Skip to search form Skip to main content You are currently offline. Some features of the site may not work correctly. Agrobacterium rhizogenes causes hairy root disease in plants. These hairy roots are genetically stable and grow rapidly. Transformed hairy roots of Hyoscyamus muticus induced by the bacterium can produce tropane alkaloids in trace amounts of intact plant tissues.
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Plants remain a major source of new drugs, leads and fine chemicals. Cell cultures deriving from plants offer a fascinating tool to study plant metabolic pathways and offer large scale production systems for valuable compounds — commercial examples include compounds such as paclitaxel.
The major constraint with undifferentiated cell cultures is that they are generally considered to be genetically unstable and cultured cells tend to produce low yields of secondary metabolites especially over time. Hairy roots, a tumor tissue caused by infection of Agrobacterium rhizogenes is a relevant alternative for plant secondary metabolite production for being fast growing, able to grow without phytohormones, and displaying higher stability than undifferentiated cells.
Although genetic and metabolic stability has often been connected to transgenic hairy roots, there are only few reports on how a very long-term subculturing effects on the production capacity of hairy roots.
In this study, hairy roots producing high tropane alkaloid levels were subjected to year follow-up in relation to genetic and metabolic stability. Cryopreservation method for hairy roots of Hyoscyamus muticus was developed to replace laborious subculturing, and although the post-thaw recovery rates remained low, the expression of transgene remained unaltered in cryopreserved roots.
It was shown that although displaying some fluctuation in the metabolite yields, even an exceedingly long-term subculturing was successfully applied without significant loss of metabolic activity. Plants offer an enormous potential for humans in applications such as novel drugs, biopolymers, high-value chemical compounds, food and feed.
Each plant cell has the whole genetic potential for differentiation into e. Besides whole plants, plant cells can be cultivated as cell cultures in synthetic growth media and this way they offer a great potential for various biotechnological applications. Plant cell cultures can be divided in two main classes, differentiated and undifferentiated cell cultures. The former consist of e. Cell cultures offer advantages to produce cell biomass or metabolites in a controlled environment.
Cultivation in laboratory or in bioreactors enables production processes which are independent of climatic conditions simultaneously allowing optimization of different production parameters. Hairy root is caused by a plant disease through the infection of Agrobacterium rhizogenes carrying Ri root inducing plasmid.
Agrobacterium Rhizobiaceae is a soil bacterium, which is able to deliver a part of its own plasmid-DNA T-DNA into the nuclear genome of the plant cell. As a result of very complex genetic machinery, hairy roots are then formed in the infection site. Finally they can be detached and grown as independent hairy root clones. Infection of a plant with A. These genes can individually induce the secondary metabolite biosynthesis, with rolB probably being the most powerful inducer followed by rolC gene Sharma et al.
Although the exact mechanism of rol gene —mediated induction of secondary metabolite biosynthesis is not known, it is thought that induction involves the function of signal transduction pathways, including interaction with proteins and tyrosine phosphatase Moriuchi et al.
In this work, H. Decades of research work has turned this hairy root disease into a valuable biotechnological application Georgiev et al. For a number of desired plants, hairy roots have been induced for the commercial scale production of metabolites, often yielding even higher amounts of metabolites than that of the parent plant Jouhikainen et al.
In addition, hairy roots gain biomass rather rapidly and have simple cultivation medium requirements. Genetic stability of hairy roots is well recognized. They show high genetic stability as well as more stable metabolic production than that of undifferentiated cell cultures. In certain cases the production of secondary metabolites in plants is limited to specialized tissues or organs and the capability is lost in undifferentiated cells.
This pattern is also recognized with tropane alkaloids, probably due to the root-specific localization of the tropane alkaloid biosynthetic pathway Hashimoto and Yamada, Much of this stability has been related to chromosomic stability displayed by hairy roots Weber et al. Hairy roots usually carry the same chromosomic number and karyotype as the parent plant.
Similarly, Mano et al. However, this is a short period as the adaptation time to culture conditions is always required. Only few reports show the accumulation of metabolites produced by hairy roots during a long subculturing. Peebles et al. Similarly Maldonado-Mendoza et al. In this work, we present, for the first time, the genetic and metabolic activities of engineered hairy roots maintained for 16 years by continuous subculturing.
After the establishment of the hairy root line 16 years ago Jouhikainen et al. Biotechnology relies to a great part on working with cell cultures and continues to offer a future beyond depletion of natural resources. Establishment and maintenance of cell cultures is laborious and time-consuming process and so far only few successful methods exist for long-term storage for these cultures. Plant cells growing in undifferentiated state are known to be genetically unstable. During continuous subculturing, various rearrangements in chromosomal or gene level may take place, which can be of genetic or epigenetic origin Larkin and Scowcroft, ; Kaeppler et al.
However, cryopreservation of plant cells still mainly relies on the fully empirical testing, which makes the method development very laborious. Basically, cryopreservation methods are divided in three categories, those based on slow freezing of the cells by using temperature gradients The two latter ones do not require special freezing equipment but cells are immersed directly into liquid nitrogen after treatment with vitrification solutions or encapsulated with e.
Intracellular water expands during crystallization and may lead to bursting of cellular membranes. In addition, ice formation increases the solute concentration of remaining liquid phase in intracellular space which may cause toxicity during slow freezing.
Prevention of intracellular water concentration may be overcome by several ways. Osmotic pretreatment means using non-permeable agents such as sugars and sugar alcohols which cause dehydration of cells. Cryoprotection on the other hand covers the use of permeable agents which function through a colligative effect and usually result in the increase in membrane fluidity.
Alternatively, when working with alginate bead protection, desiccation by air flow or silica gel is often used. However, vitrification solutions cause sometimes toxicity to plant cells and therefore are not suitable in all cases. Even though successful cryopreservation methods for a number of plant species have been established, a considerable experimentation is always needed to optimize all the steps in cryopreservation procedure Reed, So far, there exists no routine method available for different species and cultures.
Even though earlier reports of cryopreservation of hairy roots of Beta vulgaris and Nicotiana rustica Benson and Hamill, , Artemisia annua Teoh et al. Here, we present an easy method for cryopreservation of H.
The hairy root clone KB7 or formerly 13A7 of H. The hairy roots were routinely grown and subcultured every 4 weeks in solid modified B50 medium Oksman-Caldentey et al. Total RNA from H. The 18S gene was used as housekeeping control. Data were analyzed using the LightCycler Software release 1. The statistical analysis was performed with the computing environment R R Core Team, The student T -test was used for statistical comparisons.
A p -value of less than 0. Briefly, lyophilized roots were weighed 50 mg and the lipids were removed with 2 ml petroleum ether. After vortexing and centrifugation rpm, 10 min the solvent phase was discarded and the sample residue was dried under nitrogen flow. The tropane alkaloids from the culture medium were extracted correspondingly from 2 ml medium. Helium was used as the carrier gas on constant flow mode at 1.
Identification of the compounds was based on retention times, GC-MS library comparisons and literature data. Several cryopreservation methods were tested for long-term storage of H.
Experimental layout is shown in Figure 1. Experimental layout of cryoprotection. Methods which eventually showed post-thaw recovery are shown in patterned color.
Slow freezing method Teoh et al. Root tips were cut 0. Root tips were transferred into cryo-vials and incubated at room temperature together with cryoprotectant for 1 h. After that the vials were transferred into liquid nitrogen. Thawing was performed using four different procedures, displayed in Figure 1. After incubation in culture medium for 1 h, root rips were placed on the solid medium with sterile filter paper and incubated in darkness.
After 3 days the root tips were transferred on the solid medium without filter paper. The third method was as described above but root tips were transferred on solid medium without filter paper after 3 h incubation.
Fourth, cryovials were thawed as described above; root tips were washed with original culture medium and plated on the solid medium without filter paper. Slow freezing method described by Schmale et al. Slow freezing procedure described by Ogawa et al. Hairy roots were cultivated either on the solid medium for 4 weeks or in liquid medium for 6 days.
After that the root tips were cut and cultivated on fresh solid plate for 24 h. Approximately 10 tips were placed on the cryo-vial together with 1 ml LSP solution 2 M glycerol, 0.
After 4 h incubation, vials were placed in liquid nitrogen. Hairy roots were cryopreserved along a modified method essentially described for banana apical meristems by Panis et al. Hairy roots were cultivated for 14 days on solid medium. Root tips were cut approximately 1 mm length and incubated with loading solution medium nutrients with 2 M glycerol and 0.
Loading solution was replaced by ice cold PVS2 solution 3. Root tips were either placed on foil inside PVS2 bubble and were frozen directly in liquid nitrogen, or tips together with PVS2 were inserted in cryo-vials and immersed in liquid nitrogen. Root tips were thawed in a following way unless otherwise described. Tips were transferred on fresh solid medium without filter paper after 24 h and incubated in darkness.
PCR was performed using h6h gene-specific primers, and it was shown that both transgene and root phenotype responsible rolB region were present in H. As expected, virD amplification, indicating the presence of the bacterial genome was negative. PCR of h6h bp , rolB bp , and virD bp amplifications performed of Hyoscyamus muticus hairy roots and Agrobacterium samples.
Engineering tropane biosynthetic pathway in Hyoscyamus niger hairy root cultures.
In one out of 8 clones established, an unusual root tip formation was observed after transfer of cultures from half-strength Murashige and Skoog to White's medium This phenomenon was associated with the production of a fine brownish cell suspension culture. Hairy root development resumed after transfer of the root tips from White to half-strength Murashige and Skoog medium. After plating the isolated brownish cells on hormone-free half-strength Murashige and Skoog or White solid medium, callus proliferation was observed, and then redifferentiation of hairy roots occurred. The polymerase chain reaction analysis of the H. The growth and the production of five tropane alkaloids by this clone were examined.
Exploring the Metabolic Stability of Engineered Hairy Roots after 16 Years Maintenance
The cDNA from Nicotiana tabacum encoding Putrescine N-methyltransferase PMT , which catalyzes the first committed step in the biosynthesis of tropane alkaloids, has been introduced into the genome of a scopolamine-producing Hyoscyamus niger mediated by the disarmed Agrobacterium tumefaciens strain C58C1, which also carries Agrobacterium rhizogenes Ri plasmid pRiA4, and expressed under the control of the CaMV 35S promoter. Hairy root lines transformed with pmt presented fivefold higher PMT activity than the control, and the methylputrescine MPUT levels of the resulting engineered hairy roots increased four to fivefold compared to the control and wild-type roots, but there was no significant increase in tropane alkaloids. However, after methyl jasmonate MeJA treatment, a considerable increase of PMTase and endogenous H6Hase as well as an increase in scopolamine content was found either in the transgenic hairy roots or the control. The results indicate that hairy root lines over-expressing pmt have a high capacity to synthesize MPUT, whereas their ability to convert hyoscyamine into scopolamine is very limited.