Shoyama Y and Nishioka I, Natural Medicine 48 (1), 92-94(1994)
Summary
Murashige and Skoog (MS) medium supplemented with 2.2 µM 6-benzylaminopurine (BAP) and 2.9 µM indole-3-acetic acid (IAA) promoted the growth and development of isolated meristem tips of Swertia japonica. Addition of 4.4 µM BAP and 11.4 µM IAA to the medium promoted axillary shoot proliferation and up to 18 shoots were produced from a single shoot, after 50 days. The shoots were rooted on MS medium with 11. 4 µM IAA or 2.5 µM indole-3-butyric acid (IBA), and the plantlets were transferred to vermiculite.
Objectives
Micropropagation of S.japonica by meristem tissue culture.
Materials
Vegetative shoots (10-20 mm long) were removed from field-grown plants, which had been grown from seeds collected in May in Fukuoka in Fukuoka prefecture.
Explant
Shoots were de-leafed and surface-disinfected by keeping them in 1% sodium hypochlorite
for 10 min with gentle agitation. They were then rinsed first with 70% ethanol for 30 s and then washed twice with sterile water. Meristem tips (0.5- 1.0 mm long, with 2 primordia) were dissected out of the shoot tips and axillary buds under a binocular microscope and immediately placed on a semisolid medium.
Initial culture
Shoot multiplication
For shoot formation the addition of 2.9 µM IAA and 2.2 µM BAP to MS medium was more effective than the addition of 5.7 µM IAA and 4.4 µM BAP. In order to obtain clonal plantlets, the shoots regenerated from shoot tips were tested for their ability to form multiple shoots on MS medium containing cytokinins (BAP, kinetin) singly or in combination with auxins (IAA,
NAA) or GA. The multiple shoot forming ratio was not high on the MS medium supplemented with BAP singly [4.4 µM (83%), 22.2 µM (60%), 44 µM (50%)].But on the MS medium supplemented with 11.1 µM BAP and 5.7 µM IAA, 13 shoots were produced per culture, and on the medium supplemented with 4.4 µM BAP and 11.4 µM IAA approximately 18 shoots were produced per culture. However, the presence of 22.2 µM BAP in the medium inhibited the multiple shoot formation regardless of IAA concentrations. The addition of 22.8 µM IAA to the medium clearly inhibited the shoot multiplication. An addition of NAA (2.7-10.7 µM) or 2,4- D(1.1 or 2.3 µM) to the medium containing BAP was formed to have no effect. The medium supplemented with 11.1 µM BAP and 1.4 µM or 2.9 µM GA favored shoot propagation to produce 27 shoots per culture, However, the rate of successful multiple shoot formation was 63% for 1.4 µM GA-medium and 87% for 2.9 µM GA-medium, and the effects of combined use of GA and BAP need further investigation . In view of these results, the medium containing 4.4 µM BAP and 11.4 µM IAA may be routinely used for shoot proliferation. However, when this medium was used, almost all shoots bloomed under a short photocycle, resulting in a decrease in the shoot propagation ratio.
Rooting
The medium containing 11.4 µM IAA or 2.5 µM IBA induced roots most efficiently. Callus formed at the base of shoots when NAA was added to the medium.
Acclimation
The regenerated plantlets were transferred to vermiculite and cultivated for 5 months.