CALLUS CULTURE ; Plant callus (plural calluses or calli) is a mass of undiff erentiated cells derived from plant tissue

(explants) for use in biological rese arch and biotechnology. In plant biology, callus cells are those cells that cove r a plant wound.[1] To induce callus formation, plant tissues are surface steril ized and then plated onto in vitro tissue culture medium. Plant hormones, such a s auxins, cytokinins, and gibberellins, are supplemented into the medium to init iate callus formation or somatic embryogenesis. Callus tissue initiation has bee n described for a number of plant taxonomic divisions: A callus cell culture is usually sustained on gel medium. Callus induction mediu m consists of agar and a mixture of macronutrients and micronutrients for the gi ven cell type. There are several types of basal salt mixtures used in plant tiss ue culture, but most notably modified Murashige and Skoog medium,[13] White's me dium,[14] and woody plant medium.[15] Vitamins are also provided to enhance grow th such as Gamborg B5 vitamins.[16] For plant cells, enrichment with nitrogen, p hosphorus, and potassium is especially important. USES : Callus cells are not necessarily genetically homogeneous because a callus is often made from structural tissue, not individual cells. Nevertheless, callu s cells are often considered similar enough for standard scientific analysis to be performed as if on a single subject. For example, an experiment may have half a callus undergo a treatment as the experimental group, while the other half un dergoes a similar but non-active treatment as the control group. Plant calli can differentiate into a whole plant, a process called regeneration, through addition of plant hormones in culture medium. This ability is known as totipotency. Regeneration of a whole plant from a single cell allows researchers to recover whole plants that have a copy of the transgene in every cell. Regene ration of a whole plant that has some genetically transformed cells and some unt ransformed cells is called a chimera. In general, chimeras are not useful for ge netic research or agricultural applications. Genes can be inserted into callus cells using biolistic bombardment, also known as a gene gun, or Agrobacterium tumefaciens. Cells that receive the gene of inte rest can then be recovered into whole plants using a combination of plant hormon es. The whole plants that are recovered can be used for experiment to determine gene function(s), or to enhance crop plant traits for modern agriculture. Callus tissue is of particular use in micropropagation where it can be used to g row genetically identical copies of plants with desirable characteristics.

SUSPENSION CULTURE : Suspension Culture The cultivation of cells suspended in the medium rather than adhering to a surfa ce. Suspension culture is common for microorganisms but less so for the culture of t he cells of most multicellular organisms. When referring to mammalian cells, sus pension culture is used for the maintenance of cell types, which do not adhere, including some types of blood cells, or in order to have cells express character istics, which are not seen in the adherent form. Sometimes it is necessary to prevent adhesion by choosing a hydrophobic surface, which does not encourage cell adhesion. The absence of serum components from th e medium will also help to prevent adhesion.

IMMOBILA~~ of cell culture

Immobilized Cell Culture

The properties of microcarrier beads used for cell immobilization are that they develop charged surfaces, either positive or negative charges, for example, DEAE sephadex (diethyl -aminoethyl type of cross-linked dextran) developed positive

charges, polysysteine negative charges, gelatin beads slightly positive or negat ive charges. The beads require surface coating for adhesion of sufficient number of cells, because energy is required for cell adhesion. In addition glass and c eramic materials are used as good carriers as they contain high surface energy. In different types of bioreactors different materials such as glass spheres are packed for cell attachment. The materials increase surface area for cultivation of cells en masse. This type of bioreactor is called packed bed bioreactor. For a bioreactor of 100 liter capacity about 50 kg of 3 mm diameter spheres are used . This gives a total surface area of about 20m2.

SINGLE TEA ; ; Single cell culture is very important for the fundamental and m utation studies and it has a wide industrial application. i.Single cell culture can be used successfully to obtain single cell clones. ii.Plants can be regenerated from the callus tissue derived from the single cell clones. iii.The occurrence of high degree of spontaneous variability in the cultured tis sue and their exploitation through single cell culture are very important in cro p improvement programmes. iv.Isolated single cells can be handled as a microbial system for the treatment of mutagens and for mutant selection. Many cell lines resistant to amino acid an alogues, antibiotics, herbicides, fungal toxins etc. have been selected by the s implest method. v.Single cell culture in large scale could become a valuable technique for indus trial production of such and important natural compound. vi.Biotransformation means the cellular conversion of an exogenously supplied su bstrate compound not available in the cell or the precursor of a particular cell ular compound to a new compound to a new compound or the known compounds in high er amounts.

ORGANOOOOO In animal development, organogenesis (organo-genesis, compound of the Greek word s ???a??? "that with which one works",[1] and ???es?? "origin, creation, generat ion"[2]) is the process by which the ectoderm, endoderm, and mesoderm develop in to the internal organs of the organism. Internal organs initiate development in humans within the 3rd to 8th weeks in utero. The germ layers in organogenesis di ffer by three processes: folds, splits, and condensation. Developing early durin g this stage in chordate animals are the neural tube and notochord. Vertebrate a nimals all differentiate from the gastrula the same way. Vertebrates develop a n eural crest that differentiates into many structures, including some bones, musc les, and components of the peripheral nervous system. The coelom of the body for ms from a split of the mesoderm along the somite axis.[citation needed] In plants, organogenesis can occur from totipotent callus cells. ] EMBRYO CULTURE ; ; ; ; Chapter 9: EMBRYO CULTURE Collecting and conservation of coconut genetic resources are important prioritie s for breeding programmes (IBPGR 1985). The coconut seednut is characterized by considerable weight and volume. The lack of dormancy renders its transport condi tions very difficult and costly, and poses phytosanitary problems. The use of in

vitro techniques can facilitate the transport and offer some phytosanitary guar antees (Assy Bah et al. 1987). In addition, the cryopreservation of zygotic embr yos can also play a major role in the conservation of coconut germplasm and in t he exchange of genetic resources (Bajaj 1984; Assy Bah and Engelmann 1992b). A summary of the 10 years of research work conducted by Institut de Development de Foret/Department du Plante Oleagineous (IDEFOR/DPO) in collaboration with ORS TOM and CIRAD, for collecting and culturing zygotic embryos is presented. Collecting fruits using in vitro techniques Fruit collecting through zygotic embryos involves sampling, disinfection and tra nsferring of embryos to the culture medium. The ideal age of the fruit is about 10-11 months. Embryos of over-ripe nuts may grow prematurely and disinfection be comes difficult at this stage. Sampling - Sampling consists of cutting the albumen cylinder with the embryo. Th is operation is done in non-sterile conditions. The nut without the husk is spli t open. The part with the embryo is placed on the table and a cylinder of albume n around the embryo is cut using a 2.0-cm cork borer. Gouging of the albumen aro und the embryo serves to protect this organ during storage and disinfection. Thi s piece of albumen is removed just before culturing. Disinfection - The cylinders are put in a 500-ml flask containing calcium hypoch lorite solution (8% active chlorine). During the imbibition, stir the disinfecta nt in the flask once or twice. After the disinfection, the cylinders can either be stored for subsequent transfer to the growth medium or the embryos can be cul tured right away. Embryo transfer in the field - In the laboratory, the transfer is done in a lami nar flow cabinet. In the field, the different operations are conducted inside a field inoculation box (fig. 37) equipped with an alcohol lamp for flame-disinfec tion of the scalpel. The scalpel is heated on the flame, then cooled off inside the jar containing the albumen cylinders with the calcium hypochlorite solution. Using the scalpel, the albumen core or cylinder surrounding the embryo is carefu lly cut to isolate the embryo which is then placed in a sterile petri dish, wash ed with distilled water, and placed in a sterilized 30-ml flask containing the p repared culture medium. The tubes of cultured embryos can either be put directly inside a culture room (at 25-27C), or conserved under a shade before the transfe r to the culture room. If a tissue/embryo culture laboratory is near the collecting site, the albumen c ylinders can be sampled in the field and brought to the laboratory. The cylinder s should then be put temporarily in a KCl solution, before the excision of the e mbryos. Storage of albumen cylinder After the disinfection, the albumen cylinder with the embryo is transferred into a 30-ml flask of storage solution containing 16 g/L KCl. The scalpel should be regularly disinfected by heating. The different operations are conducted over a flame to avoid any contamination by microorganisms. The cylinder with the embryo can be transferred this way to the laboratory for culturing. After the storage period, maximum 14 days, the cylinder is disinfected with calcium hypochlorite s olution for 20 minutes. After the excision, the embryo is then washed with steri le distilled water and put in the culture medium (Table 1). Direct culturing in the field gives a higher contamination rate (10%) than cultu ring after storage of the cylinders (5%). Both methods are acceptable since thei

r contamination rates vary only slightly from that of the control cultured in th e laboratory. The latter has a contamination rate of 3-8%. Table 1. Equipment and materials needed in collecting coconut embryos in the fie ld Material Storage of albumen cylinders with embryos Direct culturing in the field 4 liters of hypochlorite solution (8% chlorine) + + 2 scalpels + + 20 sterile petri dishes + Plastic film + 100 tubes containing 20 ml of culture medium + 100 pc - 30 ml flasks containing 15 ml sterile water + 100 pc - 30 ml flasks containing 15 ml KCl solution + The basic equipment is composed of: 1 sponge, 1 small camping gas burner, 1 hamm er, 2 pairs of forceps (30 cm), 2 cork borers, 1 soap, 4 - 500 ml bottles, 1 por table table and one box. For the culture of 100 embryos, the complementary equip ment detailed above is necessary. Culture medium and condition applied A very simple culture medium that ensures adequate development of the embryo in

vitro and enhances its subsequent growth in natural condition can be used. This medium is composed of the mineral solution of Murashige and Skoog (1962) enriche d with 41 mg/L of iron-EDTA, 100 mg/L of sodium ascorbate, 60 g/L of sucrose, 2 g/L of active charcoal and 7 g/L of agar at pH 5.5. The culture is placed in the dark at 27 1 C until the appearance of the plumule. It is then transferred to an other room and cultured under the light (35EMm-2S-1) with a 12-hr photoperiod. S ubculturing is done every month. Acclimatization One month before the transfer into the nursery, the in vitro plantlets are subcu ltured on a medium with a higher dose of sucrose (60, 90, 120 g/L). After 1 mont h in this medium, the plantlets are washed with tap water and transferred by bat ches to sand which was previously sterilized by autoclaving. The use of a plasti c cover during the first 2 weeks allows a saturated moisture condition. The plan tlets in the batches are watered regularly (3 times/week) to keep the sand moist for 1 month and then they are uncovered and exposed in the open air. A nutritiv e solution (Table 2) is added to the batch every 2 days. After 3 months, plantle ts are transferred from the sandy batches to polybags with compost. The nutritiv e solution is regularly applied to the plantlets to stabilize growth. Table 2. Nutritive solution used for acclimatization of plantlets (mg/L) KNO3 274 Ca(NO3)2 4H2O 1095 KH2PO4 137 MgSO4 7H2O 274 (NH4)2SO4 137 KCL 2.74 H3BO3 3 MnSO4 H2O 1.7 ZnSO4 7H2O 2.74 (NH4)62MO7O24 4H2O

2.74 H2SO4 0.137 CuSO4 5H2O 1.37 EDTA 26.1 FeSO4 7H2O 24.9