STUDY ON RESULTS OBTAINED BY DIFFERENT RESEARCHERS ON IN

VITRO PROPAGATION OF HERBACEOUS PEONY

George Nicolae CAZAN1, Florin TOMA1
1
University of Agronomic Sciences and Veterinary Medicine of Bucharest, 59 Marasti Blvd,
District 1, Bucharest, Romania

Corresponding author email: [email protected]

Abstract

These researches highlights the advances made on various aspects of herbaceous peony (Paeonia lactiflora Pall.) tissue
culture, including the in vitro culture of underground buds, leaves and petioles, the induction of cluster buds, and callus
induction. Tissue culture has the ability of speeding up the rate of propagation, reducing breeding time and meeting the
needs of mass production. Problems that are currently being faced in herbaceous peony tissue culture are highlighted
and possible viable solutions are provided. More research is needed to make mass production of peony more
commercially successful. Optimization of procedures are necessary, from selection of explants, decontamination,
screening of medium, application of plant growth regulators (PGRs), induction of callus and shoots, subculture,
rooting, and to final transplanting. The major goal of these studies is to evaluate shoot induction ability of peony
explants and PGRs. The development of micropropagation methods for peonies is necessary to not only overcome this
problem but also accelerate peony breeding progress. Tissue culture is one of the most effective approaches for rapid
propagation of plants and is also providing a new approach for plant breeding in crops, ornamentals, fruits and
vegetables. Effect of Gelling Agents on Tissue Culture of Herbaceous Peony. Comparison of Basal Medium in Tissue
Culture of Herbaceous Peony. Effect of Pretreatment of Underground Buds on Tissue Culture of Herbaceous Peony.
Investigation of Callus and Shoot Induction Ability of Flower Tissue. Effect of PPM on Decontamination and Rescue of
Explants. Root Induction and Transplanting of Herbaceous Peony In Vitro.

Key words: herbaceous peony; Paeonia lactiflora Pall.; tissue culture; in vitro propagation.

INTRODUCTION (Steward, 1968; Krikorian and Berquam,

1969).
Peonies can be propagated by division, cutting, Micropropagation of peony began in the middle
grafting, and layering to obtain true-to-type 1960s. During the last 40 years, much research
plants. on micropropagation of peony has been
There are multiple traditional choices for conducted with plants successfully produced
propagation of peony. However, the limited from tissue culture labs. Planteck
number produced by these traditional methods Biotechnologies Inc., a company based in
can not meet the increasing demands in the Quebec, Canada, produced mass numbers of
market, especially for a quick release of a new herbaceous Itoh peonies and made the tissue
cultivar and massive production of a specified cultured plants available in the market for the
variety. The development of micropropagation first time in 2006 (Whysall, 2006).
methods for peonies is necessary to not only However, there is still much work to be done in
overcome this problem but also accelerate order to make mass production of peony more
peony breeding progress. commercially successful. Optimization of
The field of plant tissue culture is based on the procedure for each stage or step should be studied
in depth, including selection of explants,
premise that plants can be separated into their
decontamination, screening of medium,
component parts (organs, tissue, or cells),
application of plant growth regulators (PGRs),
which can be manipulated in vitro and then induction of callus and shoots, subculture,
grown back to complete plants. The first rooting, and finally transplanting.
successful plant tissue and cell culture was In peony, callus was induced successfully for
accomplished by Gottlied Haberlandt near the the first time by Yamada and Sinotô (1966)
turn of the 20th century when he reported the from petal culture of P. japonica. Large
culture of leaf mesophyll tissue and hair cells variation in chromosome numbers and
characteristics within cells was observed during the original research resources published by
culture. Demoise (1967) and Demoise and non-native languages.
Partanen (1969) induced callus from tree peony More research is needed to make mass
and investigated the effects of subculturing and production of peony more commercially
physical conditions of culture on the mitotic successful. Optimization of procedures are
cycle kinetics of a population of cells, necessary, from selection of explants,
particularly in relation to the degree of decontamination, screening of medium,
heteroploidy. Since then, many original studies application of plant growth regulators (PGRs),
on in vitro culture of peony have been reported. induction of callus and shoots, subculture,
Several review papers have also been rooting, and to final transplanting. The major
published. The first review on goal of this study is to evaluate shoot induction
micropropagation of peony was published by ability of peony explants and PGRs.
Buchheim and Meyer (1992), where research
on in vitro culture of peony before 1989 on has
been mostly discussed. A summary of in vitro EFFECT OF GELLING AGENTS
studies conducted on several species of ON TISSUE CULTURE OF
Paeonia was included in a table consisting of HERBACEOUS PEONY.
plant species, inoculum, medium, growth
response, and reference. This review is useful During this researches made in 2007 Torres
for a researcher working on peony tissue et al., used as material and method the
culture, although some medium formulations follouing:
have not been cited correctly. The second Four types of agar products were used to
review on in vitro culture peony was written by compare responses of nodal stem explants of
Gabryszewska (2004). The regeneration ability ‘Yang Fei Chu Yu’ (‘YFCY’) and ‘Xi Shi Fen’
of different organs of both herbaceous and tree (‘XSF’) in ½ MS + 1 mg l-1 TDZ medium with
peony was reported in this paper. The role of (1) A111 (Phytotechlab) 5 g l-1; (2) A296
exogenous and endogenous PGRs in (Phytotechlab) 6 g l-1; (3) A133 AgagellamTM
differentiation and growth of shoots, roots, and (Phytotechlab) 4 g l-1; and (4) A20020 (high gel
somatic embryos was also discussed. strength) 5 g l-1 according to the recommended
In 2007, Beruto and Curir summarized tissue rates.
culture of tree peony under a book chapter title, After this researches made in 2007 Torres et
‘In vitro culture of tree peony through axillary al., obtained the follouing result and
budding’, which included most of the results discutions:
published between 1969 and 2004. The review Explants responded to the types of agar gelling
was well organized by introduction, agents differently. Generally A111 (5 g l-1) and
experimental protocol consisting of stages of A133 (4 g l-1) were much better than A296 (6 g
tissue culture, and conclusion. During this time, l-1) and A20020 (5 g l-1) for all indexes.
five other review papers on peony tissue culture Response was also genotype dependent. A133
were published by Chinese researchers and A111 significantly increased explant
(Buchheim and Meyer, 1992; Gabryszewska, growth of ‘YFCY’ as compared to A296 and
2004; Li and Luo, 2004; Li et al., 2006a; Jia et A20020 but this effect did not occur in ‘XSF’.
al., 2006b; Meng et al.2007, Beruto and Curir, Similar results were noted on induction of
2007 2007; Zhao et al., 2007). However, each callus and shoots. The highest rates of shoot
of these papers is only 2 to 4 pages long and initiation and callus production were seen on
did not cover as much as information of explants with treatments of A111 and A133,
previous work on micropropagation of peony. respectively, in ‘YFCY’, but no significant
With the fast development of internet and difference was observed in ‘XSF’. For both
database construction in the world and the cultivars, more phenolic compounds exuded
benefit of language translation software, it is into media with A296 and A20020, and explant
becoming much easier to access and understand color looked abnormal. Agar has long been
used to solidify media for plant tissue culture.
There are lots of brands of agar gelling agents. while explants of ‘FLHZ’ produced the lowest
The type of agar or other gelling agents shooting rate in ½ MS. Compared with the data
significantly influences plant growth in tissue collected on 24 and 40 days after inoculation,
culture. Differences of results are even seen in the difference in the initiation rate among basal
the same product made at different times or medium types was consistent.
locations (Torres et al., 2007).
EFFECT OF PRETREATMENT OF
COMPARISON OF BASAL MEDIUM UNDERGROUND BUDS ON TISSUE
IN TISSUE CULTURE OF CULTURE OF HERBACEOUS PEONY.
HERBACEOUS PEONY.
During this researches made in 2008 Daike
During this researches made in 2008 Daike Tian used as material and method the
Tian used as material and method the follouing:
follouing: Responses of underground buds to three
The choice of type of basal medium is pretreatments were evaluated in tissue culture
important for tissue culture. MS, ½ MS and of two varieties of herbaceous peony.
WPM have been mostly used in peony tissue Underground buds (Fig. 1) from containerized
culture for shoot induction. In this experiment, stock plants of ‘Da Fu Gui’ (‘DFG’) were
effects of MS (1/4, half, full strength) and treated in three ways: (1) plants remained
WPM (half, full strength) medium with 1 mg l-1 untreated; (2) plants were treated in cooler for
TDZ + 0.1 mg l-1 BA + agar (A111, 4 g l-1) was 20 d beginning Nov. 20, 2006; and (3) plants
evaluated on callus and shoot induction of from outside were washed by tap water and
nodal stems from three herbaceous cultivars: repotted in container with perlite, then directly
‘Bin Shan’ (‘BS’), ‘Fen Ling Hong Zhu’ moved to greenhouse to break dormancy and
(‘FLHZ’) and ‘Yang Fei Chu Yu’ (‘YFCY’). force growth.
After this researches made in 2008 Daike
Tian obtained the follouing result and
discutions:
The choice of type of basal medium is
important for tissue culture. MS, ½ MS and
WPM have been mostly used in peony tissue
culture for shoot induction. Explants generally
performed best in full strength medium MS
followed by half strength MS, and worst in half
strength WPM. Nodal stems of all three
cultivars remained green in full strength MS
medium, while explants turned pink and
showed slight abnormalities in other MS Figure 1. Underground buds of herbaceous peony,
media. Explants cultured in WPM turned pink, (after Daike Tian, 2008).
then red over time. MS was the most favorable In this treatment, the stock plants were also
medium for explant growth of ‘BS’. Explants rinsed with 1% Zerotol for two times, once per
cultured in ½ WPM had the least increase in week, to decrease contamination. After surface
growth for all three varieties. Browning was sterilization of 20 sec in ethanol (75%)
more visual in WPM than MS. MS medium following by 25 min in 10% Chlorox bleach,
was more favorable than WPM for callus the buds (tips) were inoculated in test tubes
induction but results of shoot induction were with medium ½ MS + 1 mg l-1 BA + 0.1 mg l-1
complicated. Explants cultured on full strength GA3 or ½ MS + 0.1 mg l-1 BA + 0.1mg l-1 GA3
WPM produced the highest shoot initiation on Dec 10, 2007. Shoot multiplication medium
rates for ‘BS’ and ‘FLHZ’ but the lowest rate was ½ MS + 0.1 mg l-1 BA + 0.1 mg l-1 TDZ +
for ‘YFCY’. Explants of ‘BS’ generated the 0.1 mg l-1 GA3.
lowest shooting rate on half strength WPM
After this researches made in 2008 Daike
Tian obtained the follouing result and
discutions:
A previous experiment produced 100%
contamination of ‘DFG’ and ‘Xi Shi Fen’
(‘XSF’) dormant underground buds with a
sterilization treatment of 10 sec in ethanol
(75%) following by 10–20 min in 10% Chlorox
bleach. After 15 d of culture, no callus formed
and buds did not grow. In this experiment,
contamination and phenolic exudation 281
were visual within one day of culture and
increased over the time of the 15-d experiment.
Figure. 2. After culture in ½ MS + 1 mg l-1 BA + 0.1 mg
All explants (100%) treated in greenhouse were l-1 GA3, the cooler treated buds (left) grew fast and
contaminated within 3 d of culture. The cooler produced elongated shoots (main and lateral). After
treated explants performed better. Only 2 of 16 cutting of shoots and subculture in ½ MS + 0.1 mg l-1
explants were contaminated within three days BA + 0.1 mg l-1 TDZ + 0.1 mg l-1 GA3, the number of
although contamination rate went up to 75% shoots was multiplied, each bud eye region formed one
to several new shoots (middle). While bud explants from
after 15 d culture. More explants were outside stock plants (right) grew slow and shoot stem did
contaminated by bacteria than fungi. not elongated much, (after Daike Tian, 2008).
Contamination with bacteria also occurred
earlier. There was significant difference in If the buds were treated in cooler,
fungi caused infection among treatments. At contamination decreased significantly. Shoot or
the end of experiment, only one bud from the bud explant could readily grow and axillary
outside treatment and 4 buds from the cooler shoots were easily induced after bud dormancy
treatment were sterile for use of shoot induction was broken.
after transfer. Before culture, buds treated in
the cooler for 20 d were already slightly INVESTIGATION OF CALLUS
elongated (dormant release) and looked better AND SHOOT INDUCTION
in quality than both outside and greenhouse ABILITY OF TISSUE.
treated buds. After inoculation these buds grew
very fast and main shoots as well as some During this researches made in 2008 Daike
lateral shoots elongated (Fig. 2). These Tian used as material and method the
elongated shoots could be cut into nodal follouing:
sections and transferred to fresh medium for Callus and shoot induction ability of ‘Da Fu
shoot multiplication. Buds from the outside Gui’ (‘DFG’) and ‘Paula Fay’ (‘PF’) were
also elongated but did not grow as fast as the studied in this experiment using petals, anthers,
cooler treated buds. The shoots were much filaments, and pistils as explants from the tight
shorter than those generated from the cooler flower buds (TFB), one week before blooming,
treated buds (Fig. 2). The greenhouse perlite newly opening flowers (OF) with a small
medium treated buds performed worst and all mouth or the fully open flowers (FOF). The
nearly stopped growing. These buds finally petals of TFB, OF and FOF were from ‘DFG’,
died and no axillary shoots generated. For all and the petals, anthers and filaments of TFB
treated buds, no callus formed in the early were from ‘PF’. Three media were tested: A: ½
stages. Only very little green callus was MS (PGR-free); B: ½ MS + 1 mg l-1 TDZ; and
induced at the cutting side of buds after more C: ½ MS + 1 mg l-1 TDZ + 0.1 mg l-1 GA3.
than 15 d of culture. A more effective After this researches made in 2008 Daike
sterilization approach must be developed. Tian obtained the follouing result and
Dormant buds were not effective for shoot discutions:
induction. After 14 d of culture, contamination occurred
on the petals but not on either anthers or
filaments. Petals from FOF had higher browning, the callus and shoot induction rate,
contamination rate than those from TFD and and shoot growth of posttransferred explants
OF in ‘DFG’. Browning was obvious in all between PPM treatment and the control
treatments and it was more severe on the petals explants. This indicated application of PPM at
of FOF followed by OF in ‘DFG’. Petals from low level in medium had no side effect on
TFB had the least browning problem. However, tissue culture of peony.
in ‘PF’ nearly all of the petals from TFB turned
brown. The least browning rate was seen on ROOT INDUCTION AND
anther culture of this cultivar. No difference in TRANSPLANTING OF HERBACEOUS
browning was found among PGR treatments. PEONY IN VITRO.
There were large differences in growth of
explants among types of petals. Petal sections During this researches made in 2008 Daike
from both TFD and OF grew fast, whereas Tian used as material and method the
those from FOF grew slow and lost their follouing:
original color within two days of culture. Several trials on root induction were conducted
Callus generated very slowly and no callus on vitroshoots from the varieties: ‘Xi Shi Fen’,
formed within 15 d. Following 30 d of culture, ‘Da Fu Gui’, and ‘Cytherea’. The treatments
very little callus was induced from the base included following variables: light and
cutting side of the petals from TFB and OF in darkness; liquid (paper bridge method, Hosoki
both B and C media. et al 1989), half solid and solid ½ MS 285
medium; activated charcoal (AC); temperature;
EFFECT OF PPM ON and IBA with different concentrations or a
DECONTAMINATION AND quick soak in high concentration of IBA (10
RESCUE OF EXPLANTS. mg l-1).
After this researches made in 2008 Daike
During this researches made in 2008 Daike Tian obtained the follouing result and
Tian used as material and method the discutions:
follouing: Following 7–10 d of culture in rooting medium,
Effect of PPM on decontamination of nodal all vitroshoots generated callus. Callus even
stem segments from ‘Da Fu Gui’ (treated 3 formed sometimes on the petioles.
months in the cooler) and rescue of Plantlets rooted following about 20 d of culture
contaminated explants was investigated in and up to 20 roots developed on a shoot. Roots
media with ½ MS + 1 mg l-1 BA + 0.2 mg l-1 grew up to 3 cm after 45 d of shoot inoculation.
GA3 with supplement of 0, 0.05, 0.1, and 0.2% The vitroshoots grew very fast in semi-solid
(v/v) of PPM, respectively. The explants were rooting medium possibly benefiting from an
surface sterilized with a 12–15 sec quick soak easy absorption of nutrients (Fig. 3). AC
in 70% Ethanol followed by 20 min in 10% treatment increased the shoot length but not
Bleach. For rescue of contaminated explants, significantly. IBA shock treatment (10 mg l-1)
12 of bacteria contaminated explants were did not influence growth of in vitro shoots on
cleaned by a soft brush under running tap water rooting medium. IBA at 1 mg l-1 in a
following by a 12 min soak in 50% PPM and continuous treatment was too strong for small
then were inoculated on fresh medium. younger vitroshoots and caused their petioles to
After this researches made in 2008 Daike calluse. It remains not clear how long
Tian obtained the follouing result and vitroshoots should be treated in medium with
discutions: high concentration IBA before transfer to root
PPM had effect on minimizing contamination growth medium with either lower concentration
of explants and the contamination rate of IBA or no IBA. High-quality shoots are the
decreased in 0.2% PPM-treated medium. basic requirement to obtain high rate of rooting.
However, lower concentrations (<0.2%, v/v) of Several studies have reported treatment of
PPM only delayed occurrence of darkness and chilling was beneficial for rooting
contamination. No difference was observed in of peony in vitro (Kunneman and Albers 1989;
Habib and Donnelly 2001; Beruto et al 2004; necrosis after more than two months culture
Chen 2005). but the later formed young leaves remained
healthy. It was not clear if necrosis problem
could be avoided or minimized by an increase
of transfer frequency. Limited number of
shoots with roots or root primordia was
transplanted to jars with non-sterile peat moss.
These vitroplants grew in the first week but
quickly died because of infection with fungi
(Fig. 6).

Fig. 3. Shoots grew fast in half-solid medium and there

was no difference in shoot length between treatments
(with or without IBA shock, AC),
(after Daike Tian, 2008).

Roots were successfully induced from plantlets

in solid medium (Fig. 4) or using paper bridge
method (Fig.5) in some cases.

Fig. 5. Roots were induced from vitroshoots of ‘Da Fu

Gui’ by paper bridge (white material) method with liquid
medium: ½ MS + 1 mg 1-1 IBA. (after Daike Tian,
2008).

Figure. 4. Roots were only induced on the shoots with a

10-min pretreatment in high concentration of IBA (10
mg l-1) before inoculation on medium with ½ MS + 1 mg
l-1 IBA ± 5% AC (w/v), (after Daike Tian, 2008).
The present experiment showed that the shoots
with a 40-d darkness treatment at 25ºC nearly
stopped growth and completely died later. The
shoots with a 40-d darkness treatment at 10ºC
survived with chlorotic tissue. However, no
roots were obtained in both treatments. For Figure. 6. After transplanting, rooted shoots grew in the
both of the rooted and non-rooted shoots, the first week but then died with contamination of fungi or
rot of roots and stem, (after Daike Tian, 2008).
earlier developed leaves showed obvious
 CONCLUSIONS sterile peat-moss mix died quickly with rot of
roots and stem base.
As a result of their researches made in 2007
Torres et al., reached follouing conclusion:
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