Cell cultures

Quit Smoking Magic

How to Quit Smoking Cigarette

Get Instant Access

Alkaloids can be produced by using cell culture techniques in the same or a similar way as with other molecules624. Alkaloids such as ajmalicine, serpentine (C. roseus) and berberine (Coptis japonica) can produce remarkable quantities of alkaloids in in vitro cultures625 626. Generally, cell cultures produce species-specific alkaloids. In some cases cell cultures can produce more alkaloids than the whole plant, in relation to dry weight. This occurs, for example, in the cases of nicotine, ajmalicine and berberine. According to estimations, cell cultures of Nicotiana tabacum can produce up to 3.40% alkaloids of cell dry matter when the plant of this species can produce only 2.5% of its dry matter. In a similar manner, ajmalicine in C. roseus can be produced as 0.26% of whole plant dry matter, but as 1.3% of the dry matter of cell cultures. This is possible by the addition of enzymes to the cultures. These enzymes catalyse the biosynthesis of alkaloids by cells. Furthermore, cell suspension cultures offer the possibility to produce alkaloids in a truly biological method quite rapidly, on a large scale in an artificial environment. The research of cell physiology, molecular biology, gene engineering, genetics and other branches of biology often depends on cell cultures. They have also an important role in plant breeding and vegetative reproduction of crops and decorative plants. The term "cell cultures" is very similar with the previously widely used term "tissue cultures." Cell culture production is a routine laboratory technique from the 1950s, when it was common in animal cell and later in plant cell research. The idea of the possibility of maintaining and growing living cells was established in 1885 by Wilhelm Roux, who had experimented with parts of an embryonic chicken. However, the first mass-produced products using cell-culture techniques were polio vaccines in the 1950s. The cell-culture technique is based on the maintenance of cultures with nutrients and growth hormones in the medium (Figure 96). Sterility is a basic requirement in the cell culture. Presently, cell cultures can be stored in vitro for a long time using cryopreservation techniques627.

Cell-culture technique use in alkaloid production contains the following stages: (1) solid medium preparation and sterilization of the container for growing, (2) explantation of cells or tissues from plant and the start of growth, (3) primary callus formation, (4) isolation of growing callus and (5) initiating organogenesis of callus directly or using a protoplast cultivation technique by way of hybrid to initiate organogenesis (Figure 97).

Figure 96. Diagram of alkaloid production by cell culture. Abbreviations: A - alkaloid synthesis.
Figure 97. Cell-culture techniques in the organogenesis stage. Arrows indicate medium. (Photo: T. Aniszewski)

The hybrid is able to produce more alkaloids than the basic callus, which is an undifferentiated mass of cells. Alkaloid production in cell cultures can be more successful with the immobilization of plant cells and enzymes and by using bioreactor systems628'629'630. Alkaloid produced in cell cultures can be isolated directly from this culture or from young plants grown from this culture. More than 250 alkaloids are reported to be produced by cell-culture techniques. Only a limited number of species have been researched in this respect. The species studied are known to produce alkaloids with special use in applications. The most researched alkaloids produced by cell cultures are mentioned in Table 25.

Alkaloids can be produced in batch suspension cultures, semi-continuous and continuous cultures. Batch suspension cultures multiply the cells in a liquid medium by using a closed system in which only gases are changed. Generally,

Alkaloids - Secrets of Life Table 25 Some alkaloids produced by cell cultures

Alkaloid Name

Cell Origin

Ajmalicine

Rauvolfia serpentina

Catharanthus roseus

Akuammicine

Catharanthus roseus

Catharanthus ovalis

Rhazya stricta

Anabasine

Duboisia hopwoodii

Duboisia hopwoodii

Nicotiana tabacum

Nicotiana rustica

11-Allylcytisine

Cytisus canariensis

Berbamine

Stephania cepharantha

Berberine

Berberis wilsoniae

Coptis japonica

Thalictrum minus

Biscoclaurine

Stephania cepharantha

Caffeine

Coffea arabica

Camptothecin

Camptotheca acuminata

Canthin-6-ones

Ailanthus altissima

Brucea javanica

Catharanthine

Catharanthus roseus

Cephalotaxine

Cephalotaxus harringtonia

Cepharanone

Stephania cepharantha

Cinchonidine

Cinchona ledgeriana

Cryptopine

Papaver somniferum

(+)-Eburnamine

Tabernaemontana divaricata

Rhazya stricta

Ellipticine

Ochrosia elliptica

Emetine

Cephaelis ipecacuanha

Harringtonine

Cephalotaxus harringtonia

Quinine

Cinchona ledgeriana

Cinchona pubescens

Harman

Peganum harmala

(—)-Lupanine

Genista pilosa

Cytisus scoparius

Magnoflorine

Coptis japonica

Corydalis incisa

Eschscholzia californica

Fumaria capreolata

Papaver bracteatum

Papaver somniferum

Thalictrum minus

Applications Table 25 (Continued)

Alkaloid Name

Cell Origin

N-Methylcoclaurine

Fumaria capreolata

Morphine

Papaver somniferum

Papaver setigerum

Nicotine

Nicotiana tabacum

Nicotiana rustica

Duboisia hopwoodii

Duboisia myoporoides

Noscapine

Papaver rhoas

Papaver somniferum

Rutacridone

Ruta graveolens

Sanguinarine

Chelidonium majus

Corydalis ophiocarpa

Eschscholzia californica

Fumaria cordata

Macleaya cordata

Skimmianine

Choisya ternata

Solasodine

Solanum dulcamara

Solanum nigrum

Solanum laciniatum

Stephanine

Tinospora caffea

Tinospora cordifolia

Strictosidine

Rauvolfia serpentina

Catharanthus roseus

(—)-Tabersonine

Catharanthus ovalis

Stemmademia tomentosum

Voacanga africana

Theobromine

Theobroma cacao

(—)-Tinctorine

Cytisus canariensis

Tomatidine

Lycopersicon esculentum

Vinblastine

Catharanthus roseus

Vincristine

Catharanthus roseus

Vindoline

Catharanthus roseus

Voafrine A

Voacanga africana

Voafrine B

Voacanga africana

this method contains the following phases: (1) inoculation of cells into the medium, (2) a lag phase in which cells slowly grow, (3) a rapid division phase and (4) slower division. The duration of Phase 2 is from 0 to 9 days and of Phase 3 is 9 days. In semi-continuous cultures, the medium is removed after Phase 3 and replaced by a new medium. In this way cell growth is continuous, although there are cells from different mediums. In continuous cultures, a fresh medium is continuously added. There are two open systems in the arrangement of continuously growing cultures: chemostat and turbidostat. In the chemostat arrangement a steady state is achieved, controlled and monitored. The turbidostat arrangement is based on the maintenance of a constant cell concentration. Plant cells in cultures typically form cell aggregates. These aggregates can form small groups of cells.

In cell suspension cultures producing alkaloids, it is possible to choose and grow high-yielding cell lines. Cells and their metabolic capacity may differ as a result of genetic variability, the explant determination and cell culture acting adaptation631'632,633,634,635. Therefore clones and hybrids are generally used in cell cultures because of their higher capacity to produce alkaloids. They also have more stable and similar cells. Such clones and hybrids have been developed with nicotine (N. tabacum) and in hyoscyamine production (Duboisia leichhardtii)636. Moreover, there is evidence that the conversion of nicotine to nornicotine occurs in tobacco cell cultures637.

One of the problems of alkaloid production in cell cultures is not only the different capacities of diverse cells resulting from somaclonal and cell-to-cell variation or the necessity to use clones and hybrids for this purpose, but also low alkaloid yield. This problem is necessary in avoiding enzyme addition and the establishment of specific cells. In the case of quinolizidine alkaloids, especially in lupins, there are also some problems with cell cultures establishing and growing beyond the callus. According to recent observations and measurements it is difficult to obtain quinolizidine alkaloids from L. polyphyllus cell cultures without special treatment. However, in the cell cultures of L. polyphyllus Lindl., the production level of lupanine is only 0.5% of the level found in the growing plant638'639. Hybrids and enzymatic treatments of cell cultures increase this rate if the cells explanted for inoculation in cultures do not come from alkaloid-poor plants, which genetically block the production of lysine, a precursor of the quinolizidine alkaloids7'348'640'641. Morphine production in P. somniferum cell cultures without special treatment is known to be low. However, there is also data which shows that the yield of morphine from P. somniferum suspension cultures was 2.5mgg-1dw and that of codeine was 3mgg-1dw642. According to this research, the removal of hormones increases the alkaloid production rate. However, it is necessary to mention that despite some problems in alkaloid production in cell cultures, there is evidence that some alkaloids (e.g. serpentine, nicotine, canthin-6-one etc.) can be produced in cell cultures at higher levels than in whole plants.

The special treatment of cell cultures for increasing the yield of alkaloids can be linked to improvements in medium composition, the addition of enzymes and alkaloid precursors, the addition of the so-called "elicitors" and changes in environmental factors. The medium can be improved by changes in the relations between its components: sucrose and carbon components, nitrogen, phosphorus and hormones639'643'644'645. Higher alkaloid yields have been achieved by using this method642. The addition of enzymes and alkaloid precursors improves alkaloid yields263'264'646. The addition of tryptophan to Cinchona ledgeriana cell cultures increased the production of quinine and quinidine by up to 25%. Similarly, the addition of tryptophan to C. roseus cell cultures increases ajmalicine and serpentine production by more than 20%. Moreover, by immobilizing cells and enzymes it is possible to increase alkaloid yield in cultures and improve the culture system activity. Matrices such as agarose, alginate or polyacrylamide are used in immobilization techniques. It is known that sanguinarine, chelery-thrine and macarpine production in the cells of Eschscholtzia californica was enhanced by sodium alginate and by entrapment in Ca2+-alginate647. Tyrosine decarboxylase, which is a key enzyme for alkaloid biosynthesis in this species, has especially been induced by both sodium and calcium alginate. Moreover, it is also known that the immobilized cells of C. roseus produced ajmalicine and serpentine over 4 months629'648'649. Immobilized cells can also influence alkaloid conversion reactions such as the transformation of (—)-codeinone to (—)-codeine (P. somniferum). Alginate-entrapped cells can enhance alkaloid biosynthesis in cell cultures even up to 800-fold647.

Better yields of alkaloids in cell cultures have been obtained by the addition of elicitors, that is micro-organisms. This had been reported already in the 1980s and 1990s650 651'652'653 654. The detailed mechanism of an elicitor's influence on alkaloid synthesis is a challenge for future studies. However, in cell cultures producing alkaloids, environmental factors are of great importance. For example, dissolved oxygen concentrations up to 50% in mediums increased berberine production in cultures of Thalictrum minus655. The influence of light on alkaloid production in cell cultures of C. roseus is also noted656, though its influence is considered to be more cross the temperature and energy connected with the light. There is evidence that alkaloid production occurs in the dark in quinolizidine alkaloids, although older literature notes that their formation is associated with chloroplasts638. If all other alkaloids are produced in the dark, quinolizdine alkaloids should be also produced in such environments. No reason why quinolizidine alkaloids should be an exception exists. There is evidence that the transfer of cell cultures from dark to light environments causes a decrease in alkaloid production, and the movement of cell cultures from light to dark environments influences the increase of alkaloid production657.

Was this article helpful?

0 0
Quit Smoking Today

Quit Smoking Today

Quit smoking for good! Stop your bad habits for good, learn to cope with the addiction of cigarettes and how to curb cravings and begin a new life. You will never again have to leave a meeting and find a place outside to smoke, losing valuable time. This is the key to your freedom from addiction, take the first step!

Get My Free Ebook


Post a comment