Wu HJ et al., New Zealand Journal of Crop and Horicultural Science 39: 271-278 (2011)
The underground buds of herbaceous peony (Paeonia lactiflora Pall.) ‘Zhong Sheng Fen’ were used to investigate the effects of indole-3-acetic acid (IAA) on culture initiation, benzyladenine (BA) on axillary shoot induction, and three auxins (IAA, indole-3-butyric acid [IBA] and 1-naphthyleneacetic acid [NAA]) on axillary shoot proliferation. In addition, methods to rejuvenate hyperhydric microshoots were established. Our results showed that the best initiation medium for ‘Zhong Sheng Fen’ was half-strength Murashige and Skoog (MS) medium supplemented with double the concentration of Ca2+ (880 mg L-1), 1 mg L-1 BA, 0.5 mg L-1
gibberellic acid (GA3) and 0.1 mg L-1 IAA. Axillary shoots were successfully induced by
2.0 mg L-1 BA. Several methods allowed the successful rejuvenation of hyperhydric
microshoots, allowing them to develop normally. These included the addition of 3 g L-1 activated charcoal, the removal of ammonium nitrate from the medium, doubling the concentration of Ca2+ or eliminating BA from the medium.
Induction of axillary shoots and exploration of methods to optimize the rejuvenation of hyperhydric microshoots
P. lactiflora ‘Zhong Sheng Fen’ from the Society of Forestry Experimental
The well-developed underground buds of
P. lactiflora ‘Zhong Sheng Fen’ were collected
in winter from the Society of Forestry Experimental
Base and used as the explants.
Before inoculation, the underground buds were washed in tap water for 30 min. The outer scales were peeled off and buds were soaked in 75% ethanol for 30 s, immediately followed by 10 min sterilization with a dilute solution of HgCl2 (0.1% w/v). Plant material was rinsed five times (5 min/rinse) in autoclaved distilled
water. All sterilized explants (i.e. underground buds) were placed on agar (0.7%) solidified medium. The base medium was half-strength (macro- and micronutrients) MS medium (Murashige and Skoog 1962) with doublestrength calcium chloride (Ca2+ ) supplemented with 30 g L-1 sugar. Buds were cultured in 100 ml Erlenmeyer flasks containing 30 ml agar solidified medium with one bud per flask. The effects of indole-3-acetic acid (IAA) (0.0, 0.1, 0.3 and 0.5 mg L-1) combined
with 1 mg L-1 6-benzyladenine (BA) and 0.5 mg L-1 gibberellic acid (GA3) on culture
initiation of ‘Zhong Sheng Fen’ were tested. The highest lateral shoot initiation
(92.8%), number of lateral shoots (2.13) and
shoot length (4.2 cm) and the lowest rate of
yellowing shoot tips (17.43%) were observed in medium containing 0.1 mg L-1 IAA + 1 mg
L-1 BA + 0.5 mg L-1 GA3.
After initiation of clture, the effects of BA (0.5, 1.0, 1.5 and 2.0 mg L-1) combined with 0.5 mg L-1 kinetin (KT) on axillary shoot induction were tested. The induction of axillary shoots was favoured by a higher concentration of BA (2.0 mg L-1). The effects of different auxins (IAA, IBA, NAA) on axillary shoot proliferation were tested. The addition
of an auxin to the medium caused a significant
increase in the incidence of hyperhydricity
compared with the control. The highest multiplication
rate (2.40) and the lowest incidence of
hyperhydricity (7.01%) were observed on auxin-
free medium (0.2 mg L-1 BA + 0.1 mg L-1 GA3). Even a low concentration of BA (0.2 mgL-1) aggravated hyperhydric microshoots. In this state, no hyperhydric microshoots reversed to normal shoots and the proliferation rate of hyperhydric microshoots also decreased. Hyperhyfricity was strongly linked to BA, and even a low concentration of BA (0.2 mgL-1) did not benefit the growth of hyperhydric microshoots. Several methods allowed the successful rejuvenation of hyperhydric microshoots, allowing them to develop normally. These included the addition of 3 g L-1 activated charcoal, the removal of ammonium nitrate from the medium, doubling the concentration of Ca2+ or eliminating BA from the medium.