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A comprehensive study was carried out on in vitro multiplication and rooting using the medium enriched with different plant growth regulators and acclimatization of pineapple cv. ‘Smooth Cayenne’ using different soil growing substrates. The significantly highest shoot buds (Avg. 16.7) were obtained on the medium comprising 2.0 mg L−1 BA (6-Benzylaminopurine). Results showed that 1.0 mg L−1 IBA (Indole-3-butyric acid) increased the thickness and length of white adventitious roots and resulted in a significantly highest number of roots (Avg. 8) and root length (6.15 cm). Plantlets with healthy, multiple roots were transplanted in several soil combinations of river silt, bolhari (yellow sand), and peat moss. However, the significantly highest survival (100%) of plantlets in the greenhouse was obtained on the soil medium containing only peat moss. Furthermore, soil mixtures of bolhari and peat moss (1:1) and river silt alone exhibited 98.9% and 95.1% survivability of plantlets, which was also considered equally significant (at 5% probability level). The in vitro and ex vitro protocols optimized in the current study can be applied commercially for pineapple crop worldwide.

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