2014). At the same time, the population density of mother culture and the quality of the final products are directly related (Stephens and Raskm, 2000). As the inoculum consists of a strain, the relationship among the microorganisms interaction in the rhizosphere have to be understood. In the case of more than one microorganisms, the efficiency depends on the various mode of action by various microorganisms which might overlap with antagonistic acitivities (Vassilev et al., 2001, 2006 b). The relationship of microorganisms with plants is not based on the taxonomic diversity, but their functional diversity (Maherali and Klironomos, 2007).

The efficiency of biofertilizers could be assured by enhanced shelf life of the inoculant and biological traits which are a challenge for developing a formulation (Bashan, et al., 2014). Hence, either single or consortia of microorganisms formulation with or without additives determines the efficacy of the biofertilizers. The formulation determines shelf-life during storage and transport, which enhances the persistence of the strain in soil and get maximum benefits to the host plants after inoculation (Manikandan et al., 2010; Schoebitz et al., 2012). Different kinds of carriers are used in the formulation process which affects the overall quality and efficacy of the biofertilizers (Bashan et al., 2014; Herrmann and Lesueur, 2013). Granular inoculants showed good results under stressed soil conditions (Clayton et al., 2004; Lupwayi et al., 2006). Encapsulation of the microbial strain into polymers allow diverse compositions and structures (Vassilev et al., 2005) but, have limitations for commercial production (Bashan, et al., 2014; John et al., 2011). Use of additives might improve the shelf life of the product (Bashan et al., 2014; Malusà et al., 2012; Herrmann and Lesueur, 2013) with introduction of specific substances to enhance the efficacy of biofertilizers.

Biofertilizers improve the plant growth due to changes in the microbial community of rhizosphere by production of several substances. Normally, biofertilizers promote plant growth either by facilitating nutrient absorption or by secretion of plant hormones. Biofertilizers also cause inhibitory effects by acting as biocontrol agents on various pathogens so that the growth of plant is enhanced. The mode of action could be either direct or indirect methods

2.1 Nitrogenous biofertilizers

These biofertilizers augment nitrogen to the plants through fixation of atmospheric nitrogen, either symbiotically or asymbiotically. The major nitrogenous biofertilizers are:

2.1.1 Rhizobium

The word Rhizobium is a Greek word where “rhiza” refers to root, and “bios” refers to life. Rhizobium was first named by Frank (1889) which means ‘root living’ in Latin (Fig. 3). Some species of Rhizobium were later moved to new genera based on phylogenetic analysis. It consists of 49 rhizobial species and 11 non-rhizobial species.

In modern agriculture, use of synthetic chemical fertilizers are essential in agriculture for sustainable yield but, these have resulted in environmental pollution. The use of synthetic fertilizer could result in serious human health issues and also affects the soil microflora, besides being costly due to high production cost. Further, excess use affects soil processes and result in nutrient imbalances in plants with increased susceptibility to pests and diseases and also affects the symbiotic associations. Under these circumstances, biofertilizers could be an alternative for integrated nutrient management system in horticulture. The entire crop production system depends on soil beneficial microflora and their activities, which makes them highly potential alternatives to synthetic chemical fertilizers.

Biofertilizers or microbial fertilizers are essential for eco-friendly and sustainable cultural practices. The term “biofertiliser” has been defined in several ways due to its unique relationships between the plant and rhizosphere microflora. Biofertilizers mainly include nitrogen fixing bacteria, phosphate solublizing and potash solubiling bacteria.