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Micropropagation
of carnivorous plants
in vitro culture

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Photo-gallery in vitro

There is a special section of micropropagation of carnivorous plants: cultivation in sterile environment on special media. The principle of these technologies is based on transferring plant material into sterile, germless environment where no other organisms are present. Transferring into sterile environment is vital for undisturbed cell-process and when influenced by hormones, genetic disposition of a given plant is used for its broad propagation. Fibrillation of new plants proceeds in such a degree that it would not be possible in natural conditions. This way we can get within one year 70000 plants on the area of 1 squared meter. For Venus flytrap even 100000 ordinary sundews etc. These are great advantages of these technologies. In a relatively short time, it is possible to multiply very rare or in nature endangered species of plants and get them into collections of herbalists without violating their natural biotopes whether by changes caused by human activities or by changes of climate. And that can be done just by taking an axillary bud from a mother plant or by using several seeds for obtaining sterile culture.
It is necessary to be concerned with sterility of used plant material, sterility of the environment in which we work with explants and with sterility of culture media in the whole process of cultivation. Cultivation in aseptic environment has its own disadvantages (expensive apparatuses, chemicals, work difficulty and discomfort in the box) but it is essential considering that there are mainly sugar and vitamins in the medium. In such environment bacteria and fungi grow faster than explants themselves and are able to damage the whole culture in a short time. The first and the most important step is to get primary sterile culture. Part of the plant released from all heterogeneous organisms.

pasáž

Preparation of the passage
Containers with medium after sterilization in autoclave

Process of micropropagation can be divided into several phases. First is selection of a type of explant (a part of mother plant that we will evaluate also from the viewpoint of its health and condition. According to the species we will chose a principle of micropropagation and then we will take off the appropriate segment. The whole leaf, stem, corolla and from the growing top it can be the active part which is called meristem. It is an accumulation of cells sized c. 0.2mm which has no connection to vascular bundles, the main ways of dissemination of pathogens. This technique has a high importance in sanitation of plants from viruses and bacterial and sponge contagion. From germinated seeds it can be a part of hypocotyl – the first undercotyledonous article or isolation of embryo without endosperm which can inhibit its progress.
The second phase is derivation of sterile culture. Perfect disinfection of explant is the first step to success. Here is used quite a number of chemicals starting with antibiotic, ethanol, commercial bleach liquids. These are combined and repeated if needed.
The third phase is micropropagation. Here with the help of combination of hormones (cytokinin and auxin) occurs fibrillation of new stems in large. These are then divided – put into other containers on a fresh medium till the required number.  Multiplication coefficients are different for different plants. Every species can have different requirements regarding basic medium composition – concentration micro and macro elements, vitamins and types and combination of hormones.

Composition of basic medium

Culture medium is usually composed from micro elements, macro elements, vitamins, hormones, sugar and gelling substance. Regulating pH solution according to requirements – usually 5.5 – 5.8
The mostly used medium is MURASHIGE & SKOOG which was developed for tobacco culture in the 60th of the last century and it is used in different modifications up to the present day.

Comparing values of concentration of micro and macro elements for some media
M&S medium mg/1
Knudson C orchid medium/Morel modifikation/ mg/1
Anderson´s
Rhododendron
medium mg/1
Schenk&Hildebrandt
medium mg/1
Mikro elementy
CoC12.6H20
0.025
0,025
0,1
FeSo4.7H2O
25,00
CuS04.5H20
0.025
0,025
0,2
FeNaEDTA
36.70
73,40
19,8
H3B03
6.20
6,2
5,0
KI
0.83
0,3
1,0
MnSO4.H20
16.90
5,68
16,90
10,0
Na2MoO4.2H20
0.25
0,25
0,1
ZnS04.7H20
8.60
8,60
1,0
Makro elementy
CaCI2
332.02 
332,02
151,0
Ca(NO3)2
241,30
KCI
250,00
KH2PO4
170.00
250,00
KN03
1900.00
480,00
2500,0
MgSO4
180.54
122,15
180,54
195,05
NH4N03
1650.00
500,00
400,00
(NH4) H2PO4
300,0
(NH4)2SO4
500,00
NaH2PO4
330,60
celkem
4302,09
1894,13
1828,86
3183,25

From vitamins which are added to media, thiamin is essential. It is used in different concentration from M&S medium 0,1 mg/l, Nitsch medium 0,5 mg/l, Schenk&Hildebrandt  5mg/l, to Gamborg B5  10 mg/l. Other used vitamins myo-inositol, nicotinic acid, piridoxin etc. are supportive for growth stimulation but in many cases it is possible to do without them. Next important component for composition of a medium are plant hormones. We differentiate five basic categories: auxin, cytokinin, giberelin, abscis acid and ethylene. We will concern mainly about first three of them.

From the category of auxin we can mention:


INDOLE-3-ACETIC ACID                      IAA
INDOLE -3-BUTYRIC ACID                   IBA
a-NAPHTALENE ACETIC ACID             NAA

2,4-DICHLOROPHENOXYYACETIC ACID              2,4-D,
4-AMINO-3,5,6,-TRICHLOROPICOLINIC ACID       picloram

Auxins support cell lengthening, cause apical dominance, stimulate progression of vascular bundles and growth of roots and also support founding adventitious roots.

Cytokinins that are often used:

(furfurylaminopurin)             Kinetin
Benzylaminopurin                BAP
Thidiazuron
zeatin
izopentenyladenin

Some of the important qualities of cytokinins are stimulation of cell division/cytokineze, suppression of apical dominance and initiation of adventitious buds.
Today we know more than 80 types of Giberelins, marked as GA3, GA7 etc. Their important function is rupture of dormancy buds and seeds, mobilization of storage liquids in seeds during germination, induction of seed germination, blooming induction, lengthening of internodes connected with growth support.
Agar is very often used for hardening media. For production of agar is used thallus red wrack from families of Gracilariceae and Ceramiaceae. They affect in the warm area of The Pacific and Indian oceans. Generally are used the following concentrations of agar: 0,6-1,0%. Next to function of supporting the structure it also has another function – the chemicals are equally dispersed in the matter and do not settle as they do in fluid state media. Fluid state media have the advantage of a better contact of explant with medium but it is necessary to move them continually. We can also name Gerlite or Phytagel as gelling substances.

Every cell contains a whole set of genetic information which are vital to create the whole plant. Gene expression is exactly regulated for differentiation of different organs in time and in space. When working with in vitro culture, we have to stop this process and substitute it by another program. To start somatic embryogenesis it is the program of sympathogonia. Somatic embryogenesis we can divide into direct where sympathogonia are already on explant and require only good conditions for expression. And indirect somatic embryogenesis where occurs redetermination of differentiated cells and in the part of risen calus will induce embryogenely determinated state. Culture derivation with calus and following differentiation of adventitious buds is from the viewpoint of immunity very delicate because of a possible creation of undesireable mutants. The process goes via restitution and propagation where already existing basics regenerate.
The fourth phase is stem lengthening and stabilization of plants. At this phase it is necessary to eliminate hormones concentration impacts, to stop setting-off and prepare strong active plant for transmission to creation of root system.
The fifth phase is the creation of root system. It can be either in vitro on the particular medium which might be better in fluid state in many cases, or ex vitro. Very often is used pulse souse of a low part of footstalk in auxin solution and subsequent direct placing of plants into substrate.
The final phase is plant acclimatization. This step of micropropagation requires lots of attention. Plants that are grown in enclosed places with high air humidity, isolated, without influence of stress common in open spaces are very vulnerable when being transferred to outside conditions.
The problem that plants have to face is transit from situation when though being able of partial photosynthesis they need also organic substances for development to the state of being fully autotrophic. That means to the state when the source of carbon is carbon dioxide and light delivers energy. Sugar in the medium inhibits photosynthetic apparatus and the plant when being transferred into ex vitro conditions has to level its carbonic balance gradually. At some plants occurs creation of a different inner leaf structure, insufficiently developed cuticle which covers outer sides of cuticle cells and impedes water losses and sometimes there are problems also with functionality of leaf vents.

Every new taxon in in vitro culture has to be tested on a long term basis on reactions of different ratios of medium elements so that to achieve high-quality physiological state of a plant without any anomalies and so that the plant can sensitively react to stimulation in medium as necessary.
With Droser series we evoke creation of adventitious buds on leaf segments, with flytraps we evoke their rich creation of new stems and creation of stems on all leaf surface including traps. With genus Nepenthes it is possible to incite fibrillation of axillary buds.
There are special laboratories of tissue cultures concerned with these techniques. They develop culture media and designate suitable technologies of micropropagation. For endangered species of flytraps and mostly plants that propagate with difficulty in natural conditions and in conditions of artificial cultures and are generally too little available, these methods are the only source of their distribution to herbalists and their preservation for future generations.

Dionaea muscipulaDionaea muscipula

23Dionaea muscipula

 

Dionaea muscipula
fibrillation of new plants from the whole leaf surface
from upper and lower center of insect trap organ
the phase of propagation

Drosera regiaDrosera regiaDrosera regia


Drosera regia
initiation of stem creation from a leaf surface     the phase of propagation and acclimatization

Drosera lanataDrosera lanata

Drosera lanata


Drosera lanata
start of culture of the phase of propagation and acclimatization

Drosera paucifloraDrosera pauciflora


Drosera pauciflora
creation of adventitious buds on leafs that are in contact with medium and the phase of acclimatization

Drosera praefoliaDrosera praefolia


Drosera  praefolia
rich creation of adventitious buds in a lower part of footstalk and a top view of this beautiful drosera

kultivačka

Insight view of a cultivation room

 

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