1. Strategies for gene transfer to plant cells
By associate prof. D. Yu Golikov

2. Contents Hairy root cultures Agrobacterium - mediated Gene Transfer
Infection and tumorigenesis
Tumor characteristics
Tumor-inducing principle
Structure of Ri-plasmid
Cellular process of Agrobacterium-host interaction
Production of hairy roots in vivo
Mechanism of Agrobacterium-plant cell interaction
Induction of hairy root cultures in vitro
Characteristics of the Hairy Roots Cultures
Advantages of hairy root cultures (Active form: Discussion)
Application of hairy root cultures
Plant regeneration
Tree improvement
Genetic manipulation: Engineering plants with Agrobacterium

3. ‘Hairy roots’ are produced by infecting sterile plants with a natural genetic engineer, Agrobacterium rhizogenes (gram negative soil bacterium).
Genes for auxin synthesis and sensitivity are engineered into plant cells leading to gravity-insensitive mass root production.
Very useful for products produced in roots.
Aggregation and shear sensitivity are a major problem for scale- up
This processes take advantage of the naturally occurring hairy root disease in Dicotyledons.
‘Hairy root’ cultures

4. Agrobacterium - mediated Gene Transfer
Most common method of engineering dicots, but also used for monocots
Pioneered by J. Schell (Max-Planck Inst., Cologne)
Agrobacteria
soil bacteria, gram-negative, related to Rhizobia
species:
rhizogenes- hairy root disease

6. Crown galls caused by A. tumefaciens on nightshade.
More about Galls:

7. Agrobacterium Agrobacterium (disease symptomology and host range)
radiobacter - “avirulent” species
A. tumefaciens - crown gall disease
rhizogenes - hairy root disease
rubi cane gall disease
vitis galls on grape and a few
other plant species

8. Agrobacterium tumefaciens
the species of choice for engineering dicot plants; monocots are generally resistant
some dicots more resistant than others (a genetic basis for this)
complex bacterium – genome has been sequenced; 4 chromosomes; ~ genes

9. Infection and tumorigenesis
Infection occurs at wound sites
Involves recognition and chemotaxis of the bacterium toward wounded cells
galls are “real tumors”, can be removed and will grow indefinitely without hormones
genetic information must be transferred to plant cells
Infection and tumorigenesis

10. Tumor characteristics
Synthesize a unique amino acid, called “opine”
octopine and nopaline - derived from arginine
agropine - derived from glutamate
Opine depends on the strain of A. tumefaciens
Opines are catabolized by the bacteria, which can use only the specific opine that it causes the plant to produce.
Has obvious advantages for the bacteria, what about the plant?

11. Elucidation of the TIP (tumor-inducing principle)
It was recognized early that virulent strains could be cured of virulence, and that cured strains could regain virulence when exposed to virulent strains; suggested an extra-chromosomal element.
Large plasmids were found in A. tumefaciens and their presence correlated with virulence: called tumor-inducing or Ti plasmids.

12. VirE2 may get DNA-protein complex across host PM
VirE1 chaperones VirE2 in Agro. Cytoplasm, but complex can also bind the SS T-DNA. VirE2 may help the T-DNA cross the plant cell PM, as it can form a channel in artificial bilayers by itself.
Dumas et al., (2001), Proc. Natl. Acad. Sci. USA, 98:485

13. Recognition by Agrobacterium
Wounded plant cells
Signal Molecules
Recognition by Agrobacterium
Attachment of
Agrobacterium With plant cells
Transfer of RI-plasmid to wounded plant cells
Co-Cultivation
Integration of RI-plasmid
into plant genome
Hairy Root Induction

14. Structure of Ri-plasmid

15. Cellular process of Agrobacterium–host interaction
Tzvi Tzfira and Vitaly Citovsky, 2002, Trends in Cell Biol. 12(3),

16. A. rhizogenes, the causative agent of hairy root syndrome, is a common soil bacterium (Gram negative) capable of entering a plant through a wound and causing a proliferation of secondary roots. The underlying mechanism of hairy root formation is the transfer of several bacterial genes to the plant genome. The observed morphogenic effects in the plants after infection have been attributed to the transfer of part of a large plasmid known as the Ri (root-inducing) plasmid. The symptoms observed with A. rhizogenes are suggestive of auxin effects resulting from an increase in cellular auxin sensitivity rather than auxin production.

17. Production of hairy roots in vivo:
Agrobacterium recognizes some signal molecules exuded by wounded plant cells and becomes attached to it.
The bacteria contain the Root inducing plasmid (Ri- plasmid)
The bacteria genetically transfer part of the Ri-plasmid called the transfer DNA (T-DNA) to the plant genome, where it is get expressed and make the plant cell to:

18. Ri plasmids are large (200 to greater than 800 kb) and contain one or two regions of T-DNA and a vir (virulence) region, all of which are necessary for tumor genesis. The Ri-plasmids are grouped into two main classes according to the opines synthesized by hairy roots. First, agropine-type strains induce roots to synthesize agropine, mannopine and the related acids. Second, mannopine-type strains induce roots to produce mannopine and the corresponding acids. The agropine-type Ri- plasmids are very similar as a group and a quite distinct group from the mannopine-type plasmids. Perhaps the most studied Ri- plasmids are agropine-type strains, which are considered to be the most virulent and therefore more often used in the establishment of hairy root cultures.
Ri-plasmids

19. Production of hairy roots in vivo:
Proliferate by increasing the rate of cell division (cytokinine expression) and cell elongation (auxin expression) to produce the hairy roots.
Produce the opines which is a type of unusual amino acids (octopine, agropine, nopaline, mannopine, and cucumopine) which is used by the bacterium as a carbon, nitrogen and energy source

20. Mechanism of Agrobacterium-plant cell interaction
One of the earliest stages in the interaction between Agrobacterium and a plant is the attachment of the bacterium to the surface of the plant cell. A plant cell becomes susceptible to Agrobacterium when it is wounded. The wounded cells release phenolic compounds, such as acetosyringone, that activate the vir-region of the bacterial plasmid. It has been shown that the Agrobacterium plasmid carries three genetic components that are required for plant cell transformation.
Mechanism of Agrobacterium-plant cell interaction

21. Mechanism of Agrobacterium-plant cell interaction
It has been shown that the Agrobacterium plasmid carries three genetic components that are required for plant cell transformation. The first component, the T-DNA that is integrated into the plant cells, is a mobile DNA element. The second one is the virulence area (vir), which contains several vir genes. These genes do not enter the plant cell but, together with the chromosomal DNA (two loci), cause the transfer of T-DNA. The third component, the so-called border sequences (25 bp), resides in the Agrobacterium chromosome. The mobility of T-DNA is largely determined by these sequences, and they are the only cis elements necessary for direct T-DNA processing.
Mechanism of Agrobacterium-plant cell interaction

22. Induction of hairy root cultures in vitro:
Explants are wounded and then inoculated with Agrobacterium rhizogenes.
Usually two or three days later, the explant can be transferred into solid media with antibiotics, such as cefotaxime, vancomycin or ampicillin to kill or eliminate redundant bacteria.
The hairy roots will be induced within a short period of time, which varies from one week to over a month depending on different plant species.
The decontaminated hairy roots can be subcultured on phytohormone-free medium.

23. hairy root cultures:

24. Characteristics of the Hairy Roots Cultures
Hairy roots are fast growing and laterally highly branched, and are able to grow in hormone-free medium. Moreover, these organs are not susceptible to geotropism anymore. They are genetically stable and produce high contents of secondary metabolites characteristic to the host plant. The secondary metabolite production of hairy roots is stable compared to other types of plant cell culture. The alkaloid production of hairy roots cultures has been reported to remain stable for years. The secondary metabolite production of hairy roots is highly linked to cell differentiation. Alkaloid production decreased clearly when roots were induced to form callus, and reappeared when the roots were allowed to redifferentiate. An interesting characteristic of some hairy roots is their ability to occasionally excrete the secondary metabolites into the growth medium. However, the extent of secondary product release in hairy root cultures varies among plant species.

25. Characteristics of the Hairy Roots Cultures
The average growth rate of hairy roots varies from 0.1 to 2.0 g dry weight/liter/day. This growth rate exceeds that of virtually all- conventional roots and is comparable with that of suspension cultures. However, the greatest advantage of hairy roots compared to conventional roots is their ability to form several new growing points and, consequently, lateral branches. The growth rate of hairy roots may vary greatly between species, but differences are also observed between different root clones of the same species. The pattern of growth and secondary metabolite production of hairy root cultures can also vary. Secondary production of the hairy roots of Nicotiana rustica L. was strictly related to the growth, whereas hairy roots of Beta vulgaris L. exhibited non-growth-related product accumulation. In the case of the hairy roots of Scopolia japonica Jacq. and H. muticus, the secondary products only started to accumulate after growth had ceased. Secondary metabolite synthesis dissociated from growth would be desirable for commercial production, as it would allow the use of continuous systems.

26. Advantages of hairy root cultures:
The hairy root system is genetically and biosynthetically stable
High production of secondary metabolites
The culture can grow under phyto-hormone-free conditions.
The culture shows fast growth which reduce the culture time and easy the handling
Compare with other technologies

27. Application of hairy root cultures:
Functional analysis of genes
Expressing foreign proteins
Production of secondary metabolites
The culture may produce compounds which is not found in untransformed roots
The culture may change the composition of metabolites
The culture can be used to regenerate a whole plants

28. Hairy root cultures are characterized by a high growth rate and are able to synthesize root derived secondary metabolites. Normally, root cultures need an exogenous phytohormone supply and grow very slowly, resulting in poor or negligible secondary metabolite synthesis. However, the use of hairy root cultures has revolutionized the role of plant tissue culture for secondary metabolite synthesis. These hairy roots are unique in their genetic and biosynthetic stability. Their fast growth, low doubling time, ease of maintenance, and ability to synthesize a range of chemical compounds offers an additional advantage as a continuous source for the production of valuable secondary metabolites. To obtain a high-density culture of roots, the culture conditions should be maintained at the optimum level. Hairy root cultures follow a definite growth pattern, however, the metabolite production may not be growth related.
Hairy roots also offer a valuable source of root derived phytochemicals that are useful as pharmaceuticals, cosmetics, and food additives. These roots can also synthesize more than a single metabolite and therefore prove economical for commercial production purposes. Transformed roots of many plant species have been widely studied for the in vitro production of secondary metabolites. Transformed root lines can be a promising source for the constant and standardized production of secondary metabolites. Hairy root cultures produce secondary metabolites over successive generations without losing genetic or biosynthetic stability. This property can be utilized by genetic manipulations to increase biosynthetic capacity.

29. Transformed roots are able to regenerate whole viable plants; hairy roots as well as the plants regenerated from hairy roots are genetically stable. However, in some instances transgenic plants have shown an altered phenotype compared to controls.
Plants can be regenerated from hairy root cultures either spontaneously (directly from roots) or by transferring roots to hormone-containing medium. The advantage of Ri plasmid- based gene transfer is that spontaneous shoot regeneration is obtained avoiding the callus phase and somaclonal variations. Ri plasmid-based gene transfer also has a higher rate of transformation and regeneration of transgenic plants; transgenic plants can be obtained without a selection agent thereby avoiding the use of chemicals that inhibit shoot regeneration; high rate of co-transfer of genes on binary vector can occur without selection.
Plant regeneration

30. These hairy roots can be maintained as organ cultures for a long time and subsequent shoot regeneration can be obtained without any cytological abnormality. Rapid growth of hairy roots on hormone-free medium and high plantlet regeneration frequency allows clonal propagation of elite plants. In in vitro cultures, the hairy root regenerated plants show rapid growth, increased lateral bud formation, and rapid leaf development, these regenerants are useful for micropropagation of plants that are difficult to multiply. Altered phenotypes are produced from hairy root regenerants and some of these have proven to be useful in plant breeding programs. Morphological traits with ornamental value are abundant adventitious root formation, reduced apical dominance, and altered leaf and flower morphology. Dwarfing, altered flowering, wrinkled leaves, or increased branching may also be useful for ornamentals. Dwarf phenotype is an important characteristic for flower crops such as Eustoma grandiflorum and Dianthus.

31. A major limitation of tree improvement programs is their long generation cycle. Classical breeding programs in trees are slow and tedious and it is difficult to introduce specific genes for genetic manipulation by crossing parental lines. Agrobacterium rhizogenes mediated transformation can be a useful alternative, as a rapid and direct route for introduction and expression of specific traits. The ability to manipulate tree species at cellular and molecular level shows great potential and in vitro transformation and regeneration from hairy roots facilitates application of biotechnology to tree species. This significantly reduces the time necessary for tree improvement and gives rise to new gene combinations that cannot be obtained using traditional breeding methods. In some tree species root initiation limits vegetative propagation; by using A. rhizogenes rooting of cuttings from recalcitrant woody species have been improved.
Tree improvement

32. Transformed roots provide a promising alternative for the biotechnological exploitation of plant cells. A. rhizogenes mediated transformation of plants may be used in a manner analogous to the well-known procedures employing A. tumefaciens. A. rhizogenes mediated transformation has also been used to produce transgenic hairy root cultures and plantlets have been regenerated. With the exception of the border sequences, none of the other T-DNA sequences are required for the transfer. The rest of the T-DNA can be replaced with the foreign DNA and introduced into cells from which whole plants can be regenerated. These foreign DNA sequences are stably inherited in a Mendelian manner. The A. rhizogenes mediated transformation has the advantage that any foreign gene of interest placed in binary vector can be transferred to the transformed hairy root clone.
Genetic manipulation

33. Engineering plants with Agrobacterium:
Important: Put any DNA between the LB and RB of T-DNA it will be transferred to plant cell
Engineering plants with Agrobacterium:
Two problems had to be overcome:
(1) Ti plasmids large, difficult to manipulate
(2) couldn't regenerate plants from tumors

34. References:
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