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WO1997043430A1 - Procede de regeneration in vitro rapide de plants de coton compatible avec une transformation effectuee par l'intermediaire de l'agrobacterie - Google Patents

Procede de regeneration in vitro rapide de plants de coton compatible avec une transformation effectuee par l'intermediaire de l'agrobacterie Download PDF

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Publication number
WO1997043430A1
WO1997043430A1 PCT/US1997/008242 US9708242W WO9743430A1 WO 1997043430 A1 WO1997043430 A1 WO 1997043430A1 US 9708242 W US9708242 W US 9708242W WO 9743430 A1 WO9743430 A1 WO 9743430A1
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Prior art keywords
shootlet
explant
cotton
shootlets
plants
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PCT/US1997/008242
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English (en)
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Kent D. Chapman
John K. Hemphill
Camelia G. A. Maier
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University Of North Texas
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Priority to AU30075/97A priority Critical patent/AU3007597A/en
Publication of WO1997043430A1 publication Critical patent/WO1997043430A1/fr

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/008—Methods for regeneration to complete plants
    • 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)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
    • C12N15/8205—Agrobacterium mediated transformation

Definitions

  • This invention relates to a method for regenerating whole cotton plants from transformed or nontransformed cotton tissues.
  • cotton (Gossypmm) plants produce seeds with a potential multi-product base such as hulls, oil, linters and meal, each of which has a number of uses in a wide variety of markets National Cottonseed Products Association, Cottonseed and Its Products, 2nd Edition, pp 12-13 (1990). Per ton of seed crushed, cottonseed yields 540 lbs of hulls (27%), 320 lbs of crude oil (16%), 160 lbs of linters (8%) and 900 lbs of meal (45%) National Cottonseed Products Association, Cottonseed and Its Products, 2nd Edition, p. 16 (1990). These cottonseed products enter markets that are highly competitive.
  • the basic medium used consisted of Linsmaier and Skoog (LS) salts, vitamins, and the growth regulators ⁇ -naphthaleneacetic acid (NAA) and kinetin Linsmaier, E M and F Skoog, Physwl. Plant 18 100-127 (1965)
  • the basal medium consisted of MS salts, the vitamins inositol and thiamine, sucrose, and the growth regulators NAA, 2,4-dichlorophenoxyacetic acid (2,4-D), and kinetin Hypocotyls were used as the original tissue source Even though embryos were recovered and cultured onto BT medium, no plants were recovered
  • the first method involves the regeneration of cotton plants by somatic embryogenesis which relies on the induction of embryo formation from callus tissue as described above
  • the second method involves the recovery of plants by culturing the apices of shoots
  • somatic embryogenesis typically requires relatively long periods in culture (8-12 months) which are labor intensive and contribute to infertility and genetic changes arising from somaclonal variation Stelly, et al , Genome 32 762-770 (1989) More recently, there have been reports of the application of somatic embryogenesis to a broarder range of cotton lines Firoozabady, E and L DeBoer, In Vitro Cell Devel. Biol. 29P 166-173 (1993) Koonce, L , and N.L Trolinder, Beltwide Cotton
  • Transgenic cotton plants have been regenerated from embryogemc cells or calli derived from hypocotyls (Umbeck, et al , Bio/Technology 5 263 (1987), U S. Patent No 5,004,863) or cotyledonary tissue (Firoozabady, et al , Plant Molecular Biol.
  • the established technology used today for cotton is to transform embryogenic cells of the Coker or other responsive lines, regenerate plantlets through somatic embryogenesis, collect T, seeds from T 0 plants; and advance the desired trait into an agronomic background by conventional plant breeding techniques
  • This methodology requires an additional 6 to 10 years with 2 to 3 crosses per year to transfer the added-value traits into the more agronomically superior cultivars
  • plants regenerated from an embryogenic callus phase are often sterile and/or show signs of genetic change through somaclonal variation which affects both the phenotype and genotype of the plant Firoozabady, E. and D.L DeBoer, In Vitro CellDev. Biol.
  • the regeneration system disclosed in the '375 patent uses explants of cotyledonary nodes, the genetic requirements for whole plant regeneration from soybean cotyledons differ from those of cotton.
  • the '375 patent also discloses that a critical feature of the system is the division of the node tissue into multiple pieces. By contrast, the subdivision of cotton nodes results in abnormal shoot formation.
  • the system of the '375 patent also differs from the system disclosed herein for cotton in that it produces callus tissue, and it uses a benzyladenine concentration 5-50 times greater than that found necessary for optimum shootlet formation from cotton nodes.
  • U.S. Patent No. 5,416,011 and International Application No. PCT/US93/07009 are directed to methods for Agrobacterium-mediated transformation of soybean explants and regeneration using a cotyledonary regeneration system similar to that described in the '375 patent.
  • This regeneration system can also be used to regenerate whole cotton plants from genetically transformed cotton cells or tissues, allowing for the first time the rapid genetic engineering of improved commercial cotton varieties by clonal propagation.
  • the systems disclosed herein can be used to regenerate and/or transform and regenerate dicotyledons other than cotton.
  • elongated shootlets were induced to proliferate from explants of plant tissues comprising pre-existing meristems such as nodal or apical meristems on agar nutrient medium supplemented with a low concentration of benzyladenine.
  • the resulting shootlets were then rooted with high efficiency.
  • Regenerated plants of both glandless and glanded cotton varieties were in soil as early as six weeks after initiating cultures and matured plants that were advanced were all phenotypically normal and fertile.
  • This regeneration system provides the capability to introduce genes directly into cultivars of commercially important varieties both rapidly and efficiently to produce cotton plants with added-value traits.
  • this regeneration system is compatible with existing transgene technology such as introduction of foreign DNA via Agrobacterium (Firoozabody, et al., Plant Mol Biol. 10: 105-116 (1987)), and should also prove useful with other transgene technologies such as particle bombardment (Chlan, et al., Plant Mol. Biol. Reporter 13(1) 31-37 (1995)).
  • the present invention involves the regeneration of whole cotton plants from explants of plant tissues comprising pre-existing meristems such as nodal or apical meristematic tissues.
  • the present invention involves the transformation of explants of nodal or apical meristematic tissues and the subsequent regeneration of these transformed tissues into whole cotton plants.
  • this invention relates to cotton plants produced using the procedures disclosed herein, seeds produced from these plants, and cotton plants germinated from these seeds.
  • the invention relates to the regeneration or the transformation and regeneration of dicots other than cotton.
  • Figures la and lb are exemplary schematic representations of the preferred regeneration/transformation systems disclosed herein.
  • a rapid, direct clonal propagation system has been developed to regenerate plants, preferably mature cotton (Gossypium hirsutum L.) plants, from explants of plant tissues comprising pre-existing meristematic tissues including the nodal and apical meristems of in vitro grown cotton seedlings or plants (Figs la and lb)
  • This system has been found to be useful with a wide variety of cultivars (both glanded and glandless) including Stoneville 7A (glandless), Stoneville 474 (glanded), Paymaster HS-26 (glanded), CA-3050 (glanded), CA-3066 (glanded), CA-3076 (glanded), CA-3084 (glanded), and Stovepipe (glanded) (Table 3)
  • the Stoneville 7A seeds were provided by Dr Rick B Turiey, USDA-ARS, Stoneville, MS
  • the Paymaster HS-26 seeds were provided by Dr John J Burke, USDA-ARS, Lubbock, Texas
  • Apical meristematic tissue is preferably excised from seedlings or plants less than about 28 days old, more preferably from seedlings or plants less than about 21 days old, even more preferably from seedlings or plants less than about 14 days old and most preferably from germinated seeds about 1 or 2 days old As before, although cotton plants of the ages described above are preferred, apical meristematic tissue can be isolated from any plant bearing such tissue at any time after the radicle breaks the seed coat
  • the explants are placed vertically (basal end down) on a solid Murashige and Skoog ("MS”) nutrient medium, Murashige, T and F. Skoog, Physwl.
  • MS Murashige and Skoog
  • Plant. 15 473-497 (1962) (inco ⁇ orated herein by reference), supplemented with a carbon source, preferably sucrose, and a low concentration of cytokinin, preferably benzyladenine (BA)
  • a carbon source preferably sucrose
  • cytokinin preferably benzyladenine
  • BA benzyladenine
  • the supplemented medium as described above is hereinafter referred to as the Shooting Medium. (Table 1).
  • Meristematic explants from seedlings or plants preferably less than about 28 days old can be cultured on the Shooting Medium and regenerate elongated shootlets directly without callus formation suitable for rooting generally in about 21 days
  • the concentration of BA found to be effective in producing elongated shootlets, i e , shootlets greater than 2-3 cm in about 21 days, without callus formation from explants of nodal meristematic tissue is less than about 1 ⁇ M, preferably from about
  • Optimum formation of elongated shootlets from explants of the apical meristematic tissue from seedlings or plants is obtained with about 1.0 ⁇ M whereas optimum formation of elongated shootlets from explants of the apical meristematic tissue from germinating seeds is obtained with about 0 to about 1.0 ⁇ M
  • BA In the absence of BA, shooting has been observed for some cultivars, however, formation of elongated shootlets is neither consistent nor reproducable for explants other than those comprising apices isolated from 1 to 2-day old seedlings Concentrations of BA higher than about 1.0 ⁇ M are to be avoided as they are increasingly toxic to the explants
  • shootlets can be proliferated from nodal explants cultured on the Shooting Medium containing BA, preferably 0 3 ⁇ M Furthermore, new shootlets can continue to proliferate from these explants after elongated shootlets are harvested. In addition, shootlets can be proliferated from excised nodes, i.e., explants, of the elongated shootlets induced to proliferate in vitro This provides a means to multiply the germline by clonal propagation Other explants, including cotyledon pieces, leaf pieces, epicotyl segments and hypocotyl segments produce callus at the same concentrations of BA but do not produce shootlets (Table 2)
  • shootlets After shootlet proliferation, i e , shootlet elongation from nodal or apical meristems, the shootlets are matured and transferred to an appropriate medium to foster or induce root formation preferably as described in Example 3
  • Maturation Medium I Table 1
  • Maturation Medium II for an additional 14-21 days
  • Shootlets about 3-4 cm tall after about 21 days are transferred to
  • a preferred Rooting Medium is MS media supplemented with about 1 0 ⁇ M indole-3 -butyric acid (IB A) (Table 1) It has been found that shootlets treated in this manner typically form roots in about 6 weeks, after which the plantlets are transferred to soil, typically in 3 inch pots Preferably, the shootlets are transferred directly to soil, e.g , in 3 inch pots after application of naphthalene acetamide to the bottom of the stem to induce rooting Naphthalene acetamide is available commercially as RootoneTM powder (Greenlight Co , San Antonio, Texas) Shootlets treated in this manner typically form roots in about 2 to 3 weeks Preferably, transgenic shootlets are rooted
  • the shootlets which are rootless, or plantlets (rooted shootlets) are preferably exposed to a hardening process
  • the hardening process begins simultaneously with the initiation of the rooting process
  • the hardening process takes approximately 2 to 3 weeks depending on the height of the elongated shootlets at the time the process is begun
  • the potted shootlets/plantlets are initially enclosed, preferably within plastic bags to generate the humid environment necessary for hardening purposes
  • the shootlets/plantlets are preferably nurtured for about 2 weeks in a humid environment with nutritional supplements
  • the cotton shootlets/plantlets are uncovered daily to add water
  • a nutritional water solution, preferably Miracle GroTM (0 75 g/gallon) is added every third day (Stern's Miracle Grow, Port Washington, N Y )
  • the shootlets/plantlets are gradually removed from within the enclosed bags in stages over a 2 week period to allow for continual adaptation and plantlet growth
  • the plastic bags are completely removed Smaller shootlets (approxi)
  • the Ti plasmid has the natural ability to transfer a segment of itself, referred to as the transfer DNA (T-DNA) region, into the genome of infected plant cells
  • T-DNA transfer DNA
  • the Agrobacterium tumefaciens can transform plant cells with the foreign gene
  • the foreign gene construction is then included in the cells of a whole plant regenerated from the transformed cells and is then inherited in a simple Mendelian manner The construction can thus be treated as any inheritable trait for crop breeding purposes
  • Transformation can be performed with either the apical or nodal meristematic explants
  • Explants are transformed by co-cultivating the apical or nodal meristematic tissue with Agrobacterium tumefaciens strain LBA4404 harboring the binary vector pBI 121 preferably as described in Example 5 While the Agrobacterium tumefaciens strain LBA4404 harboring the binary vector pBI121 is preferred for transformation, other vectors or Agrobacterium strains known to those of skill in the art can be used In addition, other transformation procedures known to those of skill in the art can be used
  • the pBI121 vector carries both a selectable marker, i e., the NPT II gene, for kanamycin resistance, and a GUS-reporter gene within the left and right borders of the T-DNA region (Clontech Laboratories, Ine , Palo Alto, CA) Following the co-cultivation period, the explants are sequentially transferred to Shooting Medium supplemented with increasing amounts of kanamycin ("KAN") - KAN Selection Media - to select for transformed shootlets preferably as described in Examples 6 and 7 While selection using kanamycin resistance is preferred, insertion of gene sequences coding for resistance to other antibiotics such as neomycin, hygromycin, or chloramphenical or to other selectable genes known to those skilled in the art can be used Following co-cultivation of explants from 1 or 2-day old germinating seeds, the shootlets obtained exhibit three phenotypes during kanamycin selection - green, mottled green, or tan The green phenotype is exhibit
  • the KAN- resistant shootlets should be carefully moved through the above-described KAN selection system Essential to this success is the use of the cut/trim method in which the shootlets are cut and the cotyledonary leaves, and other lateral nodes trimmed or removed as necessary such that the apices of the shootlets are constantly less than about 2 cm from the antibiotic source If desired, the excised cotyledonary and other lateral nodes can be clonally propagated To maintain the apices of the elongating shootlets within about 2 cm of the source of antibiotics, the shootlets are evaluated on a daily basis to determine their height For apical meristems, transfer to media with 50 ⁇ g/ml
  • T 0 plants are chimerically transformed, i e , some of the pre-existing meristems are transformed and others are not
  • shootlets can be rechallenged on kanamycin rather than rooted This is done by excising nodal or apical meristematic explants from these shootlets and culturing them on fresh kanamycin supplemented medium to identify transgenic meristems within the shootlets.
  • Successful transformation can also be verified by GUS enzymatic activity in the leaves of KAN selected plantlets as described in Example 9. (See Table 15).
  • Stable or germline transformation can be verified by GUS enzymatic activity in the pollen grains (Tables 16-19) of the transformed plants as described in
  • Example 10 or by Southern-blot analysis as described in Example 12.
  • Seeds from diverse germplasm were sterilized, germinated, and grown in vitro for different time periods.
  • seeds (approx. 150) were placed in ultrapure water (MilliQ plus UF) with two drops (approximately 100 ⁇ l) of Tween-20 per 100 ml of H 2 O and washed with a brush gently for several minutes. This procedure was repeated until no more bubbles arose from the seeds during brushing, but in no case less than three times.
  • the seeds were then wrapped in cheesecloth and submerged in running Dl-water for about 3 hours. Next, the seeds were placed under sterile conditions in 70% ethanol containing Tween-20 (2 drops per 100 ml) for 60 seconds.
  • the seeds were then collected in a sterile strainer and rinsed with sterile ultrapure water (MilliQ plus UF) for 3 minutes and placed in sterile 20% commercial bleach plus Tween-20 (2 drops per 100 ml) for 20 minutes with continuous mixing. The seeds were then thoroughly rinsed (3X) with sterile ultrapure water (Milli-Qplus UF) for 3 minutes. Following the rinsing step, the seeds were allowed to imbibe water until needed for experimentation. Approximately 4 hours was required for the complete sterilization/imbibition period.
  • the cotton seedlings of the cultivars After the 14- to 28-day period, the cotton seedlings of the cultivars, containing 3 to 5 nodal meristems, had grown to approximately 7 to 10 inches in height.
  • the following explants were then excised from the 14- to 28-day old seedlings: apical meristems and/or nodal meristems including, primary leaf nodes and cotyledonary nodes. These explants were harvested, placed directly on Shooting Medium (Table 1) and utilized as described herein.
  • Stage 2 Medium 2 50 ⁇ g/ml Kanamycin; and 500 ⁇ g/ml Carbenicillin
  • Stage 3 Medium 3 75 ⁇ g/ml Kanamycin; and 500 ⁇ g/ml Carbenicillin
  • BA ⁇ -benzyladenine
  • IBA Indole-3 -butyric Acid
  • AC Activated Charcoal
  • MES 2-(4-Morpholino)-Ethane Sulfonic Acid
  • KAN Kanamycin.
  • MS medium Murashige & Skoog major & minor salts, MS vitamins; sucrose 15 g/1, Phytagel (Sigma) 2.2 g/1; pH 6.0 (except as otherwise noted).
  • CPS nodes refers to nodes of clonally propagated shootlets.
  • BA containing Shooting Medium to induce regeneration, i.e., shootlet proliferation.
  • a benzyladenine concentration ranging from about 0 to about 1.0 ⁇ M was used.
  • a concentration of about 1.0 ⁇ M BA gave optimum results.
  • the BA was added to the Shooting Medium immediately before autoclaving. Following the proliferation of shootlets, a shootlet maturation step was used before rooting in most cases.
  • Rooting Medium consisting of l ⁇ M indole-3-butyric acid (IBA) substituted for benzyladenine in the Shooting Medium was used The IBA was added to the Rooting Medium before autoclaving Except when otherwise noted, the pH of media used was adjusted to 6 0 with NaOH(lN) or HCl(lN) prior to adding Phytagel and the media autoclaved at 1 46 kg/cm 2 for 15 minutes Petri dishes (
  • the regenerants (nontransformed shootlets) and KAN selected (putatively transformed) shootlets were placed in a growth chamber which maintained controlled environmental conditions (temperature, 30°C, light intensity, 155 ⁇ mol s 'm 2 , and a 16 hr photoperiod)
  • the light source consisted of cool white fluorescent and incandescent lamps
  • these plants were moved to a greenroom which supported continual plant growth and flowering under the following environmental conditions high temperature (90-100°F) to low temperature (65-75 °F); light intensity dependent upon the location within the room (66 to 134 ⁇ mol s 'm 2 ) and a 16 hr photoperiod
  • the light source consisted of high-pressure sodium (140 ⁇ mol s 'm 2 ) and metal halite lamps (100 ⁇ mol s 'm 2 )
  • Example 4 Plant Regeneration Results The regeneration method disclosed herein was found to stimulate shootlet proliferation in a variety of diverse germplasms or cultivars (Table 3)
  • the method described herein for cotton plant regeneration was first observed in a preliminary experiment where BA was tested for its capability to generate shootlets from excised cotyledonary nodes of 14-day old seedlings (Stoneville 7A)(Table 4)
  • Benzyladenine at 0 3 ⁇ M when applied to the explants as part of the Shooting Medium, initiated elongated shootlets from excised cotyledonary nodes after a 3 week culture period.
  • higher concentrations of BA (3.0 ⁇ M and greater) suppressed shootlet formation after a 3 week culture period and were often toxic to the explants. (Table 4).
  • Stoneville 474 d + "Stoneville 7 A seeds were provided by Dr. Rick B. Turiey, USDA-ARS, Stoneville, MS.
  • TX c CA-Series and Stovepipe seeds were provided by Dr. John Gannaway, Texas
  • the optimal BA concentrations for inducing shootlet formation from nodal meristems cultured on Shooting Medium was 0 3 ⁇ M based on the development of elongated shootlets (2-3 cm in height) after a 3 week culture period (Tables 5, 6 and 7)
  • the apical meristems isolated from 14 to 21 -day old cotton seedlings also were optimally induced to form elongated shootlets (Tables 8 and 9)
  • a BA concentration of 0 to 1 0 ⁇ M was optimal
  • hormone-free medium failed to support elongated shootlets of apices excised from seedlings other than the 1
  • Week 3 2/2 (0/2) 2/4 (0/4) 3/8 (0/8) 4/6 (0/6) 3/3 (3/3) a The number of shootlets/number of apical meristems.
  • HS-26 a The number of shootlets that rooted / number of attempts.
  • the elongated shootlets were cultured on the Rooting Medium for about 6 weeks until rooting had occurred The plantlets were then transferred to soil in 3 inch pots and hardened as described.
  • the base of the elongated shootlets were first dipped into RootoneTM and then placed into 3" pots and hardened for 2 to 3 weeks. Once the roots were established, all regenerants and KAN selected plantlets began to elongate and develop new leaves. The plantlets were moved to 6 to 10 inch pots at the 4-leaf stage for plant maturation and flowering/seed set.
  • the soil consisted of 3 parts potting soil and 1 part vermiculite. All regenerated plants that were advanced to soil were normal, and all of the matured plants regenerated to date have initiated flowers and set viable seed under greenroom conditions as previously described in Example 3. (Table 12). The establishment of plantlets via rooting in agar required approximately 3 months; however, complete plantlet formation by rooting directly in soil took half that time, i.e., approximately 6 weeks.
  • Explants consisting of meristematic tissue from 1 or 2-day old seedling (apices of isolated seed embryos) (Fig. la) and 14 to 28-day old seedlings (nodal meristems, i.e., leaf nodes and cotyledonary nodes) (Fig. lb) were co-cultivated with Agrobacterium tumefaciens strain LBA4404 harboring the binary vector pBI121 ("Agro+”) at a bacterial concentration of approximately 8 x 10* cells/ml.
  • the binary vector pBI121 carries the selectable marker (NPT II gene) for kanamycin resistance and a GUS- reporter gene (Clontech Laboratories, Inc., Palo Alto, CA).
  • LBA4404 without the binary vector pBI121 (“Agro-") was used. Bacteria were grown at 27°C in liquid LB (Luria-Bertani) medium (PH 6.6-7.0) and maintained in liquid medium with weekly transfers to fresh medium. For transformation experiments, bacteria were allowed to grow to an O.D 550 of approximately 1.7-2.0. Alternatively, an O.D. 5S0 of approximately 0.1 to 0.8 is used. Appropriate antibiotics, streptomycin (25 ⁇ g/ml) and kanamycin (50 ⁇ g/ml) for Agro+ or streptomycin (25 ⁇ g/ml) for Agro- were present throughout the vegetative growth of the bacteria.
  • the explants were blotted on sterile filter paper and transferred to Shooting Medium plus antibiotics, i.e., a KAN Selection Medium for KAN selection.
  • the explants are washed with LB medium + 500 ⁇ g/ml carbenicillin before they are blotted on sterile filter paper and transferred to KAN Selection Medium.
  • the KAN selection procedure for the putatively transformed explants of the 1 or 2-day old and the 14- to 28-day old seedlings involved a cut/trim step procedure and a KAN 37 to 50 to 75 ⁇ g/ml or a KAN 37 to 50 ⁇ g/ml selection process, respectively.
  • the co-cultivated explants were placed on Shooting Medium (0.3 ⁇ M BA) + 37 ⁇ g/ml KAN (+ 500 ⁇ g/ml carbenicillin) (KAN Selection Medium I - Table 1) to allow for shootlet formation.
  • the developing explants were monitored daily for Agrobacterium contamination and elongated growth of either the hypocotyl or stem portion of the source materials utilized.
  • apices from 1 or 2-day old seedlings grew at a faster rate than the other explants, therefore, the hypocotyl of the germinating apices were usually cut/trimmed after about 5 days and, then, transferred to Shooting Medium (0.3 ⁇ M BA) + 50 ⁇ g/ml KAN + 500 ⁇ g/ml carbenicillin (KAN Selection Medium II - Table 1).
  • KAN Selection Medium II the developing shootlets derived from explants of 28- day old seedlings and clonally propagated shootlets grew at a slower rate and, therefore, were transferred to KAN Selection Medium II after about 7 days and were cut/trimmed when necessary.
  • KAN Selection Medium II Following selection with KAN Selection Medium II, putative transgenic shootlets derived from cultured explants from 1 or 2-day old seedlings required an additional KAN selection pressure. These shootlets were therefore transferred to Shooting Medium (0.3 ⁇ M BA) + 75 ⁇ g/ml KAN + 500 ⁇ g/ml carbenicillin (KAN Selection Medium III - Table 1) and were cut/trimmed when necessary.
  • the developing explants were kept close (less than about 2 cm) to the kanamycin source for continual selection; also, the cutting kept the vascular tissues in direct contact with the MS medium (+ kanamycin)
  • the KAN-selection procedure described above involved a step-wise increment of 37 ⁇ g/ml KAN (5-7 days) to 50 ⁇ g/ml (4 weeks) to 75 ⁇ g/ml (4 weeks), each step containing carbenicillin at 500 ⁇ g/ml.
  • lateral meristems required only the first two levels (37 to 50 ⁇ g/ml) of kanamycin selection while apices from the 1 or 2-day old seedlings required the complete KAN-selection procedure (37 to 50 to 75 ⁇ g/ml) All developing shootlets subjected to this selection procedure were cut/trimmed as follows hypocotyl/epicotyl from germinating seedlings were cut to 2 cm on a regular basis and the stems with lateral meristem growth were trimmed when necessary before transferring to the next medium combination. Shootlets that were advanced through this KAN-selection procedure [50 ⁇ g/ml (4 weeks) to 75 ⁇ g/ml (4 weeks)] were transferred on a weekly basis Following a maturation step with Maturation Medium I or
  • Kanamycin-resistant shootlets obtained after transformation of apical meristematic tissue from 1 or 2-day old germinating seeds were of two types - green and mottled green.
  • the green phenotype was exhibited by early emerging shootlets, which were generally evident after about five days and which did not show any phenotypic stress.
  • the mottled green phenotype was exhibited by later emerging shootlets, which were generally evident after about 5 days to 2 weeks and which showed various degrees of stress such as bleached leaves, white leaf tips and edges, and green sectors and white vascular tissue in cotyledonary leaves.
  • the mottled green phenotype grew more slowly during the KAN selection procedure (37, 50 and 75 ⁇ g/ml).
  • the cotton cultivars used herein exhibited maximum selection at incremental kanamycin concentrations of 37 to 50 ⁇ g/ml for nodal and greater than 2-day old apical meristematic tissues and 37-50-75 ⁇ g/ml for 1 or 2-day old apical me ⁇ stematic tissue These kanomycin selection levels were selected using a conventional antibiotic screen
  • DNA transfection oi Agrobacterium tumefacians was accomplished by the triparental mating procedure Ditta, et al , Proc. Natl Acad. Sci. U.S.A. 11 7347-7351
  • E. coli HB101 - pRK2013 and Agrobacterium LBA4404 Two parental lines, E. coli HB101 - pRK2013 and Agrobacterium LBA4404, were obtained from Clontech Laboratories, Ine , Palo Alto, CA
  • the third parental line, E. coli HB101 RecA ' was made competent and transformed (plasmid pBI121 - Clontech Laboratories, Ine ) by the calcium chloride procedure Sambrook, et al , Molecular Cloning: A Laboratory Manual, 2d ed, Cold Spring Harbor, 1 1 74 (1989) The E.
  • coli HB101 RecA cells were provided by Dr Dan Kunz (Department of Biological Sciences, University of North Texas, Denton, TX) These cells can also be purchased from Gibco BRL (BRL Laboratories, Gaithersberg, MD) Example 9 - Fluorometric Analyses of GUS Activity in Developing Shootlets
  • T1 indicates progeny (seeds or plants) of T 0 plants
  • HS-26T0- 01 Tl-03 designates one of several T, progenies of a T 0 plant
  • the GUS gene was shown to be incorporated into the cotton genome of the T 0 plants, i e , the transformants, by Southern blot analyses Following digestion with Hind III and subsequent resolution by electrophoreses, the GUS probe hybridized to a genomic DNA band at approximately 9 kb for the HS-26T 0 -03 plant and a corresponding band was not found for the HS-26NT (nontransformed) plant This was smaller than the linearized pBI121 vector (13 kb) indicating that the GUS fragment was not in the free plasmid or in Agrobacterium, but rather was integrated into the genomic DNA of the cotton plant
  • T j seeds from individual flowers of the T 0 plants were germinated in the presence of 50 KAN (50 ⁇ g/ml) to screen for the KAN-selected seeds of the next generation
  • 50 KAN 50 ⁇ g/ml
  • KAN-resistant T seeds were transferred to soil and three phenotype growth patterns were evident in the T, plants after a 21 -day growth period These phenotypes were as follows Large (epicotyl growth of 15 cm in height), Medium (7 cm in height) and Small (little or no epicotyl development - these usually died) Seeds within each boll and from different bolls of the same T 0 plant showed different seed germination rates and seedling phenotpyes which is consistent with the concept that these T 0 plants are chimeric As demonstrated in Tables 17, 18 and 19, these KAN-selected T, plants also were found to be GUS positive by the pollen assay This strategy allows the rapid screening of T 0 and T, progeny to identify likely transformants
  • Table 20 Summary of transgenic screens shootlet KAN-selection procedure for T 0 s (1), GUS positive in the leaf fluorescence assay for T 0 s and T,s (2), GUS positive in the leaf mid-vein/petiole histochemical assay for T 0 s and T j S (3), GUS positive in the pollen histochemical assay for T 0 s and T,s (4), seedling KAN-selection procedure for T,s Large (L), Medium (M) and Small (S) plant development phenotypes (5) and Southern blot assay (6)

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Abstract

La présente invention porte sur un procédé polyvalent de régénération rapide de plants de coton à partir d'explants de tissus méristématiques apicaux et/ou nodaux, pouvant être associé à des procédés connus de transformation tels que la transformation effectuée par l'intermédiare de l'agrobactérie, en vue de la production rapide de variétés de coton mises au point par génie génétique et revêtant une grande importance sur le plan agronomique.
PCT/US1997/008242 1996-05-16 1997-05-15 Procede de regeneration in vitro rapide de plants de coton compatible avec une transformation effectuee par l'intermediaire de l'agrobacterie WO1997043430A1 (fr)

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WO1999041975A1 (fr) * 1998-02-19 1999-08-26 Cotton Incorporated Procede de production de plantes transgeniques utilisant des meristemes apicaux
WO2000071733A1 (fr) * 1999-05-19 2000-11-30 Aventis Cropscience N.V. Technique amelioree de transformation de coton induite par agrobacterium
WO2000077230A1 (fr) * 1999-06-11 2000-12-21 Institute Of Molecular Agrobiology Transformation a haut rendement de coton induite par agrobacterium et effectuee a l'aide d'explants de petiole
WO2001000785A2 (fr) 1999-06-28 2001-01-04 Zeneca Limited Regeneration de cotonniers
US6483013B1 (en) 1999-05-19 2002-11-19 Bayer Bioscience N.V. Method for agrobacterium mediated transformation of cotton
US6900057B2 (en) 2001-01-16 2005-05-31 Monsanto Technology Llc Multiple shoot proliferation and regeneration system for plants
US7498428B2 (en) 2003-06-19 2009-03-03 Evogene Ltd. Nucleotide sequences for regulating gene expression in plant trichomes and constructs and methods utilizing same
EP2080805A1 (fr) 1998-08-19 2009-07-22 Monsanto Technology LLP Vecteur d'expression de plante
EP2339005A2 (fr) 1999-12-16 2011-06-29 Monsanto Technology LLC Nouvelles constructions d'expression de plantes
US8129514B2 (en) 2003-06-19 2012-03-06 Evogene Ltd. Nucleotide sequences for regulating gene expression in plant trichomes and constructs and methods utilizing same
US8536406B2 (en) 2008-04-28 2013-09-17 Michigan Technological University COMT1 gene fiber-specific promoter elements from poplar

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WO1995006741A1 (fr) * 1993-08-30 1995-03-09 Biocem Procede de production de plantes transgeniques, entierement transformees en generation to, a partir de meristemes
WO1996026639A1 (fr) * 1995-02-28 1996-09-06 Calgene, Inc. Modification de coton a l'aide de facteurs de transcription de tissu ovarien
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US5164310A (en) * 1988-06-01 1992-11-17 The Texas A&M University System Method for transforming plants via the shoot apex
WO1992015675A1 (fr) * 1991-03-06 1992-09-17 Agracetus, Inc. Transformation du coton induite par des particules
WO1995006741A1 (fr) * 1993-08-30 1995-03-09 Biocem Procede de production de plantes transgeniques, entierement transformees en generation to, a partir de meristemes
WO1996026639A1 (fr) * 1995-02-28 1996-09-06 Calgene, Inc. Modification de coton a l'aide de facteurs de transcription de tissu ovarien
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BAJAJ, Y., ET AL .: "MICROPROPAGATION AND GERMPLASM PRESERVATION OF COTTON (GOSSYPIUM spp.) THROUGH SHOOT TIP AND MERISTEM CULTURE", INDIAN JOURNAL OF EXPERIMENTAL BIOLOGY, vol. 24, September 1986 (1986-09-01), pages 581 - 583, XP002041383 *
CHLAN, C.A., ET AL.: "A PROCEDURE FOR BIOLISTIC TRANSFORMATION AND REGENERATION OF TRANSGENIC COTTON FROM MERISTEMATIC TISSUE", PLANT MOLECULAR BIOLOGY REPORTER, vol. 13, no. 1, 1995, pages 31 - 37, XP002041977 *
GOULD, J., ET AL .: "REGENERATION OF GOSSYPIUM HIRSUTUM AND G. BARBADENSE FROM SHOOT APEX TISSUES FOR TRANSFORMATION", PLANT CELL REPORTS, vol. 10, 1991, pages 12 - 16, XP002041978 *
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Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7122722B2 (en) 1998-02-19 2006-10-17 Cotton Incorporated Methods for producing transgenic cotton plants using chilled apical shoot tips
AU747514B2 (en) * 1998-02-19 2002-05-16 Cotton Incorporated A method for the production of transgenic plants using apical shoot tips
WO1999041975A1 (fr) * 1998-02-19 1999-08-26 Cotton Incorporated Procede de production de plantes transgeniques utilisant des meristemes apicaux
EP2811024A1 (fr) 1998-08-19 2014-12-10 Monsanto Technology LLC Vecteur d'expression de plante
EP2080805A1 (fr) 1998-08-19 2009-07-22 Monsanto Technology LLP Vecteur d'expression de plante
WO2000071733A1 (fr) * 1999-05-19 2000-11-30 Aventis Cropscience N.V. Technique amelioree de transformation de coton induite par agrobacterium
US6483013B1 (en) 1999-05-19 2002-11-19 Bayer Bioscience N.V. Method for agrobacterium mediated transformation of cotton
CN100370031C (zh) * 1999-06-11 2008-02-20 淡马锡生命科学研究院有限公司 利用叶柄外植体进行的土壤杆菌介导的高效棉花转化
WO2000077230A1 (fr) * 1999-06-11 2000-12-21 Institute Of Molecular Agrobiology Transformation a haut rendement de coton induite par agrobacterium et effectuee a l'aide d'explants de petiole
WO2001000785A2 (fr) 1999-06-28 2001-01-04 Zeneca Limited Regeneration de cotonniers
EP2339005A2 (fr) 1999-12-16 2011-06-29 Monsanto Technology LLC Nouvelles constructions d'expression de plantes
EP2339006A2 (fr) 1999-12-16 2011-06-29 Monsanto Technology LLC Nouvelles constructions d'expression de plantes
EP2944695A1 (fr) 1999-12-16 2015-11-18 Monsanto Technology LLC Nouvelles constructions d'expréssion végétales
US6900057B2 (en) 2001-01-16 2005-05-31 Monsanto Technology Llc Multiple shoot proliferation and regeneration system for plants
US7498428B2 (en) 2003-06-19 2009-03-03 Evogene Ltd. Nucleotide sequences for regulating gene expression in plant trichomes and constructs and methods utilizing same
US8129514B2 (en) 2003-06-19 2012-03-06 Evogene Ltd. Nucleotide sequences for regulating gene expression in plant trichomes and constructs and methods utilizing same
US8536406B2 (en) 2008-04-28 2013-09-17 Michigan Technological University COMT1 gene fiber-specific promoter elements from poplar

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AR007163A1 (es) 1999-10-13
ZA974176B (en) 1998-09-01

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