Makowczyńska J and Andrzejewska-Golec E, Acta Soc. Bo t. Pol., 72: 191-194 (2003)
Summary
Shoot-tip multiplication of the medicinal species-Plantago asiatica was carried on MS medium with IAA and BAP or kinetin. Best results in micropropagation were achieved by adding 0.1 mg/l IAA and 1 mg/l BAP. After 6 weeks shoots were transgerred to MS medium for rooting. The resulting plantlets were transferred after 8 weeks into pots and after a period of adaptation into the ground (field culture). The species Plantago asiatica was propagated in vitro by shoot-tip multiplication for the first time.
Objectives
Micropropagation of P. asiatica by use of shoot-tip culture.
Materials
Seeds of Plantago asiatica L. from Botanical Garden Faculty of Science, University of Tokyo
Explant
Seeds
Initial culture
Seeds of Plantago asiatica L. were surface sterilized by immersion in 2.0% sodium hypochlorite for 10 min, then rinsed 3 times in sterile water. Then, they were germinated in aseptic conditions on Murashige and Skoog medium supplemeted with 2.0 mg/l kinetin and 1 mg/l giberellic acid and 0.7% Difco Bacto Agar. The cultures were placed in the dark and after germination they were transferred into light. The source of the explants were 2- and 4-week seedlings, from which 1 cm fragments of the shoot-tip were taken.
Shoot multiplication
The shoot-tips were placed on the MS medium （0.7% agar）containing 1 or 0.1 mg/l indole-3-acetic acid (IAA) and diggerent concentration of kinetin or 6-benzylaminopurine (BAP). All culture were done in growing chamber at 25±2℃, in 80-90% humidity and light intensity 40 μmol/m2s. At the beginning of the second week of shoot-tip culture in vitro roots started to appear, and at the end of the second week the first buds appeared. The age of the seedling (2 or 4 weeks), from which explants were sampled, did not affect their ability to form shoots or roots. The best micropropagation (multiplication rate) results were obtained for MS medium enriched with 0.1 mg/l IAA and 1 mg/l BAP (5.2±0.6shoots/shoot-tip).
Rooting
The resulting shoots were transgerred after six weeks for rooting onto an agar MS medium without growth regulators. The plants did not require any additional media for elongation. After 8 weeks well-rooted plants were obtained, which were able to develop further in pot culture.
Acclimation
After a rooting period of two weeks the plantlets were washed in sterile water to remove agar and transferred into pots with a sterilized mixture of soil, sand and peat (3:4:4 v/v). The plants were watered every two days with sterile water. Glass covers were used to ensure high humidity around the plants at the initial steps of growth. After one week the time of contact with non-sterile air was increased gradually. Three weeks later the glass covers were removed and the plants were watered with normal tap water.
Planting
Following adaptation the plants were transferred into the ground in the Garden of Medicinal Plants, Department of Pharmacognosy, Medical University of Lodz.
Cultivation conditions
Traints of regenerants
Plant derived from in vitro clonal propagation by shoot-tip multiplication were normal in appearance. They did not significantly differ in morphology from the plants obtained in our climate during conventional culture in the soil. The plants showed vigor. They developed a normal root system. Plant cultures in vitro blossomed between the 3rd and 5th months of the pot culture. In room conditions, they gave fruit and normal fertile seeds. The plants were transplanted into the soil (field culture) and survived winter well.