Chitosan Nanoparticles Alleviate Salinity‐Induced Growth Inhibition in in Vitro Shoot Cultures of Tobacco ( Nicotiana tabacum L.)

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Abstract
Salinity stress is a major environmental constraint that limits plant growth and productivity by disrupting ionic balance, inducing osmotic stress, and promoting oxidative damage. Chitosan (Ch), a naturally derived biopolymer, enhances plant tolerance to abiotic stresses, but its effectiveness may be limited by poor solubility and reduced interaction with plant tissues. Chitosan nanoparticles (ChNP) may overcome these limitations through improved bioavailability and cellular interaction. This study evaluated the effectiveness of Ch and ChNP in alleviating salinity stress in in vitro shoot cultures of tobacco ( Nicotiana tabacum L.), a valuable model system. Shoot cultures maintained on shoot multiplication medium (Murashige and Skoog (MS) medium, 0.2 mg L −1 NAA (1‐Naphthaleneacetic acid) and 2.0 mg L −1 BAP (6‐Benzylaminopurine)) were cultured on medium containing 150 mM NaCl with or without 20 ppm Ch or ChNP for 30 days. Salinity stress significantly reduced shoot length, shoot number, and shoot fresh weight by 33.3%, 39.7%, and 40%, respectively, compared with non‐stressed shoots. Both Ch and ChNP alleviated these effects, with ChNP showing greater efficacy by increasing shoot length, shoot number, and shoot fresh weight by 3.6‐, 2.0‐, and 3.2‐fold, respectively, relative to untreated stressed shoots. ChNP also reduced hydrogen peroxide, superoxide radical, and malondialdehyde levels while enhancing catalase, superoxide dismutase, and peroxidase activities. Furthermore, ChNP increased chlorophyll a, chlorophyll b, total chlorophyll, and proline contents by 77.9%, 167.5%, 86.8% and 27%, respectively. These findings demonstrate that ChNP effectively enhanced salinity tolerance by strengthening physiological and biochemical stress‐response mechanisms in tobacco under in vitro conditions.

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