In vitro mutagenesis in banana (Musa paradisiaca L.) cv. Grand Naine for its improvement against salinity
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Date
2021-01
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Department of Horticulture (Fruit Science), BAU, Sabour
Abstract
The present investigation entitled “In vitro mutagenesis in banana (Musa paradisiaca L.) cv. Grand Naine for its improvement against salinity” was carried out in the Division of Horticulture (Fruits and Fruit Technology), BAU, Sabour during 2017-2020 with the aim to develop salinity tolerant mutant in banana cv. Grand Naine using gamma irradiation and chemical mutagens. Six different experiments were laid out under in vitro and in vivo condition. The irradiation experiment was carried out in 5×5 factorial complete randomized design consisting days of subculture (DAS) (4 DAS, 7 DAS, 10 DAS, 13 DAS and 16 DAS) and irradiation doses (0 Gy, 10 Gy, 20 Gy, 30 Gy and 40 Gy) replicated five times. The chemical mutagen experiment was conceded in 5×7 factorial completely randomized design comprising days of subculture (4 DAS, 7 DAS, 10 DAS, 13 DAS, 16 DAS) and chemical mutagen doses (Control @ 0 mM, EMS @ 3 mM, EMS @ 5mM, DES @ 3 mM, DES @ 3 mM, NaN3 @ 1mM, NaN3 @ 2 mM) replicated three times. The observations with regards morphological characters were taken up to M1V4 generation (up to 120 days from M1V1 to M1V4 at 30 days interval). Under in vivo condition, ten mutant lines (SM1, SM2, SM3, SM4, SM5, SM6, SM7, SM8, SM9, SM10, SM11 and SM12) were used for evaluating salinity tolerance with the application of NaCl (0, 100 and 250 mM) under factorial completely randomized design. Results revealed that 39.5 Gy as the LD50 dose among gamma irradiated shoot tips. 10 Gy treated over 16 DAS explants was found most effective in terms of number of new shoots (8.86), survival (80-90 %), shoot length (5.54 cm), root length (5.44 cm). The LD50 of EMS, DES and NaN3 for shoot tip culture of banana was recorded at 5.5, 5.1 and 2.1 mM respectively. EMS and DES retained dormant condition until M1V2. EMS was found most effective in new shoot development. Morphological characters like shoot length (4.40 cm) and root length (5.30 cm) found maximum in NaN3 @ 1 mM and DES @ 3 mM respectively. All the morphological characters increased with increase in DAS. Explant of 16 DAS exposed with EMS @ 3mM improved survival rate in the range of 60-90 % and rooting by 70-75 %. EMS @ 3mM and EMS @ 5 mM performed best in the development of visible mutant when subculture was done at 10 and 13 DAS.
As many as seven microsatellite markers were selected for identification of salinity, three have been found most suitable for screening of mutant tolerant to salinity. Ma2/3 showed no amplification whereas Ma1/3 and STMS 1fp/1rp showed amplification at 160 bp and 250 bp were selected for screening of salinity. 10 Gy when exposed over 13 DAS explants distinguished positive for markers whereas higher doses i.e. 20, 30 and 40 Gy were found screened positive at 4 DAS and 7 DAS. EMS @ 3 mM, EMS @ 5 mM and DES @ 5 mM at the 4, 7 and 10 DAS was found positive for salinity markers while NaN3 generated explants didn’t identified salinity tolerant line using molecular markers. There was no evidence of salinity positive lines in all the treatments at above13 DAS.
Out of 1100 putative mutant line derived, 134 lines confirmed through molecular markers. Out of molecularly confirmed line 22 mutant lines were found effective based on survival, plant height and root length for further screening under in vitro salinity condition. The 12 most effectively performed lines were again screened for in vivo salinity experiments. Under in vivo condition among irradiated mutant lines SM1 and SM2 obtained from 10 Gy and 20 Gy and chemical mutagen treated lines SM7 and SM8 obtained from EMS @ 3 mM, EMS @ 5 mM respectively were found most effective treatments among mutant lines with respect to per cent change in plant height, leaf area, necrosis, number of leaves, MSI, RWC and proline. SM11 and SM12 obtained from treatment of NaN3 @ 1 mM and NaN3 @ 2 mM performed higher in terms of per cent change in salinity damage, chlorophyll, MSI and proline. SM9 (DES @ 3 mM) showed the improved per cent change in no. of leaves, proline and chlorophyll then other mutant lines.
The entire set of experiment suggested that under in vitro condition lower gamma irradiation doses (10 Gy, 20 Gy) exposed over 13 DAS explants was found to be most effective to generate salinity tolerant mutant lines. Among chemical mutagens, EMS provided maximum number of visible mutant and salinity tolerant mutant line from 4 to 10 DAS. Under in vivo condition, 10 Gy and 20 Gy derived mutant lines were most effective to improve plant height, leaf area, necrosis, no. of leaves, MSI, RWC and proline under salinity condition. However, EMS @ 3 mM was found effective in improving plant height, leaf area, no. of leaves, MSI and proline. Also, DES @ 3 mM improved RWC and NaN3 @ 1 mM enhanced chlorophyll content of the mutant lines against salinity.