Determination of minimum sample size for testing proportion of non-Bt seeds under refuge-in-bag (RIB) for Bt cotton

Bt cotton has played a great role in bollworm management since its approval for commercial cultivation in India. To ensure the longevity of Bt technology, structured refuge was advocated wherein, the non-Bt seeds were provided as separate pack along with Bt seeds. Poor compliance of refuge planting is considered as a major factor for resistance development in Pink bollworm against Bt toxins and therefore, an alternate concept of refuge-in-bag (RIB) was recommended for Bt cotton. This strategy ensures automatic compliance of refuge, since a prescribed range (5–10%) of non-Bt seeds are blended with Bt seeds within each seed packet sold in the market. Since, Bt and non-Bt seeds are mixed and indistinguishable, assessment of Bt trait purity and ascertaining the proportion of refuge within each seed packet becomes critical for the success of RIB. A multi-phased study was conducted to determine the minimum seed sample size to be drawn and tested for RIB compliance in seed lots. Initially, the probable estimate to detect 5% non-Bt in blended seed lot was calculated theoretically which was later analyzed through visual observation of differentially colored seeds on various sample sizes. The minimum seed sample size thus arrived was validated through ELISA testing on a custom-built seed lot in which prescribed level of non-Bt seeds (5%) were blended with the Bt seeds. This was later revalidated using commercial Bt seed packets available in the open market. Based on the results, we propose a minimum sample size of 180 seeds which shall be tested individually for presence/absence of transgenes to determine proportion of non-Bt seeds in the Bt cotton seed packets. The need for strict care and critical cognizance by the seed producer organizations during the seed blending and packaging is specifically emphasized so as to ensure homogeneity of seed lot and prevent its possible misrepresentations. The proposed sample size may be used for ascertaining RIB compliance in the commercially available Bt cotton seed lots.