Isolation of mesophyll protoplasts of nicotiana rustica and their regeneration into plants flowering in vitro

Gill, R.; Rashid, A.; Maheshwari, S.C.

Physiologia Plantarum 47(1): 7-10

1979


ISSN/ISBN: 0031-9317
DOI: 10.1111/j.1399-3054.1979.tb06502.x
Accession: 005770987

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Abstract
Protoplasts were isolated from leaves of in vitro grown plants of N. rustica L. by a 1 step enzymatic method. With 3% cellulase in 0.5 M mannitol at 25.degree. C and pH 4.6, within 10-12 h about 1/2 the total cells were transformed into protoplasts. The enzyme activity had 2 pH maxima, one at pH 3.5 and the other at pH 6.5, indicating the presence of isoenzymes. A time-course study at different temperatures indicated that at 30.degree. C protoplast liberation was quicker but resulted in subsequent bursting. The protoplasts were cultured in liquid and on agar-jelled medium of Ohyama and Nitsch supplemented with 2,4-D and benzylaminopurine, each at 1 mg/l and 14% sucrose. Regular divisions could be seen only on agar-jelled medium. Colonies on transfer to Murashige and Skoog (1962) medium, containing IAA and kinetin, differentiated into plantlets. These plants, within 2 months, flowered in vitro and set seed of which about 20% germinated.

Isolation of mesophyll protoplasts of nicotiana rustica and their regeneration into plants flowering in vitro