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The research work “Media improvement for in vitro culturing and hardening of tissue culture banana cv. Nendran” was carried out 1) to study the effect of nitrogen sources (ammonium and nitrate) on shoot and root regeneration in tissue cultured Nendran banana and 2) to standardize methods to enhance the growth and survival of plantlets during primary and secondary hardening stage. Experiments were conducted at the Regional Agricultural Research Station, Pilicode, Kasargod during 2017-2019. First experiment consisted of in vitro culture with five treatments- T1 – MS media (control) (NH4NO3 1650mg/l and KNO31900mg/l), T2 - Modified MS media (2850mg/l KNO3 + 825mg/l NH4NO3), T3 - Modified MS media (1900mg/l KNO3), T4 -Modified MS media (3800mg/l KNO3) and T5 - Modified MS media (1900mg/l KNO3 + 1361mg/l (NH4)2H2PO4). In the first experiment, normal MS media T1 (control) was observed to be superior one compared with other treatments (modified MS media). It recorded maximum number of multiple shoots per explant after 4th, 5th and 6th subculture and highest shoot length, number of leaves, number of roots, root length and maximum fresh and dry weight of shoot after the rooting stage. Second experiment on primary hardening was carried out with nine treatments such as T1- sand (control), T2 - coco peat, T3 - vermicompost, T4 - sand + coco peat (1:1), T5 - sand + vermicompost (1:1), T6 - coco peat + vermicompost (1:1), T7 - sand + coco peat + vermicompost (1:1:1), T8 - sand + coco peat + vermicompost (1:2:1) and T9 - sand + coco peat + vermicompost (1:1:2) . Primary hardening study was carried out with different hardening media. Plant growth and survival were effectively influenced by hardening media. T1, T2, T6, T7 and T9 showed 100 percent survival but T3 (47.33 %) and T5 (18.00 %) had lower survival rates. With respect to various growth parameters T6 recorded highest 117 plant height, pseudostem girth, number of leaves, leaf length, leaf width, leaf area, root length and highest shoot fresh and dry weight and T2 had recorded maximum number primary and secondary roots and root weight which was on par with T6. Third experiment on biotization was done with nine treatments consisting of T1 - Arbuscular mycorrhizal fungi, T2 - Azotobacter, T3 - Azospirillum, T4 - PSB (Pseudomonas fluorescens), T5 - AMF + Azotobacter, T6 - AMF + Azospirillum T7 - PSB + Azotobacter, T8 - PSB + Azospirillum and T9 - Control (Potting mixture only). Investigation on biotization of plants, revealed significant effects on plant growth and development through enhanced nutrients uptake. All treatments showed 100 % survival. Among treatments, T8 recorded highest plant height at 30 and 60 DAT, maximum pseudostem girth was found in T1 and T6 at 30 and 60 DAT. With respect all leaf characters T7 was significantly superior which recorded maximum leaf length, leaf width and leaf area at both stages. T5 recorded more number primary and secondary roots, primary root length and root weight and shoot fresh and dry weight compared to control. Chlorophyll content was maximum in T7 and T4 at 30 and 60 DAT respectively. In the nutrient status of plants, T1 recorded maximum content of P and K and N content was more in T3. Plants of T5 recorded highest uptake of Zn, Fe and Mn. Cu and B content were more in T7 and T1 respectively. For successful in vitro culture of banana cv. Nendran, both ammoniacal nitrogen and nitrate nitrogen are required as per the results obtained. For successful transplantation of tissue cultured plants, acclimatization is an integral process in tissue culture technique, which involves primary and secondary hardening. For primary hardening, T6 - mixture of coco peat and vermicompost (1:1) was the best for successful survival and growth of plantlets. Biotization was found to be an important technique in secondary hardening stage, which enhanced the growth and development of plants through increased uptake of nutrients from media. Among the treatments, T5 - AMF and Azotobacter combination was the best for secondary hardening.