SHORT COMMUNICATION

In Vitro Regeneration and Micropropagation of Some Liverwortsfrom Vegetative Ex Plants

Vishal Awasthi • Virendra Nath • A. K. Asthana

Received: 23 June 2011 / Revised: 17 August 2011 / Accepted: 17 August 2011 / Published online: 27 March 2012

� The National Academy of Sciences, India 2012

Abstract In order to minimize dependency on sporo-

phytic material for establishing axenic culture of bryo-

phytes, in vitro regeneration and multiplication of three

medicinally potential liverwort taxa viz., Conocephalum

conicum (L.) Lindenb., Reboulia hemispherica (L.) Raddi

and Marchantia paleacea Bertol. have been carried out by

inoculating hormone free inorganic media with their apical

vegetative thallus parts. Concentrations: 2, 1, 0.5 and

0.25% of sodium hypochlorite solution for 2–4, 8–10,

15–30 s and 1 min were tested in order to find optimal

method of surface sterilization. The best out come resulted

from the application of 1% sodium hypochlorite solution

for 8–10 s. All the three species grew well in half strength

Knop’s macronutrients ? Nitsch’s trace elements with

10 ppm freshly prepared ferric citrate under the continuous

illumination of 4,500–5,000 lux at 20 ± 2�C temperature.

Ex plants (apical thallus part) directly regenerated into well

developed thalli, while spontaneous regeneration via callus

formation was observed in presence of certain contami-

nating microbes, in which aseptic condition eventually

achieved by repeated sub culturing.

Keywords Culture � Liverworts � Regeneration �Sterilization � Vegetative apical part

Introduction

In recent years, bryophytes have not only been proved as

favourable model system for morphogenetic, genetic,

physiological, biochemical, molecular and metabolic stud-

ies [1, 2] but also emerged as potential source of many

biologically active novel compounds pertaining to phar-

maceuticals [3–10]. Application of plant tissue culture in

bryophytes have also been encouraging for the isolation and

production of secondary metabolites or pharmaceutically

interesting substances [11–15]. But the available data on

culture of bryophytes is still far from the need keeping the

fact of huge diversity in this group (nearly 22,750 species)

[16]. Only a very few species have been attempted in culture

studies and in excess of 95% of all bryophytes have never

been cultured [17]. This situation is chiefly due to difficulty

in availability of ripened and undehisced sporophytic

material in a large number of bryophytes while spore culture

is the safe and easiest mode of establishing axenic culture, as

the capsule wall is protective enough to protect spores from

the toxic effect of surface sterilizing agents during the trial

and make the spores remain viable. The major problem in

culturing bryophytes is surface sterilization of the explants

while dealing with gametophytic tissue or vegetative prop-

agule as explants. As these ex plant materials are very del-

icate, devoid of any covering cuticle layer and only a few

celled thick, often killed during surface sterilization and thus

added to the dilemma of over-sterilization versus contami-

nation. Although some workers [17, 18] generalized the

concentration of surface sterilizing agents and exposure time

for different types of bryophytic tissue, these information

seems insufficient and also need extreme care and critical

attention during the trial. Hence only a limited number of

species in which sporophytic material is commonly avail-

able were attempted in culture studies, even those studies in

which regeneration achieved via callus route the explants

were usually spores [1, 19, 20]. But a large number of bry-

ophytes never or rarely produce sporophytes. The life cycle

of bryophytes reveals certain limitations which impose

V. Awasthi � V. Nath � A. K. Asthana (&)

Bryology Laboratory, CSIR-National Botanical Research

Institute, Lucknow 226001, India

e-mail: drakasthana@rediffmail.com

123

Natl. Acad. Sci. Lett. (January–February 2012) 35(1):7–12

DOI 10.1007/s40009-011-0001-y

constraints on sexual reproduction such as requirement of

water for fertilization, limited durability of the sporophyte,

dioecism etc. [21]. As an ‘‘escape’’ from these limitations

asexual or vegetative reproduction is heavily relied upon

majority of bryophytes, which often leads genetic stenotypy

[22]. Such species inherently adapt somatically, rather than

genetically in response to various environmental factors, any

major change in their habitat may lead to the extinction of

entire biotype as a whole [23, 24]. Such species call for

efficient method of culturing vegetative part from the con-

servation point of view. Efficient protocol for the in vitro

propagation of Lunularia cruciata Dum. has been estab-

lished recently from gemmae [25], which is the only mean of

reproduction in this species. Hence, in order to reduce

dependency on sporophytic material, development of cul-

ture protocols from vegetative ex plants becomes pertinent

for bryophytes particularly for those species in which spo-

rophyte is not commonly available or species purely rely on

asexual or vegetative reproduction from the conservation as

well as bioprospection point of view.

In the present contribution, in vitro regeneration and

multiplication of three medicinally potential liverwort taxa

viz., Conocephalum conicum (L.) Lindenb., Reboulia hemi-

spherica (L.) Raddi and Marchantia paleacea Bertol. have

been achieved by inoculating defined hormone free inorganic

media with apical vegetative parts. The selected liverwort

taxa are of medicinal value as these are used as antipyretic,

antidotal, diuretic, for cure of cuts, fracture, snake bite, burns,

scalds, haemostasis, external wounds and bruises [26].

Marchantin–A a bis bibenzyl isolated from M. paleacea and

R. hemispherica, while Riccardin-C isolated from R. hemi-

spherica [27] display cytotoxic activity against KB cells [26].

These species also display significant antimicrobial activity

against several human pathogenic bacteria [3, 28] while

C. conicum has been reported to have antibacterial activity

against burn infection [29] and antifungal activity against the

growth of Macrophomina phaseolina causing charcoal rot of

soybean [30]. Klebsiella pneumoniae, a gram negative human

pathogenic bacteria, refractory to many drugs is inhibited

strongly by the extract of C. conicum, R. hemispherica and M.

paleacea [3]. The mature and undehisced capsules of these

species are not commonly available due to highly seasonal

behaviour of sporophyte production. Hence the described

method of culturing these liverwort species may be useful as a

model for establishing axenic cultures of not only other

potential liverwort taxa having economic or medicinal value

but also rare, endangered and threatened taxa.

Materials and Methods

Plants of C. conicum, R. hemispherica and M. paleacea

were collected from their natural habitats in western

Himalaya and brought in polythene bags. The pure patch of

these species along with their substratum soil were kept in

humid chambers under low temperature (20 ± 2�C) and

sprinkled with water once in every day. After about

7–10 days several innovations emerged out from the thalli

that tended to grow vertically upward. These actively

growing innovations were detached from the thalli with

taking precaution that no or minimum soil particles came

with these innovations in order to minimize chance of

contamination. Now these innovations were washed thor-

oughly with running tap water followed with double dis-

tilled water. Before inoculation of media, these washed

innovations were immersed into 2, 1, 0.5, 0.25% sodium

hypochlorite solution for 2–4, 8–10, 15–30 s and 1 min in

order to determine optimum concentration and exposure

time in sodium hypochlorite solution for the surface ster-

ilization and subsequently washed with sterilized double

distilled water twice. The apical part about 2 mm in length

of these surface sterilized innovations were cut aseptically

and inoculated with the culture media in a Laminar Air

Flow Cabinet. The culture medium used was half strength

Knop’s macronutrients ? Nitsch trace elements with

10 ppm freshly prepared ferric citrate as it suited well for

the micropropagation of some other liverworts of Mar-

chantiales viz., Lunularia cruciata, Marchantia paleacea

[25, 31]. Addition of 1% sucrose also tried as external

carbon source.

All media were gelled with 0.8% agar (bacto–grade) and

pH was maintained at 5.8 before autoclaving. Culture media

and glasswares were sterilized by autoclaving at 15 lb/sq in

for 15 min. After inoculation cultures were maintained

under controlled and aseptic conditions. Cultures were

provided continuous illumination of 4,000–5,500 lux as well

as alternate light and dark period of 14 and 10 h respectively

with the help of a combination of fluorescent tubes. Tem-

perature was maintained at 21 ± 2�C.

Results and Discussion

The best outcome in reference to surface sterilization

resulted from application of 1% sodium hypochlorite for

8–10 s. The higher concentration (2% or more) even for

short exposure (1–2 s) proved toxic to plant material and

insufficient to kill all microbes, as a result necrotic tissue

developed within 2 days and an aggregation of microbes

(bacteria and fungi) developed on it, hence the material had

to be discarded. On the other hand longer exposure (30 s,

1 min or more) even in dilute hypochlorite solution (50 or

25%) although sufficient to kill microbes but also proved

equally detrimental to explant tissue and death of the

explants occurred within 4–5 days of inoculation. The

optimum concentration of sodium hypochlorite (1%) and

8 Natl. Acad. Sci. Lett. (January–February 2012) 35(1):7–12

123

short exposure (8–10 s) proved promising as the explant

remained alive and maximum microbes killed or if

remained alive not proved detrimental to explant instead

beneficial in regulation of morphogenesis in some ways.

After 6–7 days of inoculation, the explant (innovation)

of C. conicum turned into brownish green mass of

deformed tissue in which brown part represented the

necrotic area (Fig. 1a). The alive and green mass of tissue

subsequently regenerated into 2–3 young thalli in

10–15 days (Fig. 1b). These young thalli grew continu-

ously and developed into dichotomously branched thalli

after about 20–25 days (Fig. 1c). At this time a few

microbial contamination was appeared at the site of ex

plant inoculation along with few green callus like droplets.

Subsequent sub culturing of the apical part of growing

thalli (while taking precaution it should not have carried

adhering rhizoids that were in contact with medium sur-

face), resulted into establishment of aseptic culture that

subsequently multiplied and bulked up by repeated sub

culturing (Fig. 1d, e). In culture of R. hemispherica the

explants disintegrated into dark green coloured callus like

tissue after 7–10 days of inoculation. From this green mass,

several new regenerants appeared into next 10 days (Fig. 1,

f). Some microbial growth was also appeared at the site of

ex plant inoculation but that did not affect adversely the

growth of regenerants instead it enhanced the formation of

callus like growth of cells, while regenerants grew con-

tinuously into healthy vigorous dichotomously branched

thalli (Fig. 1g, h). In 1 month old culture the apical part of

the growing thalli were sub cultured (Fig. 1i) that gave rise

another population of healthy thalli. By repeated subcul-

turing in 2–3 generations the aseptic conditions were

achieved and thalli could be multiplied and bulked up

aseptically (Fig. 1j). However, in culture of R. hemi-

spherica, it was observed that the thalli raised in contam-

inated cultures were more vigorous and healthy in

comparison to that raised in aseptic cultures. The explants

of M. paleacea disintegrated into its component cells and

turned into bright green coloured undifferentiated liquified

mass of tissue after 6-10 days of inoculation (Fig. 1k),

from which several regenerants emerged out (Fig. 1l).

Bacterial growth was also appeared at this time. Subse-

quently profuse growth of green cells occurred that dis-

tributed evenly throughout the mediums. At several sites on

medium these cells clumped each other to form undiffer-

entiated callus like structure. In about 1 month old culture

several regenerant emerged from the callus like tissue as

well as from small clumps of green cells. These regener-

ants grew subsequently to develop young thalli (Fig. 1m)

that were differentiated into mature dichotomously bran-

ched thalli in about 50–60 days of inoculation (Fig. 1n).

The apical growing part of the thalli were detached (while

taking precaution it should not have carried rhizoids that

were in contact with medium surface) and placed in fresh

medium where they regenerated into several new healthy

thalli and only a few contaminations occurred at later stage.

By repeated sub culturing of 2–4 generations healthy and

axenic cultures of M. paleacea were established (Fig. 1o)

for the multiplication and bulking up. In culture of all the

three species it was observed that in medium supplemented

with 1% sucrose, the microbial growth as well as callus

like growth of green cells was prominent while regenera-

tion of thalli occurred scarcely. Temperature at 20 ± 2�C

and continuous illumination of 4000-5500 lux proved

favourable for the rapid growth and micropropagation of

thalli. Temperature above 30�C not only restricted the

regeneration process but also proved fatal for existing thalli

even for short time (1 h).

The gametophytic tissue of bryophytes are often water

repellant, hence the adhering air bubbles, when these were

immersed in hypochlorite solution act as contaminant

pockets preventing complete surface sterilization [17]. This

may be the reason that at optimum concentration of

hypochlorite and short exposure time in which explants

remained alive microbial contamination also occurred,

however at minimum level. Longer exposure in hypo-

chlorite even in dilute concentrations proved hazardous for

explants as the tissue of ex plants are only a few celled

thick and devoid of cuticle, hence sensitive towards

hypochlorite in longer duration through absorption by the

plant body surface. If we just washed thoroughly with

sterile distilled water, the microbial growth was too high to

be fatal for ex plants. Further many hepatics and hornworts

contain endophytic microbes [32] those can be killed only

when their surrounding gametophytic tissue also killed.

Hence, it seems to be prudent to establish growth of the

explant along with minimum microbial contamination

through optimum surface sterilization, from which axenic

state can be achieved through repeated sub culturing, a

some what analogous to serial dilution technique of

microbial isolation. An interesting aspect regarding to

regulation of morphogenesis through microbial contami-

nation was also observed during the experiment. The dis-

integration of the explants of M. paleacea and R.

hemispherica into their component cells, development of

callus like tissue and spontaneous regeneration of thalli

from such callus like tissue revealed the role of contami-

nant microbes into these morphogenetic process via

secretion of macerozymes like substances for deterioration

of cell walls of the explants and synthesis of certain auxins

and kinetin like growth hormones by the contaminating

microbes. Microbial interaction with the explants including

phytostimulation and circumvention of basal plant defence

mechanism might be elicited after surface sterilization trial

in response to chemical stress. Some scattered reports have

recently been published that revealed the role of

Natl. Acad. Sci. Lett. (January–February 2012) 35(1):7–12 9

123

Fig. 1 a–e In vitro growth of Conocephalum conium (L.) Lindenb. a,

b Growth of regenerant from the ex plant; c dichotomously branched

thalli developed from regenerant; d, e well developed thalli in aseptic

culture after repeated sub culturing. f–j In vitro growth of Rebouliahemispherica (L.) Raddi. f growth of young thalli from regenerant

emerged from dark green callus like tissue; g, h growth of

dichotomously branched thalli; i an apical portion of cultured thalli

after 7 days of sub culturing; j well developed thalli in aseptic

cultures. k–o In vitro growth of Marchantia paleacea Bertol.

k Degeneration of ex plant in its component cells and formation of

callus like droplets; l development of regenerants on callus like

growth on explants; m profuse growth of undifferentiated green cells

and regeneration of thallus; n development of thalli from callus like

growth; o well developed thalli in aseptic culture after repeated sub

culturing

10 Natl. Acad. Sci. Lett. (January–February 2012) 35(1):7–12

123

contaminating microbes in regulation of morphogenesis.

Kutschera and Koopman [33] reported that the methylo-

bacteria microbes that inhabit the surface of the liverwort

Marchantia and Lunularia secrete phytohormone cytokinin

and promote the growth of isolated gemmae cultivated on

agar plate. They concluded that normal development in

these taxa is dependent on (and possibly regulated by)

epiphytic microbes. Lata et al. [34] identified IAA pro-

ducing endophytic bacteria Pseudomonas stutzeri during

the micropropagation of Echinacea plant and found endo-

phytes are generally beneficial to plants in situ and may

affect culture growth under modified controlled conditions.

Kalyaeva et al. [35]. demonstrated that methylobacteria

actively promoted growth and morphogenesis in several

dicot and monocot plant species. Inoculation of the embryo

of Triticum aestivum with the strains of methylotrophic

bacteria led to their stable colorization with bacteria that

stimulated the formation of morphogenetic calli, shoots and

also promoted development of regenerant plants. In many

liverworts, the callus formation is induced by the applica-

tion of auxins and cytokinin [19, 36]. In the present work

callus like growth of the cells occurred in the presence of

contaminating microbes. Production of IAA from common

contaminating microbes and their potential application for

cell cultures of a medicinal plant Alternanthera sessilis

have recently been demonstrated [37]. Contaminating

microbes use IAA to interact with plant including phyt-

ostimulation and circumvention of basal plant defence

mechanism.

Thus common contaminating microbes can be utilized

for plant growth regulators and for further cell culture

studies in order to enhance the production of secondary

metabolites from the potential liverwort taxa. Benevolence

of the culture medium and controlled conditions for these

taxa was in congruence with our previous attempt to cul-

ture Marchantia, Lunularia from gemmae and Cryptomi-

trium himalayense from spore. Such strategy to raise the

bryophyte taxa from their vegetative part will certainly

enhance the scope of culturing wide range of species for

bioprospection and their potential utilization in secondary

metabolite production, apart from the conservation of many

bryophyte taxa including rare, endangered and threatened

taxa at the time of collection without waiting for sporo-

phytic phase. Aseptic conditions may also eventually be

achieved by subculturing of apical part of regenerant thalli

as these actively growing apical meristematic regions are

usually free from endophytic microbial contamination [38,

39].

Acknowledgements Authors are grateful to the Director, CSIR-

National Botanical Research Institute, Lucknow for encouragement

and providing facilities. Thanks are due to the Ministry of Environ-

ment & Forests, Govt. of India, New Delhi for providing financial

assistance.

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