In vitro cultivation and conservation of bryophytes

1. CULTIVATION AND
CONSERVATION OF
BRYOPHYTES

2. BRYOPHYTES
• Bryophytes are second largest group of land plants.
• Rich in terpenoids, phenols, glycosides and fatty acids.
• Bryophytes can be used to produce medicines but does not have
enough quantities of same species.
• In-vitro cultivation of bryophytes is the most appropriate way of
large biomass production and isolate numerous useful
compounds showing biological activities.
• In-vitro cultivation not only used in cellular, developmental
and molecular research.
• Also show vital role in juvenile stages in reproductive biology.

3. IN-VITRO CULTURE TECHNIQUE
Cultures, initiated from surface sterilized spores, gemmae or
vegetative fragments.
Are handled easily in petri-dishes on media containing
inorganic salts.
Soldification of medium with phytogel or gelrite.
Dilution of nutrients are beneficial to the growth of some taxa.

4. In-vitro culture technique cont…
• Prior knowledge of seasonality of sporophytic production is
vital for establishing cultures from spores.
• Period of spore availability can be increased by storing ripe
capsules at 4 degree celsius.
• Irradiances in culture are sufficient for good growth.
• Temperature above 25 degree celsius are damaging.
• Cryopreservation techniques are being developed for long term
preservation of endangered taxa.
• In-vitro cultivation of hepatics and hornworts are complicated
by presence of fungal and cyanobacterial endophytes.

5. OBJECTIVES OF IN-VITRO CULTURE
OF BRYOPHYTES
• Further understanding cellular, genetic and molecular basis for
morphogenesis embracing phenomina such as tip growth,
gravitropism and mode of action of growth regulators.
• Elucidating roles of juvenile stages, particularly moss
protenemata and propagules they produce.
• Assessing significance of juvenile characters in systematics and
phylogeny.
• Maintaining rare and endangered taxa for their conservation,
cryopreservation and genetic analysis.
• To determine functional and ecological significance of fungal
and cyanobacterial association in hepatics and hornworts.

7. BASIC HARDWARE AND SOFTWARE
A. Culture vessels
• Disposable petri dishes are more convenient for maintainig
bryophyte culture.
• Where sporophytes, which exceed height of deepest dishes are
required-plants will be grown in test tubes or boiling tubes.
• Petri dish cultures dry out in 4-6 weeks.
• Their lonevity may be extended by ringing with parafilm.
• This may change morphology and growth rate of plants.
• Sealing of culture with micropore tape allows gaseous exchange
and increase rate of drying.
• Thus produces more natural protonemal growth.

8. Basic hardware and softwares
B. Light and temperature
• Most bryophytes, be they tropical, temperate or high latitude
will grow in-vitro with temperature 5-25 degree celsius.
• For tropical taxa- 20 to 25 degree celsius optimal.
• Those from cooler places-10 to 15 or at 5 to 10 degree celsius.
• Prevention of overheating is crucial.
• Temperature higher than 30 degree celsius –for a short period
can be fatal to most taxa.

9. Basic hardwares and softwares
C. Media
• Organic supplements may stimulate development and sugars
can be used in some taxa.
• It replace light requirement for growth.
• Dark-grown protenemal cultures –essential prerequisite for
gravitropism studies.
• Auxins –to stimulate regeneration from tissue fragments.
• Cytokinins-induce bud formation.
• Abscisic acid-induce protonemal brood cells.

10. Media cont...
• In higher plants- callus formation induced using media
containing sugar, growth regulators and coconut milk.
• Increase in ph and accumulation of metabolites-lead to death of
cultures.
• Agar are commonly used.
• Recently, phytogel or gelrite are widely used.
Advantages of gelrite over agar:
i) lack of organic compounds.
ii) being completely clear when solid.
iii) allow easy observation of samples and signs of
contaminations.

11. Media cont…
Disadvantages of gelrite over agar:
i) gelrite require cations to soldify.
ii) water in gelrite soldified medium is loosely bound.
iii) results in loss of structure over time and medium
become liquified.

12. SOURCES OF MATERIALAND
STERILIZATION PROTOCOLS
• Culture- surface sterilized for 1-2 min in either 1.5% sodium
hypochlorite.
• Contain a drop of detergent as wetting agent or sodium
dichloroisocyanurate (NaDCC).
• NaDCC- effective against vegetative bacteria, bacterial spores,
fungi, algae, protozoa and viruses.
• NaDCC- reduce effectiveness of sterilization resulting tissue
death.
• Thus not used in sterilizing solutions.

13. TECHNIQUE
1) Collect bryophytes plants with or without sporophytes.
2) Collect plants with healthy immature sporophytes that contain
spores.
3) Wash them thoroughly in running tap water for 5 min.
4) Surface sterilize unopened sporophytes in 10-19% sodium
hypochlorite solution and 8% active chlorine for 5-10 min.
5) Time can vary depending on species.
6) Rinse them 3 times in cold, sterile distilled water for 5 min.
7) Open sporophytic capsules aseptically.
8) Transfer spores with a sterile needle to petri dish containing
20ml solid nutrient MS medium.

14. Technique cont…
9) MS media contain micro and macro salts, without sugar, and
phytohormones, pH-6.0.
10) Incubate cultures at 25+/- 2 degree celsius for 1-3 week in
long day conditions.
11) Within 1-3 week spores start germination.
12) Protonema – develop on same media within 1-4 week.

15. Tehnique cont..
When plants are collected without sporophytes:
1) Take apical shoots of plants.
2) Remove them from soil.
3) Wash them thoroughly in running water for 30 min.
4) Surface sterilize small gametophyte apical shoots in 7-10%
sodium hypochlorite solution containing few drops of liquid
soap for 5 min.
5) Concentration of sodium hypochlorite vary depending of
species.
6) Thus for sterilization , use different commercial bleach
solutions.

16. Tehnique cont…
7) Rinse sterilized apical shoots 3 times in cold, sterile distilled
water.
8)Transfer 10-20 apical shoots using sterile forceps to petri
dishes containing 20ml MS medium.
9) Incubate culture at 25+/-2 degree celsius for 4-8 week in long
day.
10) Protonema develop on same medium within 4 weeks.

17. REFERENCE
1)https://www.tandfonline.com/doi/abs/10.1179/174328213X137
89822578469.
2) https://pubmed.ncbi.nlm.nih.gov/19521840/
3)https://www.researchgate.net/publication/233595578_In_vitro_
cultivation_of_bryophytes_A_review_of_practicalities_problems
_progress_and_promise
4) https://link.springer.com/protocol/10.1007/978-1-60327-287-
2_10