Suitability of seed esterases for establishing distinctness, uniformity and stability of pearl millet genotypes

Plant morphological characters have been the univer- sally undisputed descriptors applied for testing distinct- ness, uniformity and stability (DUS) of crop va rieties. However, these descriptors, other than being limited in number, make the process time-consuming and also less reliable owing to its interaction with the environ- ment in which the variety is grown and subjectivity in decision-making. The potential of biochemical de- scriptors such as seed esterases has been well estab- lished for diversity analysis and varietal identification. However, their suitability as DUS test characters merits investigation. In the present study, seed esterases were used to examine distinctness of 45 pearl millet genotypes comprising 14 hybrids and their parental lines. Among 45 genotypes studied, 36 could be differentiated from each other and 11 were grouped into four categories at 2.29 × 10 -3 probability of identical match by chance. The utility of this marker to study uniformity and sta- bility was analysed using two genotypes, 841B and D- 23. Analysis of single-earhead seeds from 100 individ- ual plants, which are morphologically similar, revealed no variations within a variety in 841B and one variant in D-23, which is attributed to the residual heterozy- gosity. The possibility of esterase markers for testing the DUS of new pearl millet genotypes to give protec- tion under the Protection of Plant Varieties and Farmers' Rights Act, 2001 has been discussed. IN India, pearl millet is an important dry-land cereal crop grown over an area of about 9.39 m ha. More than 70 genotypes, of which about 60 are hybrids, have been re- leased in the last 35 years and their number will increase in future due to the ever-changing breeding objectives to meet the current and future demands of the producers and consumers. Apart from this, the Government of India under the obligation of the TRIPS agreement has r ecently passed the Protection of Plant Varieties and Farmers' Rights Act, 2001 (PPV&FR Act) to encourage public/private invest- ment in research and development of new plant varieties by giving protection to the new plant genotypes against unauthorized multiplication of seeds or propagating mate- rials for a specified period. The purpose of the Act will be achieved only when the new varieties are given proper protection under it. The new pearl millet genotypes will be protected under the PPV&FR Act after confirming the distinctness, uni- formity and stability (DUS) of new genotypes through DUS testing 1 . The DUS of the new genotype will be es- tablished by growing the new and existing varieties side by side and comparing them with respect to a set of morpho- logical characters throughout the plant growth period. This will be time-consuming and expensive, requiring large areas of land and skilled personnel often making subjective decisions. Added to this, many of the characters used are multigenic or quantitative and their expression is altered by environmental factors, which essentially require replication of observations. It also demands costly cold storage facilities to store, and seeds of all protected and extant varieties. Further, the number of characters may not be sufficient for discrimination of all the extant and new varieties. There are thus compelling reasons to find more rapid and cost-effective procedures to augment this approach. Biochemical markers assay variation in the polypep- tides of proteins and isozymes based on size and/or charge differences. Such differences remain unaffected across different seasons, locations and agronomic practices. Varie- tal identification and differentiation therefore become more reproducible and objective. The characteristics of a new variety based on electrophoretic patterns of proteins/ isozymes can therefore be compared with those commonly known in any part of the world for establishing its unique- ness. The working group on Biochemical and Molecular Techniques (BMT) of the International Union for the Pro- tection of New Varieties of Plants (UPOV) has in fact identified isozymes markers that could be used as com- plementary characteristics in maize and soybean DUS testing 2,3 , and protein markers in wheat and barley 4,5 . How- ever, it is necessary that prior to the use of such markers in DUS testing, it would be essential to evaluate the uni- formity and stability of the markers as used for distinct- ness. In pearl millet, when compared to other isozymes, esterases were found to have good resolution and wide variability in the entire Pennisetum gene pool 6 . Hence this marker was extensively used for diversity analysis,