Tian D et al., Scientia Horticulturae 123: 385–389 (2010)
Summary
Shoot induction ability of explants of herbaceous peony was investigated in semisolid MS medium containing BA, TDZ and GA3. Callus was readily induced from stem without node and petiole explants within 2 days of culture but failed to generate shoots. Adventitious shoots were successfully produced from meristematic regions only: bud eyes on nodal stem sections, and junctions of petioles and petiolules. No shoots were induced from internode sections, petiole without junctions, or leaf sections. Nodal sections were the most efficient explants. There were up to 20 shoots in one explant generated within 20 days of culture. TDZ was more effective than BA to induce shoots. The 100% shoot induction rate was obtained in medium containing 0.1–3 mg/L of TDZ. However, higher concentrations of TDZ inhibited shoot elongation and only large leaf clusters were produced. Combinations of BA and TDZ failed to increase shoot induction rates but caused shoots shorter. The 2–60-min pretreatment of explants with 20 mg/L TDZ solution was very effective to induce adventitious shoots directly, but both shoot number and shoot length tended to decrease as treatment time increased. GA3 was beneficial for shoot and stem elongation.
Objectives
Themajor goal of this study is to find the best combination of young vegetative explants and PGR’s for shoot induction from P. lactiflora. This research would be an enabling step in propagation of true-to-type cultivars in commercial laboratories.
Materials
Three cultivars of herbaceous peony were investigated in this study: ‘Xi Shi Fen’ (‘XSF’), ‘Yang Fei Chu Yu’ (‘YFCY’) and ‘Fen Ling Hong Zhu’ (‘FLHZ’).
Explant
Five types of explants from elongated young shoots were evaluated including stem sections (5–8 mm long) with or without nodes, petioles (5 mm long) without junction, fork petioles (5–8 mm long) consisting of top main-petiole section and basal sections of three petiolules, and sections (16–25 mm2) of young leaves (unexpanded or just expanded).
Initial culture
Young elongated shoots were washed in tap water three times and cut into 2-4 cm long sections. Sections were soaked in 70% ethanol for 8-10s followed by 15 min of sterilization with 10% commercial Bleach, to which Tween 20 (1 drop per 100 mL) was added. Plant material wes rinsed three times for 5 min in autoclaved distilled water. After sterilization, explants were cut into shoeter sections (3-8 mm); inoculated on Petri dishes, and moved to 25 mm × 95 mm tubes or small plastic culture jars after initial culture. Culture vesseles were maintained under cool fluorescence light, 60 lx, with a 16 h light cycle at 25 ± 1℃. Experiment 1: the response of five explant types of 'XSF' to different plant growth regulators (PGRs) were evaluated on medium (1) a half strength Murashige and Skoog medium (1/2MS) + 1 mg L-1 benzylaminopurine (BA) + 0.1 mg L-1 gibberellic acid (GA3); (2) 1/2MS + 0.1 mg L-1 BA + 1 mg L-1 GA3; (3) 1/2MS + 1 mg L-1 BA + 3 mg L-1 thidiazuron (TDZ); and (4) 1/2MS + 1 mg L-1 BA + 3 mg L-1 TDZ + 1 mg L-1 GA3. Exreriment 2: four types of explants (except petiole with junction) from 'YFCY' and 'FLHZ' were used to evaluate their shoot induction ability in 1/2MS medium with the following concentrations of TDZ: (1) 0 mg L-1 as control; (2) 0.1 mg L-1; (3) 0.5 mg L-1; (4) 1 mg L-1; and (5) 3 mg L-1. After 12 days of culture, explants were transferred to fresh medium with 1/2 MS + 1 mg L-1 GA3. Experiment3: four explants (except leaf) of 'XSF' was pretreated with high concentration TDZ at 20 mg L-1 (90 µM) for (1) 2 min; (2) 15 min; or (3) 60 min at 25℃. The explants were then inoculated onto full strength PGR-free MS medium. After 15 days of culture, explants were transferred to the fresh medium with 1/2MS + 0.1 mg L-1 BA + 1 mg L-1 GA3 or medium with 1/2MS + 1 mg L-1 GA3.
Shoot multiplication
Culture conditions were indicated above. Young nodal stems were the most efficient explants for shoot induction with nearly 100% shoot induction rate obtained. Each node can produce several to over 20 shoots in 20 days. Addition of BA to medium resulted in shoot induction of peony. A higher concentration (1 mg L-1) of BA was more effective than a lower concentration (0.1 mg L-1). Application of TDZ to BA-containing medium did not significantly increase shooting rate but made shoots visually shorter and stronger. GA3 had a significant effect on shoot elongation, but shoots induced on medium with high GA3 concentrations were thin and too weak for root induction. TDZ showed very strong shoot induction ability and 100% shooting rate (the number of explants with induced shoots/the number of inoculated explants) was obtained on the nodal segments of 'YFCY' with treatments of TDZ ranging from 0.1 to 3 mg L-1. Shooting rates of 'FLHZ' were low. Shooting rate of 'FLHZ' also decreased at TDZ concentration larger than 0.5 mg L-1. Pretreatment with high concentration of TDZ inhibited stem elongation of induced shoots significantly and resulted in larger leaf clusters after transfer to shoot elongation medium (1/2MS + 0.1 mg L-1 BA + 1 mg L-1 GA3). TDZ shock treatment (a short-time treatment with very high concentration of TDZ compared with the normal usage) of 'XSF' resulted in same shoot induction rates for the same type of explants. Treatments with 2, 15 or 60 min of 20 mg L-1 TDZ soak resulted in 100% shooting rates for explants of both nodal stems and folk petioles. Although fork petioles also showed higher shoot-induction ability after shock treatment with high concentrations of TDZ, the induced shoot primordia were difficult to form normal shoots, and only three shoots at most could be produced on a segment. No differences in shoot number and shoot length of 'XSF' was observed between shock treatments although longer shock treatment of 20 mg L-1 TDZ tended to inhibit the number and elongation of adventitious shoots. Other experiments showed that normal elongated shoots could be obtained from nodal stem sections of 'XSF' even if explants were treated with 20 mg L-1 TDZ at 8℃ for 10 h.
Rooting
The roots were obtained on semisolid medium or by paper bridge method (liquid medium) with IBA in later experiments.