WO1999015001A1 - Method for producing stable chlorophyll deficient plants and plants obtained with such a method - Google Patents

Method for producing stable chlorophyll deficient plants and plants obtained with such a method Download PDF

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Publication number
WO1999015001A1
WO1999015001A1 PCT/GB1998/002803 GB9802803W WO9915001A1 WO 1999015001 A1 WO1999015001 A1 WO 1999015001A1 GB 9802803 W GB9802803 W GB 9802803W WO 9915001 A1 WO9915001 A1 WO 9915001A1
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plant
plants
chlorophyll
plastid
protein synthesis
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PCT/GB1998/002803
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French (fr)
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Anil Day
Mikhajlo Konstantinovitch Zubko
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The Victoria University Of Manchester
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Priority to GBGB9719880.8A priority patent/GB9719880D0/en
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Publication of WO1999015001A1 publication Critical patent/WO1999015001A1/en

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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10Cells modified by introduction of foreign genetic material
    • C12N5/12Fused cells, e.g. hybridomas
    • C12N5/14Plant cells
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01HNEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H3/00Processes for modifying phenotypes, e.g. symbiosis with bacteria
    • A01H3/04Processes for modifying phenotypes, e.g. symbiosis with bacteria by treatment with chemicals

Abstract

A method of producing stable chlorophyll deficient plants and plant cell lines comprises subjecting plant material to a treatment which inhibits plastid protein synthesis, and growing plants and/or plant cell lines from the treated plant material under conditions in which said treatment is not applied.

Description

METHOD FOR PRODUCING STABLE CHLOROPHYL DEFICIENT PLANTS AND PLANTS
OBTAINED WITH SUCH A METHOD
The present invention relates to plants and more particularly to chlorophyll deficient plants and plant cell lines.
Chlorophyll-deficient plants and plant cell lines are useful in several areas of industry. For instance, chlorophyll-deficient mutants of higher plants and plant cells are widely used as easily detectable genetic markers in experiments on plant genome mutability and cytoplasmic genetics. Furthermore in plant breeding chlorophyll deficiency is a marker that can be used when combining cytoplasm from one cell with the nuclear material of another cell.
Variegated plants (the white areas of the plant being Chlorophyll-deficient) are also very popular ornamental plants and there is a need in the horticultural industry to develop such plants.
Conventionally chlorophyll-deficient plants and plant cell lines are developed by mutation which may occur spontaneously with low frequency or may be induced by mutagenic agents. A disadvantage associated with such mutational procedures is that it is not possible to control mutagenic changes to the plant genotype. In most cases, induction of chlorophyll-deficient plants by mutagenesis is associated with a high rate of random genome mutations at different loci which alter the genetic composition of plant populations. Mutagenesis procedures also take a long time and often use reagents which may pose a hazard to the operator.
Alternatively chlorophyll-deficient plants and plant cells may be derived by targeted mutation using genetic engineering techniques. However such techniques can be prohibitively expensive and require sophisticated laboratory apparatus, materials and expert workers. Chlorophyll deficiency can also be induced using non-mutagenic agents. However plants and plant cell lines developed using known non-mutagenic techniques revert to a chlorophyll sufficient ("green") phenotype when the agents are withdrawn and/or the cells are incapable of cell division whilst maintaining the chlorophyll deficient phenotype.
It is an object of the present invention to provide a method for producing stable chlorophyll-deficient plants and plant cell lines as well as variegated plants.
According to a first aspect of the present invention, there is provided a method of producing stable (as defined herein) chlorophyll deficient plants and plant cell lines comprising subjecting plant material to a treatment which inhibits plastid protein synthesis, and growing plants and plant cell lines from the treated plant material under conditions in which said treatment is not applied.
According to a second aspect of the present invention, there is provided a plant in which plastid protein synthesis has been inhibited whereby the plant is chlorophyll deficient and is stable (as defined herein).
According to a third aspect of the present invention, there is provided a plant in which plastid protein synthesis has been inhibited whereby the plant is variegated and is stable (as defined herein).
According to a fourth aspect of the present invention, there is provided a plant cell line in which plastid protein synthesis has been inhibited whereby the plant cell line is chlorophyll deficient and is stable (as defined herein).
By "stable" we mean that a chlorophyll deficient phenotype is maintained by the plant or plant cell line following: (i) cell division (i.e. daughter cells, generated by mitosis and/or meiosis, from cells in which plastid protein synthesis is, or was, inhibited remain chlorophyll deficient); and (ii) removal of the inhibition of plastid protein synthesis.
By "plastid" we mean the group of plant intracellular organelles which are usually pigmented and may be typified by the chloroplast.
By "chlorophyll deficient" we mean that the plant or plant cell line has at least partially reduced chlorophyll content compared to a normal green plant. The term also encompasses variegated plants which may contain some cells which have reduced chlorophyll levels and other cells with normal levels of chlorophyll.
We have found that inhibitors of plastid protein synthesis may be used to generate stable chlorophyll deficient and/or variegated plants and stable chlorophyll deficient plant cell lines without the need for genetically manipulating plant cells. Plastid protein synthesis should be inhibited such that the capacity to form chloroplasts is irreversibly blocked.
Since the method of the invention does not require mutagenesis it has considerable advantages in terms of speed of production, safety and the elimination of undesirable mutations in genes that could introduce undesirable traits into plants. Additionally the method does not require sophisticated laboratory apparatus or expertise in molecular biology.
A known correlation exists between the accumulation of plastid ribosomal RNAs and chlorophyll levels in plant cells. It is also known that physical and chemical agents that block plastid protein synthesis result in an inability to accumulate chlorophyll in plant cells. Plants treated with such agents become bleached (i.e. they turn from green to white) after which the plants or plant cells subsequently die. However we have conducted experiments which have surprisingly established that agents which inhibit plastid protein synthesis can generate genetically unmanipulated and stable chlorophyll deficient and/or variegated plants .and genetically unmanipulated and stable chlorophyll deficient plant cell lines which may be maintained in vitro (or in vivo as appropriate) in a suitable growth medium. Furthermore such plants and cells do not revert to a green phenotype (even when the agent used to inhibit plastid protein synthesis is withdrawn).
Light and electron microscopic investigations showed that the plants and plant cell lines treated according to the method of the invention contain a high proportion of reduced plastid-like structure (relative to mature chloroplasts) than is normally found in green plants. Molecular analyses of stable chlorophyll-deficient plants showed that they were deficient in plastid ribosomal RNAs and plastid proteins. Although we do not wish to be bound by any hypothesis, we believe that the chlorophyll-deficiency and variegation is caused by a deficiency of an intact plastid expression apparatus which prevents the plant material from re-greening since without this apparatus the plastid genome cannot be expressed.
An important feature of the invention is that we have produced genetically unmanipulated chlorophyll-deficient plants and cell lines which have a stable phenotype. The plants and cells can be propagated without reverting to a chlorophyll sufficient ("green") phenotype even when the inhibition of plastid protein synthesis has been removed.
The inhibition may be by chemical or physical means although the inhibition should not result in a mutation in the plants genome (nuclear, mitochondrial or plastid).
A suitable means of inhibiting plastid protein synthesis is to treat the plants or cell lines with an inhibitor of plastid gene expression or plastid protein synthesis. Preferred inhibitors are those which inhibit the function of plastid ribosomes. Most preferred inhibitors are antibiotics such as Spectinomycin and Streptomycin and extreme temperatures..
The method of the invention is suitable for producing a wide range of plants according to the second and third aspects of the invention and plant cell lines according to the fourth aspect of the invention. The method according to the first aspect of the invention is highly suitable for inducing high frequency (up to 100%) chlorophyll deficiency and also represents any easy and quick procedure relative to known mutagenic methods. Furthermore, the method is particularly useful for providing an abundant and easily generated source of stable chlorophyll-deficient and variegated phenocopies.
Many different type of plant material may be treated according to the invention. The material may be a growing plants, a plant cell line, a seed or any other suitable source of plant material which is capable of growth. If desired plant tissue may be removed from the treated plant material for further cultivation. This tissue may be the form of a growing shoot tip, a bud or a root tip from a plant. Such tissue may be cultivated in soil or may be grown in vitro. In vitro grown tissue may be grown under suitable conditions to give rise to a plant or alternatively the tissue may be grown to develop a plant cell line.
Examples of types of plants which may be treated according to the method of the invention (and which may be manipulated to derive plants according to the second and third aspects of the invention) include Brassicaceae (e.g. Brassica napus, Brassica rapa, Brassica oleracea, Brassica nigra and Arabidopsis thaliana), Graminae (e.g. Zea mays, Hordeum v lgare and Triticum aestivum) Solanaceae (e.g. Lycopersicon esculentum and Solanum tuberosum) and Compositae (Q.g.Helianthus annus). Cell lines according to the fourth aspect of the invention may be developed from such plants. The plant material, chlorophyll deficient plants and chlorophyll deficient plant cell lines should be maintained or propagated in a suitable growth medium. When plants are grown in the ground, the environment may represent a suitable medium, although supplementation with phytohormones, vitamins and/or carbohydrate (because of the lack of photosynthesis) etc may be required.
Plant material, plants and plant cell lines propagated in vitro require a suitable growth medium to sustain the cells. Conventional plant growth medium containing MES may be used if supplemented with phytohormones.
The treatment regime required for inducing chlorophyll-deficiency and/or variegation depends upon a number of factors including:
(a) how the inhibition is being effected;
(b) how long plastid protein synthesis must be carried out before a stable chlorophyll deficient plant or plant cell has been generated;
(c) the efficacy of any inhibitor of plastid protein synthesis which is being used;
(d) the type of plant or plant cell being used; and
(e) the desired effect (e.g variegation or complete chlorophyll deficiency).
The extent of chlorophyll deficiency and/or variegation may be controlled by altering the treatment regime of the method of the invention. Generally speaking lower concentrations of inhibitor and shorter treatment times are required for variegation than for inducing complete chlorophyll deficiency.
Purely by way of example, an effective treatment for inducing chlorophyll deficiency in Brassicaceae is to soak seeds in Spectinomycin for approximately 6-18 hours (for instance overnight). Such a treatment time has been found to be sufficient to generate chlorophyll deficient seedlings which may be subsequently maintained in a growth medium in the absence of inhibition of plastid protein synthesis. The concentration of Spectinomycin required also varies between different members of the Brassicaceae family. In this respect 2-5 mg/ml of Spectinomycin was ideal for B. napus and 5-10 mg/ml of Spectinomycin was ideal for B. rapa and B. oleraceae. Following such treatments the seeds may be propagated in a medium, such as RMOP (Svab et al. (1990) Proc. Natl. Acad. Sci. USA, 87, p8526) (supplemented +ccordingly) to produce plant cells (which may be grown up as plants or developed into cell lines) with stable chlorophyll deficient phenotypes and an unaltered genetic make-up.
By way of further example, Graminae (cereals) usually require longer inhibition of plastid protein synthesis to generate stable chlorophyll deficient plants and cells. A preferred way by which stable Graminae plants and cells may be generated is by soaking seeds in Streptomycin for approximately 6-18 hours (for instance overnight) and then also propagating the seeds in medium containing Streptomycin at inhibiting concentrations for at least 2 days, preferably for between 1 and 8 weeks, and most preferably 3-5 weeks. Subsequently bleached seedlings may be isolated and maintained in a medium without antibiotic and be stably propagated.
An alternative method is to germinate seeds on wet filter paper in darkness following which they are propagated in vitro at extreme temperatures. This temperature shock for two to four weeks produces stable albino plants that can be propagated in vitro.
The chlorophyll-deficient plants and cell-lines according to the second, third and fourth aspects of the invention may be derived from chlorophyll sufficient plants and plant cell lines which are treated according to the method of the first aspect of the invention.
Chlorophyll-deficient cell lines may also be obtained directly from any plant material treated with an inhibitor of protein synthesis. In this way we have generated chlorophyll-deficient cell lines of Arabidopsis. The plant cell lines according to the fourth aspect of the invention may be developed from plant tissue explanted from plants according to the second or third aspects of the invention using standard plant cell culture techniques.
Plants and plant cell lines according to the second, third and fourth aspects of the invention are useful in several ways.
For instance, the variegated plants according to the third aspect of the invention are valuable to the horticulture industry because they represent attractive plants for retail. Variegation in plants is normally produced by natural or artificial mutation. The production of natural mutations is difficult to control while artificial mutations involve mutagenesis procedures that are hazardous, costly and time consuming. The method of the first aspect of the invention is quick and does not utilise known mutagens. EP 0,257,845 describes the use of chlorophyll inhibitors to generate variegated plants using chlorophyll inhibitors which are not mutagens. However the chlorophyll-deficient plant parts disclosed in EP 0,257,845 require continual exposure to the chlorophyll inhibitors to remain white and the plants and plant cell lines are therefore not stable. The method of the first aspect of the present invention requires the inhibition of plastid protein synthesis which allows the stable propagation of chlorophyll-deficient plants and plant cells when inhibition of plastid protein synthesis is withdrawn. This means that permanent or repeated applications of inhibitors of plastid protein synthesis are not necessary.
New cell lines according to the fourth aspect of the invention may be developed with altered metabolism. Such cell lines are be particularly useful in the development of new pharmaceuticals. Plant metabolites accumulate at altered levels in chlorophyll-deficient plants and plant cell lines. Such metabolites include biologically active compounds that have therapeutic value. Therefore the method according to the first aspect of the invention allows the rapid generation of stable chlorophyll deficient plants and cell lines which may be used for the production of plant metabolites which are therapeutically useful compounds. For example, induced chlorophyll deficiency in Brassica napus leaves results in increased production of anthocyanin from the plant. By way of further example, induced chlorophyll deficiency in the medicinal plant Tanacetum parthenium results in high levels of production by the plant of anti-migraine parthenolides. This cell lines according to the fourth aspect of the invention represent a useful source for the establishment of cultured in vitro cells for producing altered spectra of secondary metabolites which may be used directly or as precursors for pharmacologically important substances.
The plants and cell lines according to the second, third and fourth aspects of the invention may also be used in fundamental studies on plant metabolism (e.g. analysis of photosynthesis and fatty acid metabolism).
An important use of the cell lines according to the fourth aspect of the invention is for producing plastome genetic markers which are useful in somatic hybridization (cell fusion) and especially for one-step generation of hybrids which possess cytoplasmic traits (conferred by plastid, and mitochondrial genes) from one cell and nuclear genes from a different cell.
Thus somatic hybridisation represents an ideal means by which traits encoded by the cytoplasm such as cytoplasmic male sterility (CMS), herbicide resistance and viral resistance or even genetically engineered organelle genomes (which may comprise genes encoding for any desired protein) can be introduced into a cell with a particular nuclear genome. For instance, cytoplasm conferring herbicide resistance could be combined with the nuclear genes from a cereal which has otherwise favourable genetic characteristics (so called elite germplasm). It is also possible to use the abovedescribed methods to generate new traits like CMS de novo by somatic hybridisation. Genetically transformed organelle genomes can only be produced in plants which have a high regeneration capacity in vitro. Somatic hybridisation allows these engineered genomes to be introduced into crop plants whose organelle genomes cannot be manipulated in situ.
A crucial stage in somatic hybridisation of plant cells is a selection of true cytoplasmic hybrids (cybrids). To maximize an output of real cybrids, different selective systems have been proposed, of which the most reliable systems are based on the exploitation of cytoplasmic genetic markers. A favoured selection procedure is the use of a parental species (recipient of cytoplasm) which is a chlorophyll-deficient mutant (in which the mutation is controlled by a cytoplasmic gene). The other parent (donor of cytoplasm) may be protoplasts from a normal green plant. Following fusion between protoplasts from the chlorophyll deficient mutant and green parent inactivated in cell divisions, all green colonies are real cytoplasmic hybrids due to their green colour. Further morphological and chromosome/ploidy analyses may be required to sort out real cybrids containing nuclei of recipient and cytoplasm from the donor.
A problem with this selection method is the difficulty in providing suitable chlorophyll deficient recipients because conventional means for inducing chlorophyll deficiency are time consuming and often inappropriate. For instance, it is undesirable to cause mutations (with the aim of conferring selectability for somatic hybridisation) in a cell which has a favoured genetic make up because the mutagen may corrupt favoured nuclear genes.
Somatic hybridisation utilising chlorophyll-deficient cell lines according to the fourth aspect of the invention provides an improved method for forming fusion products with desirable characteristics. Such a method represents an important feature of the invention and according to a fifth aspect of the invention there is provided a method of forming a fusion cell comprising combining cytoplasm from a donor cell with the nuclear genome from a cell according to the fourth aspect of the invention or a cell derived from plants according to the second or third aspects of the invention.
The cytoplasm from the donor cell is selected such that it contains a heritable property (such as male sterility or herbicide resistance) which can be inherited by and detected within the products of fused cells. The cytoplasm from the donor cell is ideally green (i.e chlorophyll sufficient) and preferably contained within the protoplasts. Such protoplasts are derived from the donor cell and should be inactivated chemically or by irradiation to prevent cell division.
Such protoplasts are preferred sources of cytoplasm for fusion with a cell according to the fourth aspect of the invention because one step selection is possible (only colonies of green dividing cells are true cybrids).
The present invention will be further described, by way of examples, with reference to the accompanying drawings in which:
Figure 1 represent photographs of plants derived from seeds of Brassica napus in Example 1 ;
Figure 2 represent photographs which illustrate altered ultrastructure of plastids in yellow-green and albino plants of Brassica napus in Example 1 (Bars = 0.25μm);
Figure 3 is a photograph of SDS-PAGE gel (A) and an immunoblot experiment (B) of proteins in green and chlorophyll deficient plants of Brassica napus .and Arabidopsis thaliana in Example 1 ;
Figure 4 is a photograph of an agarose gel illustrating plastid ribosomal RNA levels in plant cells of Example 1 ;
Figure 5 is a absorption spectrum illustrating anthocyanin and chlorophyll levels in plant cells of Example 1 ; and
Figure 6 represent photographs of plants treated with Spectinomycin in Example 5; and Figure 7 represents a photograph of (A) stable and (B, C) variagated cereal plants (barley) treated with Streptomycin.
EXAMPLE 1
In vitro experiments were conducted in which chlorophyll deficiency was induced in Brassicaceae using Spectinomycin as an inhibitor of plastid protein synthesis.
1.1 METHODS
1.1.1 Sterilisation of seeds.
Seeds were immersed into 10% (W/V) sodium hypochlorite and gently shaken in jars at room temperature for 20 minutes. The seeds were then washed five times in sterile distilled water. Each wash lasted for 10 minutes.
1.1.2 Preparation of Growth Media
Spectinomycin free RMOP medium (Svab et al., 1990 supra) was used for the growth of Brassicaceae in vitro with some modifications: (1) the addition of 2-[N-Morpholino]-ethane-sulfonic acid (MES).
The composition of the final medium used was:
Macro- MS salts (me/1): Micro- MS salts (mε/1):
NH4NO3 1650 H3BO3 6.2
KNO3 1900 MnSO4 x 4H2O 22.3
CaCl2 x 2H2O 440 ZnSO4 x 4H2θ 8.6
MgSO4 x 7H2O 370 KI 0.83
KH2PO4 170 Na2Moθ4 x 2H2O 0.25
Na2-EDTA 37.3 CuSO4 x 5H2O 0.025
FeSO4 x 7H2O 27.8 C0CI2 x 6H2O 0.025
Organic constituents (mg/1): Phytohormones (mε/1): sucrose 30000 N^-benzyladenine 1 inositol 100 1 -naphthaleneacetic acid 0.1 thiamine 1
2-[N-Morpholino]- ethane-sulfonic acid (MES)* 500 After adjusting pH to value 5.7 with KOH, 0.6% of bactoagar (Difco) was added and the medium was autoclaved for 20 minutes at 120OC.
1.1.3 Seed Treatment and propagation
Sterilized seeds of Brassica napus, B. rapa, B. oleracea, B. nigra and Arabidopsis thaliana were soaked in solutions of Spectinomycin (0, 1, 2. 5 and 10 mg/ml) overnight at room temperature and plated (without washing) on to RMOP medium (see 1.2.1).
Every 3 weeks, white shoots appeared and were dissected and placed flat on fresh medium (solidified or liquid). This approach allowed chlorophyll deficient and variegated phenocopies to be obtained in 3-6 weeks.
1.2 RESULTS
1.2.1 Phenotypic changes following treatment with Spectinomycin
Control cells treated with vehicle only (Omg/ml Spectinomycin) remained green.
The most effective Spectinomycin concentrations for inducing chlorophyll deficiency were 2-5 mg/ml for B. napus and 5-10 mg/ml for B. rapa and B. oleraceae.
Within 3-6 weeks the plants were segregated out into three different phenotypes: green, yellow-green and white (albino type) which were subsequently propagated with stable phenotypes.
Figure 1 represent photographs of plants derived from seeds of B. Napus in which (A) represents an untreated green plant, (B) a yellow-green plant treated with spectinomycin and (C) an albino (white) plant treated with spectinomycin. TABLE 1 shows a correlation between regime of treatment and extent of chlorophyll-deficiency in plants grown in vitro from the treated seeds.
TABLE !
Species Treatment Phenotypes
B. napus 1 mg/ml of Spectinomycin White plants (about 5%); variegated (white and yellow sectors) plants (10-20 %); green plants (70-85%)
2 mg/ml of Spectinomycin White plants (about 15%); variegated (white and yellow sectors) plants (20 -40 %); green plants (35-65%)
5 mg/ml of Spectinomycin White plants (about 40%); yellow plants (about 10%);variegated (white and yellow sectors) plants (50%)
10 mg/ml of Spectinomycin Significant inhibition of growth was observed. White plants (about 10%); variegated (white and yellow sectors) plants (20%)
B. rapa 1 mg/ml of Spectinomycin White plants (about 2-5%); variegated (white sectors) plants (10-15%); green plants (about 80%)
2 mg/ml of Spectinomycin White plants (about 12%); variegated (white sectors) plants (15-25 %); green plants (about 75%)
5 mg/ml of Spectinomycin White plants (about 35%); yellow plants (about 5- 8%); variegated (white and yellow sectors) plants (55%) 10 mg/ml of Spectinomycin Significant inhibition of growth. White plants (about 5-10%); variegated (white and yellow sectors) plants (15-20%)
B. oleracea 1 mg/ml of Spectinomycin All plants were green
2 mg/ml of Spectinomycin variegated (white sectors) plants (5%); green plants (about 95%)
5 mg/ml of Spectinomycin variegated (white sectors) plants (10%)); green plants (about 90%)
10 mg/ml of Spectinomycin Significant inhibition of growth. White plants (about 5%); variegated (white sectors) plants (20%)
1.2.2 Characteristics of Spectinomycin treated plants, (i) Ultrastructure of plastids and mitochondria
Transmission electron microscopy revealed that plastids in (chlorophyll- deficient) plants were smaller and less differentiated than in green plants (Fig 2)
Fig. 2 represent photographs which illustrate altered ultrastructure of plastids in yellow-green and albino plants:
(A) represents normal morphology (top) and ultrastructure (middle and bottom) of chloroplasts from a control green plant (untreated); grana (g) are well developed, plastid ribosomes (pr) are clearly visible;
(B) aggregations of electron-opaque material (eom) instead of grana are observed in plastids of a yellow-green plant; cytosolic ribosomes (cr) outside plastids are clearly visible, plastid ribosomes are not clearly visible within the plastid matrix (m); and (C) perturbed poorly formed, incompletely differentiated plastid-like organelle from albino plant with an external envelope which is not resolved.
(ii) Plastid proteins
Molecular analyses revealed a deficiency in plastid translation products.
Analysis of proteins expressed from green and chlorophyll deficient plants of Brassica napus and Arabidopsis thaliana was performed by SDS-PAGE analysis and immunoblot analysis (see Fig 3).
In Fig 3(A) Coomassie blue stained total soluble proteins were fractionated by SDS/PAGE on a 10% (W/V) gel. Extracts were prepared from 300 mg of frozen tissue homogenised in 600 μl of buffer (80 mM Tris-HCl pH 7.5, 10% (V/V) glycerol, 10% (W/V) SDS, 0.5% (V/V) β-mercaptoethanol), boiled for 3 minutes and centrifuged. 15 μl of extract were loaded per lane (GR=green, AL=albino). The Arrow in Fig 3 shows the position of the most abundant plastid specific protein (the large subunit of ribulose bis phosphate carboxylase-oxygenase). This was absent from the Spectinomycin treated cells (AL) indicating that plastid protein synthesis was inhibited in the chlorophyll deficient cells.
Fig 3(B) shows an immunoblot analysis. SDS-PAGE fractionated proteins were transferred to nitrocellulose (Hybond-ECL, Amersham), incubated with antibodies to Dl (a plastid specific protein of Photosystem 2 encoded by the ps6A gene) and bound antibodies detected by chemiluminescence (Amersham ECL Western blotting detection Kit). Only GR plants expressed Dl, whereas there was no antibody binding to AL protein extracts. This indicates that protein synthesis was specifically inhibited in the plastid. (iii) Plastid ribosomal RNA
Plastid ribosomal RNA (rRNA) was measured in chlorophyll-deficient plants of B. napus (AL) and untreated Green plants (GR) by Northern-blot analysis of total RNA (3 μg of RNA fractionated on a 2% (W/V) agarose gel in 1 x TBE and visualised by ethidium bromide fluorescence (EtBr)). The top half of Fig 4 is a photograph of an agarose gel illustrating plastid ribosomal levels RNA in the plant cells. The photograph illustrates that there was only a slight qualitative decrease in total RNA in albino cells. However, when the RNA was blotted and probed with pHvcP8 (specific for chloroplast (cp) rRNA ), the probe only bound to RNA from the green cells (bottom half of Fig 4). This demonstrates that rRNA is absent in the Spectinomycin treated albino cells because plastids failed to differentiate and plastid protein synthesis was inhibited. This suggests that inhibition of plastid ribosome formation leads to the subsequent breakdown of any transcribed rRNA in the plastid.
(iv) Pigment content
Little chlorophyll was detected in albino Brassica napus plants using spectrophotometrical methods or microscopy relative to green Brassica napus plants. Such albino plants were shown to synthesize significantly more anthocyanins than green plants (Fig 5).
EXAMPLE 2
Experiments were conducted in which chlorophyll deficiency was induced in Graminae using Streptomycin as an inhibitor of plastid protein synthesis.
2.1 METHODS
Seeds were sterilised (1.1.1) and growth medium used (1.1.2) as described above. 2.1.3 Seed Treatment and propagation
Sterilised seeds of Zea mays, Hordeum vulgare andTriticum αestivum were soaked in solutions of Streptomycin (2 and 5 mg/ml) overnight at room temperature and plated (without washing) on RMOP medium (see 1.1.2) which was supplemented with 500 mg/1 of Streptomycin.
After 3-6 weeks, bleached plants were plated on Streptomycin free RMOP medium. Completely Chlorophyll-deficient and variegated plants were collected in the next 3-5 weeks.
2.2 RESULTS
H. vulgαre and T. aestivum (treated with both 2 and 5 mg/ml Streptomycin) only generated variegated plants with a frequency of 10-15% in the first six week period. However during the following cultivation period (the next 3-5 weeks) completely white plants formed and were segregated out from variegated plants.
Maize {Zea Mays) was treated with both 2 and 5 mg/ml Streptomycin according to the methods of 2.1.3. Completely albino (chlorophyll deficient) plants were obtained.
All resulting plant cells were stably propagated in vitro.
EXAMPLE 3
Experiments were conducted in which chlorophyll deficiency was induced in Solanaceae. Treatment of Lycopersicon esculentum and Solanum tuberosum with spectinomycin produced chlorophyll-deficient plant material that could be propagated in vitro. EXAMPLE 4
Experiments were conducted in which chlorophyll deficiency was induced in Compositeae. Treatment of Helianthus annus with spectinomycin produced chlorophyll-deficient plant material that could be propagated in vitro.
EXAMPLE 5
Chlorophyll deficiency was induced in vivo after application of Spectinomycin solutions (5-20 mg/ml) on the top buds of oil-seed rape, linden tree, climbing ivy and holly-tree. New growth from the treated buds produced chlorophyll deficient and variegated leaves.
The chlorophyll-deficiency (albino phenotype) was transmitted from the treated plants to seedlings derived therefrom.
Fig 6 illustrates stable chlorophyll deficiency induced by Spectinomycin in vivo.
Brassica napus were rendered chlorophyll deficient using 20-50 μl of Spectinomycin solution (20 mg/ml) dropped on the apical buds of 4 week old plants growing in soil. After 5 days the procedure was repeated. Bleaching was apparent in new growth after 1 week and increased at two weeks (Fig 6 A). During flowering anthers were removed and flowers fertilised with pollen from non-treated plants. Fertilised flowers produced variegated pods (Fig 6 B) which gave rise to seed. Seeds germinated on RMOP medium produced some variegated seedlings which segregated out yellow and white shoots (Fig 6 C,D)
EXAMPLE 6
Germinated seeds of barley exposed to high or low temperatures produced bleached plants. Bleached plants propagated in vitro produced stable albino plants. EXAMPLE 7
Variagated plants of barley produced by Streptomycin were successfully grown in soil (Fig. 7).
CONCLUSIONS
We conducted experiments which showed that exposure of any part of a plant to Spectinomycin, streptomycin and extreme temperatures resulted in the bleaching of plant tissues in vitro or in v/vo.This was consistent with known literature data. However we have established that subsequent removal of the antibiotics (by transferring bleached tissues onto media without antibiotics) surprisingly led to restoration of growth and produced stable (Chlorophyll-deficient) plants (completely white or variegated) according to the second and third aspects of the invention and from which cell lines according to the fourth aspect of the invention were easily derived.
The abovedescribed experiments represent what we found, after significant modification of preliminary experiments, to be effective procedures for producing chlorophyll deficient and/or variegated plants and plant cell lines.
Our data show that the frequency of white plants and the extent of variegations was dependent on the concentration of antibiotics and time of exposure.
For Brassica species, best results were obtained by treating sterilised seed with highly concentrated water solutions of antibiotics for relatively short exposure times and subsequent germination and cultivation of seeds on media without antibiotics.
For cereals, the most efficient approach was to combine soaking seeds in the Streptomycin solutions, following which the treated seeds were generated on medium containing Streptomycin at inhibiting concentrations. Subsequently bleached seedlings were transferred onto medium without antibiotic and were stably propagated. Exposure of germinated seeds to a heat or cold shock also produced albino plants that could be propagated in vitro..

Claims

ZjCLAIMS
1. A method of producing stable (as defined herein) chlorophyll deficient plants and plant cell lines comprising subjecting plant material to a treatment which inhibits plastid protein synthesis, and growing plants and/or plant cell lines from the treated plant material under conditions in which said treatment is not applied.
2. A method as claimed in claim 1 wherein said plants and/or plant cell lines are grown in a medium containing phytohormones.
3. A method of producing stable (as herein defined) chlorophyll deficient plants and plant cell lines comprising subjecting plant material to a treatment which inhibits plastid protein synthesis, and growing plants and/or plant cell lines from the treated plant material in a growth medium containing plant phytohormones.
4. The method according to any one of claims 1 to 3 wherein the inhibition of plastid protein synthesis is such that the capacity to form chloroplasts is irreversibly blocked.
5. The method according to any one of claims 1 to 4 wherein the inhibition of plastid protein synthesis is effected by an inhibitor of plastid genome expression or plastid protein synthesis.
6. The method according to claim 5 wherein the inhibitor disrupts the function of plastid ribosomes.
7. The method according to claim 6 wherein the inhibitor is Spectinomycin or Streptomycin or extremes of temperature.
8. The method according to any preceding claim wherein the plant material is a Brassicaceae plant.
9. The method according to claim 8 wherein the plant is one of Brassica napus, Brassica rapa, rassic. oleracea, Brassica nigra or Arabidopsis thaliana.
10. The method according to any one of claims 1-8 wherein the plant material is a Graminae plant.
11. The method according to claim 10 wherein the plant is one of Zea mays, Hordeum vulgare or Triticum aestivum)
12. The method according to any one of claims 1-8 wherein the plant material is a Solanaceae plant.
13. The method according to claim 12 wherein the plant is Lycopersicon esculentum or Solanum tuberosum.
14. The method according to any one of claims 1-8 wherein the plant material is a Compositeae plant.
15. The method according to claim 14 wherein the plant is Helianthus annus.
16. A plant in which plastid protein synthesis has been inhibited whereby the plant is chlorophyll deficient and is stable (as defined herein).
17. A plant in which plastid protein synthesis has been inhibited whereby the plant is variegated and is stable (as defined herein).
18. A plant cell line in which plastid protein synthesis has been inhibited whereby the plant cell line is chlorophyll deficient and is stable (as defined herein).
19. A method of forming a fusion cell comprising combining cytoplasm from a donor cell with the nuclear genome from a cell according to claim 18 or a cell derived from plants according to claims 16 and 17.
PCT/GB1998/002803 1997-09-19 1998-09-18 Method for producing stable chlorophyll deficient plants and plants obtained with such a method WO1999015001A1 (en)

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JP2000512397A JP2001516596A (en) 1997-09-19 1998-09-18 Method for producing stable chlorophyll-deficient plant and plant obtained by the method
EP19980944041 EP1014779A1 (en) 1997-09-19 1998-09-18 Method for procucing stable chlorophyll deficient plants and plants obtained with such a method
AU91719/98A AU9171998A (en) 1997-09-19 1998-09-18 Method for producing stable chlorophyll deficient plants and plants obtained with such a method
NO20001344A NO20001344L (en) 1997-09-19 2000-03-15 Process for the production of stable chlorophyll-deficient plants and plants obtained by such a method

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EP0257845A2 (en) 1986-08-01 1988-03-02 Masayasu Taguchi Method of producing variegated plants

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CN1279580A (en) 2001-01-10
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NO20001344L (en) 2000-05-16
GB9719880D0 (en) 1997-11-19
EP1014779A1 (en) 2000-07-05

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