Hosoki T. et al., Plant Cell Reports 8: 243-246 (1989)
Summary
A procedure for the clonal propagation of
Paeonia lactiflora Pall. cvs. Takinoyosooi and Sarah Bernhardt through shoot tip culture is described. Half strength Murashige and Shoog (1962) medium supplemented with 0.5 mg/l 6-benzylaminopurine plus 1 mg/l gibberellic acid promoted formation and growth of axillary buds. Continuous shoot multiplication was achieved by vertically splitting the shoot axis and subsequent division of elongated axillary shoots every 36 days. High frequency (57- 100%) of rooting was obtained on paper- bridge liquid medium supplemented with 1 mg/l indole-3-butyric acid. Half of the rooted plantlet
were established on porous soil. Thus, 700 and 300 plants of cv. Takinoyosooi and Sarah Bernhardt could be theoretically obtained from a single bud in one year.
Objectives
A rapid multiplication method for herbaceous peony
Materials
Herbaceous peony cultivars Takinoyosooi and Sarah Bernhardt
Explant
early spring buds on the rhizomes
Initial culture
A few scale leaves were removed and the
buds were sterilized in a diluted solution of sodium hypochlorite (active chlorine 0.7%) and rinsed with a sterile water two times. Main shoot tips and axillary shoot tips (2-3 mm long) were excised after removal of small scale leaves under a dissecting microscope. The explants were placed on agar (0.8%) solidified medium (15 ml) in test tubes (2 cm ø, 15 cm long). The nutrient medium consisted of half strength Murashige and Skoog (MS) medium and Fe-EDTA, Ringe and Nitsch minor elements and vitamins and 3% sucrose at pH 5.6. BAP (0.5 mg/l) plus GA (1 mg/l) were supplemented as growth regulators for the initial culture. All the test tubes were placed at 20 ºC under 16 hr illumination from 52 µEm-2 S-1 cool white fluorescent lamps. Excised shoot tips elongated and formed axillary buds at most nodal positions.
Shoot multiplication
BAP (0.5 mg/l) plus GA (1 mg/l) or only 0.5 mg/l BAP were tested for the subculture. The elongated shoot axes with 3 to 5 axillary buds were then longitudinally split into halves left. Some of the long shoots were further sectioned transversely into two so that four-stem sections were obtained. Here, each section should contain at least one axillary bud. Otherwise, no more mutiplication occurred. The average numbers of split sections obtained in cv. Takinoyosooi was 3.8 shoots, and in cv. Sarah Bernhardt 3.3 shoots. From the next division, 0.5 mg/l BAP single treatment as well as GA and BAP combination treatment was used. Within 35 days after culture, each section developed into shoots (2-3 cm long) and again produced secondary axillary buds at the nodes. Some of the axillary buds elongated and looked like branching shoots in BAP and GA combination treatment in both cultivars. From this time on, multiplication was achieved only by division of axillary shoots. About 33 days after culture, axillary buds elongated to 2-4 cm long with new axillary buds in both cultivars. Again, shoot division was achieved. This continued division was possible at 36
day intervals at least upto 5 times without losing the capacity of axillary bud formation in both cultivars. The number of divided shoots and the number of elongated axillary shoots were somewhat greater in BAP and GA combination medium than in BAP single treatment. In BAP treated shoots, many minute axillary buds were observed around the nodal area at the lower stem position. These bud aggregates were morphologically similar to those on underground rhizome or crown grown in the field. If this division was repeated in BAP plus GA medium at 36 day intervals for one year including the time for
initial shoot elongation (2-3 months), more than 5000 shoots could be theoretically obtained from a single bud. This number is similar to that in multiplication of Japanese horseradish for which a similar shoot split method was developed. This multiplication method is much easier and more practical, compared with that of somatic embryogenesis from callus of seed embryo.
Rooting
For rooting, shoot (2-3 cm long) was transferred to 0.1 mg/l or 1 mg/l of NAA and IBA supplemented agar solidified medium. The 0.1 mg/l of both auxins produced roots only in 0-40% of the shoots in both cultivars whereas 1 mg/l of them produced roots in 40-80% of the shoots but it took over 3 months (data not shown). Therefore, paper-bridge method on liquid culture was adopted to expose stem-base to more oxygen. One mg/l of NAA or IBA was again used. Within 2 months. 100% IBA treated shoots produced roots in cv. Takinoyosooi and 57% in cv. Sarah Bernhardt. NAA produced roots in 40% and 70% of the plants, respectively, but it also produced callus at the stem-base, which caused tissue decay by fungus attack after ex vitro culture. IBA treated plantlets with roots were transferred to a porous soil medium under plastic cover at 18- 20ºC under 16 hr
illumination from 52 µEm-2s-1 cool white fluorescent lamps. More than half of shoots were established. The remaining plants turned yellow. Such an incomplete establishment may be due to lift-up of the shoots above paper- bridge as the roots grew down during culture. Therefore, nutrient absorption became insufficient. Such a problem is being resolved by placing shreds of gauze about 4mm thick on the paper- bridge where roots penetrate into medium and good aeration is maintained. Although more efficient acclimatization after ex vitro culture must be established, we have demonstrated a
clonal propagation system of commercial cultivars of Paeonia lactiflora by a shoot splitting method and subsequent division of axillary shoots.