in vitro Culture of Pinguicula and Drosera

By Dr. Laurent Legendre

Email : l.legendre@uws.edu.au

(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.

Details for in vitro culture of these plants are now provided.

Preparation of culture media

Drosera 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).

 One 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.

 Minerals 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 refrigerator.

The 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.

When 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.  

Choice of the explants

 In 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.

Disinfection of seeds and other parts. 

Many 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.

For 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).

Working in sterile conditions

 As 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. 

In vitro culture conditions

The 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).

Acclimatisation of plants from in vitro culture 

In 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 respected.

Murashige T. et Skoog F. (1962) Physiologia Plantarum, 15, 473

Linsmaier E.M. et Skoog F. (1965) Physiologia Plantarum, 18, 100