Ketolactose Test For Agrobacterium Biotype 1

OBJECT: To identify specific Agrobacterium strains.

MATERIALS:

MEDIA: yeast-extract indicator medium (YI).

REAGENTS: Benedict's reagent

EQUIPMENT AND SUPPLIES: Inoculating loop, Agrobacterium biotype 1 culture, Agrobacterium biotype 2 culture, Escherichia coli culture.


PROCEDURE:

1. With the help of waterproof marker or wax pencil, draw lines on the bottom of a YI plate to divide it into three pie-shaped sections of equal size.

2. Use flame-sterilized inoculation loop, streak one third of the plate with Agrobacterium biotype 1 cells, one third with biotype 2 cells and the last third with E.coli cells.

3. Mention your name, the type of cell in each section of the plate and the date on the bottom of the plate.

4. Incubate the plate in an inverted position for two days at 28-29 'c.

5. Flood the YI plate with 4 ml of Benedict's reagent.

6. Incubate the plate at room temperature for one hour.

Following is a simple technique for isolating and precipitating high molecular nuclear DNA from plant cells. A mortar and pestle are used to break cells open mechanically and to disrupt the plasma membrane. The plasma membrane is further degraded by the use of an extraction buffer containing a detergent that dissolves membranes. Their combined action produces a homogenate containing cell wall and plasma membrane fragments as well as intact nuclei, chloroplasts and mitochondria. Low-speed centrifugation is used to separate the nuclei from the smaller organelles.

OBJECT: To demonstrate how DNA is isolated from plant tissues.

MATERIALS: REAGENTS: Cauliflower homogenization solution, sodium citrate solution, sodium chloride
solution, Absolute ethanol.

           EQUIPMENT AND SUPPLIES: Centrifuge, Graduated cylinder, Funnel, Cheese-cloth, Centrifuge bottles, Centrifuge tubes, Chilled mortar and pestle, Glass rod, Razor blade, Fresh cauliflower head.

PROCEDURE:

1. Using a razor blade, remove 25g of the outer 2-3 mm of the cauliflower surface.

2. Place the tissue in a mortar and add 25 ml of sodium citrate solution. Grind the mixture until it becomes a smooth slurry.

3. Add 150 ml of cauliflower homogenization solution to the mortar. Continue grinding the mixture an additional 30 min.

4. Filter the homogenate through a funnel lined with two layers of cheese-cloth. Squeeze the cloth to recover any additional liquid. Transfer the liquid two centrifuge bottles.

5. Add 2 volumes of absolute ethanol to each bottle while stirring continuously. Balance the bottles and centrifuge them for 5 min at 200 x g at 4'c.

6. Pour off the supernatant, saving the pellet containing nuclei at the botton of the bottles.

7. Add 1.5 volumes of sodium chloride solution and stir the mixture.

8. Transfer the mixture to a pair of clean centrifuge tubes. Centrifuge them at 10,000 x g for 25 min at 20'c.

9. Save the supernatant in a clean beaker.Resuspend the pellet in 15 ml of sodium chloride solution.   Centrifuge the mixture at 10,000 x g for 25 min at 20'c.

10.Add the supernatant to the beaker containing supernatant from the previous centrifugation. Slowly add an equal volume of absolute ethanol while slowly stirring with a glass rod.

11. Fibrous DNA strands will collect on the rod. continue stirring until DNA no longer adheres to the rod.

Booroola Gene

Gene mapping is essential as the foundation for genetic manipulation. Thus far, however, few specific genes of significance to animal agriculture have been identified, isolated or mapped. one example of a gene that is beginning to be understood, although it has not been specifically isolated, is he BOOROOLA GENE from Australian merino sheep. This gene boosts the incidence of twinning and triplets in sheep, giving an overall 20-40 percent increase in the number of lambs weaned. Introducing the booroola gene into other sheep and cattle could offer a fast, reliable way to increase the productivity of ewe and cow herds. Although the gene could be crossed into some breeds by sexual breeding, its introduction by molecular gene transfer would be faster and more important, it would allow the trait to be passed to a wider range of livestock. Mapping of the booroola gene is helping scientists determine more precisely how the gene operates and is also aiding in its cloning. Scientists may then attempt to transfer the gene to other valuable livestock species.

DNA Chip Technology

DNA chip technology, a marriage of the semi-conductor manufacturing industry and molecular genetics, will transform genetic analysis because it allows us to analyze tens of thousands of genes simultaneously on a single chip. The manufacturing process of microchips and DNA chips is similar, in principle, but instead of shining light through a series of masks to each circuits into silicon, automated DNA chip-makers use a series of masks to lay down an array of DNA fragments on a glass slide. DNA chip technology is being used for:

1. detect mutations in disease-causing genes.

2. monitor gene activity.

3. diagnose infectious diseases and identify the best antibiotic treatment.

4. identify genes important to crop productivity.

5. improve screening for microbes used in bioremediation.

DNA chip will be essential for converting the raw genetic data provided by the Human Genome Project into useful products.

Germplasm Conservation (Gene Bank)

Tissue culture methods offer the opportunity for in vitro collecting, rapid multiplication and distribution of important elite, or rare plants that are threatened with extinction. The two major in vitro  storage strategies are slow growth and cryo-preservation. Since the first results of seibert (1976), who was able to initiate shoots from carnation shoot apices frozen to -196'c. This technique is now successful for many of horticultural species. Dereuddre et al. (1991) have provided a very simple technology to freeze encapsulated meristems in dried alginate beads. It works for pear, strawberry, eucalyptus, potato. The international Potato center (CIP) in Lima, Peru has a large word potato collection. Germplasm of sweet potato and cassava is at the International Institute of Tropical Agriculture (IITA), Ibadan, Nigeria.

The movement of germplasm involves the risks of accidentially introducing plant quarantine pests along with the host plant material. To limit these risks, the plant material should be transferred from one country to another as in vitro cultures through a transit centre, where it should be indexed. For bananas, in the framework of INIBAP, the transit centre is the catholic university of Leuven in Belgium where a very large in vitro germplasm exists. The germplasm conservation (vitro preservation) of asexually propagated horticultural crops for storage under conditions closer to ambient temperature to suit the local conditions are important. An in vitro storage slow growth and cryopreservation have been standardized for many horticultural crops. Freezing of encapsulated meristems is possible in pears, strawberry and potato. For the movement of germplasm, in-vitro technique is very handy. The molecular taxonomy (like RAPD and RFLP) in genomic analysis and classification and development of molecular tools for disease indexing in in vitro propagated materials, in checking clonal fidelity, germplasm classification and identification of markers linked are important priority areas in this field. Molecular characterization of indigenous germplasm, application of DNA markers for identification of cultivars and molecular linkage maps for developing new varieties are important aspects of biotechnological studies.

Prime Numbers Less Than 1000

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97 101 103 017 109 113 127 131 137 139 149 151

157 163 167 173 179 181 191 193 197 199 211 223

227 229 233 239 241 251 257 263 269 271 277 281

283 293 307 311 313 317 331 337 347 349 353 359

367 373 379 383 389 397 401 409 419 421 431 433

439 443 449 457 461 463 467 479 487 491 499 503

509 521 523 541 547 557 563 569 571 577 587 593

599 601 607 613 617 619 631 641 643 647 653 659

661 673 677 683 691 701 709 719 727 733 739 743

751 757 761 769 773 787 797 809 811 821 823 827

829 839 853 857 859 863 877 881 883 887 907 911

919 929 937 941 947 953 967 971 977 983 991 997

...and they just keep going on and on forever...

Cartagena Protocol On Biosafety

Overview of the Biosafety Protocol:

The Biosafety Protocol seeks to protect biological diversity from the potential risks posed by living modified organisms resulting from modern biotechnology.

The Biosafety Protocol makes clear that products from new technologies must be based on the precautionary principle and allow developing nations to balance public health against economic benefits. It will for example let countries ban imports of a genetically modified organism if they feel there is not enough scientific evidence the product is safe and requires exporters to label shipments containing genetically altered commodities such as corn or cotton.


Objective of the Protocol:

In accordance with the precautionary approach, contained in Principle 15 of the Rio Declaration on Environment and Development, the objective of the Protocol is to contribute to ensuring an adequate level of protection in the field of the safe transfer, handling and use of 'living modified organisms resulting from modern biotechnology' that may have adverse effects on the conservation and sustainable use of biological diversity, taking also into account risks to human health, and specifically focusing on transboundary movements.

Living modified organisms (LMOs):

Living modified organisms (known as LMOs) resulting from modern biotechnology are broadly equivalent to genetically modified organisms. 'Modern biotechnology' is defined in the Protocol to mean the application of in vitro nucleic acid techniques, or fusion of cells beyond the taxonomic family, that overcome natural physiological reproductive or recombination barriers and are not techniques used in traditional breeding and selection.


The Protocol and the Precautionary Approach:



One of the outcomes of the United Nations Conference on Environment and Development (also known as the Earth Summit) held in Rio de Janeiro, Brazil, in June 1992, was the adoption of the Rio Declaration on Environment and Development, which contains 27 principles to underpin sustainable development. Commonly known as the precautionary principle, Principle 15 states that "In order to protect the environment, the precautionary approach shall be widely applied by States according to their capabilities. Where there are threats of serious or irreversible damage, lack of full scientific certainty shall not be used as a reason for postponing cost-effective measures to prevent environmental degradation."
Elements of the precautionary approach are reflected in a number of the provisions of the Protocol, such as:

• The preamble, reaffirming "the precautionary approach contained in Principle 15 of the Rio Declaration on environment and Development";
• Article 1, indicating that the objective of the Protocol is "in accordance with the precautionary approach contained in Principle 15 of the Rio Declaration on Environment and Development";
• Article 10.6 and 11.8, which states "Lack of scientific certainty due to insufficient relevant scientific information and knowledge regarding the extent of the potential adverse effects of an LMO on biodiversity, taking into account risks to human health, shall not prevent a Party of import from taking a decision, as appropriate, with regard to the import of the LMO in question, in order to avoid or minimize such potential adverse effects."; and
• Annex III on risk assessment, which notes that "Lack of scientific knowledge or scientific consensus should not necessarily be interpreted as indicating a particular level of risk, an absence of risk, or an acceptable risk."

What does the Protocol cover?

The Protocol applies to the transboundary movement, transit, handling and use of all living modified organisms that may have adverse effects on the conservation and sustainable use of biological diversity, taking also into account risks to human health.

Parties and non-Parties to the Protocol:

The governing body of the Protocol is called the Conference of the Parties to the Convention serving as the meeting of the Parties to the Protocol (also the COP-MOP). The main function of this body is to review the implementation of the Protocol and make decisions necessary to promote its effective operation. Decisions under the Protocol can only be taken by Parties to the Protocol. Parties to the Convention that are not Parties to the Protocol may only participate as observers in the proceedings of meetings of the COP-MOP.

The Protocol addresses the obligations of Parties in relation to the transboundary movements of LMOs to and from non-Parties to the Protocol. The transboundary movements between Parties and non-Parties must be carried out in a manner that is consistent with the objective of the Protocol. Parties are required to encourage non-Parties to adhere to the Protocol and to contribute information to the Biosafety Clearing-House.

Relationship between the Protocol and the WTO:

A number of agreements under the World Trade Organization (WTO), such as the Agreement on the Application of Sanitary and Phytosanitary Measures (SPS Agreement) and the Agreement on Technical Barriers to Trade (TBT Agreement), and the Agreement on Trade-Related Aspects of Intellectual Property Rights (TRIPs), contain provisions that are relevant to the Protocol. The Protocol states in its preamble that it:

• Recognizes that trade and environment agreements should be mutually supportive;
• Emphasizes that the Protocol is not interpreted as implying a change in the rights and obligations under any existing agreements; and
• Understands that the above recital is not intended to subordinate the Protocol to other international agreements. 

Main features of the Protocol:

The Protocol promotes biosafety by establishing rules and procedures for the safe transfer, handling, and use of LMOs, with specific focus on transboundary movements of LMOs. It features a set of procedures including one for LMOs that are to be intentionally introduced into the environment called the advance informed agreement procedure, and one for LMOs that are intended to be used directly as food or feed or for processing. Parties to the Protocol must ensure that LMOs are handled, packaged and transported under conditions of safety. Furthermore, the shipment of LMOs subject to transboundary movement must be accompanied by appropriate documentation specifying, among other things, identity of LMOs and contact point for further information. These procedures and requirements are designed to provide importing Parties with the necessary information needed for making informed decisions about whether or not to accept LMO imports and for handling them in a safe manner.



The Party of import makes its decisions in accordance with scientifically sound risk assessments. The Protocol sets out principles and methodologies on how to conduct a risk assessment. In case of insufficient relevant scientific information and knowledge, the Party of import may use precaution in making their decisions on import. Parties may also take into account, consistent with their international obligations, socio-economic considerations in reaching decisions on import of LMOs.



To facilitate its implementation, the Protocol establishes a Biosafety Clearing-House for Parties to exchange information, and contains a number of important provisions, including capacity-building, a financial mechanism, compliance procedures, and requirements for public awareness and participation.




Procedures for moving LMOs across borders:

Advance Informed Agreement:

The "Advance Informed Agreement" (AIA) procedure applies to the first intentional transboundary movement of LMOs for intentional introduction into the environment of the Party of import. It includes four components: notification by the Party of export or the exporter, acknowledgment of receipt of notification by the Party of import, the decision procedure, and opportunity for review of decisions. The purpose of this procedure is to ensure that importing countries have both the opportunity and the capacity to assess risks that may be associated with the LMO before agreeing to its import. The Party of import must indicate the reasons on which its decisions are based (unless consent is unconditional). A Party of import may, at any time, in light of new scientific information, review and change a decision. A Party of export or a notifier may also request the Party of import to review its decisions.

However, the Protocol's AIA procedure does not apply to certain categories of LMOs:

• LMOs in transit;
• LMOs destined for contained use;
• LMOs intended for direct use as food or feed or for processing.

LMOs intended for food or feed, or for processing:

LMOs intended for direct use as food or feed, or processing (LMOs-FFP) represent a large category of agricultural commodities. The Protocol, instead of using the AIA procedure, establishes a more simplified procedure for the transboundary movement of LMOs-FFP. Under this procedure, A Party must inform other Parties through the Biosafety Clearing-House, within 15 days, of its decision regarding domestic use of LMOs that may be subject to transboundary movement.

Decisions by the Party of import on whether or not to accept the import of LMOs-FFP are taken under its domestic regulatory framework that is consistent with the objective of the Protocol. A developing country Party or a Party with an economy in transition may, in the absence of a domestic regulatory framework, declare through the Biosafety Clearing-House that its decisions on the first import of LMOs-FFP will be taken in accordance with risk assessment as set out in the Protocol and time frame for decision-making.

Handling, Transport, Packaging and Identification:

The Protocol provides for practical requirements that are deemed to contribute to the safe movement of LMOs. Parties are required to take measures for the safe handling, packaging and transportation of LMOs that are subject to transboundary movement. The Protocol specifies requirements on identification by setting out what information must be provided in documentation that should accompany transboundary shipments of LMOs. It also leaves room for possible future development of standards for handling, packaging, transport and identification of LMOs by the meeting of the Parties to the Protocol.

Each Party is required to take measures ensuring that LMOs subject to intentional transboundary movement are accompanied by documentation identifying the LMOs and providing contact details of persons responsible for such movement. The details of these requirements vary according to the intended use of the LMOs, and, in the case of LMOs for food, feed or for processing, they should be further addressed by the governing body of the Protocol.

Biosafety Clearing-House:

The Protocol established a Biosafety Clearing-House (BCH), in order to facilitate the exchange of scientific, technical, environmental and legal information on, and experience with, living modified organisms; and to assist Parties to implement the Protocol. It was established in a phased manner, and the first meeting of the Parties approved the transition from the pilot phase to the fully operational phase, and adopted modalities for its operations.