To use cultured cells rather than plant is one other possible alternative production method. Genetic manipulation in plant cell cultures by delivering foreign DNA into plant cells allows stable transgenic integration into the recipient genome and permits not only sustained cell proliferation but also production of natural plant products of interest. Four different bacterial strains were used in our experiments - TR 10-5, R 1601, ATCC 15834, and LBA 9402. A variety of experimental approaches have been used to increase the efficiency of the production of saponins, polysaccharides and alkaloids in hairy roots of different plants. Rapidly growing root lines initiated from Astragalus, Datura, Hyoscyamus and Althaea plants were selected for cultivation in an air-lift bioreactor.
Due to their complex structure alkaloids, saponins, polysaccharides and flavonoids are still most efficiently produced by the plants. Production of these compounds has been reported for several genera (1, 2, 3). However there are several problems connected with this production method (3) Variable quantities and qualities of the plant material, plants that need to grow several years before they are ready for harvesting and over collecting of endangered species are just a few of the problems connected with the production of these natural products. Therefore, cultured cells rather than plants are as a possible alternative production method.
It has been shown that, on many plant cell culture the growth without differentiation is incompatible with the expression of a secondary metabolic pathway (3) or that the use of differentiated cultures does result in considerably higher and stable production of the compounds of interest (1). Since the root is mostly the site of saponin, alkaloids and polysaccharide biosynthesis, in vitro root cultures from different Astragalus, Datura, Hyoscyamus and Althaea species have been able to produce large amounts of secondary metabolites (1, 2, 3).
In order to produce highly productive transformed root cultures it is essential to use plant material selected for its interesting metabolite spectrum and with a high content of biologically active substances relative to other plants of the same genus. The selection of plants (3) showing desirable properties was the first step in obtaining hairy root cultures.
Transformation procedure
Hairy root cultures have been established from a variety of species.The bacteria were maintained on solid YMB media as described earlier (2, 3). Before the infection the bacteria were grown for 48 hours at 26°C, 80 ppm in liquid YMB. The bacterial concentration may to effect the infection and in this way to have an effect on the number roots formed. Concentration below or highly the optimum (0.9ˇ108 cellsˇml-1) may result in a lower availability of bacteria for transforming the plant cells.
Excess bacteria were eliminated on agar-solidified MS medium, supplemented with 0.5 mg/ml Claforan. After 3 weeks of culture, the tips of newly formed roots were excised and placed on fresh medium of the same composition. The roots were cultured at 25°C in the dark, at 80 ppm and subcultured at 4 weeks.
It was observed, that the infection should be done immediately after wounding in order to get the maximal number of roots as have been reported for other plants (3).
Young leaves or stems of sterile Asragalus, Hyoscyamus and Althaea plants were wounded with a sterile needle and the different bacteria from the liquid media were speed onto the midrib in order to generate transformed root cultures. The hairy root cultures were maintained on an MS medium without phytochormones, containing 2-6% (w/v) sucrose in 300 ml flasks on a gyratory shaker at 25°C in darkness as already described (1). The elimination of Agrobacterium has been sometimes difficult. Generally, it was possible to establish transformed roots in axenic culture after subculturing a few times at 4-week intervals in liquid media containing antibiotic Claforan.
The whole experiment was repeated three times. Statistical calculation (regression analysis and one factor analysis of variance) was carried out on a Hewlett Packard computer using statistical program.
Effect of bacterial strains
Four different bacterial strains were used in our experiments - TR 105, R 1601, ATCC 15834, and LBA 9402. The phenotypic response results from the insertion into the plant genome of T-DNA (transfer DNA) was different (Fig. 1, 3). In some of the more recalcitrant species, successful transformation was achieved with a variety of Agrobacterium rhizogenes strains showing different host specificities. However, different strains of bacteria showed various abilities to induce hairy roots on the leaf explants on the same species. The difference in virulence could be explained by the plasmids harbored by bacterial strains.
Stability, growth kinetics and confirmation of transformation ofhairy roots
The hairy root cultures (HR) from different Astragalus, Datura, Hyoscyamus and Althaea species grown rapidly in simple media without phytochormones (1) were stable in their growth rate, alkaloids, flavonoids, polysaccharide and saponine (Fig.2) production over a period of more than 5 years in cultures.
HR has a profusion of root hairs, a high degree of lateral branching and absence of geotropism, resulting in high growth rates. After a short lag phase the HR started to grow rapidly and the fresh weight increased 35-40 fold for A.mongholicus, 14-15-fold for A.gummifer, 20-25-fold for A.membranaceus, for example, till day 30.
Using HR line of A.membranaceus effect of the nitrogen source on growth and saponin production was studied. They were investigated in MS medium with or without ammonium nitrate over a time period of 30 days. The resulting dry weight is higher in the absence of ammonium nitrate than in its presence. However, unexpectedly, in both media similar saponin contents were observed. Saponin production in HR cultures of A.membranaceus was not impaired by NH4NO3. This is in contrast to the results for polysaccharides in HR of Althaea officinalis, (1).
Saponins and polysaccharide content of the hairy root clones
The hairy root clones, transformed with different bacterium, were cultured individually. Agrobacterium strain had a some effect on growth and content of biologically active compounds. The clones infected by ATCC 15834 and LBA 9402 grew the fastest and the biomass increased 2-3 fold more than the other clones after 28 days of cultures. Moreover, different Agrobacterium strains have an effect on total saponin content in older hairy roots. Maximum saponin content was found in HR of A. mongholicus, transformed with LBA 9402 bacteria. Our results are similar with these, obtained for some Solanaceous species as well (3).
The saponin production by the hairy root cultures is typical of those found in many species of Astragalus (2). The transformed roots produce cycloartane-saponins, which do not differ significantly from those produced by the parent plants.
HR cultures of Astragalus spp. released part of the saponin product (about 16-20% of the total saponin) into the medium. This is essential, in order to establish continuous production of saponins. The most apparent effect in saponin production was seen when sucrose levels of the medium were modified. A medium (MS) containing 2% sucrose increased overall saponin yield, but the growth was very low. The optimal medium for both yield and growth was supplemented with 4% sucrose. The saponin content was not impaired significantly at high levels of nitrogen. These results suggest that biosynthetic regulation is affected by altered cell organization in roots, as might be expected. HR cultures of Astragalus spp. can therefore be an interesting system by which fast growth of the biomass as well as relatively high saponin production can be achieved, facilitating further studies on tritrpene saponin biosynthesis.
Monosaccharide compositions of the macromolecules in various cultures and parent plants have been investigated. Differences in the sugar composition of polysaccharides between HR and intact plants (Althaea, Astragalus) have been found. Furthermore, the amounts of each neutral monosaccharide produced in the various HR cultures were not uniform, and composition varied widely. The polysaccharides of the mother plant contained more compounds different to the mucilage of HR. We suggest that this difference can be attributed to the different stages of development in the transformed roots.
Tropane alkaloid production of transformed roots of H. reticulatus
Infection of Hyoscyamus plant cells by Agrobacterium rhizogenes usually results in "hairy root" disease at the sites of infection. This morphogenic event is du to the transfer of genetic information carried out by Ri plasmids from bacteria to the plant cells. The integration and expression of the transferred DNA (T-DNA) of Ri plasmids cause metabolic changes mainly determined by genes involved in auxin syntheses. Two regions, TL- and TR-DNA, were found to be integrated and stable conserved in the plant genome.
From the 24 clones, transformed with LBA 9402, clone - Hr-L12, producing high levels of tropane alkaloids (1.44% dw) was selected. The root cultures transformed with the two different strains of A. rhizogenes showed different characteristics. We presume that the Ri-plasmids induced various clones with different genotypes inducing variation of the alkaloid productivity.
Some differences were found in the contents of these alkaloids among the clones. In contrast with the parent plant, many of established clones tended to synthesize scopolamine preferentially rather than hyoscyamine. The both alkaloids were presented in the culture media of many of the hairy root clones. There was no clear relationship between growth and alkaloid production.
Clone Hr-3 produced the largest amount of tropane alkaloids from all ATCC-clones. The spectrum is dominated by scopolamine (46%), hyoscyamine (35%), acetiltropine (9%), littorine (3%), 6-hydroxyhyoscyamine (1.3%) with a further 5 compounds. The ratio, between scopolamine and hyoscyamine in normal root was 0.9. In contrast with the parent plant the production of scopolamine in this clone was 1.3 times greater than that of hyoscyamine. These differences in tropane alkaloid metabolite production between normal and transformed roots could be due to the different state of development in the transformed roots, to the absence of long-distance transport and/or a high hyoscyamine-6-b-hydroxylase activity. Only in clone Hr-21 no scopolamine was detected. The differences in scopolamine levels of H. reticulatus - plant and hairy roots however showed that the role of aerial tissue in epoxidation here is minor. The ratio scopolamine/hyoscyamine and alkaloid spectrum in the transformed roots of H. reticulatus was found no similar to those in the parent plant, which seems to indicate that Ri-T-DNA does have some effect on qualitative and quantitative changes in the alkaloid composition.
A major problem associated with organized cultures remains their growth on a large scale. The morphological structure of roots presents a unique reactor engineering challenge for industrial-scale application of root culture. It is well recognized that plant system has a slow growth rate and that their cell and tissue chemical transport system is unique and different from that of typical microorganisms. Their air or oxygen requirements are lower than those of microorganisms and may amply be supplied by air-lift fermentors. A rapidly growing root line of Astragalus membranaceus, Althaea officinalis, Hyoscyamus reticulatus was selected for cultivation of roots in an air-lift fermenter. It was observed that the inner glass tube air-lift type restricted the free movement of growing roots. The meristem-dependent growth of hairy root cultures of A. membranaceus established a distinct spatial and temporal order. They are relatively insensitive to oxygen levels. Growth in liquid medium results in a root "nucleus" with young, rapidly growing roots on the periphery and a core of older tissue inside. Restriction of nutrient and oxygen delivery to the center of the mass gives rise to a growing pocket of senescent tissue. In our experiments, partial success has been achieved, leading to the formation of dense beds of roots with root hairs, which provide a large surface for nutrient absorption and release of products into the medium. The biomass concentrations showed a high final density above 17 g dry roots/l. The saponins were released by A. membranaceus transformed roots in growth medium, reaching a maximum after 35 days, while the hairy roots retained high internal saponin levels and remained viable. The possibility of controlling this release of root saponins into the medium is important for Astragalus HR scale-up.
In our studies with H. reticulatus it was shown that in tropane alkaloid production, the Agrobacterium-mediated transformationmay be favorable for the full expression of a line's potential biosynthetic capacity. In a screening program a number of cell lines are set up and the production of the compounds of interest was determined. The best producing lines are selected. Transformation with different Agrobacterium strains might have an effect not only on total alkaloid content, but also on the ratio of the alkaloids.
A variety of experimental approaches have been used to increase the efficiency of the production of saponins, polysaccharides and alkaloids in this Astragalus, Hyoscyamus, Datura and Althaea roots. The excretion of these compounds to the liquid medium offers possibilities for continuous cultures. In general HR are preferable to conventional cultures, because of the higher genetic stability, higher growth rates, and production of pharmaco-logically active compounds.
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