An effective and consistent in vitro propagation technique for Boerhaavia diffusa L. was developed using different types of explants and media compositions. Shoot apex and nodal explants of field grown plants were aseptically cultured on Murashige and Skoog (MS) medium supplemented with different concentrations and combinations of auxins (IAA and NAA) and cytokinins (BAP and Kn). The maximum number of multiple shoot buds (6.65 ± 0.09) obtained from nodal explants on MS medium containing 2.0 mg/l BAP + 0.5 mg/l IAA. Multiple shoot buds underwent rapid elongation (4.24 ± 0.06 cm) on MS media fortified with 3.0 mg/l BAP + 0.5 mg/l NAA. Half strength MS media supplemented with 1.0 mg/l IBA + 0.5 mg/l IAA was better for induction and proliferation (6.42 ± 0.07) of roots and 75% of plantlets were successfully acclimatized to ex vitro condition, exhibiting a normal development. Somatic chromosome number of in vivo and in vitro grown plants were confirmed to be 2n = 26. Chromosome length ranged from 1.40 to 2.43 μm in the mother plants and 1.34 to 2.31 μm for in vitro grown plants. The total form percent (TF%) of mother and in vitro grown plants was 42.91% and 41.16%, respectively. The karyotype formula of in vivo grown plants was 2n = 26 = 4Lsm + 6Msm + 2Mm + 14Sm, whereas that of the in vitro grown plants was 2n = 26 = 8Lsm + 4Msm + 2Mm + 12Sm. The frequency of the chromosome having arm more than 2:1 was 0.08 for in vivo grown plants and 0.15 for in vitro raised plants. Thus, according to Stebbins classification (1971) the karyotype of both plants falls into 2A symmetrical type.
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