Plant biotechnology

biotechnology

obligatory courses

Classroom

The exercise program is divided into three autonomous modules operated by three Departments at the Faculty of Biology. The first module presents algae transformation techniques on the example of C. merolae and C. subellipsoidea. Students will learn techniques for mutant selection and verification of mutagenesis as well as methods used for long-time storage of algal cells. In the second part students will learn about both genetic engineering techniques and modern biology –based methods used for upgrading growth parameters of crops. Students will compare phenotypic effects of mutations introduced using different methods (mutagenesis, gene edition), as well as learn how chemical growth regulators can be developed.

In the third module, students generate transgenic tobacco using A. tumefaciens. They learn the procedures used in the selection of transgenic lines. They also perform molecular analysis of obtained transgenic plants and phenotypic analysis of lines owned by department.

The practical part will be preceded by a theoretical introduction.

*) Exercise program in first module

Theoretical introduction:

The use of algae in biotechnology - the advantages and disadvantages of using algae in comparison to higher plants and prokaryotes in the production of food, biofuels, pharmaceuticals, cosmetics, valuable chemicals, environmental protection. Advantages and disadvantages of nuclear versus plastid (chloroplast) transformation. Algae transformation methods: electrotransformation, osmotic shock, biolistic bombardment (gene shotgun). Selection markers and methods of selecting obtained algae mutant lines, methods of verifying the introduced mutation / transgene expression. Methods for long-time storage of algal cells and methods of DNA isolation from algal cells.

Practical part:

Classes 1. Transformation of the red algae C. merolae by osmotic shock using polyethylene glycol.

Classes 2. Transformation of C. subellipsoidea by electroporation (electrotransformation). The influence of voltage and electrode distance parameters to determine their practical impact on transformation efficiency. Plating transformed cells cultures on solid substrates.

Classes 3. Transformation using a gene gun of the green algae C. subellipsoidea. The influence of pressure and distance parameters on transformation efficiency. Plating transformed cultures on solid substrate.

Classes 4. Methods of DNA isolation from algae cells and methods of long-term storage of algae cells. Counting colonies. Summury

*) Exercise program in module two:

-Theoretical introduction:

Selection of new crop varieties by marker-assisted breeding, analyses of polymorphisms underlying natural variation of important traits for agriculture (GWAS, genome-wide association studies). Generation of transgenic plants and editing of plant genomes using CRISPR/Cas9 system. Application of transgenic plants and CRISPR/Cas9-modified crops. Chemical biology in plant biotechnology – classic growth regulators (e.g. phytohormones and their inhibitors) and development of new active compounds. Small molecule library screening and development of targeted protein inhibitors.

Practical part:

Classes 1. Students will compare Arabidopsis insertion mutant line with the line generated using CRISPR/Cas9. Students will analyse these lines by means of genotyping T-DNA transgene and CRISPR deletion, analysis of transcript levels of selected genes, and assessing protein levels by western blot.

Classes 2. Growth regulators analysis. Classical regulators: gibberellins and gibberellin biosynthesis inhibitors. Phenotypic analysis of gibberellin-deficient Arabidopsis mutants, as well as mutants with hyperactive gibberellin signalling pathway. Demonstration of phenotypic screen developed to find new compounds capable of regulating plant growth.

*) Exercise program in third module

Theoretical introduction:

Transgenic higher plants applications in biotechnology and science. Plasmids used in higher plants transformation,techniques used in obtaining desired genetic constructs. Methods used to generate transgenic higher plants, detailed presentation of Agrobacterium-mediated techniques. Rules to obtain independent transgenic lines and their analysis.

Practical part:

Classes 1:(a) Construction of plasmid used in plant transformation (Gateway system using DNA recombination). (b) E.coli transformation with product of recombination reaction. (C)

Agrobacterium-mediated transformation of tobacco – co-cultivation with bacteria.

Classes 2: (a) Colony PCR(continuation of classes 1a and 1b).(b) Phenotype analysis of selected transgenic lines owned by department - start of culture under conditions differentiating phenotype.

Classes 3:(a) Agrobacterium-mediated transformation of tobacco - transfer of explants to fresh medium (continuation of classes1c). (b) Analysis of colony PCR products (continuation of classes 2a).(c) Comparison of phenotype of transgenic and non-transgenic plants - analysis of differences (continuation of classes 2b). (d) RNA isolation from plants subjected to phenotype analysis (cDNA synthesis will be carried out by tutors).

Classes 4: (a) Analysis of tissue-specific promoter activity-GUS reporter gene assay - an example of transgenic plants used in science.(b) Differences in endogenous genes expression between transgenic and non-transgenic plants - real-time PCR (continuation of classes3d).

Classes 5:(a) Agrobacterium-mediated transformation of tobacco -transfer of explants/regenerants to fresh medium with collection of tissue for molecular analysis (continuation of classes3a). (b) Determination of transgene presence in the obtained regenerants– isolation of DNA from the collected fragments of regenerants (PCR will be carried out by the tutors). (c) Transgene segregation test -sowing seeds of various transgenic lines on selective medium.

Classes 6: Determination of transgene presence in the obtained regenerants – PCR product analysis by agarose gel electrophoresis (continuation of classes 5b). (b) Results of the transgene segregation test (continuation of exercise 5c). (c) Endogenous genes expression in transgenic and non-transgenic plants – analysis of the results (continuation of classes4b). (d) Analysis of tissue-specific promoter activity - GUS reporter gene assay – analysis of the results (continuation of classes4a).

Books:

• Biotechnologia roślin, PWN, redakcja S. Malepszego, 2014.

• Plant Transformation Technologies. Neal S.C., Touraev A., Citovsky V., Tzfira T., Wiley-Blackwell, 2010.

• Plant Biotechnology and Genetics: Principles, Techniques, and Applications 2nd Edition. Stewart C. Neal Jr., Wiley-Blackwell 2016.

• Recombinant Gene Expression: Reviews and Protocols. Balbás P., Lorence A., Springer, 2012.

• Genetic Transformation of Plants. Jackson J. F., Linskens H.F., Springer, 2003

• Advances in New Technology for Targeted Modification of Plant Genomes. Zhang F., Puchta H., Thomson J. G., Springer, 2015.

• Transgenic Plants: Methods and Protocols. Peña L., Springer, 2005.

• Algae Biotechnology: Products and Processes. Bux F., Chisti Y. Springer, 2016

• Molecular Cloning: A Laboratory Manual (Fourth Edition). Green and Sambrook, Cold Spring Harbor Laboratory Press, 2012.

Once you have mastered the material covered in lecture and exercises the student:

Demonstrates knowledge of basic techniques and tools in the study of natural phenomena and understands the importance of experimental work in biotechnology. Student can describe the importance of molecular analysis in research in the field of plant biotechnology (K_W04)

Has knowledge of the use of technical and technological aspects of biotechnology (K_W05)

Knows the basic laboratory, measurement and imaging techniques used in chemical, microbiological, genetic and molecular biology researches and used in plant biotechnology (K_W14)

Has elementary knowledge of plant biotechnology and understands the relationships between various disciplines (K_W01).

Has basic knowledge in the field of intellectual property protection (K_W10).

Uses basic techniques appropriate for plant biotechnology (K_U01).

Carries out simple research tasks or expertise under the supervision of a supervisor (K_U04).

Uses basic techniques and research tools of experimental biology and is able to explain the principles of their operation (K_U01).

Demonstrates the ability to correctly infer based on data from various sources (K_U06).

Social competence.

Student:

Demonstrates the ability to work effectively in a team (K_K04).

Demonstrates understanding of biological phenomena and processes in nature (K_K01).

Demonstrates responsibility for his own work and entrusted equipment; shows respect for own and other work (K_K03).

Understands the need to provide information on new developments in biotechnology to the public and is able to convey this information in an understandable way (K_K06).

The criteria of evaluation of the lab:

(i) participated in at least 85 percent of classes;

(ii) worked in a way that positively assess the knowledge, skills and social competence, which in the course of the activities obtained (described in the syllabus as a subject learning outcomes).

Detailed criteria of evaluation of the lab:

(i) exercise activity ;

(ii) Preparing a presentation on a selected/random topic in groups of 2-3 people and presenting it to the lecturers and students taking part in the exercises. Obtaining a minimum of 51% of the total number of points for the presentation.

The condition for taking the exam is a positive assessment of the exercises

The conditions for passing the exam are:

(i) taking the written examination;

(ii) obtaining at least 61% of the total number of points.

The grade from the exam is also the grade from the exercises.

Not applicable