Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
  • C12N15/8287—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for fertility modification, e.g. apomixis
  • C12N15/8289—Male sterility
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/04—Plant cells or tissues

Definitions

• the present invention particularly relates to the thinning of a large variety of fruit plants, the reduction of seeds in ci trus plants and the reduct ion of the incidence of shotberries in grapes using activated RNase, inactivated RNase or mixtures of these.
• the series of events that occur between pollination and fertilization include processes of specific recognition of pollen grains and pollen tubes by the pistil tissues.
• S-protein a major stylar protein
• the gene tha t encodes f or t he protein was c loned and sequenced and was found to share homology wi th fungal RNase. It was established that this protein i s indeed RNase, and was found to arrest pollen tube growth (B. A. McClure et al. , Nature, 1989, pp. 955-957).
• the RNase was al so found in s t yl ar di f fusates of sel f-compatible species such as Nicotiana tabacum (McClure et al.
• RNase is a known enzyme obtainable from a large variety of sources.
• One main source i s the fermentat ion of various fungi.
• the present invent i on there i s provided a method for the biological control of pollen in plants, compris ing applying an ef fec t ive amount of activated or inactivated RNase or a mixture thereof.
• the present invent ion resul t s in the thinning of a large variety of fruit plants, the reduction of seeds in citrus plants and, in the case of grapes, the reduction of the incidence of shotberries.
• RNase may be obtained from sources such as fungi , bacteria, plants, animal and viral . When obtained from fungal sources, the RNase may easi ly be obtained by the fermentation of various fungal sources. Examples are Aspergi l lus niger , Aspergi l lus clari tus, Aspergi l lus oryzae and Rhisopus miveus, preferably Aspergillus niger and Aspergil lus oryzae. A mos t preferred fungus i s Aspergillus niger BKCMICC Number 324,626).
• This strain was deposi ted w i t h t he Commonwea l t h Myco l og i c a l Institute, Ferry Lane, Kew, Richmond-upon-Thames, Surrey TW9 3A5, Uni ted Kingdom, on 20 May, 1988.
• the plants where the method of the biological control of pollen is chosen from the group consisting of fruit trees, ornamental trees, vegetables, field crops, plantations, ornamentals and the like.
• the trees where the method of thinning was found to be applicable are chosen from the group consisting of deciduous plants, tropical plants, sub-tropical plants, citrus plants, nut tree and grape vines. Examples of deciduous plants are stone fruit, pears, apples, quince, peaches, plums, nectarines, cherries and apricots.
• Examples of ornamental trees are olive and Fica trees.
• Examples of field crops are cereals, especially hybrids.
• citrus plants where the RNase in use in the thinning and the reduction of seeds are orange, lemon, grapefruit, Clementina, mandarin, ci tron, pomela and their hybrids.
• the method of the prevention of fruit set of field crops of the present invention was found to be especially useful in preventing fruit set of ornaments whose fruits and flowers cause environmental problems.
• the RNase is added to the pollen in the form of crude or purified extract of the RNase in the form of spray, powder and the like.
• a really novel way to apply the RNase is to powder bees and let them distribute the RNase powder during their feeding on the pollen in the flowers.
• the RNase is applied at a rate of 1 to 10,000 uni ts per hectare.
• Aspergillus niger was grown in medium that contained 1 % wheat flour and 0.059. (NH 4 ) 2 SO 4 in distilled water. The solution was brought to pll 3.5 wi th HCl and then auto-claved. An inoculum of 10 6 spores of A. niger were suspended into 100 ml of the growth medium and the suspension was incubated at 30°C and shaken at 150-200 rpm.
• RNase act ivi ty in the growth medium reached i t s peak after 5-7 days of incubation.
• the medium was then passed through a millipore filter of 0.2 ⁇ m to remove any traces of hypha and spores.
• the medium was ultrafi ltered to concentrate the enzyme.
• RNase activi ty in the growth medium of A. ni ger was determined by diffusioti plate assay, using 0.1% torula yeast RNA (Sigma) in 1% agarose as a substrate. A drop of 10 ⁇ l was applied on the substrate in a 15cm petri dish and t hen was incuba ted a t 37 ° C for one hour . The substrate was then stained with 0.02?. toluidine blue in water. RNase activity was indicated as a white halo on a blue background. A quantitative spectrophotometric assay of RNase was conducted as a modification of the procedure described by Brown & Ho (1986). Each sample contained 100 ⁇ l of the examined enzyme mixed with and 4 ⁇ g torula yeast RNA and 1 ml of 20 mM buffer of choice. The sample was incubated in 37° for 30 minutes.
• the reaction was stopped by putting the tube into ice and adding 200 ⁇ l "stop reagent" (0.7..% uranyl sulphate in 251 HCIO 4 ). The solution was centrifuged in 12,000 rpm for 5 minutes. The supernatant was diluted to 1/20 in distilled water and OD in 260 nm was examined.
• stop reagent 0...% uranyl sulphate in 251 HCIO 4
• the blank solut ion contained the same component and incubated by the same procedure, only the enzyme was added after the reaction was stopped.
• the results were given in units/ml, equivalent to standards of bovine pancreatic RNase.
• the optimal pll for RNase activity in the growth medium was examined using 20 mM citrate buffer at the pH value of: 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, and 7.0.
• the above spectro photometric procedure showed that the optimal pH for the fungal RNase activity was 3.5 (Fig. 1).
• the effect of presence or lack of metal ions on RNase activity was also examined.
• Each of the following salts: MgCl 2 , FeCl 3 or CaCl 2 was supplemented into the reaction mixture to a final concentration of 10 mM. Additional samples contained the same concentration of DTT as a reductant and EDT ⁇ that has strong affinity to metal ions.
• RNase activity was examined by the above diffusion plate assay. No difference in the diameter of the white halo (1cm ) on the blue background was observed between the different treatments and the control.
• Pollen grains of peach cv. Texas were germinated in vitro in growth medium containing 15% (w/v) sucrose, 100 ⁇ g/mlH 3 BO 3 , 200 ⁇ g/ml MgSO 4 .7H 2 O and 200 ⁇ g/ml Ca(NO 3 ) 2 ⁇ 4H 2 O (Galletta, 1983).
• approximately 1000 pollen grains were suspended by the edge of a needle into 100 microl i ter s of growth medium.
• RNase was suspended in t he pol len growth medium to a f inal concentration of 0.05, 0.5, 5, 50 or 500 units/ml.
• the RNase produced by A. niger was examined in the field as a thinning agent of deciduous fruit trees.
• peach Prunus persica
• plum Prunus salicina
• Table 2 shows that RNase caused a significant reduction of about 40% ( P ⁇ 0.01 ) of frui t-set .
• the repeated treatments showed better results than one treatment but interestingly, the low concentration was most effective in this case.
• Vikson branches were sprayed wi th 1, 10 or 100 units/ml of RNase, dissolved in water (R1, R10 and R100, respectively, supplemented with 0.025% Triton X-100 and applied once at 50% blooming (I) or three t imes, starting from 10% blooming (M).
• Table 4 shows that the combination of high concentration of RNase and repeated applications during anthesi s were most ef fect ive in reducing fruit-set.
• R Spraying with RNase (extract from Aspergillus niger)
• the vineyard tested did not receive any sort of spraying of gibberellin to increase the size of the grapes.
• the vine was sprayed by hand during flowering on 6/5/93. On 11/7/93 the bunches were weighed and the number of shot berries counted. The results are shown in Table 5.
• RNase samples from the fermentation of Aspergillus niger were loaded on 15% polyacrylamide gel electrophoresis (SDS PAGE). The proteins were resolved and after the run the proteins were renatured in situ by removing the SDS using an isopropanol buffer. After the run the gel was divided into two lanes. One lane was subjected to RNase zymogram detection (overlayed on 0.1% yeast RNA and 1.5% agarose for several hours) and then stained with 0.1% toluidine blue. RNase bands were visualized as evident by a whi te halo around an active band at approximately 15KDa and 30 KDa.
• SDS PAGE polyacrylamide gel electrophoresis

Landscapes

• Life Sciences & Earth Sciences (AREA)
• Health & Medical Sciences (AREA)
• Genetics & Genomics (AREA)
• Engineering & Computer Science (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Organic Chemistry (AREA)
• Chemical & Material Sciences (AREA)
• Zoology (AREA)
• Biomedical Technology (AREA)
• Wood Science & Technology (AREA)
• Biotechnology (AREA)
• General Engineering & Computer Science (AREA)
• Microbiology (AREA)
• Cell Biology (AREA)
• Biochemistry (AREA)
• General Health & Medical Sciences (AREA)
• Molecular Biology (AREA)
• Plant Pathology (AREA)
• Biophysics (AREA)
• Physics & Mathematics (AREA)
• Botany (AREA)
• Breeding Of Plants And Reproduction By Means Of Culturing (AREA)

Priority Applications (2)

Applications Claiming Priority (2)

Publications (1)

Family

ID=11065295

Family Applications (1)

Country Status (9)

Families Citing this family (2)

Family Cites Families (2)

• 1993
  • 1993-09-23 IL IL107080A patent/IL107080A0/xx unknown
  • 1993-11-04 US US08/145,512 patent/US5552139A/en not_active Expired - Fee Related
• 1994
  • 1994-09-01 ZA ZA946701A patent/ZA946701B/xx unknown
  • 1994-09-08 CA CA002172540A patent/CA2172540A1/en not_active Abandoned
  • 1994-09-08 EP EP94928037A patent/EP0739213A1/en not_active Withdrawn
  • 1994-09-08 NZ NZ273792A patent/NZ273792A/en unknown
  • 1994-09-08 WO PCT/US1994/010082 patent/WO1995008346A1/en not_active Ceased
  • 1994-09-08 AU AU77226/94A patent/AU678289B2/en not_active Ceased
  • 1994-09-16 TR TR00936/94A patent/TR28111A/xx unknown

Non-Patent Citations (4)

Also Published As

Similar Documents

Legal Events