全 文 ：植 物 学 报
Acta Botanica Sinica 2003, 45 (12): 1475-1480
Received: 2003-03-10 Accepted: 2003-06-20
Supported by the Natural Science Fund of Beijing (5982007).
* Author for correspondence. Tel: +86 (0)10 68902374; E-mail: .
http://www.chineseplantscience.com
Establishment of In Vitro Regeneration System of the Atrichum Mosses
GANG Yong-Yun1, DU Gui-Sen1 *, SHI Ding-Ji2 , WANG Mei-Zhi2 , LI Xue-Dong1 , HUA Zhen-Ling1
(1. Department of Biology, Capita Normal University, Beijing 100037, China;
2. Institute of Botany, The Chi ese Academy of Sciences, Beijing 100093, China)
Abstract: In vitro regeneration systems of Atrichum mosses, Atrichum undulatum(Hedw.) P. Beauv. and
A. undulatum var. minus(Hedw.) Par. were established. After one month, soft, friable and green calli were
induced successfully from inoculated protonema of Atr chum mosses on MS medium containing glucose
(4%) and 6-BA (0.2-2.0 mg/L). The suitable culture medium for the callus induction and regular subculture
was MS medium with 1.0-2.0 mg/L 6-BA and 4% glucose. The calli of Atric um mosses developed into
protonema, when it was transferred to phytohormone-free MS m ium with 4% glucose. Meanwhile, the
calli developed into erect gametophytes through protonema phase on carbohydrate-free Benecke medium.
Key words: mosses; protonema; callus; redifferentiation
Bryophytes are unique plant organisms whose cultured
cells contain active chloroplasts and can grow under pho-
toautotrophic conditions among green land plants
(bryophytes, pteridophytes and seed plants) (Katoh, 1983).
Cells of bryophytes, especially in suspension culture, have
been noted as ideal materials for morphogenetic, genetic,
physiological, biochemical and molecular studies (Ono et
al., 1988). Moreover, bryophytes possess the following
characteristics: (1) relatively simple structure compared to
other higher plants, (2) haploid gametophyte of dominant
vegetative phase, and (3) less chromosome numbers (Glime,
1988).
Bryophytes callus induction began in the 1950-1960s.
Allsopp (1957) and Word (1960) reported respectively
calli of liverworts and mosses in first (Wu, 1998) , Takami et
al. (1988) reported firstly that the callus of hornworts was
induced and the system of suspension culture was estab-
lished (Takami et al., 1988). According to Felix (1994) ,
there were 31 liverworts, 18 mosses and one hornworts cal-
lus inductions of bryophytes. Moreover, systems of sus-
pension culture from 27 liverworts, 16 mosses and one horn-
worts of bryophytes have been established. However, the
progress in bryophyte tissue culture did not go as fast as
with cell culture techniques of the other higher plants and
the number of cases achieved still does not satisfy suffi-
ciently the demands of various research fields (Felix, 1994).
Bryophytes tissue culture appeared later, fewer and
slowly in China. Li (1990) studied firstly in vitro
propagation, dedifferentiation and redifferentiation of
Marchantia polymopha in 1990. No paper has focused on
tissue culture research of mosses up to now. Atrichum in
Polytrichaceae, such as Atrichum undulatum (Hedw.) P.
Beauv. and A. undulatum var. minus (Hedw.) Par., more
later in evolution of mosses, were selected and their callus
induction and redifferentiation studied, and the regenera-
tion system of their in vitro tissue culture was established.
1 Materials and Methods
1. 1 Collection of Atrichum mosses
Plant materials, Atrichum undulatum (Hedw.) P. Beauv.
and A. undulatum var. minus (Hedw.) Par., were collected
from the habitats given in Table 1. We adopted some special
humidity methods to keep them alive, then, cultivated them
under the light culture cabinet (illumination of 1 500-2 000
lx , at (16±1) ℃) for tissue culture experiments. The
collected plant materials were identified by WANG Mei-Zhi
senior engineer in Institute of Botany, The Chinese Acad-
emy of Sciences. Voucher specimens were deposited in the
plant herbarium, Department of Biology, Capital Normal
University.
1. 2 Culture media
The culture media for proliferation, dedifferentiation and
redifferentiation used in this study included MS (Murashige
and Skoog’s, 1962) and Benecke medium (BM) (Benecke,
1903) that usually solidified with 0.8% agar. The pH of MS
was adjusted to 5.7-5.8 before autoclaving. MS40 media
contains 4% glucose and phytohormone-free. For callus
induction, different concentrations of 6-BA and 2,4-D (0.2-
2.0 mg/L) were added to MS40 media. BM used consists of
NH4NO3 (0.2 g/L), MgSO4·7H2O (0.1 g/L), KH2PO4 (0.4 g/L),
植物学报 Acta Botanica Sinica Vol.45 No.12 20031476
CaCl2 (0.1 g/L), and very little FeCl3·6H2O.
1. 3 Surface sterilization and sterile inoculation of ex-
plants
Healthy grown gametophyte tips of A. undulatum were
selected and surface sterilized as follows: (1) Washing
with distilled water five times to scour off superficial dust,
one minute per time; (2) Sterilizing with 0.025% HgCl2
solution for 50 s; (3) Washing with sterile distilled water
five times to eliminate remaining HgCl2 solution, one minute
once; (4) Transferring the sterilized gametophyte tips to a
sterilized Petri dish containing wet filter paper and cutting
into 5-10 mm in length by sterilized knife for inoculation.
Mature capsules were more resistant to HgCl2 solution than
fresh gametophyte tips. Spores used for inoculation were
released from mature capsules of A. undulatum var. minus
that had been surface-sterilized with 0.05% HgCl2 solution
for 60 s. Using standard sterile technique, the explants of
surface-sterilized were inoculated onto the surface of pre-
pared sterilized culture media under aseptic conditions.
1. 4 Cultivation methods
1. 4. 1 Proliferation Excised aseptic gametophyte tips
and dispersed sterile spores were inoculated onto MS40 or
BM , cultivated under an illumination of 1 500-2 000 lx for
14 h/d, at (20±1) ℃. The protonema and gametophyte
tips of A. undulatum and A. undulatum var. minus, ob-
tained from gametophyte tips and spores, were inoculated
onto MS40 or BM under the same cultivation conditions,
respectively.
1. 4. 2 Dedifferentiation The protonema of A. undulatum
and A. undulatum var. minus, obtained from above
proliferation, were cut into 4-7 mm × 4-7 mm in size, and
inoculated onto MS4 (4% glucose) media containing dif-
ferent concentrations of phytohormones, cultivated under
an illumination of 1 500-2 000 lx for 14 h/d , at (25±1) ℃.
Calli induced from protonema were routinely subcultured
at every 20-day interval on MS4 (4% glucose) media con-
taining 1.0-2.0 mg/L 6-BA under the same conditions.
1. 4. 3 Redifferentiation After five times subculture, the
green calli, induced from protonema of Atrichum mosses,
were transferred onto the culture media (MS40 or BM) with-
out phytohormone and carbon source. Culture conditions
were the same as shown in 1.4.2.
2 Results and Discussion
2.1 Surface sterilization of explants
Most scientists use the Washing Machine Technique
invented by Basile to surface-sterilize explants from bryo-
phytes (gametophyte tips and unopened capsules) (Glime,
1988). Using Clorox (9%) for one minute was suitable for
most species, but the time and concentration have to be
modified according to delicate and tough explants.
A procedure of surface sterilization for explants of
Atrichum mosses used in this study was developed from
the Washing Machine Technique. When the NaClO solu-
tion was used, the rates of contaminated or dead explants
was above 70%. However, when the low-concentrated
HgCl2 solution was used, the rate of decontaminated or
survived explants was over 80%.
2.2 Proliferation of Atrichum mosses
Green protonema of A. undulatum and A. undulatum
var. minus, developed from inoculated gametophyte tips
and spores on MS40 after one month. After 20 d, dispersed
spores of A. undulatum var. minus on BM produced many
erect green gametophytes. About 10 to 15 erect green ga-
metophytes formed around each inoculated gametophyte
tip after one month.
Gametophytes of Atrichum mosses obtained from the
above cultures, were regularly subcultured every 20 d on
MS40 and BM. Green protonema occurred around inocu-
lated gametophyte tips on MS40 after one month (Fig.1).
About 10 to 15 erect green gametophytes emerged around
inoculated gametophyte tips on BM after one month
(Fig.2).
Protonema of Atrichum from inoculated protonema or
gametophyte tips on MS40, were proliferated by a regular
subculture at 20-day intervals on MS40. The number of
gametophytes, derived from inoculated protonema or ga-
metophyte tips on BM, increased by regular subculture
every 30 d on BM.
Thus, the proliferation of Atrichum mosses was suc-
cessfully achieved with enough, purified and sterile plant
material.
2.3 Callus induction of Atrichum mosses and factors
affecting callus induction
In th is experimen t, the cal li from protonema of
Table 1 Atrichum mosses used for tissue culture
Species Habitats Al t i t u d e（m） Collector Time
Atrichum undulatum Wuling Mt., Hebei Province 1 400 GANG Yong-Yun June, 2000
A. undulatum var. minus Wunü Mt., Jilin Province 1 450 LI Xue-Dong September, 1999
1477GANG Yong-Yun et al.: Establishment of In Vitro Regeneration System of the Atrichum Mosses
A. undulatum and A. undulatum var. minus were success-
fully induced on MS medium containing glucose (4%) and
6-BA (0.2-2.0 mg/L) , the frequency of induction ranged
from 81.5% to 95.8% (Table 2). However, calli induction
from protonema of A. undulatum and A. undulatum var.
minus failed on MS medium containing glucose (4%) and
2,4-D (0.2-2.0 mg/L) (Table 2).
Soft, friable and green calli of A. undulatum and A.
undulatum var. minus were successfully induced around
inoculated protonema on MS medium containing glucose
(4%) and 6-BA (0.2-2.0 mg/L) after one month (Fig.3). The
calli sizes were larger on MS4 (4% glucose) media contain-
ing 1.0-2.0 mg/L 6-BA than on MS4 (4% glucose) media
with 0.2-0.5 mg/L 6-BA. When subcultured repeatedly on
MS4 (4% glucose) media containing 1.0-2.0 mg/L 6-BA at
15-day intervals, calli grew rapidly and maintained the dedi-
fferentiation states (Fig.4). Under the light microscope (LM),
each of the cells of calli contained many oval, spherical
green chloroplasts (Fig.5).
At the early research stage on bryophytes callus
induction, most of calli were induced from spores on cul-
ture media containing several organic additions; factors
affecting callus induction were not clear (Wu, 1998). Since
Ono (1973) successfully induced callus of Marchantia
polymopha from gemmae, it could be inferred from many
bryophytes callus induction studies that a high concentra-
tion of sugar and high culture media osmolarity have been
regarded as the main factors for bryophytes callus induc-
tion (Ono et al., 1988).
Ono et al. (1988) reported that callus induction was
achieved on eight species of liverworts by culturing spores
or thalli on MS medium containing 2%-8% glucose with-
out any phytohormones. The addition of 10-6 mol/L 2,4-D
to this medium was necessary in inducing calli of
Polytrichum commune (a mosses) and Anthoceros
punctatus (an anthocerote) (Ono et al., 1988). Katoh (1988)
considered that the concentration and mutual ratio of glu-
cose and 2,4-D in the culture media varied over a wide range
for bryophytes callus induction. Best results were obtained
from a combination of 4% or 6% glucose and 10-6 mol/L 2,
4-D. However, it may vary depending on the species of
cultured bryophytes (Glime, 1988).
In the present study, when transferring Atrichum mosses
protonema to MS medium with 4% glucose and 1.0-2.0
mg/L 2, 4-D, after one or two months, protonema was still
green, and callus induction of Atrichum mosses failed.
Moreover, when Atrichum mosses protonema was trans-
ferred to MS4 (4% glucose) containing 1.0-2.0 mg/L 6-BA
and 1.0-2.0 mg/L 2, 4-D, callus induction of Atrichum
mosses still occurred, but the frequency decreased. Maybe
it needs further research to explain the phenomena.
Word (1960) obtained callus from spores of A.
undulatum on Knudson’ media containing sucrose,
casamino acid and coconut milk, along with X-ray, after
two months. Ono et al. (1987) obtained callus of A.
undulatum on MS culture media (0.8% agar) with 10-6
mol/L 2,4-D and 4% glucose, still from spores.
Definitive composition of callus induction culture media,
high induction frequency, short culture cycle and simplify
induced processes are the prominent achievements of this
experiment, in contrast to Word’s study. Moreover, the
culture media used in Ono’s study has not induced callus
successfully in A. undulatum and A. undulatum var. minus,
and it requires further research to elucidate the reasons.
The hormones in culture media for callus induction of
Atrichum mosses in this experiment were distinctly differ-
ent from what have been using aboard. And high frequency
of induction was observed. In addition, the culture media
Table 2 Frequencies of callus induction of Atrichum undulatum/A. undulatum var. minus in different concentrations of phytohor-
mones
Phytohormones No. of No. of No. of Frequency of callus induction
（mg/L） explants calli protonema （% ± S D）
2 , 4 - D（0 .2） 94 / 93 0 / 0 94 / 93 0 / 0
2 , 4 - D（0 .5） 96 / 98 0 / 0 96 / 98 0 / 0
2 , 4 - D（1 .0） 95 / 93 0 / 0 95 / 93 0 / 0
2 , 4 - D（2 .0） 97 / 95 0 / 0 97 / 95 0 / 0
6 - BA（ 0 .2） 92 / 94 75 / 77 17 / 17 （8 1 . 5 ± 0 .0 2）/（8 1 . 9 ± 0 .0 2）
6 - BA（ 0 .5） 93 / 95 78 / 80 15 / 15 （8 3 . 9 ± 0 .0 0）/（8 4 . 2 ± 0 .0 0）
6 - BA（ 1 .0） 95 / 94 85 / 86 10 / 8 （8 9 . 5 ± 0 .0 2）/（9 1 . 5 ± 0 .0 2）
6 - BA（ 2 .0） 97 / 96 91 / 92 6 / 4 （9 3 . 8 ± 0 .0 0）/（9 5 . 8 ± 0 .0 2）
6 - BA（1 .0）+ 2 , 4 - D（1 . 0） 93 / 93 83 / 85 10 / 8 （8 9 . 2 ± 0 .0 2）/（9 1 . 3 ± 0 .0 2）
6 - BA（1 .0）+ 2 , 4 - D（2 . 0） 97 / 95 86 / 86 11 / 9 （8 8 . 7 ± 0 .0 2）/（9 0 . 5 ± 0 .0 3）
6 - BA（2 .0）+ 2 , 4 - D（1 . 0） 98 / 97 91 / 92 7 / 5 （9 2 . 9 ± 0 .0 2）/（9 4 . 8 ± 0 .0 2）
6 - BA（2 .0）+ 2 , 4 - D（2 . 0） 96 / 95 88 / 89 8 / 6 （9 1 . 7 ± 0 .0 2）/（9 3 . 7 ± 0 .0 2）
植物学报 Acta Botanica Sinica Vol.45 No.12 20031478
Figs.1-6. 1.Green protonema occurred around inoculated gametophyte tips of Atrichum undulatuv r. minuson MS40. 2. Many
erect green gametophyte emerged from inoculated protonema of A. undulatum n BM. 3. Green calli induced from inoculated protonema
of A. undulatum. 4. Subcultured calli of A. undu atum var. minus. 5.Cell of calli of A. undulatum nder a light microscope. ×460. 6 Many
erect green gametophyte developed from inoculated calli of A. undulatum va . minuson BM.
1479GANG Yong-Yun et al.: Establishment of In Vitro Regeneration System of the Atrichum Mosses
used for callus induction of Atrichum mosses were far sim-
pler to that for Marchantia polymorpha (Li, 1990).
2.4 Redifferentiation of calli
Calli of Atrichum mosses were repeatedly subcultured
five times, and then, transferred to MS40 or BM,
redifferentiated into protonema or many erect gametophyte
through protonema development stages after one month
respectively (Table 3; Fig.6). Protonema and gametophyte
redifferentiated from calli were able to proliferate on MS40
and BM as shown above.
All studies confirmed that the redifferentiation and plant
regeneration of bryophytes calli were easier than those of
higher plants. The calli which have been subcultured sev-
eral times could redifferentiate and regenerate easily when
it was transferred to MS culture medium without phytohor-
mone or the culture medium without carbon source. (Ono
et al., 1988; Li, 1990). In this study, calli of mosses achieved
the redifferentiation and plant regeneration successfully
and developed into protonema and gametophytes with rates
above 95%.
In conclusion, using MS medium with 0.2-2.0 mg/L
6-BA and 4% glucose, the induction of callus from inocu-
lated protonema of A. undulatum and A. undulatum var.
minus was successful. Also, in vitro tissue culture regen-
eration system was established. Atrichum mosses used in
this study merit further research due to their traditional
medical values. Moreover, in vitro tissue culture regenera-
tion system of mosses establishment can provide sufficient,
purify and sterile experiment materials for cell suspension
culture, morphogenetic, genetic, physiological, biochemi-
cal and molecular biological studies within a short time.
Furthermore, this study may be useful for tissue culture of
other mosses.
Acknowledgements: The authors thank Prof. CHEN
Wei-Lun, WU Peng-Cheng and JIA Yu in Institute of
Botany, The Chinese Academy of Sciences, and Dr. ZHANG
Fei-Yun of Department of Biology, Capital Normal
University, for their help.
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Beneke W. 1903. Über die Keimung der Brut-Knospen von
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Table 3 Redifferentiation of Atrichum moss calli on MS40 / BM

Species
No. of No. of Redifferentiation frequency
calli redifferentiation (%±SD)
Atrichum undulatum 95 / 94 91 / 90 (95.8±0.02) / (95.7±0.02)
A. undulatum var. minus 98 / 96 94 / 93 (95.9±0.02) / (96.9±0.00)
(Managing editor: WANG Wei)
植物学报 Acta Botanica Sinica Vol.45 No.12 20031480
中图分类号: Q944 文献标识码: A 文章编号: 0577-7496(2003)12-1475-06
收稿日期:2003- -10 接受日期:2003- 6-20
基金项目:北京市自然科学基金(5982007)。
*通讯作者。Tel: 010-68902374; E-mail:。
(责任编辑: 王 葳)
仙鹤藓属(Atrichum)藓类植物组织培养再生体系的建立
刚永运1 杜桂森1* 施定基2 汪楣芝2 李学东1 华振玲1
（1.首都师范大学生物系，北京 100037；2.中国科学院植物研究所，北京 100093）
摘要： 报道了仙鹤藓(Atrichum undulatum (Hedw.) P. Beauv.)和仙鹤藓小形变种(Atrichum undulatum var.
minus (Hedw.)Par.)的组织培养再生体系的建立。为研究仙鹤藓属(Atrichum)藓类愈伤组织的诱导和再分化，将
仙鹤藓和仙鹤藓小形变种原丝体接种在含有4%葡萄糖和0.2~ .0 mg/L 6-BA的MS培养基上，培养一个月后，成
功地诱导出疏松、易碎的绿色愈伤组织。愈伤组织诱导和常规继代培养较适合的培养基为含4%葡萄糖和1~2 mg/L
6-BA的MS培养基。当将继代培养5次的脱分化藓类愈伤组织转移到含4%葡萄糖但无任何激素的MS培养基上时，能
再分化形成原丝体，而在无任何碳源的Benecke培养基上培养时，能再分化形成经原丝体阶段发育来的直立配子体。
关键词： 藓类; 原丝体； 愈伤组织; 再分化