Mustafa Abul Kalam Azad et al., Journal of Society of High Technology in Agriculture 16: 122-130 (2004)
Summary
Hypocotyl and internodal explants were collected from in vitro grown seedlings and in vitro proliferated shoots, respectively. Callus induction and subsequent plant regeneration were established from those explants. Friable callus with somatic embryo-like structure (ELS) developed from hypocotyl and internodal explants on MS medium supplemented with 0.89-4.44 µM BAP and 2.26-9.05 µM 2,4-D or 2.69-10.74 µM NAA. The maximal frequency of callus and ELS formation were obtained when the MS medium was amended with 0.89 µM BAP and 4.52 µM 2,4-D. After callus induction, they were cultured on the MS medium supplemented with 0.98-4.44 µM BAP and 0.54-2.69 µM NAA or 0.49-2.46 µM IBA for the regeneration of the shoots. Among different hormonal conditions, a combination of 2.22 µM BAP and 1.07 µM NAA produced the highest percentage of shoot differentiation from calli. In vitro grown shoots were rooted on the MS medium with either of 0.5-4.0 µM IBA, NAA or IAA. Regenerants were transferred to Kanuma soil and successfully established under ex vitro environment.
Objectives
The present paper reports on development of a new micropropagation protocol throgh callus of P.amurense.
Materials
Fruits were collected from a 50 year-old tree growing at the Medicinal Plant Garden of Kumamoto University. Seeds were stirred for 15 min in a household detergent solution (1 ml/l in tap water) and then riced for 20 min, then were surface-sterilized with 70% ethanol for 30 min and 3% sodium hypoclorite solution for 20 min. Then the seeds were washed at least 3 times using sterilized distilled water.
Explant
Sterilized seeds were germinated on MS medium supplemented with 2.22µM BAP
Initial culture
Hypocotyls were plated on MS medium supplemented with 0.89µM BAP + 4.52µM 2,4-D for inducing ELS. The culture were grown at 25±1oC under the illumination of a cool-white floresent tubular lamp with a light intensity of 50 µmol･m-2/s-1 for 16h photoperiod.
Shoot multiplication
The highest percentage of adventitous shoots were obtained the combination of 2.22 µM BAP + 1.07 µM NAA from ELS.
Rooting
2.0 µM IBA in MS medium
Acclimation
Plantlets were transffered to plastic pots (9cm diameter) containing Kannuma soil, covered with a transparent plastic cup to ensure high humidity during an acclimatization period of 20 days. The potted plants were irrigated with MS basal salt solutions (1/4 strength) devoid of sucrose and mio-inositol every 4 days for 3 weeks. Plastic pots were removed after 3 weeks in order to acclimatize plants yo lab. room conditions.
Planting
Acclimatized plants were transffered to larger pots (24cm diameter) and maintained in a greenhouse for 3 months, and then placed outdoors under the full sun in Spring.