Barnase and bartar system

Male Sterility is a condition in which the males produce unviable pollens. Barnase-barstar is a type of nuclear male genetic sterility

1. By Alicia Tiny 20LS401001 Msc Biotechnology B arnase and Barstar Sytem

2. Table of Contents H ybrid seed production Advantages and Disadvantages M ale sterility in plants B arnase – bartsar system 04 02 03 01

3. Male sterility is characterized by the production of non- functional unviable pollen grains while female gametes function normally. Koelreuter in 1763 first reported male sterility in flowering plants. Male sterility results from the action of nuclear genes or cytoplasmic genes or both . Nuclear genetic male sterility is governed by single recessive gene ms . Barnase-barstar system is a type nuclear genetic male sterility Types of male sterility Genetic male sterility Cytoplasmic male sterility Transgene male sterility Male sterility occurs in nature through spontaneous mutations as well as can be induced artificially by chemical or physical mutagens. Ethidium bromide has been found effective in inducing cytoplasmic male sterility in some plants . Male sterility

4. Barnase – barstar system is a type of nuclear genetic male sterility. The first success in developing genetically engineered male sterility in crop plants was achieved by Mariani in 1990 by transforming tobacco and rapeseed plants with the barnase - barstar system. Barnase and barstar are two small bacterial proteins, one a ribonuclease and the other its specific inhibitor. Barnase , an extracellular enzyme secreted by Bacillus amyloliquefaciens strain H (IAM1521), is one of a family of small microbial ribonucleases. It was first reported and purified as an extracellular ribonuclease of Bacillus subtilis H by Nishimura and Nomura. Its small size (110 amino acids, 12,382 Da) and does not contains disulfide bonds. Barnase-barstar system

5. Barstar is an even smaller protein, with 89 amino acids (10,213 Da). It is produced intracellularly by B.amyloliquefaciens . It is a specific inhibitor of barnase ribonuclease. B arstar inhibits barnase by forming with it a tight, one-to-one, noncovalent complex, prohibiting access of substrates to the active site of the enzyme. The male fertility of barnase male sterile is restored by barstar

6. A Line - It refers to genetic male sterile line ( barnase ) which is used as female parent in hybrid seed production. It is a heterozygous line ( barnase /-). B Line- It is a maintainer line. It is crossed with A line to maintain the male sterility . R Line ( Barstar ): This line is used to store fertility in the F 1  hybrid. Male fertility is restored to a male-sterile line by introducing the fertility restorer gene in the ms line Components of Barnase-barstar system

7. Chimeric RNase and barnase gene is driven under the control of tapetum specific promoter TA 29 T he tissue specific TA 29 gene is highly regulated and specifically expressed in tapetum cells of anther. Expression of TA 29 and barnase fused gene consequently acts as cytotoxic and selectively destroys the tapetal cell layer by complete elimination of RNA molecule. B arstar was fused to tapetum specific TA 29 gene upstream frag­ment that comprises all regulatory segments targeted for tapetum specific expression.  Both barnase and barstar gene co-expressed specifically in the tapetal cells of anther led to the formation of barstar-barnase stable complex. In the Fi progeny ratio of fertile to sterile would be 2:1 ratio.  Mechanism

8. Male sterile plants ( barnase -bar/) are crossed with male fertile plants( R line). Pollens will move from male fertile plants to male sterile plants and pollinate the male sterile plants. The seeds from restorer lines must be harvested to compare male sterile plants. After fertilization process two genetically diverse seeds will be formed. Hybrid Seed Production

9. It is not dependent on natural source of male sterility.  Transgenic male sterility can be used for the production of hybrid seed in both seed propagated crops and vegetatively propagated species. Transgenic male sterility is stable and provides a complete and usable male sterility- restorer system. Transgenic male sterility generally does not have undesirable agronomic characters Congenial conditions like temperature, photoperiods is not a requirement in Barnase - barstar system. This method produces pure hybrid seeds. Advantages of Barnase - barstar system

10. Fifty percentage of the population is lost, so more amount of female parent seeds are needed for hybrid seed production. It is an expensive method. The method requires more area, labour, because the breeder has to maintain many populations. Since the seeds are transgene they face many ethical, social , environmental and biological issues. Disadvantages Barnase - barstar system

11. In nature the availability of male sterility and fertility restoration sources are limited. Therefore Barnase - barstar system provides us with a method to develop male sterile plants. The method can be used for production of pure hybrid seeds, that are stable and provide complete and usable male sterility restorer system. Conclusion

12. https://www.botanylibrary.com/plant-breeding-2/transgenic-male-sterility/transgenic-male-sterility-meaning-features-plant-breeding-botany/14026 Robert W. Hartley, Barnase and barter, National Institute of health , 1997, pg-51-57 Subhash Chand, Om Prakash Patidar, Vijay Kamal Meena , Barnase-barstar system : an inedible technique to produce hybrid seeds in self- pollinated crops, International Journal of Farm Sciences 2018, 109-113 Bibliographical References