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(11) Patent Number: KE 143
(45) Date of grant: 04/03/2002
(12) PATENT
 
(51) Int.CL6:A 01H I/06, 5/00, C 12N 15/29
(21) Application Number: 1998/ 000011
(22 Filing Date: 20/07/1998
(30) Priority data: P08174 22/07/1997 AU
(86) PCT data
PCT/AU98/00573 20/07/1998
W099/04620    04/02/1999
 
(73) Owner: VIRGIN COTTON COMPANY of, AUSTRIA, l PERCIVAL ROAD, STANMORE, NSW 2048, Australia
(72) Inventor: FURSOV, VIKTOR and HULMAN, KAMILA
(74)    Agent/address for correspondence: W.R.McA SPENCE, P.o.Box 43984 Nairobi
 
(54) Title:SELF-DEFOLIATING PLANT.
(57) Abstract:
This invention relates to a cotton plant which self defoliates, and to the progeny, reproductive material, seeds, cuttings, seedlings, protoplast, leaves, stems, flowers and cotton thereof. It also relates to fibres and textiles made using cotton from such a plant, and to nucleic acid molecules comprising a sequence associated with the self-defoliation characteristics.
 

SELF-DEFOLIATING PLANT
This invention relates to plants of cotton (Gossypium hirsutum L) having new and improved
characteristics, and in particular to cotton plants which have the property of self-defoliation. The invention also relates to the [gene or gene] which determines the self-defoliating properly.
BACKGROUND OF THE INVENTION
The production of cotton is a major industry in many countries, including Australia. All cotton fibre is produced from plants of the genus Gossypium. The most commonly grown cotton plants are varieties of Gossypium hirsutum (American Upland cotton), which produces fibres of, medium staple length, and these are grown in the United States, Australia, Pakistan and other countries where extensive irrigation is available. Egyptian cotton, which has a finer, longer fibre, is produced from Gossypium barb dense, grown extensively in Egypt and Sudan. Gossypium herbaceousand Gossypium arboreum are grown in unirrigated areas of India, Pakistan and other Asian countries, and produce coarser, shorter fibres.
The purity of commercial seed stocks is carefully controlled to avoid problems resulting from crossing between varieties or seed mixing.
However, the cultivation of cotton plants traditionally has required high-intensity agricultural practices, including heavy irrigation and the application of a number of pesticide Cotton plants are prone to disease andto infestation by a variety of insect pests, such as Heliothis caterpillar, and the various species of cotton boll-worms, and hitherto control of these pests has required intensive use of chemical insecticides. Furthermore, because of the requirements of mechanical harvesting, defoliants are applied just before harvest in order to remove the leaves from the plant so as to render the cotton bolls easily accessible to the harvesting machinery.
Consequently the cotton-growing industry has been the cause of considerable Environment-al pollution and the industry is under great pressure to reduce release of chemicals into the environment. Integrated pest-management practices are increasingly being used, and cotton plants genetically engineered to be resistant to disease or which express Bacillus thuringiensis toxin, a natural insecticide of bacterial origin, are becoming available to commercial cotton growers. However, hitherto there has been no alternative to the use of Chemical defoliants before harvest.
Whilst wild species of perennial cotton such as G. aridum genom D4 G. gossypiodes D6 and G trilobum D are said to lose leaves acquired during the rainy season when the dry season arrives, this self-defoliation to date has not been found in cotton strains grown commercially.
For many years, traditional breeding methods have been used in an endeavor to identify and select strains of cotton which have improved resistance to the major insect and fungal pests which attack these plants, or which have other desirable characteristics. In parallel, breeding programs have also been directed to the production of self coloured cotton, which does not require the use of chemical dyes during textile processing.
Professor Victor Fursov, a member of the Academy of Technological Science of the former Soviet Union, commenced cotton plant development programs in March 1962.
The program between 1962 and 1993 involved the development of a strain of cotton with specific characteristics, bred and tested under commercial conditions. Theseincluded trains of cotton in varying colours of green beige, brown and “snow white”. Surprisingly the beige and brown self-coloured strains were found to have superior resistance to major insect and fungal pests and had bactericidal properties.
 
Further development, generation of strains and selection carried out in Australia identified certain strains which have the property of self-defoliation, and which do not need application of chemical defoliation agents.
SUMMARY OF THE INVENTION
In one aspect, the invention provide self-defoliating cotton plant. In one embodiment, the plant has a gene or functional fragment thereof which is activated to effect self-defoliation of the cotton plant.
In a particularly preferred embodiment, there is provided a strain of cotton (Gossypium hirsutum) characterized in that the plants self-defoliate at the stage of boll opening.
The present invention further provides a self-defoliation slant which includes a nucleic acid or functional fragment thereof which is activated to effect self-defoliation of the cotton plant.
 In a second aspect, the invention relates to a self-defoliating cotton plant having a DNA fingerprint as shown in Figure (3). The plant comprises a nucleic acid sequence which determines self-defoliation of cotton (defoliating gene), which gene can be activated by chemical
treatment and irradiation. Preferably the nucleic acid sequence comprises the sequence set out in SEO ID NO: 2. More preferably, the gene is activated by treatment with ethylene imine (Aziridine) and ionizing radiation.
In a third aspect, the invention provides a method of activating a defoliating gene in cotton,
comprising the step of treating the seed of said cotton with ethylene imine and ionizing radiation. Preferably, hybrid seeds produced by crossing of parent cotton plants which have been selected for desired characteristics or traits are treated with 0.1% v/v aqueous ethylene imine for 10 hours, followed by gamma-irradiation of said seeds with 20 kiloroentgens absorbed dose, with a preferred dose of 4 kilo roentgen for 50 seconds. The irradiation may be suitably effected by exposure of the seeds to a Cobalt 60 gamma-ray source, such as MPX-gamma 3.
In a fourth aspect the invention relates to cotton plants which exhibit self-defoliation. The gene for self-defoliation may be activated by chemical and radiation methods, or may be inherited from a parent plant.
Preferably the cotton fibres are of a colour selected from the group consisting of beige, snow-white, brown and green. Also preferably the cotton plants are resistant to one or more diseases caused by Thielaviopsisbabicola, Fusarium vasinfectum and/or Bemisia tabaci.
In a particularly preferred embodiment, the plant is of a variety selected from the group consisting of Rainbow 34, Rainbow 39, Rainbow 38 and Rainbow 37, as herein described. It will be clearly understood that these varieties have colours of the cotton fibres as the principal characteristic differentiating between them.
The whole plants, seeds, and other reproductive material derived from the plants, including cuttings and protoplasts, all form part of the invention. In addition, products derived from the cotton plants, including cotton fibres and textiles produced therefrom, also form part of the invention.
In a separate aspect, the invention provides cotton plants which have been transformed with the self-defoliation gene of the invention. In particular, genetically-engineered strains of cotton and methods for their production are known. There are a number of patents and literature publications by workers from Agracetus and Monsanto describing methods for transformation of cotton, and transgenic cotton plants expressing exogenous proteins such as Bacillus thuringiensis crystal protein. Such transgenic cotton plants have been widely field tested, and some strains are in commercial production.
For the purposes of this specification, the term "self-defoliation" is to be understood to mean the self-shedding of foliage and/or leaves from the lower sections of the plant to the higher points, between the period of the growth cycle from 110 days to 135 days, at which time watering can delay the cycle.
Full boll opening occurs at approximately 110 to135 days.
The terms "activated" and "activating" are to be understood to mean the conversion of the dormant gene for defoliation to one the expression or expression product of which contributes to self-defoliation of a plant containing such a converted gene. The activation includes unblocking of a dormant gene by mutation or by removal of a blocking agent, or by inhibition of an activity thereof. It also includes the inheritance of a gene that was previously activated in the manner described above.
Throughout the description and claims of this specification, the word "comprise" and variations of the word, such as "comprising" and "comprises", means "including but not limited to" and is not intended to exclude other additives, components, integers or steps.
Detailed Description of the Invention
The invention will now be described in detail by way of reference only to the following non-limiting examples, and to the figures, in which: Figure 1  comprises photographs of plants of the preferred embodiments of the invention, “Rainbow 34" (Figure 1A) and "Rainbow 39" (Figure 1B).
Figure 2 illustrates the manner in which Rainbow 39 self-defoliates at the time of opening of the cotton bolls, compared with a non-self-defoliating comparator strain, Sicala-34.

Figure 3 shows the DNA fingerprint of Rainbow 39 and the comparator strain, Sicala-34.
Figure 4 is a bar graph showing the results of one way analysis of variance of Rainbow 34, Rainbow 39 and Sicala-34.
 
Example 1    Production of Hybrid Seeds for Activation of the Gene for Defoliation.
Analysis of the genealogical data on cotton plants suggested that crossing of the initial parental varieties, 5476I and 7631 I, was likely to produce white and light beige fibre with high spinning and technological parameters in the hybrid progeny.
The selected elite or P seeds were first subjected to reciprocal hybridization of not less than
1000-1500 pollinated flowers at 75 per cent setting of seeds in each variant, and F2 plants were crossed with F3 plants. In the second hybrid generation, desired characteristics were selected by sowing seeds and subjecting the plants to the progeny test pedigree method which is familiar to one skilled in the art. Separate sibs were picked from the heterogenic complex, and posterities were developed, with strict determination of the principal characteristics. In particular, resistance to fungal and bacterial disease and to insect pests, and colour of the cotton fibre, were used as selection criteria. This selection program was carried out in Russia from 1978 to 1993, and in Australia from 1995 to 1996 under a contract arrangement with the University of Sydney.
Uniform families were crossed to form a new variety, "Genetic 1" bred by Professor Fursov at the Russian Academy of Technological Science, which was deposited in the selection catalogue of the Institute of Cotton Selection of Turkmenistan in 1978. This variety was selected for the technological and spinning qualities of the cotton fibre which it produces.
Example 2Treatment of Hybrid Seeds and Selection for Self-Defoliation.
The hybrid seeds produced as described in Example 1 were treated by exposure to 0.1 per cent aqueous ethylene imine for 10 hours.
 
Ethylene imine was used in concentrations of 0.025 - 0.05, and 0.1 - 0.4 6 (v/v) in water.
Application of various concentrations of this mutagen resulted in different degrees of mutation. After treatment, the quantitative analysis and qualitative analysis of the degree of mutation showed that the mutagen had the greatest effect in "young", recently selected varieties, but hybrids also gave an especially high yield. The more heterogeneous a genotype, the more mutations were observed at a given level of mutagen. The optimum concentration of ethylene imine was 0.1%.
Following treatment with 0.1% ethylene imine for 10 hours, the hybrid seeds were gamma-irradiated using a Co 60 source at a dose of 20 kiloroentgens using the gamma-ray source, MPX-gamma 3-, with 4 kiloroentgen for 50 seconds. The cotton seeds were also irradiated with multiple doses of gamma-irradiation, at levels of -0.25 - 0.05, -1.0 -2.0, - 3.0 - 5.0. Doses of 10.0 - 20.0 were semi-lethal, and doses of 30.0 - 60.0 were superlethal. In storage, however, the semi-lethal doses were not totally harmful, and the recovery of the seeds was carefully observed.
The treated seeds were placed in gauze sacks and stored for 18 months at ambient, indoor temperatures.
After 18 months of storage, the selected family seeds were sown into a third hybrid generation, and the elite plants including those manifesting the ability to self-defoliate were selected and crossed between each other. The stored seeds were sown in summer and picked in autumn, with each plant contained in separate sacks. The percentage of deformation was determined as the frequency of phenotypic change, modification of morphology, teratism and other non-hereditary, new growths due to the gamma radiation. After selecting and recording variations in phenotype and any visible changes resulting from mutation, the seeds which gave rise to each phenotype were individually ginned and kept as separate families for sowing the following year.
 
The genealogical scheme for producing a cotton plant in accordance with the invention is summarized as follows:-
F3 (7631-I x 5476-1)x F2 (5476-I x 7631-I) Natural hybrid progeny of these parents were then
treated with 0.1 per cent ethylene imine for 10 hours. Seeds were selected from the treated population to form seminal M2 and M3 generations. The best lines from these were then crossed.
Example 3    Reproduction of Self-Defoliating Cotton Producing Coloured Fibres; the "Rainbow" Series.

The best lines of seeds treated in the manner described in Example 2 were top crossed and the most significant maternal form of 4 strains were selected. The rest of the strains were hybridized as paternal forms.
The first generation genetic characteristics of self-defoliation were determined by phenotypic domination. All were gathered by individual selection. In the second generation, the population heterogeneity or heritability coefficient was determined. The polygenicity resulted in-display of the dominant phenomenon in the first generation hybrid.
This selection method was used to produce a variety of cotton with natural white and light beige fibre with full self-defoliation, such as Rainbow 34 [light beige] and Rainbow 39 [white].
Two other strains, respectively designated Rainbow 38 and Rainbow 37, were also selected for development. The characteristics of these varieties are set out in Table 1.
 
 
Plants of strains Rainbow 34 and Rainbow 39 are illustrated in Figure 1, and the manner in which Rainbow 39 loses its leaves at the time of boll opening is illustrated in Figure 2.
It is practicable to repeat the invention using current state of the art techniques to carry out the number of trials necessary to achieve the desired result. Samples of seed Rainbow 39 were deposited under the provisions of the Budapest Treaty with Australian Government Analytical
Laboratories on 2 July 1998 and accorded the accession number NM 98/06259. These seeds are also held in the depositary of The University of Sydney, Plant Research and Quarantine Station at Cobbitty, N.S.W., Australia.
The general principles of the method of selecting white and mutant coloured and self-defoliating cotton and methods of defining self-defoliation were as follows:-
First Year
To reproduce cotton in the initial form of two species G. hirsuturn and G. peruvianum Cav, the treatment described in Example 2 was applied to the progeny, and dominant mutants manifesting early natural defoliation genotypes by any character were selected.
During the first year, any initial form or variety was produced, so that the total number of seeds with the selected traits would be not less than 105-106 in the heterogeneous population of the variant. Following treatment with the mutagen 1.4 bisdiazoacetyl butane in aqueous solution of 0.1 per cent (v/v} concentration for 20 hours, seeds were washed clean in running water for 2 hours and a sample sowed by placing in highly fertile ground, with 3 seeds in each hole at a depth of 4-5 cm. The soil temperature at a depth of 4-5 cm is preferably 12-15°C. The plot desirably contains 3 plants in a plot 60 cm x 20- 25 cm or 3 plants in a plot of 90 cm x 15 cm.
 
After shoots appeared, only 2 plants were left in the hole. When two real leaves appeared, the plot was further thinned out to only one plant in the hole.
During growth of the plant, surveys andphenol typing during development were conducted to reveal donor forms and label them for later selections.
During the period of blossoming and ripening of the bolls, all morphological variations of flower, leaf, tomentum of stem, pollen colour, and boll shape were compared with those from non-treated plants, and findings were recorded.
When bolls opened, all clonal forms of mutation were chosen by individual selections. The collected material was analyzed, ginned and stored until the next year.
Second Year
The individual seeds showing characteristics selected in the first year were stored and then sown in the second year, with the purpose of selecting multiple mutations, caused by meiosis in comparison with normally
formed seeds not treated with mutagens. Morphological observations and self-defoliation dynamics test were then conducted.
The percentage index of timely defoliation is very important: Firstly, the phenomenon should be visually and clearly characterized.
Secondly, a quantitative index is desirable for any comparison or dispersion factor analysis.
The harvest from the selected self-defoliating plants was picked by individual selection, and analyzed for boll mass, fibre yield, wave length, fibre index, 1000 seeds mass, metric number, filament breaking strength, sinuosity, luster, colour etc. The seeds were stored until sowing in the next year.
 
Third Year
Among selected self-defoliated and other donor forms, consistency and genetic purity tests were carried out during the third year of growth. The percentage of hereditary, homologous mutant lines as determined. All of these lines were chosen by individual selection, and a laboratory grade quality test was also performed.
Fourth And Following Years
After the final study of prospective and competitive new lines, comprehensive varietal grade testing was conducted, and stable forms were carefully examined to ensure that the required characteristics of early, natural defoliation, white and naturally coloured fibre etc. were maintained.
These characteristics usually demonstrated consistency at the beginning of the third growth year. With precise sowing diaries, the degree of any weak character or contaminated species was marked in families.
 Then the breeding study (selection) was carried out using two subsequent grading’s of excellent characteristics which were displayed. Families with any poor characteristics or which were susceptible to attack by whiteflies were rejected, and further selection of these terminated.
Remaining plants were simultaneously selected from the most reproducible families, and used for testing of varietal purity and to ensure continued resistance to damage by whiteflies.
The most superior breeding lines were planted for grade testing and studying against an artificial
provocative background. For this purpose, 10 samples of bolls were taken for complex analysis. Selected group families then provided 2.5-3.0 kg of pure grade seeds and were frozen for future use.
The cotton defoliation percentage is defined in two ways.
 
1. The ratio of defoliated leaves on the main stem (the number of fruit node leaf ribs) to the total number of the formed ones, including already fallen leaves on the main stem by the fixed time in per cent.
These ratios can be expressed by formulae:-
      Cf
D=----     x 100 per cent (defoliation for one plant)
     F

     Cf
D------ x 100 per cent at F. Cf +con f
Cf+con f

where:
          D Defoliation
          Cf Number of leaf ribs from Latin-cicatris folii
           F Total number of leaves con f Dead leaves and preserved leaves
Self-defoliation of cotton most desirably occurs coincidentally with the time of full boll opening and ripeness, and the earlier this characteristic is detected, the more commercially useful.
In the second generation, stable, constant elite plants with early natural self-defoliation were selected.
They represented dominant and recessive mutations, which can be progenitors of self-defoliating cotton varieties, without the unblocking of the gene. Those exhibiting early natural self-defoliation were designated as "Fc", from the Latin folium caolucus (fallen leaf).
The chemical-mutagenesis method of the invention permits rapid, ordinary selection of progressive mutations.
 
Furthermore, the greatest probability of efficient induction of self-defoliation is ensured by chemical action of agents having chemical affinity for DNA. Without wishing to be bound by any proposed mechanism for the observed advantages, it is believed that the affinity of a chemical such as ethylene imine together with irradiation reverses the blockage of the dormant or "sleeping" defoliating gene (dominant as well as recessive), thereby activating it.
It is practicable to repeat the invention using current state of the art techniques to carry out the number of trials necessary to achieve the desired result.
Additional data of the invention are given in Tables 2 to 5.
     
      Table 5
COTTON
Gossypium hirsuium
`Rainbow - 39' synonym: 'Genetic 39'.
Description: Plane spreading; medium to tall height; dense foliage; the fruiting branches are long. Leaves: palmate with pubescent midrib and also have gossypol glands and nectarines and are deciduous at maturity. Flowers: cream. Bolls are elliptic with long 26.24 mm peduncles. Fibre length is 1.26 ins when ginned with the "shark-skin" method. Fibre: uniformity index 89.26%, elongation 5.9 %, strength 32.24 g/tex and micronair value is 2.7. Origin: Induced mutation by radiation used on seed of breeding line 'Turkmenistan Genetic 1'. Breeder: Professor V. N. Fursov, Ashgabat, Turkmenistan. In the following generations pedigree method was used to select early maturing, self defoliating plants with long staple length until the stable variety was established.
Comparative Trials. Comparator: Sicala-34. Conducted in 1994/95 in the greenhouse of the Commonwealth Quarantine Station, Rydalmere and in 1995/96 at The University of Sydney, Plant Breeding Institute, Narrabri. Measurements were taken from 95 plants selected at random from a trial arranged in randomised complete blocks in four replicates. The fibre quality data were acquired from lint obtained by 'shark-skis" ginning and the tests were replicated five limes. Greenhouse-grown plants also displayed long fibre lingth.
Adaptation
Rainbow- 39' can be grown in any district where cotton could be produced
 
Example 4    Other Embodiments.
Interspecific hybrids - previously in Fl -treated with ethylene imine in 5 concentrations of aquatic solution with control variant, were sown in an area of 0.25 hectares.
Then in laboratory trials, F3-Chem3, the breeding strains/progenies of the newly obtained variety were grown, re-pollinated by the top cross method for the best mutant line, combining one each genotype of white or beige colour with 100 per cent self-defoliation.
To define the genetic nature of the defoliation trait of top crosses in Fl, the phenotypic domination degree-P was determined.
These Fl hybrids showed dominance of the self- defoliation characteristics of initial forms when crossing the most superior selected self-defoliation mutant lines, breeding the variety herein designated 'BD", obtained as the result of the selection method.
In F2 the heritability of the self-defoliation characteristic of hybrids is defined. The degree of
heterogeneity in mutation generations and in hybrid F2 -F3 populations was determined by the genetic variability or hereditary ability co-efficient which was calculated by the Allard formula:
h2          hereditary ability coefficient of any character
G2 P1 first parent dispersion
G2 Fl hybrids Fl dispersion
G2 P2 second parent dispersion
G2 F2 hybrids F2 dispersion
G2 F2 - G2 Fl 4- G2 P1    G2 P2
    h2 =                                  3
   G2                         F2
 
Example 5    DNA Fingerprint of Rainbow 39
DNA extracted from Rainbow 39 was compared to DNA of Sicala-34 (the comparator strain) in DNA fingerprint assays performed under contract by the Australian Government Analytical Laboratories, Molecular Biology Laboratory.

Samples of genomic DNA were extracted from two Gossypium hirsutum varieties (Rainbow 39 and Sicala 34) by the cetyltrimethyl ammonium bromide (CTAB) method. CTAB is a detergent which disrupts cell walls and forms a complex with nucleic acids. The CTAB-nucleic acid complex can then be purified and separated from carbohydrates (Brian et al 1994, Scott et al 1994). The DNA extracted using the CTAB was then subjected to further purification steps to eliminate inhibitors of DNA polymerase. Firstly, polyvinyl-pyrrolidone (PVP) was added to the CTAB extraction buffer. PVP improves the precipitation of polyphenolics and other organic substances (Kim at al 1997). Secondly, the final DNA pellets were incubated with
InstaGene Matrix. (BIO-RAD), a commercial DNA purification matrix. This resin-based matrix efficiently absorbs cell lysis products that interfere with the PCR amplification process. The results showed that a satisfactory PCR amplification was achieved using the cotton DNA extracted by this new method. Suitable 10-mer primers for the generation of DNA fingerprints were screened from 40 randomly selected 10-mer primers.
A large amount of genomic DNA is required for the generation of DNA fingerprints for cotton plants. Fresh young leaf tissues therefore are required for the DNA extraction. Cotton leaf tissues that are not fresh have very low levels of DNA, a high level of polyphenolics and other inhibitors.
Extracted DNA was assayed by the technique of Random Amplified Polymorphic DNA Polymerase Chain Reaction (RAPD PCR).
 
The DNA fingerprints of RAINBOW 39 and SICALA 34 showed significant differences when three single primers were employed in RAPD PCR assay although most primers generated identical DNA fingerprints for both RAINBOW 39 and SICALA 34. This indicated that RAINBOW 39 and SICALA 34 are very closely related cotton varieties. However, as shown in Figure 3, one primer (primer .11) generated reproducibly distinct DNA fingerprints between RAINBOW 39 and SICALA 34. The DNA fingerprint gel profiles generated with primer .11 showed the different RAPD PCR profiles (lanes 4 and 6) between RAINBOW 39 and SICALA 34. The DNA band sizes were indicated by DNA standard GENESCAN-2500.
The protocol for obtaining the DNA fingerprints is given in detail below:
1. Extraction of genomic DNA from Gossvpium hirsutum
Deoxyribonucleic acids (DNA) was extracted from fresh young leaf of cotton by the protocol described below.

(1) 250 mg of cotton tissues were ground in a 20 motar with 20 µl of 2-mercaptoethanol (SIGMA, M-3148) until the tissues were of a creamy consistency.
(2) 600 µl of 2 x extraction buffer was added to the tissues and was further ground until the tissue solution became clear (2 x extraction buffer: 2.0 8 cetyltrimethylammonium bromide, 1.4 M Nadi, 100mM Tris-HC1 pH 8.0, 20 mM ethylenediamine tetraacetic acid, 1.0 % polyvinylpyrrolidone).
(3) The tissue solution was transferred into a 1.5m1 microtube and incubated at 65°C for 5 minutes.
(4) 600 µl of chloroform / isoamyl-alcohol (24:1) was added into the tissue solution and mixed thoroughly with a vortex mixer to form an emulsion. The microtube was then centrifuged at 10,000 x g for 5 minutes.
(5) The solution from the top aqueous phases was transferred into new microtubes each containing 600 µl of isopropanol, and mixed by inversion until the white thread-like strands of DNA formed visible masses.
 
(6) The microtube was incubated on ice for 10 minutes and centrifuged at 5,000 x g for 2 minutes.
(7) The supernatant was removed with a pipette and discarded.
(8) The DNA pellet was hydrated by the addition of 400 µl of high-salt TE buffer and then incubated at 65C for 10 minutes (high-salt TE buffer: 10mM Tris pH 8.0, 1.0 mM ethylenediamine tetraacetic acid pH 8.0, 1.0 M NaCl).

(9) The DNA was precipitated by the addition of 10 800 p1 of absolute ethanol and mixed by inversion until the white thread-like strands of DNA formed visible masses.
(10) The microtube was incubated on ice for 10 minutes and centrifuged at 5,000 x g for 2 minutes.
(11) The supernatant was removed and discarded 15 with pipette.
(12) The DNA pellet was dried at room temperature by leaving the microtube open to air for 1 hour.
(13)  The DNA pellet was rehydrated by the addition of 200 µl of 10% (w/v) of Chelex® 100 Resin (BIORAD) in TE Buffer and incubated at 55°C for 30 minutes. (TE Buffer: 10mM Tris pH 8.0, 1.0 mM ethylenediamine tetraacetic acid pH 8.0).
(14) The rehydrated DNA solution was centrifuged 25 at 12,000 x g for 10 minutes.
(15)The DNA supernatant was transferred into new microtubes.
(16) The resin pellet was suspended by 200 µl of TE Buffer and incubated at room temperature for 20 minutes. 30 Steps (14) and (15) were then repeated.
(17) The ribonucleic acids (RNA) were removed from DNA solution by the addition of 2 µl of RNase (20 mg/ml) and the microtube was incubated at 37°C for 30 minutes.
(18) The DNA concentration and quality were measured by the ethidium bromide fluorescence quantitation method.
 
(19) The DNA at this stage was used for RAPD PCR assay or stored at 4°C.
2. Random amplified polymorphic DNA polymerase chain 5 reaction (RAPD PCR) for the generation of DNA fingerprints.
This protocol generated DNA fingerprints by random amplified polymorphic DNA (RAPD) technology, utilizing a single, 10-mer oligonucleotide primer of arbitrary sequence to amplify genomic DNA sequences of cotton Gossypium hirsutum varieties (RAINBOW 39 and SICALA 34). GeneScan 672 software was used to analyze the RAPD PCR products.
(I) RAPD PCR Procedure
RAPD PCR was set in a volume of 25µ1 with approximately 25 nanograms of cotton genomic DNA, 100 µM of a single 10-mer primer, primer z11:
5'-CTCAGTCGCA-3' (SEQ ID NO: 1),

2.0 mM MgC12 10 mM Tris-HC1, pH 8.3, 16.6 mM (NH4),SO4, 100 µg/m1 gelatin, 0.45% Triton-X100, 100gm dATP, 100µM dCTP, 10011M dUTP, 1001.1M tap, 0.1 µM dUTP, and 1.0 unit of Taq DNA polymerase (Perkin Elmer). The reactions were performed within a 0.5m1-microtube overlaid with mineral oil. Amplification was cycled in a thermal cycler (HYBRID, OmniGene, U.K.) preheated to 95°C. The cycler was programmed for 45 cycles of 1 minute at 94°C, 1 minute at 36°C and 2 minutes at 72°C on a for DNA denaturing, primer annealing, and primer extension, respectively. The PCR products were stored at -20°C.
 
(ii) RAPD PCR Generated DNA Fingerprints were analyzed using AB1 GeneScan 672 Softwareon a DNA Sequencer 373 System.
Electrophoresis Conditions
A 4.5% native polyacrylamide gel solution was prepared with 9 ml 40% Acrylamide: N,N’-Methylen-bis-acrylamid - 19:1 stock solution (BIO-RAD), 16 ml of 5xTBE buffer (5xTBE buffer in 1 liter: 54g Tris base, 27.5g boric acid, 20 ml of 0.5M ethylenediamine tetraacetic acid pH 8.0), 55 ml of distilled water, 400p1 of 10% ammonium per sulfate, and 45p1 of N,N,W,1T-Tetra-methy¬ethylenediamine.
The buffer was 1xTBE (280 ml of 5xTBE buffer, 1120 ml of distilled water). Electrophoresis was performed at a voltage of 700v for 18 hours. RAPD PCR products were diluted with distilled water in 1:10. 111 diluted products were combined with 1p internal lane DNA size standard GeneScan-2500 ROX and 3111 loading buffer (AB1 GeneScan kit). The combined samples then were loaded on the gels described above. Results of DNA fingerprints were automatically analysed and reported by AB1 373 automatic DNA sequencer with GeneScan 672 software, and are illustrated in Figure 3.
Example 6    Sequencing of Rainbow 39
A 227 by genomic DNA fragment was amplified from cotton Gossypium hirsutum variety (Rainbow 39) using the Polymorphic Random Amplified DNA Polymerase Chain Reaction
(RAPD PCR) assay as outlined below. This DNA fragment was not amplified by this assay from another, non-self-defoliating cotton variety (Sicala 34). The 227 by DNA fragment was cloned into a plasmid vector and sequenced using the ABI 373 automatic DNA sequences. The DNA sequence of the fragment was determined, and is shown in SEQ ID NO: 2.
 
Experimental Protocols
1. PCR by random amplified polymorphic DNA (RAPD) assay Genomic DNAs from cotton Rainbow 39 and Sicala 34 varieties were subjected to RAPD-PCR reaction. Each amplification reaction was performed in a volume of 25p1 with approximately 25 nanograms of genomic DNA, 25 µg of a single 10-mer primer, 2.0 mM MgC12, 10 mM Tris-HC1, pH 8.3, 16.6 mM (NH4)2504, 100 µg/ml gelatin, 0.45% Triton-X100, 100pm of each dNTPs, and 1.0 unit of Tag DNA polymerase.
The reactions were performed within a 0.5m1-microtube overlaid with mineral oil. Amplification was programmed for 45 cycles of 1 minute at 94°C, 1 minute at 36°C and 2 minutes at 72°C on a thermal cycler for (HYBRID, OmniGene, U.K.) DNA denaturing, annealing, and primer extension, respectively. Blank control (no DNA added in the PCR reaction and replaced with water) was included in each RAPD PCR performance. The PCR products were analysed by electrophoresis on a 1.4% agarose gel and visualized with ethidium bromide staining and photographed.
2. Purification of RAPD PCR Fragments DNA bands were separated on 1.2% low melting point agarose gels stained with ethidium bromide, and the desired fragment was sliced from the gel.
The sliced gel containing the DNA fragment was put into a 1.5 ml Eppendorf tube and covered with TE buffer and then heated for 15 minutes at 70°C to melt the gel. The melted gel solution was extracted with phenol/chloroform and then with chloroform. The DNA was precipitated by adding 0.1 volume of 3M sodium acetate and 1 volume of isopropanol and incubation at -20°C overnight. The DNA was collected by centrifugation at 10,000 g for 15 minutes and suspending the DNA pellet in sterile water. The DNA concentration was measured by running an electrophoresis gel with quantified DNA molecular size markers.
 
3. DNA Cloning
(a) Ligation

The pGEM-T vector (Promega) was also used for cloning the PCR band. Ligation of PCR products using these vectors was carried out according to the manufacturer's instructions.
(b) Transformation
10% of the ligation reaction, or 5 µg of uncut vector for control, was mixed with 100 µl of competent cells in an Eppendorf tube and incubated on ice for 1 hour. The Eppendorf tubes were heat shocked at 42°C for 2 minutes in a water bath then incubated on ice for 20 minutes.
Then 1 ml of LB was added to the tubes and the cells were incubated at 37°C for 30-40 minutes on a rotating vertical wheel. The bacterial cells were collected by centrifugation at 1,500 g on a minicentrifuge for 10 minutes and suspended in 200 µl of LB containing 30 mg/ml of X-gal and 20 mg/ml IPTG. The bacterial solution was spread on LB- agar plates containing 100 pg /m1 of ampicillin and dried in a 37°C oven for 2 hours and then the agar plates were inverted and incubated at 37°C overnight.
(c)  Selection of recombinant clones
Bacterial colonies containing the recombinant vector were white, while those with non-recombinant vector were blue. Five white colonies were selected for plasmid preparation as described below. The presence of an insert was confirmed by restriction enzyme cleavage to linearize the plasmid and running the products on a agarose gel.
(d)  Restriction digestion analysis of inserts
Restriction endonucleases: EcoR I for the pGEM"-5Zf(+) vector were used to cut out the insert from the plasmid vector. The cut insert and the vector were then separated on 1.2% agarose gel electrophoresis. A control insert and linearized vector DNA were also run on the same gel. The correct recombinant plasmid was identified by the presence of both insert and vector bands.
4. Plasmid DNA preparation for DNA sequencing
A single colony of transformed E. coli strain JM109 was cultured in 5 ml of LB broth containing  µg/ml of ampicillin at 37°C with shaking overnight (about 16 hours). Three 1.5 ml aliquots of each sample were pelleted in 1.5 ml Eppendorf tubes by micro centrifugation with 1,500 g for 10 minutes. The E. coil pellets were fully suspended in 200 µl of GET buffer (50 mM glucose/25mM EDTA/20mM Tris-HC1, pH 8.0), and were then centrifuged with 1,500 g for 10 minutes. The E. coil pellets were suspended in 50 µl of GET containing 10 mg/ml of lysozyme. After leaving on ice for 30 minutes, the E. coli was lysed by the addition of 150 µl of 0.5 M NaOH/1.0% SDS. Proteins and genomic DNA were precipitated by adding 200 µl of 3M potassium acetate pH 5.2. The supernatant was transferred to new Eppendorf tubes. Plasmids were then precipitated by the addition of an equal volume of isopropanol and then centrifuged at 10,000 g for 10 minutes. Plasmid pellets were washed with 70% ethanol and air dried for 1 hour. Plasmid DNAs were dissolved in 200 µl of TE buffer containing 10 µg/ml of RNase and incubated at 37°C for 1 hour. DNA solutions were extracted twice with Phenol:Chloroform (1:1). Plasmid DNAs were then precipitated by adding 0.1 volume of 3M sodium acetate pH 5.2 and 2.5 volume of absolute ethanol. After incubating on ice for 30 minutes, plasmid DNAs were then centrifuged at10, 000 g for 10 minutes. DNA pellets were washed with 70% ethanol and dried as described above. Plasmid DNAs were finally dissolved in 50 µl of water and stored at -20°C.
5. DNA Sequencing
Dideoxy chain termination sequencing was performed on an AEI automatic DNA sequencer (Model 373, UAS) using an AmpliTaq DNA polymerase Dye Terminator Cycle Sequencing Kit according to the conditions recommended by Perkin Elmer. DNA sequence data were obtained using ABI sequencing analysis software.
PREFERRED EMBODIMENT OF THE INVENTION
In a preferred embodiment, the cotton plant of the invention is the variety designated "BD", which has snow-white cotton fibres, and exhibits timely self-defoliation by the end of vegetation.
 The variety shows sympodial branching, is a shrub of pyramidal form with 0-2 monopodia shrub heights 100-110 cm, and boll mass about 5.5 g. The shrub is high yielding,
resistant to beating down, tomentose and stem sun-burn intensified by autumn. Leaves are medium light green, 3-5 lobed. The flower is pentagonal, medium sized, without an
anthocyanin spot. Raw fibres are white, flesh-coloured or have sandy tints, and are kept firm in the boll without falling out and are suitable for hand and machine picking.
Fibre yield is 34-36% and length 36/38 mm.
By the end of vegetation growth, the self-defoliation reaches 100 per cent. The plant has a tendency to drying in the apical or top section, leading to some "self-embossing" of plants.
The white and coloured fibres are unaffected by sunlight.
The fibre is ecologically favorable, i.e. it does not contain toxins on its surface, and the variety has 95-100% early natural self-defoliation.
The invention has been described in detail for the purposes of clarity and understanding of the invention. Various forms and embodiments may be made by a person skilled in the art without departing from the scope of the invention.
References cited herein are listed on the 35 following pages, and are incorporated herein by this reference.
 
References
1. Kim SC at al (1997) nucleic acids Research 25: 1085-1086.
2. Brian H et al (1994) Methods in Plant Molecular 5 Biology and Biotechnology. pp. 37-47.
3. Scott 0 et al (1994) Plant Molecular Biology Manual Dl: 1-8.
 
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(I) APPLICANT:

(A)  NAME: Virgin Cotton Company Pty Ltd
(B)  STREET: 1 Percival Road
(C)    CITY: Stanmore
(D)    STATE: NSW
(E) COUNTRY: Australia
(F) POSTAL CODE (ZIP): 2048
(ii) TITLE OF INVENTION: SELF-DEFOLIATING PLANT
(iii) NUMBER OF SEQUENCES: 2
(iv) COMPUTER READABLE FORM:
(A)    MEDIUM TYPE: Floppy disk
(B)    COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patenting Release #1.0, Version #1.30 (EPO)
(v) CURRENT APPLICATION DATA: APPLICATION NUMBER: AU P08174
(2) INFORMATION FOR SEQ ID NO: 1:
(I) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION:  /desc = "PCR primer"
(iii) HYPOTHETICAL: YES
(iv) ANTI-SENSE: NO
(v)  FRAGMENT TYPE: N-terminal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Gossypium hirsutum 10    (B) STRAIN: RAINBOW 39
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
CTCAGTCGCA  
(2) INFORMATION FOR SEQ ID NO: 2:
SEQUENCE CHARACTERISTICS:
(A) LENGTH: 227 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v)  FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Gossypium hirsutum
(B) STRAIN: RAINBOW 39
(F) TISSUE TYPE: Leaf

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
GTCTAAAATG CAGGAGGACC AGAACTAACT CAACGCCACT CAACACTATA CCTCGGATCC    60

CACAGGAGCC CTGGCTTGTC CCTCTGTGCT CACTCATCCT TTCCCGTGTC
GTTAACTAAT    120
GTCTGCCTAC AGGAGGGAGT TGTTGCAGTC AAGGGAAATG ATCCCTAAAA
TTCTCTACGC    180
TGCACCATTC CCCGATCAAG ACCATGTGAT TCATGAAAAT TATAACA 227
 
CLAIMS
1. A self-defoliating cotton plant.
2. A plant according to claim 1, wherein the cotton plant comprises a nucleic acid sequence or functional fragment thereof which is activated to effect self-defoliation of the cotton plant.
3. A cotton plant according to claim 2, wherein the nucleic acid sequence is activated by chemical treatment and irradiation.
4. A plant according to claim 3, wherein the nucleic acid sequence is activated by treatment with ethylene imine and ionizing radiation.
5. A plant according to any one of claims 2 to 4, wherein the nucleic acid sequence or fragment thereof 15 comprises the sequence shown in SEQ ID NO:l.

6. A plant according to claim 5, wherein the sequence is a functional fragment of the sequence shown in SEQ ID N0:2.
7. A plant according to any one of claims 1 to 6, 20 wherein the cotton plant is of the strain Gossypium hirsutum.
8. A plant according to claim 1, wherein the plant is selected from the group consisting of Rainbow 34, Rainbow 39, Rainbow 38 and Rainbow 37.
9. A plant according to any one of claims 1 to 8, wherein the plant self-defoliates at the boll opening stage.
10. A plant according to claim 2, wherein seeds of the plant have the biological characteristics of AGAL 30 deposit number NM 98/06259.
11. A self-defoliating cotton plant according to claim 2, having cotton fibres of a colour selected from the group consisting of beige, snow-white, brown and green.
12. A cotton plant according to claim 11, wherein the plant is resistant to one or more diseases caused by Thielaviopsis babicola, Fusarium vasinfectum and/or Bemisia tabaci.
 
13. A cotton plant according to claim 12, wherein the plant comprises a nucleic acid sequence or functional fragment thereof which is activated to effect self-defoliation of the cotton plant.
14. A cotton plant which has been transformed with a nucleic acid sequence conferring self-defoliation ability, and which comprises the nucleotide sequence shown in SEQ ID N0:2
15. A cotton plant which is the progeny of, or is derived from, a plant according to any one of claims 1 to 14, and which is self-defoliating, with the proviso that said progeny is not Rainbow 34 or Rainbow 39 as herein described.
16. A method of activating a defoliating nucleic acid 15 sequence in cotton, comprising the step of treating cotton seed with ethylene imine and ionizing radiation.
17. A method according to claim 16, wherein the seeds are hybrid seeds produced by crossing a parent cotton plant which has been selected for desired characteristics ortraits, and the seeds are treated with ethylene imine for about 10 hours followed by y-irradiation of the seeds with aboutkilo roentgens absorbed dose.
18. A method according to claim 17, wherein the y-irradiation is at a dose of 4 kilo roentgens for 50 seconds.
19.    A method according to claim 18, wherein the seeds comprises a nucleic acid sequence or functional fragment thereof, comprising a DNA segment of sequence SEQ ID N0:2.
20. A method according to any one of claims 16 to 19, wherein the seeds have the biological characteristics of 30 AGAL deposit number NM 98/06259.
21. A nucleic acid molecule comprising the associated sequence set out in SEQ ID NO: 2, with the ability to confer self-defoliation when present in a cotton plant.

22. A nucleic acid molecule according to claim 21, 35 wherein the associated sequence is expressed in the cotton plant.
 
23. A vector or plasmid comprising a nucleic acid sequence according to claim to claim 21 or claim 22.
24.  A plant cell comprising a nucleic acid sequence according to claim 21 or claim 22.
25. A plant comprising a plant cell according to claim 24.
26. A product of a cotton plant according to any oneof claims 1 to 15, claim 24 or claim 25, selected from the group consisting of reproductive material, seeds, cuttings, seedlings, protoplasts, leaves, stems, flowers, cotton fibres and textiles.
 
ABSTRACT
This invention relates to a cotton plant which self defoliates, and to the progeny, reproductive material, seeds, cuttings, seedlings, protoplasts, leaves, stems, flowers and cotton thereof. It also relates to fibres and textiles made using cotton from such a plant, and to nucleic acid molecules comprising a sequence associated with the self-defoliation characteristics.

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