in vitro Culture of Pinguicula and Drosera
By Dr. Laurent Legendre
Email : firstname.lastname@example.org
(Translation of the french article by Serge Mallet)
Pinguicula and Drosera have similar habits in vitro and in a culture pot. If a species reproduces by leaf or root cuttings in habitat, it will do the same in vitro. Similarly, if it forms winter hibernacula (temperate pinguicula), the hibernacula will be formed also in vitro. The only difference is that in vitro, the plants are growing on media that have been optimised. Light and temperature conditions are stable and also optimal. moreover, there is no risk to loose a plant by the attack of an infectious organism (fungus, bacteria, insect or nematode…). It is also more easy to observe the plants to check what is wrong and it is possible to artificially add vegetal hormones to enhance natural phenomenon such as growing of cuttings, vegetative multiplication or tubercle formation (for tuberous Drosera ). But if a species does not reproduce in his habitat by leaf cutting or bunch division as for example P. pumila, P. sharpii or P. ionantha , such multiplication modes won't be possible in vitro.
In vitro culture is thus a good tool for the germination of precious and rare seeds or to prevent a young and weak plantlet from dying consequently to all potential accidents that may occur in a pot.
As growing and multiplication is more rapid in vitro, this method also allows you to build up a collection more rapidly. Unfortunately in vitro culture is really time consuming and this is not always compensated by the results as many species of Drosera or Pinguicula grow and multiply rapidly in a greenhouse with suitable environmental conditions. Preparing and sowing in vitro culture containers is long and fastidious. As the plants grow rapidly and the containers are small, the in vitro media are rapidly consumed and the plants have to be moved to a new container every two months approximately. Moreover, what I would like to add that is rarely indicated in the literature on the subject : in vitro cultivated plants are loosing strength with each pricking out. They are often more subject to chlorose, grow more slowly and have advanced dormancy with reduced life cycles. This problem is certainly due to the lack of an oligo-element (Zn, I, B…) not present in sufficient amounts in the media. The dose initially present in the seed is progressively diluted in the growing plant to a limiting level. Drosera seem to be less susceptible to this phenomenon contrarily to Pinguicula Which are very sensitive.
for in vitro culture of these plants are now provided.
and Pinguicula appreciate a mineral
media complemented with vitamins, sugar (saccharose
or kitchen sugar), a jellying agent and in some occasions amino acids (generally
not essential). Most of the species are readily multiplied on such media and it
is not necessary to add hormones except for
particular applications such as the formation of cell suspension or
embryo regeneration. Media derived from those of Gamborg or Murashige and
Skoog (MS - 1962) are convenient and our plants are known to be especially good
growers on various media. Personally I use the complete media of Murashige and
Skoog adapted by Linsmaier et Skoog (1965).
important point is that the components of the original media have to be diluted
(with a factor from 4 to 8) for an optimal development of our plants. More
concentrated formulations lead to a slower growing rate and abnormal elongation
of the leaves and stems (P. gigantea
for example grows like a liana…) sometimes problems of cellular
differentiation are observed as for P.
vulgaris forming callus (mass of non-differentiated and non-organised cells)
and finally dying. For practical reasons the basal formulation (minerals and
vitamins) is diluted by 5 but the level of sugar and jellying agent is
maintained unchanged. The type of jellying agent used is also important : Phytagel induces more
spontaneous multiplication than
Agar-agar alone but the development of flowers is poor
with this component. Phytagel is a little more expensive but less is
needed and finally the expense is similar.
I recommend this product for it's positive effects on the plants.
and vitamins for MS media are sold pre mixed and ready to use.
I recommend using such packets as preparing the media is long and needs
some expertise. I also recommend
not to divide the powder in the packet as
some components are present in very low quantities
(such as a micro crystal) and, lost
in a great quantity of powder, it
won't be possible to part it equally. So, it is better
preparing the amount of medium recommended by the manufacturer (generally one litre for a packet) and then to divide the
liquid in which the homogenisation of the different components is better. After
sterilisation the medium can be stored at least 3 weeks in the dark in a
water to be used have to be very pure (distilled then desionised) in order not
to introduce any unwanted minerals. Nevertheless Pinguicula and Drosera are
not too much susceptible to water impurities and I have obtained good results
with commercial demineralised water.
the minerals and vitamins base is constituted, you just have to add the sugar
and to adjust the pH (protons
concentration) to 5,9 with a 0.1 M KOH solution. Jellying agent is then added.
It's dissolution is only possible with some heat provided for example by a quick
stay in a micro-wave Oven. At this step the
medium, still hot and liquid, is poured in the different containers (1-2 cm per
pot). Then the pots are sealed and sterilised 20 min in a pressure cooker
"Cocotte Minute" . During cooking, it is important not to seal the
pots hermetically to avoid bad surprises when you open the cooker. When
the pots are out, the covers can be sealed completely. After cooling the
media will be jellied with a light film of water on the surface.
theory, each part of our plants can be introduced in vitro : Leaf and root cuttings as well as seeds. The only
limiting factor is that these explants have to be safe of any disease and then
perfectly disinfected. If this step is completed successfully, all the parts
that would have grown outside will do the same thing in vitro. Now, if it
is easy to obtain a safe decontamination of
Drosera and Pinguicula
seeds, it is more difficult with leaves and roots.
Infectious germs are present more deeply in the tissues that needs a
longer decontamination. Unfortunately a too long decontamination leads
invariably to the death of the disinfected tissue. The leaves of our plants are
particularly susceptible to such treatments as their glands allow a rapid
absorption of the disinfecting agent . Nevertheless,
if the plant is healthy, just a light surface decontamination is necessary.
disinfecting agents are available : chlorine water (Eau de Javel), 70% ethanol,
fungicides, mercuric chloride…. Personally I prefer using calcium
hypochlorite. This product is close to chlorine water as it kills also all the
micro-organisms and animals but it is less toxic on vegetal cells. I prepare a
4% saturated solution that is homogenised by a 20 min agitation and then
filtered on a filtration paper. Filtration is a slow process and the filtrate
can be conserved at least a week but be careful as this product erode glass on
the long term… A treatment with pure formic acid allows to clean back the
attacked glass. To the filtrate I add a little amount (0.1 %) of a non ionic
detergent (Tween-20). As an
alternative a teeth pick just soaked a few seconds in your usual dish washing
detergent and then in the glass containing the filtrate will provide enough
moistening agent for a good contact between the explant and the disinfecting
solution. It is necessary to add always the same amount of detergent as the more
it is concentrated the more rapidly the samples are decontaminated. Moreover, as
soon as the detergent is added, calcium hypoclorite begins to slowly precipitate
and should be used within 2 hours.
the decontamination process, the explants are placed in a small volume of
freshly prepared disinfecting solution (enough to cover the leaves or roots or
10 ml for the seeds) in the bottom of a sealed plastic tube. It is very
important for the organs to be disinfected to be very clean (without earth,
insects or dead tissues…). After a vigorous agitation, a foam is forming at
the surface. The seeds of most of the species sink at the bottom of the tube
when they are alive while dead ones are
floating or trapped in the foam. Decontamination is stopped easily by discarding
gently the disinfecting solution and replacing it by sterile water (sterilised
as the medium). Generally the seeds
sink rapidly at the bottom of the
container and it is easy to keep it inside while discarding the solution.
Unfortunately, there are some exceptions such as D.
rotundifolia and P. primuliflora.
The duration of the disinfecting period depends on the size of the seeds :
8 min for the small seeds of
Mexican Pinguicula or
tropical Drosera and 12 min for temperate Pinguicula and Drosera.
Leaves and roots are decontaminated about 8 min. After
disinfecting, the samples have to be rinsed (3 times 5 min in large quantities
of sterile water) in order to eliminate all the disinfecting agent (if not
totally eliminated, it will continue it's action).
the disinfecting pot is decontaminated with the explant, the first step of the
sterilisation can be done without particular precaution. But when adding sterile
water for rinsing, transferring the
explant in the culture container and the growing plantlet from a pot to another,
a sterile environment is necessary. Professional laboratories use appropriate
enclosures providing an air
filtrated and without any contamination. Unfortunately
such an equipment is very expensive
and needs to be checked regularly. Nevertheless, it is possible at home to work
in a pseudo sterile atmosphere with limited contamination. For this, it is
necessary to work in a room without any air circulation and on a plastic surface
easy to decontaminate (with "eau de Javel" for example). Something
like the covering of a toilet seat is a good working surface (Yes, don't smile
!). For air sterilisation, you can use a powerful flame from
a small camping gas cooker. The zone of 20 cm around the flame is
considered as sterile if the opening of the containers are directed upright
as the heat of the flame induce a movement of the air
from the bottom to the top, preventing the
microbes from entering the pots. It's also a good idea (if the pots are
not in plastic !) to pass rapidly the opening
of the culture containers in the flame to heat the air inside thus
creating an air flux out of the container.
As an alternative it is also
possible to install on the working surface a plastic cylinder
(also decontaminated) large enough for all the instruments you need and
with lateral openings for the hands. You shall always use chirurgical gloves
regularly disinfected with 70 %
ethanol as shall be also disinfected all the instruments used for the
manipulations. Take care not to manipulate ethanol
near the flame as it may be dangerous.
plants are cultivated in vitro at a
constant temperature of 25°C,
with a relative humidity of 70% and with
a 16 hour period of light under neon bulbs (industrial white).
vitro, the plants do not develop a cuticle (wax coating). So, they are
dehydrating very fast when placed outside in the air. They are also more
susceptible to diseases. It is thus necessary to place the plants in an
enclosure with a temperature of 25°C and with a 100 % relative humidity during
the 2 first weeks outside. After that, the temperature is lowered to 18°C (still
constant) for a week before changing for alternate temperatures (25°C
during the day and 18°C
at night) which is the best for an optimal growing. This acclimatisation period is generally considered as
delicate. Nevertheless, our plants like a high humidity and the acclimatisation
is thus more easy. Failures occur when the protocol of temperatures is not
T. et Skoog F. (1962) Physiologia
Plantarum, 15, 473
E.M. et Skoog F. (1965) Physiologia
Plantarum, 18, 100
. 2 H2O