WO1997043430A1 - A rapid in vitro regeneration scheme of cotton plants compatible with agrobacterium-mediated transformation - Google Patents

A rapid in vitro regeneration scheme of cotton plants compatible with agrobacterium-mediated transformation 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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shootlet
explant
cotton
shootlets
plants
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PCT/US1997/008242
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French (fr)
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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/en

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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

This invention relates to a versatile method of rapidly regenerating cotton plants from explants of apical and/or nodal meristematic tissues which can be coupled with well known methods of transformation such as Agrobacterium-mediated transformation for the rapid production of genetically-engineered cotton varieties of agronomic importance.

Description

A RAPID IN VITRO REGENERATION SCHEME
OF COTTON PLANTS COMPATIBLE WITH
A GROBA CTERIUM-MEΌIATED TRANSFORMATION
TECHNICAL FIELD OF THE INVENTION
This invention relates to a method for regenerating whole cotton plants from transformed or nontransformed cotton tissues.
BACKGROUND OF THE INVENTION
Besides producing spinnable fibers, 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. Under numerous marketing pressures, each product must stand on its merits or yield to its competitors that serve the consumer more effectively or at lower cost This dynamic business environment dictates the requirement for a highly efficient regeneration/transformation system to effectively address changes within the marketplace Therefore, there exists a need for a rapid, versatile regeneration/transformation system that targets cotton plants and which provides for the expression of seed specific added-value traits
The art of plant tissue culture has been an area of active research for decades. Over the past 10 to 20 years, this scientific research has been intensified in an effort to develop regenerable plant tissue culture procedures not only for cotton but also for other important agricultural crops such as maize, wheat, rice, and soybeans While the propagation of many of these agronomically significant crop plants in vitro is now commonplace, the routine culture and regeneration of cotton plants remains difficult. To date, a micropropagation system has not been defined for diverse cotton germlines which can be coupled with existing transgene technology Therefore, the in vitro culture systems developed to date do not offer the cotton biotechnology industry with a versatile and rapid regeneration/transformation system that provides a means to move added-value seed traits into cotton
One of the first cell culture regeneration systems for cotton was described by Davidonis and Hamilton They successfully regenerated whole plants from somatic embryos of cotton Davidonis, G. H. and R. H Hamilton, Plant Set. Lett. 32:89-93 (1984) These experimenters used immature cotyledon tissues of the cultivar Coker
310 The basic medium used consisted of Linsmaier and Skoog (LS) salts, vitamins, and the growth regulators α-naphthaleneacetic acid (NAA) and kinetin Linsmaier, E M and F Skoog, Physwl. Plant 18 100-127 (1965)
Later that same year, the regeneration of plants from several different California cotton cultivars was reported Rangan, T S et al , In Vitro 20 256 (1984) In this procedure, callus cultures were established from somatic tissues as well as immature embryos on Murashige and Skoog (MS) medium (Murashige, T and F. Skoog, Physwl. Plant. 15 473-497 (1962)) supplemented with auxin or auxin and cytokirun After the third to fifth subculture, these tissues gave rise to embryogenic callus and somatic embryos The embryos were then transferred to a low salt medium, e g , Beasley and Ting's (BT) medium (Beasley, C A and I P Ting, Amer. J. Bot. 60 130-139 (1973)) plus casein hydrolysate which permitted some of them to germinate and grow into whole plants However, plants regenerated with this method exhibited significant amounts (30%) of sterility and genetic change by somaclonal variation Stelly, et al , Genome 32 762-770 (1989) Somatic embryogenesis was also observed using the Coker line 312 and a Texas race stock called T25 Robacker, D C and T W Zimmerman, In the Ann. Mtg. of the American Society of Agronomy, November 25-30, Las Vegas, Nev , p 85 (1984) 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
Currently two cotton culture/regeneration systems have been reported 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
The currently utilized system for whole plant regeneration of cotton by somatic embryogenesis was first developed by Trolinder and coworkers in 1987 Trolinder, N and J.R Goodin, Plant Cell Reports 6.231-234 (1987), see also Firoozabady, et al., Plant Molecular Biol. 10 105-116 (1987) Unfortunately, while a wide range of cultivars were screened, only a few related varieties of cotton were amenable to this type of regeneration methodology Moreover, none of these cultivars have agronomic significance to today's farmers Therefore, any genetic engineering projects employing this embryogenesis strategy with a Coker or other responsive line must incorporate an extensive 6-to 10-year conventional breeding program to transfer added-value gene traits into agronomically useful germplasm Furthermore, 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
Conferences, Nashville, Tennessee, January 8-12 (1996) However, the procedures do not avoid the limitations associated with somaclonal variation
An alternative approach for whole plant regeneration - micropropagation via culturing of pre-existing apical meristems - was taken by Gould and co-workers which substantially decreased the time required for in vitro development of plantlets and also reduced the number of abnormal plants recovered Gould, et al , Plant Cell Reports, 10 12-16 (1991) This methodology was based upon culturing existing shoot apices which were excised from 3 day old seedlings No growth regulators were added to manipulate plant development Similar results were noted by Bajaj and Gill where excised meristems (1 mm) and shoot tips (1 cm) of two Gossypium species (G. arboreum and G. hirsutum) were vegetatively regenerated with and/or without a callus phase Bajaj, Y P S and M L Gill, Ind. J. Exp. Biol. 24 581-583 (1986) In this study, kinetin (1, 2, 4 and 6 mg/1) plus indole acetic acid (IAA) (0.5 mg/1) was reported to support shootlet formation in both Gossypium sp. The authors also reported shootlet formation in the presence of a combination of benzyladenine (2 mg/1) and NAA (0 5 mg/1), however, no supporting data was provided Neither of these clonal regeneration systems were successfully coupled with gene transfer
In U.S. Patent 5,164,310 ("the '310 patent") there is reported a method for possibly transforming and regenerating the shoot apex of two cotton cultivars, Gossypium hirsutum, var Coker 312 Tamcot CAB-C and Gossypium barbadense, var Pima 5-6
However, no data was provided confirming GUS incorporation into the plant genome There is no disclosure in the '310 patent of the ability to transform and regenerate other than the shoot apices from 5 day old seedlings In addition, the '310 teaches that regeneration should occur in the complete absence of hormones such as benzyladenine The '310 patent also appears to be genotype dependent, i e , it is limited to only the Coker and Pima cultivars Thus, to transform other cultivars would still therefore require the extensive conventional breeding programs avoided by the present invention
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. 10 105-1 16 (1987)) co-cultivated with Agrobacterium tumefaaens Unfortunately, these methods have been successfully applied to only a few regenerable cotton cultivars (e g , Coker 312) However, there are nearly one hundred cotton cultivars under cultivation in the United States, and they are not, in general, amenable to tissue culture techniques as described for Coker 312 Koonce, L , and N L Trolinder, Beltwide Cotton Conferences, Nashville, Tennessee, January 8-12 (1996); Firoozabady, E and D L
DeBoer, In Vitro CellDev. Biol. 29P 166-173 (1993)
As a result, 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 T0 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 Moreover, 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.
29P 166-173 (1993), Stelly, D M et al , Genome 32 762-770 (1989); U S Patent No 5,004,863 In addition, cotton plant regeneration through somatic embryogenesis remains germplasm-dependent Koonce, L., and N L Trolinder, Beltwide Cotton Conferences, Nashville, Tennessee, January 8-12 (1996) Thus, the cotton industry needs a rapid, reliable regeneration system that regenerates whole plants without a callus phase or somatic embryogenesis, and which works with all germlines that are currently grown commercially in the field, breeder lines, experimental lines, and foreign germlines. Furthermore, the regeneration system must work with transformed plants, preferably Agrobacterium-mediated transformants. This system would bring significant advantages to the cotton industry. Emphasis on extensive conventional breeding techniques would be reduced, somatic embryogenesis and its limitations would be avoided, and finally, it would provide the means to directly incorporate genetic traits into today's commercial lines within a short period of time. A number of regeneration or regeneration/transformation systems have been developed for plants other than cotton. However, none of these have been successfully applied to cotton. While some of these methods use benzyladenine ("BA") as a media supplement, there are no known reports of regenerating/culturing cotton using benzylamine as the sole growth regulator as is disclosed in the present application. U.S. Patent No. 4,992,375 ("the '375 patent") is directed to a method of regenerating soybeans from cultured soybean cotyledonary nodes. Although 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.
Other regeneration systems are described in Japanese Patent No. 5,176,647 which discloses a method of tissue culturing and mass propagating plants of the genus Cytisus and Japanese Patent No. 5,023,071, which discloses a method of proliferating adventitious buds of peony. To date, none of these systems have been successfully adapted for use with cotton plants. Thus, there remains a need for a regeneration method that overcomes the current problems associated with the regeneration of cotton plants - long culture periods, somaclonal variation, infertility, and applicability to a limited number of cultivars. To that end, an in vitro culture and regeneration system for the rapid regeneration of the fertile cotton plants via micropropagation has now been developed. 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. Finally, the systems disclosed herein can be used to regenerate and/or transform and regenerate dicotyledons other than cotton.
Using the regeneration system disclosed herein, 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. As demonstrated, 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)). SUMMARY OF THE INVENTION
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. In another embodiment, 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. In this manner, the present invention is useful in the rapid genetic engineering of cotton plants in general and of the commercially important cultivars in particular. In other embodiments, this invention relates to cotton plants produced using the procedures disclosed herein, seeds produced from these plants, and cotton plants germinated from these seeds.
In still other embodiments, the invention relates to the regeneration or the transformation and regeneration of dicots other than cotton.
BRIEF DESCRIPTION OF THE DRAWINGS
Figures la and lb are exemplary schematic representations of the preferred regeneration/transformation systems disclosed herein.
DETAILED DESCRIPTION
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 The CA-series and Stovepipe seeds were provided by Dr John Gannaway, Texas Agricultural Experiment Station, the Texas A&M University System, Lubbock, Texas, and the Stoneville 474 seeds were provided by Dr Catherine Houck, Calgene, Davis, California This clonal propagation system preferably utilizes different explants isolated as described in Examples 1 and 2 by excising apical (Fig la) and/or nodal (Fig lb) meristematic tissue from cotton seedlings (or plants) or germinated seeds germinated and grown under aseptic conditions or from clonally propagated shootlets ("CPS") grown under aseptic conditions (Figs la and lb) Preferably, nodal meristematic tissues, i e , leaf nodes, including primary leaf nodes, and/or cotyledonary nodes are excised from cotton seedlings or plants having an apical and at least 4 lateral or leaf nodes The period of growth needed to reach this stage depends on the seed vigor but typically takes less than or about 28 days For plants derived from seeds with greater seed vigor, nodal expants may be taken from seedlings or plants 14 to 28 days old, and even from seedlings or plants 14 to 21 days old Although cotton seedlings or plants of the ages described above are preferred, nodal meristematic tissue can be isolated from any plant bearing the appropriate tissues
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
Once isolated, the explants are placed vertically (basal end down) on a solid Murashige and Skoog ("MS") nutrient medium, Murashige, T and F. Skoog, Physwl.
Plant. 15 473-497 (1962) (incoφorated herein by reference), supplemented with a carbon source, preferably sucrose, and a low concentration of cytokinin, preferably benzyladenine (BA) 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 μM, preferably from about
0.15 μM to about 1 0 μM, even more preferably from about 0.3 μM to about 0 5 μM, and most preferably about 0.3 μM. Optimum formation of elongated shootlets from explants of the apical meristematic tissue from seedlings or plants is obtained with about 1.0 μM 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 μM
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 μM are to be avoided as they are increasingly toxic to the explants
After an approximately 3 week culture period, multiple shootlets (2-5) can be proliferated from nodal explants cultured on the Shooting Medium containing BA, preferably 0 3 μM 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)
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 Shootlets less than about 2-3 cm tall after about 21 days are transferred to Maturation Medium I (Table 1) for 14-21 days to induce additional growth and to remove any remaining phenolic compounds prior to rooting and are then transferred to Maturation Medium II for an additional 14-21 days Shootlets about 3-4 cm tall after about 21 days are transferred to
Maturation Medium II (Table 1) to remove any remaining phenolic compounds prior to rooting Shootlets greater than about 4 cm tall after about 21 days can be rooted directly without a maturation step At the end of the maturation period of 14-21 days, the shootlets are ready for rooting, either in vitro or directly in soil For in vitro rooting, a preferred Rooting Medium is MS media supplemented with about 1 0 μM 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 Rootone™ 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 by transferring the shootlet directly to soil after the application of Rootone™ It has also been found that shootlets greater than about 3 cm tall will root more quickly (about 2 weeks) than shootlets less than about 3 cm tall (about 3 weeks) when rooted
As shown in Table 11 (Example 4), the efficiency of rooting for cultivars was low in vitro (22-26% of the shootlets rooted) By contrast, rooting efficiency was greatly increased when shootlets were transferred directly to soil (50-90% of the shootlets rooted) Regenerated plants that were advanced to soil appeared morphologically similar to seed-borne cotton plants and showed no obvious chromosomal abnormalities Furthermore, these regenerants flowered, produced bolls, and set viable seeds Other high-fiber-yielding breeder lines (cvs , CA-3066, ST474, CA-3076, CA-3084, CA-3050, and Stovepipe) were also found to be adaptable to this regeneration scheme Using the in vitro propagation techniques disclosed herein, high- fiber-yielding varieties of cotton plants can be regenerated rapidly (explant-to-soil in approximately 6 weeks)
During or after the rooting process, the shootlets, which are rootless, or plantlets (rooted shootlets) are preferably exposed to a hardening process Most preferably, the hardening process begins simultaneously with the initiation of the rooting process In general, the hardening process takes approximately 2 to 3 weeks depending on the height of the elongated shootlets at the time the process is begun Generally, 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 Gro™ (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 Preferably by the end of the third week, the plastic bags are completely removed Smaller shootlets (approximately 2 cm in height) that are advanced to soil early usually require a longer exposure to the humid environment for root/plantlet formation and the hardening process After root formation, the plantlets begin to elongate and develop new leaflets Plantlets which produce new leaves during this hardening process are transplanted into larger, typcially 6 to 10 inch pots and grown in a greenroom until flowering and boll set The regeneration system described above can also be used to regenerate plants from transformed cotton explants comprising pre-existing meristems or other cotton tissues The preferred transformation technique of the present invention makes use of the Ti plasmid of Agrobacterium tumefacians. 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 By inserting a foreign gene construction into the T-DNA region, 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 exhibitied by early emerging shootlets, generally evident after about five days and showing no signs of phenotypic stress, i.e , having green leaves and stems The mottled green phenotype is exhibited by later emerging shootlets, generally evident after about 5 days to 2 weeks and showing various degrees of stress such as bleached leaves, white leaf tips and edges, and green sectors and white vascular tissue in cotyledonary leaves The third phenotype, i e , the tan phenotype, is exhibited by kanamycin-sensitive (non-transformed) explants which turn brown and die The mottled green phenotype grows more slowly during the KAN selection procedure which involves a step-wise incremental increase of the KAN concentration
(37 to 50 to 75 μg/ml) to which the putative transformants are exposed However, newly formed leaves of the mottled green phenotype which emerge after about 2 weeks, are phenotypically normal With both the green or mottled green phenotypes, 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 μg/ml
KAN (Kan Selection Medium II) from that with 37 μg/ml KAN (KAN Selection Medium I) and the cut/trim step is generally first required after about 4-6 days For nodal meristems, transfer to KAN Selection Medium II from KAN Selection Medium I and if necessary the cut/trim step is generally first required after about 7 days In either case, KAN selection is enhanced by keeping the meristem close to the antibiotic source and the vascular tissues of the shootlet in direct contact with the kanamycin supplemented medium Following KAN selection, both the green and mottled green phenotypes are matured and rooted by the methods previously described herein with respect to the non-transformed explants Following co-cultivation of explants from the 14 to 28-day old seedlings and clonally propagated shootlets, the explants first become necrotic and then develop a green shootlet after about 7 to 10 days of the KAN selection process As the shootlets grew and elongated, the necrotic explants were excised (in some cases - recultured) and the green shootlets were transferred to the fresh kanamycin medium In general, the explants from the established shootlets were more sensitive to the kanamycin medium than explants from the 28-day old seedlings
It is generally believed that the transformed (T0) plants are chimerically transformed, i e , some of the pre-existing meristems are transformed and others are not To verify kanamycin resistance/transformation or kanamycin sensitivity/non- transformation, 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.
Examples Example 1 - Seed Sterilization and Germination
Seeds from diverse germplasm (Table 3) were sterilized, germinated, and grown in vitro for different time periods. For seed sterilization, seeds (approx. 150) were placed in ultrapure water (MilliQ plus UF) with two drops (approximately 100 μl) of Tween-20 per 100 ml of H2O 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.
For seed germination, seeds were placed on sterile filter paper moistened with 2 ml of water (MilUQplus UF) within a Petri plate and allowed to germinate in the dark
(30 °C) overnight. The following day, the seed coats were removed and the shoot apex was isolated as described in Example 2 and placed directly on Shooting Medium (Fig. la). In some cases, the shoot apex was isolated after two days of germination in the dark rather than one depending on the seed vigor and the rate of germination. Alternatively, the germinated seeds (minus seed coat) were placed on Germination Medium (Table 1) in Magenta vessels and allowed to grow under tissue culture chamber conditions (30°C; light intensity 85 μmol s 'm2) for about 14 to 28 days. (Fig. lb). To allow for seedling maturation, inverted sterile Magenta boxes and couplers were added to the existing seedling vessels. 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.
Table 1 Tissue culture media utilized to regenerate and transform cotton plantlets
Days or
Stage Medium Composition Hours
Seed Germination Germination Medium Vτ MS medium4 with sucrose 14-28 (10 g/l), Phytagel (2.25 g/l),
(pH 7.0)
Shootlet Induction Shooting Medium MS medium with BA 21 Shootlet Maturation Maturation Medium I MS medium with BA and 14-21 3 g/1 AC Maturation Medium II MS Medium with 3 g/1 AC 14-21
Root Formation Rooting Medium MS medium with 1 μM/1 B3A 28-42
Co-cultivation Period Co-cultivation MS medium with Sucrose l h
Medium 1.5 g/100 ml; Acetosyringone (20 mM Stock) 200 μl; MES (20 mM Stock) 10 ml; pH 5.5; Sterilization by membrane filtration (0.22 μ)
Induction/KAN- KAN Selection MS Medium with 0.3 μM BA; 5-7
Selection Stage 1 Medium 1 37 μg/ml Kanamycin; and 500 μg/ml Carbenicillin
KAN-Seiection KAN-Selection MS Medium with 0.3 μM BA; 28
Stage 2 Medium 2 50 μg/ml Kanamycin; and 500 μg/ml Carbenicillin
KAN-Selection KAN-Selection MS Medium with 0.3 μM BA; 28
Stage 3 Medium 3 75 μg/ml Kanamycin; and 500 μg/ml Carbenicillin
Abbrev.: BA = β-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). Example 2 - Source Materials
For experiments utilizing tissue from 1 or 2-day old seedlings, seeds were surfaced sterilized by the procedure described in Example 1 Following the 1 or 2-day germination period, seeds that supported extended radicle growth (2 mm to 5 mm) were selected and their embryonic axes and portions of the attached cotyledons
(approximately 3 to 4 mm in length) were isolated The apices (approximately 2 mm to 3 mm in length) were placed vertically on the Shooting Medium (Table 1 ) in the same orientation as in the intact seedling (Table 2)
For experiments utilizing tissue from in vitro grown 14- to 28-day old seedlings, the following explants were isolated shoot apices, primary leaf nodes, leaf nodes, and cotyledonary nodes The excised explants were placed vertically on the Shooting Medium (Table 1 ) and in the same orientation as in the intact seedling (Table 2) For experiments utilizing pre-existing meristems from clonally propagated shootlets, apices and lateral meristems were isolated and placed vertically on the shootlet induction media and in the same orientation as in the intact shootlet These clonally propagated shootlets which were derived from diverse germlines (HS-26, CA- 3076, ST7A, ST474 and ST 139) were maintained for several months While cultured on MS medium with 0.3 μM BA, the established shootlets could only be cultured for only about 3 weeks The substitution of different vitamin sources for the MS vitamins improved the shootlets culturability in that they could be maintained for 5 to 6 weeks before being transferred to fresh medium The other vitamin sources were as follows: (1) B5 vitamins (Exp. Cell Res. 50 151 (1968)) and (2) modified Nitsch's vitamins (Science 163 85 (1969); Thiamine-HCl at 10 mg/1 and nicotinic acid at 0 5 mg/1) (Table 2) Table 2. Comparison of Explant Sources Derived from in vitro Grown Cotton (Stoneville 7A, Glandless) Seedlings for Their Capacity to Form Shootlets When Cultured on Shooting Medium Containing 0.3 μM BA
Explant Sources Response Cotyledon pieces Callus
Foliar leaves Callus
Hypocotyl segments Callus
Epicotyl segments Callus
Cotyledonary nodes Shootlets Primary leaf nodes Shootlets
Apices Shootlets
CPS nodes' Shootlets
"CPS nodes refers to nodes of clonally propagated shootlets.
Example 3 - Regeneration Procedures Explants of the three different source materials were placed on the benzyladenine
(BA) containing Shooting Medium to induce regeneration, i.e., shootlet proliferation. For apical meristematic tissue from 1 or 2-day old seedlings a benzyladenine concentration ranging from about 0 to about 1.0 μM was used. For apical meristematic explants from 14 to 28-day old seedlings a concentration of about 1.0 μM BA gave optimum results. For nodal meristemtic tissue from 14 to 28-day old seedlings and for meristematic tissue from clonally propagated shootlets a BA concentration of less than about 1 μM, preferably about 0.15 μM to about 1.0 μM, even more preferably about 0.3 μM to about 0.5 μM, and most preferably about 0.3 μM was used. 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. Shootlets less than about 2-3 cm tall after 21 days were matured by transferring the explant to Maturation Medium I (Table 1) for an additional period of 14 to 21 days and then transferred to Maturation Medium II for an additional 14 to 21 days Shootlets about 3-4 cm tall after 21 days were matured by transferring the explant to Maturation Medium II (Table 1) for an additional period of 14 to 21 days Shootlets greater than about 4 cm tall after 21 days were not matured, but rather, were rooted immediately In all cases, rooting was accomplished as described in Example 4 For those shootlets rooted in vitro, a Rooting Medium (Table 1) consisting of lμM 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/cm2 for 15 minutes Petri dishes (100 x 25 mm) were sealed with Parafilm and Magenta vessels were capped or enclosed with polypropylene covers All cultures were maintained at a constant light intensity (85 μmol s 'm2), temperature (30°C), and a 16 hr photoperiod The light source consisted of cool white fluorescent lamps
For rooting and initial plantlet development, 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 μmol s 'm 2, and a 16 hr photoperiod) The light source consisted of cool white fluorescent and incandescent lamps For further plant growth and flowering, 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 μmol s 'm2) and a 16 hr photoperiod The light source consisted of high-pressure sodium (140 μmol s 'm2) and metal halite lamps (100 μmol s 'm2)
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 μM, when applied to the explants as part of the Shooting Medium, initiated elongated shootlets from excised cotyledonary nodes after a 3 week culture period. In contrast, higher concentrations of BA (3.0 μM and greater) suppressed shootlet formation after a 3 week culture period and were often toxic to the explants. (Table 4).
Table 3. Shootlet Proliferation of Explants Excised from in vitro Grown Seedlings of Different Cotton (Gossypium hirsutum) Cultivars
Cultivars Shootlet Formation
Stoneville 7Aa +e
Paymaster HS-26" +
CA-30500 + CA-3066C +
CA-3076C +
CA-30840 +
Stovepipe0 +
Stoneville 474d + "Stoneville 7 A seeds were provided by Dr. Rick B. Turiey, USDA-ARS, Stoneville, MS.
"Paymaster HS-26 seeds were provided by Dr. John J. Burke, USDA-ARS, Lubbock,
TX c CA-Series and Stovepipe seeds were provided by Dr. John Gannaway, Texas
Agricultural Experiment Station, The Texas A & M University System, Lubbock, TX. d Stoneville 474 seeds were provided by Dr. Catherine Houck, Calgene, Davis, CA. e"+" sign indicates formation of elongated shootlets from explants.
Table 4 Shootlet Proliferation from Excised Cotyledonary Nodes of 14-day old Cotton Seedlings (Stoneville 7A, Glandless) When Cultured on Shooting Medium Containing Different Concentrations of Benzyladenine (BA)
BA Concentration (μM)
Culture period 0 3 1 0 3 0 10 0 30 0 Week 2 4 a 2 3 0 0
Week 3 7 3 0 0 0
* The number of shootlets / 3 explants
A range of BA concentrations (0 15 to 1 0 μM) when added to the Shooting Medium supported shootlet formation in excised primary leaf nodes and cotyledonary nodes isolated from 14- to 21 -day old cotton seedlings (Stoneville 7A) (Table 5) The optimal BA concentrations for inducing shootlet formation from nodal meristems cultured on Shooting Medium was 0 3 μM based on the development of elongated shootlets (2-3 cm in height) after a 3 week culture period (Tables 5, 6 and 7) At 1 0 μM BA, the apical meristems isolated from 14 to 21 -day old cotton seedlings also were optimally induced to form elongated shootlets (Tables 8 and 9) For apical meristems isolated from 1 or 2-day old germinated seeds a BA concentration of 0 to 1 0 μM was optimal In contrast, hormone-free medium failed to support elongated shootlets of apices excised from seedlings other than the 1 to 2-day old germinated seeds during the same culture period in a consistent or reproducible manner Similar results on shooting efficiencies and BA concentrations were found for the glanded variety, Paymaster HS-
26 (Table 6) and the other cultivars used in the study (Table 7) Table 5. Effect of Benzyladenine on Shooting Efficiencies of the Primary Leaf Nodes and the Cotyledonary Nodes Excised from 14- to 21 -Day Old Seedlings of Cotton (Stoneville 7A, Glandless)
Benzyladenine (μM)
Culture period 0.0 0.15 0.3 0.5 1.0
Week 2 3/9a 14/17 18/24 12/15 5/8
Week 3 5/9 15/17 19/24 15/15 7/8
"The number of shootlets / number of nodal meristems.
Table 6. Effect of Benzyladenine on Shooting Efficiencies of the Primary Leaf Nodes and the Cotyledonary Nodes Excised from 14- to 21 -Day Old Seedlings of Cotton (Paymaster HS-26).
Benzyladenine (μM)
Culture period 0.0 0.15 0.3 0.5 1.0 10.0
Week 2 3/351 16/22 29/35 23/26 19/28 1/13
Week 3 16/35 15/22 29/35 24/26 22/28 0/13
"The number of shootlets / number of nodal meristems.
Table 7. Effect of Benzyladenine on shootlet formation for lateral meristems, primary leaf nodes and the cotyledonary nodes derived from 28-d old seedlings of different cotton cultivars.
BA Concentrations
0.0 μM 0.3 μM 1.0 μM
HS-26 9a 83 68
46" 83 79
CA-3050 40 75 50
80 75 100
CA-3066 18 65 64
73 88 100
CA-3076 55 100 86
33 107 71
CA-3084 60 93 50
80 100 60
Stovepipe 53 76 62
71 71 62
Stoneville 474 47 44 44
77 71 61
*The number of shootlets after two weeks/number of nodal meristems expressed as a percentage.
"The number of shootlets after three weeks/number of nodal meristems expressed as a percentage. Table 8. Effect of Benzyladenine on shooting efficiencies of apical meristems excised from 14-21 day old seedlings of cottom (Stoneville 7 A, glandless).
Benzyladenine (μM)
Culture period 0.0 0.15 0.3 0.5 1.0
Week 2 0/2a(0/2)b 3/4 (0/4) 4/9 (0/9) 3/6 (0/6) 3/3 (0/3)
Week 3 2/2 (0/2) 2/4 (0/4) 3/8 (0/8) 4/6 (0/6) 3/3 (3/3) aThe number of shootlets/number of apical meristems.
"The number of elongated shootlets (> 2-3 cm tall)/number of apical meristems.
Table 9. Effect of Benzyladenine on shooting efficiencies of apical meristems excised from 14-21 day old seedlings of cottom (Paymaster HS-26, glanded)^
Benzyladenine (μM)
Culture period 0.0 0.15 0.3 0.5 1.0 10.0
Week 2 3/6a(0/6)b 4/6 (0/6) 8/9 (0/9) 6/7 (0/7) 7/7 (0/7) 2/4 (0/4) Week 3 1/8 (0/8) 6/7 (0/7) 9/9 (0/9) 4/7 (0/7) 6/8 (6/8) 0/4 (0/4)
Similar data (not shown) has been collected for the following cultivars: CA-3076; CA-
3050; CA-3066; CA-3084; and Stovepipe.
"The number of shootlets/number of apical meristems.
"The number of elongted shootlets (> 2-3 cm tall)/number of apical meristems. Using the Shooting Medium as a screening system, different explant sources derived from in vitro grown cotton (Stoneville 7A) seedlings were tested for their ability to form shootlets (See Table 2). After a 21 -day culture period, only cotyledonary nodes and primary leaf nodes formed shootlets; the remaining explants formed crystalline type callus primarily at the cut ends of the explant. Occasionally, a hypocotyl segment formed a shootlet which apparently developed from the pericycle region
High shooting efficiencies occurred for both primary leaf nodes and cotyledonary nodes derived from 14 to 28-day old cotton seedlings when cultured on Shooting Medium containing 0.3 μM BA (Table 10). Explants from both the glandless variety (Stoneville 7A) (94%) and the glanded variety (Paymaster HS-26) (97%) possessed a prolific capability to form shootlets under these cultural conditions. Both the glanded and glandless varieties generally demonstrated multiple shootlet formation after about 3 weeks of culture
Table 10 Shootlet Proliferation of Excised Primary Leaf Nodes and Cotyledonary Nodes from 14-Day Old Cotton Seedlings When Cultured on Shooting Medium Containing 0.3 μM BA.
Cultivar Number of Number of % Explants Time Required
Explants Shootlets/Explants Formed Shootlet Shootlets Formation
Stoneville 7A 46 2-5 94 7-21 days*
Paymaster 37 2-5 97 7-21 daysa
HS-26
"Twenty-one days is required for the formation of elongated shootlets (2-3 cm), however, shootlet growth is generally first visible after about 7 days. Rooting of the shootlets derived from the pre-existing meristems of clonally propagated shootlets, the nodal or apical meristems of 14- to 28-day old seedlings, or the apical meristems of 1 or 2-day old germinated seeds was accomplished in one of two ways. Shootlets were either cultured on Rooting Medium (Table 1 ) or rooted by transferring the shootlets directly to soil after treating the shootlet with Rootone™
(Table 11). Of the cultivars examined (Stoneville 7A and Paymaster HS-26), elongated shootlets demonstrated a low efficiency rate for root formation when cultured on MS medium plus 1 μM IBA. However, the glanded and glandless varieties (Paymaster HS- 26 and Stoneville 7A) demonstrated a high efficiency for rooting when placed directly into soil after the application of Rootone™ (Table 11). Elongated shootlets of non- transformed Stoneville 7A and Paymaster HS-26 as well as KAN selected shootlets of Stoneville 7A, Paymaster HS-26, Sphinx, and CA-3076 have all been rooted successfully by advancing directly to soil (with Rootone™).
Table 11. Comparison of Rooting Efficiency of Cotton Shootlets When Cultured on Shooting Medium Containing Either 1 μM IBA or Rooted Directly by Transferring to
Soil (+ Rootone™)
In vitro Culture Period Rooted Directly to Soil
Cultivar MS + 1 μM BA (+ Rootone ™)
Stoneville 7A 6/23" 12/23
Paymaster 6/27 25/28
HS-26 a The number of shootlets that rooted / number of attempts. To establish plantlets via rooting on the IBA supplemented Rooting Medium, 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. To establish plantlets via rooting directly in soil, the base of the elongated shootlets were first dipped into Rootone™ 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.
Table 12. Flowering and Seed Set of 137a Bolls From 32 Paymaster HS-26 Plants
Cultivar % Germ # Flowers Seeds # Germ
HS-26 73% 15 419 306
HS-26 74% 23 573 423
HS-26 65% 6 138 90
HS-26 69% 10 152 105
HS-26 88% 9 178 156
HS-26 84% 18 357 300
HS-26 72% 15 208 150
HS-26 87% 27 535 466
HS-26 71% 14 292 207
Totals 77% 137 2,852 2,203
"The 137 bolls generated a total 2,852 seeds for an average of 20.8 seeds/flower. Example 5 - Agrobacterium tumefacians Mediated Cotton Transformation Procedures
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). As a control for each transformation experiment, 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 μg/ml) and kanamycin (50 μg/ml) for Agro+ or streptomycin (25 μg/ml) for Agro- were present throughout the vegetative growth of the bacteria. Before transformation solid kanamycin (50 μg/ml) was added to the liquid culture (50 ml) of Agro-. Both were then diluted 1 : 1 during the co-cultivation phase. Explants from the source materials were placed into 7 ml of the Co-cultivation Medium (Table 1) under sterile conditions. To this was added an equal volume of the
LB-Agrobacterium tumefaciens strain LBA4404 solution. The explant - Co-cultivation Medium-LB -Agrobacterium tumefacians solution was then placed in a dessicator and subjected to a vacuum pressure (29" Hg) for about 5 minutes. The co-cultivation period consisted of a 15 minute period for temperature equilibrium (25°C or 30°C) and an incubation period of 1 hour under sterile conditions. Example 6 - Transformation and Selection Procedures
Following the co-cultivation period, the explants were blotted on sterile filter paper and transferred to Shooting Medium plus antibiotics, i.e., a KAN Selection Medium for KAN selection. Alternatively, following the co-cultivation period, the explants are washed with LB medium + 500 μg/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 μg/ml or a KAN 37 to 50 μg/ml selection process, respectively. Essential to the success of the KAN selection procedure was the use of the cut/trim method in which the shootlets were cut and the cotyledonary leaves and other lateral nodes trimmed when necessary to constantly maintain the apical meristem less than 2 cm from the antibiotic source. This was done by evaluating the shootlets on a daily basis to determine their height. For the apical meristems of 1 or 2-day old seedlings, trimming was generally first performed after about 4 to 7 days. For nodal meristems of the 14 to 28-day old seedlings, trimming was generally first performed after about 7 to 10 days.
First, the co-cultivated explants were placed on Shooting Medium (0.3 μM BA) + 37 μg/ml KAN (+ 500 μg/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. In general, 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 μM BA) + 50 μg/ml KAN + 500 μg/ml carbenicillin (KAN Selection Medium II - Table 1). In contrast, 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. 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 μM BA) + 75 μg/ml KAN + 500 μg/ml carbenicillin (KAN Selection Medium III - Table 1) and were cut/trimmed when necessary. Using this cut/trim method, the developing explants (shootlets) 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 μg/ml KAN (5-7 days) to 50 μg/ml (4 weeks) to 75 μg/ml (4 weeks), each step containing carbenicillin at 500 μg/ml. In all cases, lateral meristems required only the first two levels (37 to 50 μg/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 μg/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 μg/ml (4 weeks) to 75 μg/ml (4 weeks)] were transferred on a weekly basis Following a maturation step with Maturation Medium I or
Maturation Medium II (Table 1), these KAN-selected shootlets (3-leaf stage) were rooted directly by transferring to soil (+ Rootone™). For germline transformation, various tissues such as pollen grains, leaf tissues and genomic DNA were harvested from maturing plants and assayed for GUS gene expression (fluorescent and histochemical localization of GUS activity) and DNA integration (Southern-blot analysis).
To verify kanamycin resistance, a number of shootlets from the CA-3076 and Paymaster HS-26 varieties were rechallenged on kanamycin rather than rooted. Explants of nodal and apical meristematic tissues were excised from these shootlets and cultured on fresh KAN Selection Medium II (Example 6) to identify transgenic meristems within these shootlets. All of the rechallenged apical meristems and most of the nodal meristems developed green shootlets.
Example 7 - Transformation and Selection Results
For transformation, explants from 14 to 28-day old seedlings (cotyledonary nodes, primary leaf nodes, nodal meristems, and apices) were used as starting materials with the regeneration system described herein (Table 13) Also used with this regeneration system were apices (2 to 3 mm) excised from 1 or 2-day old seedlings or apical and nodal meristems from clonally propagated shootlets ("CPS") (Table 13) Explants from both these sources were harvested and maintained for 1 -2 hours in 7 ml of Co-cultivation Medium (Table 1) sterilized by filtration through a 0.22 μm Acrodisc filter (Gelman Sciences, Ann Arbor, Michigan)
Following excision of explants, an equal volume of LB-Agrobacterium tumefaciens strain LBA4404 containing pBI 121 was added to Co-cultivation Medium/explants and incubated for approximately 1 hour at either 25 ° or 30°C After the co-cultivation period, the explants were blotted and transferred onto the KAN selection procedure as described in Example 6 After two, 2-week culture periods on
KAN Selection Medium II or on each of KAN Selection Medium II and III, the kanamycin-resistant shootlets elongated to approximately 2 to 3 cm, while the kanamycin-sensitive explants ceased to grow and died (Table 13) Transformation efficiencies (based on the number of explants cultured) varied depending upon the genotype, explant source (Tables 13 and 14), and temperature (Table 14) In general, as indicated in Table 13, the 1-day system supported a higher regeneration/transformation efficiency rate (18%) than both the 28-day (11.9%) and CPS (1 4%) systems However, based on explant and temperature (Table 14), Paymaster HS-26 (glanded) demonstrated explant-independency at both 25° and 30 °C. In contrast, Stoneville 7A (glandless) demonstrated explant-dependency for both temperature and efficiency rates for regeneration/transformation (Table 14) In both cases, however, 25 °C supported a higher regeneration/transformation efficiency rate than 30 °C for co-cultivation (Table 14) Under greenroom conditions, the KAN selected plantlets matured to flowering stage and set seeds. Several KAN selected (T0) plants expressed GUS enzymatic activity in leaves and pollen. (See Tables 15-19) Table 13. Summary of KANAMYCIN-selected Shootlets and Mature Plants
KAN-selected Shootlets Mature Plants
Cultivar Experiment* # Explants
1 -day 28-day CPS 1-day 28-day CPS
CA-3076 #1 21 10
HS-26 #1 28
ST-7A #4 42 24 16
HS-26 #5 1 19 24
HS-26 #8 48
Sphinx" #12 104
ST-474 #14 62
ST-139b #15 65
HS-26 #17 25
ST-474" #18 142
HS-26 #20 50 20
ST-7A #21 240 15
Totals'1 946 70 49 28 12
"Sphinx seeds were provided by Dr. K.M. El-Zik at Texas A&M Univ., College Station,
Texas.
"Stoneville 474 and Stoneville 139 seeds were provided by Dr. Catherine Houck,
Calgene, Davis, California. cFor apical meristematic explants from 1 or 2-day old germinating seeds, a total of 392 explants have been tested, 18% of which have generated shootlets. For nodal or apical meristematic explants from 14 to 28-day old seedlings, a total of 412 explants have been tested, 11% of which have generated shootlets. For meristematic explants of clonally propagated shootlets, a total of 142 explants have been tested, 1.4% of which have generated shootlets. Table 14. Efficiency rate of KAN-selected shootlets when co-cultivated with A. tumefaciens at 25 °C vs. 30°C. Pre-existing meristems from 1 or 2-day old germinated seeds and 28-day old seedling were used as starting material.
Cultivar 25 °C KAN-selected 30 °C KAN-selected
ST-7A" 12/17° (70%) 12/25 (48%) ST-7A" 9/1 12 (8%) 6/128 (4.6%) HS-26" 14/28 (50%) 6/22 (27%) HS-26" 24/49 (48.9%) 17/58 (29%)
"Apices from 1 -day old seedlings.
"Apices, lateral and cotyledonary meristems from 28-day old seedlings.
Total number of KAN-selected shootlets/total number of pre-existing meristems.
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. In addition, the mottled green phenotype grew more slowly during the KAN selection procedure (37, 50 and 75 μg/ml). However, newly formed leaves of the mottled green phenotype which emerged after about 2 weeks, were phenotypically normal and uniformly green. The KAN-resistant shootlets of both types were carefully moved through the previously described KAN selection procedure. Following selection, both the green and mottled green phenotypes were matured and rooted easily by the methods described herein. Kanamycin-sensitive explants did not exhibit proliferation; but rather, turned brown and died.
To date, 121 KAN-selected shootlets and 40 mature viable plants have been generated by this procedure with an efficiency range of 1.4 to 57% (Table 13). Under greenroom conditions, these KAN-selected plants were morphologically normal, they flowered and developed viable pollen grains As determined by the GUS assays (Jefferson 1987, Jefferson et al 1987, Gallagher 1992, Stomp 1992), these KAN- selected plants (T0) were found to have a chimeric growth pattern for positive GUS activity within their leaves and flower parts (pollen grains, pollen sacs and pollen grain tubes) (Tables 15-19)
The cotton cultivars used herein exhibited maximum selection at incremental kanamycin concentrations of 37 to 50 μg/ml for nodal and greater than 2-day old apical meristematic tissues and 37-50-75 μg/ml for 1 or 2-day old apical meπstematic tissue These kanomycin selection levels were selected using a conventional antibiotic screen
Other cultivars, however, may require different selection procedures or conditions Appropriate selection conditions for additional genotypes can be easily determined by incorporating a "mork" transformation control — Agrobacterium tumefaciens without the pBI121 binary vector (Agro-) — and a stepwise incremental increase in KAN concentration The level at which tissue or shoots co-cultivated with Agro- die is the optimum level of KAN to be used to select transformants using Agro+
Example 8 - Introduction of Binary Vectors Into Agrobacterium tumefacians
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
(1980), Methods in Plant Mol. Biol, Cold Spring Harbor Laboratory Press pp 63-65 (1995) (both of which are incorporated herein by reference) 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
The fluorometric analysis of GUS activity was carried out as described by Jefferson (Jefferson, R A , Λ. Mol. Biol. Reporter 5 387-405 (1987), Gallagher, S R., GUS Protocols, Academic Press, Ine , pp 47-59 (1992)) (both of which are incorporated herein by reference) using 4 mM 4-methylumbelliferyl-β-D-glucuronide
(MUG) as the substrate except that 100 mM potassium phosphate buffer (pH 7 2) was used in the extraction buffer (100 mM KPO4, pH 7 0, 10 mM β-mercaptoethanol, lOmM Na2 EDTA, 0 1% sodium lauryl sarcosine and 0 1% Triton X-100) Insoluble polyvinylpyrrolidone (5%) and silicon powder was added to the extraction buffer (0 5 ml) before homogenization Leaf tissue (approximately 20 mg of fresh weight) was homogenized in Eppendorf tubes by a motorized conical teflon pestle (1100 rpm) in 0 5 ml of the extraction buffer Assays (GUS activity) of leaf extracts were performed on leaves formed after rooting Green leaves from several nodes were also analyzed to identify chimeric plants Readings were made on an Aminco Bowman Scanning Spectro- fluorometer (SLM Amino, Rochester, N Y ) with a xenon lamp at excitation wavelength
(365 nm) and emission wavelength (455 nm) GUS activity was measured at time intervals (0, 30, 60, 150 minutes and overnight) and expressed as relative fluorescence units/hour/μg protein Representative results are shown in Table 15
Table 15. Fluorometric Analysis of GUS Activity in KAN-selected Plantlets
Cultivar Fluorescence units/hour/μg protein
HS-26NT-01a 0.99
HS-26NT-02 0.40
HS-26T0-01" 0.40
HS-26T0-02 0.60
HS-26T0-03 7.40
HS-26T0-04 3.54
HS-26T0-05 0.34
CA-3076T0-01 5.70
CA-3076T0-02 0.50
CA-3076T0-04 0.90
CA-3076T0-05 0.17
""NT" refers to non-transformed plants. " TO (or T0) indicates transformants.
Example 10 - Histochemical Analysis of GUS in Pollen Grains
A histochemical localization of GUS in the pollen of non-transformed cotton plants (HS-26NT-01) and in the transformed cotton plants whose leaves exhibited the greatest GUS activity (HS-26T0-04, CA-3076T0-01, HS-26T0-03) was performed according to the technique of Jefferson, R.A. , Plant Molecular Biology Reporter
5(4):387-405 (1987) (incorporated herein by reference). The presence of insoluble, blue product inside pollen grains of transformed plants (Table 16) indicates germline transformation and transmission of the inserted GUS gene to gametes and subsequently to the progeny of the transformed plants produced by the methods discussed herein. Example 11 - Histochemical localization of GUS activity in leaf tissue and flower parts
Leaves of shootlets and small plantlets and flower parts (pollen, pollen sac and pollen tubes) were tested for GUS expression as previously described (Jefferson 1987, Stomp 1992) The histochemical localization of GUS activity (Jefferson 1987, Stomp
1992) demonstrated that many of the KAN-selected plants generated the distinguishing blue color reaction in their pollen grains (HS-26T0's-Table 17, ST7AT0's-Table 18, CA- 3076T0's-Table 19 T0 plants appeared to be chimeric because some flowers produced GUS positive pollen while others did not However, Tj plants showed a much higher percentage of flowers with GUS positive pollen, indicating that they are not chimeric
The expression of GUS in pollen indicates germline transformation (Table 19)
Table 16 Histochemical Analysis of GUS in Pollen Grains
Individual Plants Expression of GUS
HS-26NT0-01 (nontransformant) HS-26T0-03 (transformant) +"
HS-26T0-04 (transformant) +
CA-3076T0-01 (transformant) +
"The (+) indicates the presence of precipitated enzyme reaction product inside pollen grains
Table 17 Histochemical Localization of GUS Activity in Pollen Grains of Putative Transgenic Cotton Plants, c v Paymaster HS-26 Pre-existing meristems were excised from 28-day old seedlings and utilized as starting material for co-cultivation with Agrobacterium
Individual Plants Total U Flowers Total # Flowers Pollen in Sac
Tested w/+Pollen (Pollentube)
HS-26T0-O1" 9 3 1
HS-26T0-02 10 3 -
HS-26T0-03 6 5 -
HS-26T0-04 13 3 -
HS-26T0-05 12 2 -
HS-26T0-07B" 23 8 -
HS-26T0-08b 6 3 -(2 Pollentube)
HS-26T0-09 6 1 -
HS-26T0-1 1 3 - -
HS-26T0-12 12 5 -
HS-26T0-13 9 4 1(1 Pollentube)
HS-26T0-14 23 1 1 -
HS-26T0-01 Tl-03c 2 1 -
HS-26T0-01 Tl-04 2 2 -
HS-26T0-01 Tl-06 3 - -
HS-26T0-01 Tl-07 4 1 -
HS-26T0-01 Tl-09 1 - -
HS-26T0-03 Tl-03 7 4 -(1 Pollentube)
HS-26T0-03 Tl-44 2 2 -
HS-26T0-04 Tl-04 6 4 -
HS-26T0-04 Tl-08 2 1 -
HS-26T0-04.T1-23 2 1 -
HS-26T0-04 T1-29 1 1 -
HS-26T0-04B Tl-41 2 - - HS-26TO-04 T1-42 9 7 -(2 Pollentube)
HS-26T0-04 T1-43 1 1
HS-26TO-07B Tl-01 7 6
HS-26T0-07B Tl-08 1
HS-26T0-07B T1-10 3 2
HS-26T0-07B Tl-12 2 2
HS-26T0-07B T1-14 2 1
HS-26T0-07B T1-40 6 6
HS-26T0-07B Tl-44 8 8 "TO (or T0) indicates transformants "A mottled green phenotype
CT1 (or Tj indicates progeny (seeds or plants) of T0 plants For example, HS-26T0- 01 Tl-03 designates one of several T, progenies of a T0 plant
Table 18. Histochemical Localization of GUS Activity in Pollen Grains of Putative Transgenic Cotton Plants, Stoneville 7A. Apices were excised from 1-day old seedlings and utilized as starting material for co-cultivation with Agrobacterium.
Individual Plants Total # Flowers Total # Flowers Pollen in Sac Tested w/+Pollen (Pollentube)
ST7AT0-01 5 4
ST7AT0-02 3 1
ST7AT0-03 5 1
ST7AT0-04 7 3
ST7AT0-05 3 2 -(l :Sac)
ST7AT0-06 5 4 -(LPoIlentube)
ST7AT0-07 14 8
ST7AT0-08 19 8
ST7AT0-09 12 2
ST7AT0-10 10 3
ST7AT0-11 9 4
ST7AT0-12 7 2
ST7AT0-13 8 1 -(LPoIlentube)
ST7AT0-14 15 5 -(LPoIlentube)
ST7AT0-15 13 5 -(LPoIlentube)
ST7AT0-16 11 2 -(LPoIlentube)
Table 19 Histochemical Localization of GUS Activity in Pollen Grains of Putative Transgenic Cotton Plants, CA-3076 Apices were excised from 1 -day old seedlings and utilized as starting material for co-cultivation with Agrobacterium
Individual Plants Total # Total # Flowers Pollen in Sac
Flowers Tested w/+Pollen (Pollentube)
CA-3076T0-01 14 4
CA-3076T0-02 9 3
CA-3076T0-03 17 10 2"(1 Pollentube)
CA-3076T0-04 16 5 1(1 Pollentube)
CA-3076T0-09 2 -
CA-3076T0-10A" 1 1
CA-3076T0-11" 21 6
CA-3076T0-12" 2 1
CA-3076T0-0I T1-00C 16 7 -(1 Pollentube)
CA-3076T0-01 Tl-01c 9 3
CA-3076TO-01 Tl-02c 13 8
CA-3076T0-01 Tl-030 16 8
CA-3076TO-01 Tl-04c 30 18 -(LPoIlentube)
CA-3076T0-01 Tl-05c 18 11
CA-3076T0-01 Tl-06c 19 14
CA-3076T0-01 Tl-07c 21 12
CA-3076T0-01 Tl-08c 20 1 1
CA-3076T0-01 Tl-09c 10 2
CA-3076T0-01 Tl-10 3 3
CA-3076TO-01 Tl-11 3 3
CA-3076T0-01.T1-12 3 3
CA-3076T0-01 Tl-13 1 -
CA-3076T0-01 Tl-31 1 1 CA-3076T0-03 Tl-30 1 1
CA-3076T0-04 T1-11 4 4
CA-3076T0-04 T1-13 1 1
* Sac stained positive while pollen grains were negative " A "mottled green" phenotype cNo kanamycin selection of T, seeds
Example 12 - Southern Blot Analysis of genomic DNA isolated from nuclei of cotton leaves
Cotton genomic DNA was extracted from isolated nuclei of fresh young leaves (Paterson et al. 1993) Purity and yield of DNA preparations were estimated spectrophotometrically (Sambrook et al 1989) DNA (15 μg) was digested with 10 units/μl of Bam HI or Hind III and then electrophoresed in 0 8% agarose for 30 min at 20 volts and then 2 h at 60 volts The genomic DNA was transferred to nylon membranes (Zeta-Probe by BioRad) for hybridization with a random primer-labelled GUS probe (provided by Dr R Pirtle's laboratory) The location of the bound GUS probe was identified by an alkaline phosphatase-catalyzed chemiluminescent reaction
(Gene Images CDP-Star detection module, Amersham LIFE SCIENCE)
The GUS gene was shown to be incorporated into the cotton genome of the T0 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-26T0-03 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
Example 13 - In vitro Tl Seedling Assay
Tj seeds from individual flowers of the T0 plants were germinated in the presence of 50 KAN (50 μg/ml) to screen for the KAN-selected seeds of the next generation The T, seeds expressing KAN-resistant capability appeared normal in comparison to the non-transformed T, seeds which showed poor germination with yellow root tips and swollen roots without extensive root hair development The nontransformed control
(HS-26NT) also showed these stressed characteristics (Data not shown) 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 T0 plant showed different seed germination rates and seedling phenotpyes which is consistent with the concept that these T0 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 T0 and T, progeny to identify likely transformants
Example 14 - Transgenic Screens
Selected individual plants have been tabulated to demonstrate the evaulation procedure utilized to describe the transgenic T0 and T, plants (Table 20) The individual plants were scored (1 to 6) as described in Table 20 In brief, all the T0 were shootlet KAN-selected and all the T, were seedling KAN-selected for the L phenotype All putative transgenics had positive GUS activity for pollen In most cases, these plants also scored high for fluorescent GUS activity for leaf protein and histochemical GUS activity for leaf mid-vein/petiole explants To date, HS-26T0-03 showed a positive Southern test
Table 20 Summary of transgenic screens shootlet KAN-selection procedure for T0s (1), GUS positive in the leaf fluorescence assay for T0s and T,s (2), GUS positive in the leaf mid-vein/petiole histochemical assay for T0s and TjS (3), GUS positive in the pollen histochemical assay for T0s 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)
Individual Plants (1) (2) (3) (4) (5) (6)
HS-26T0-03 + + nd + na +
HS-26T0-03 Tl-03 na + - + L nd
HS-26T0-04 + + nd + na nd
HS-26T0-04 Tl-04 na - + + L nd
CA-3076T0-01 + + nd + na nd
HS-26T0-01 + nd nd + na nd
HS-26T0-01 Tl-07 na + + + L nd
HS-26T0-07B + nd nd + na nd
HS-26T0-07B Tl-08 na - + + L nd
HS-26T0-07B Tl-16 na + + + L nd nd - not determined na - not applicable to the criteria in question

Claims

WE CLAIM:
1. A method for regenerating cotton plants comprising the steps of:
(a) isolating an explant of a cotton plant wherein said explant comprises a pre-existing meristem;
(b) inducing the proliferation of a shootlet from the explant by culturing said explant with a nutrient media supplemented with a concentration of cytokinin which promotes shootlet proliferation; and
(c) rooting the shootlet.
2. The method of claim 1 wherein the pre-existing meristem is isolated from a seedling which is less than about 28 days old.
3. The method of claim 1 wherein the pre-existing meristem is isolated from a nodal or apical meristem of a seedling about 14 to about 28 days old.
4. The method of claim 1 wherein the pre-existing meristem is isolated from an apical meristem of a seedling about 1 or 2 days old.
5. The method of claim 3 wherein the nodal meristematic tissue comprises leaf nodes and cotyledonary nodes.
6. The method of claim 1 wherein the cytokinin is benzyladenine.
7. The method of claim 6 wherein the concentration of benzyladenine is no greater than about 1.0 μM.
8. The method of claim 6 wherein the concentration of benzyladenine ranges from about 0.15 μM to no greater than about 1.0 μM.
9. The method of claim 8 wherein the concentration of benzyladenine is about 0.3 μM.
10. The method of claim 1 further comprising the step of maturing the shootlet prior to rooting
11. The method of claim 1 further comprising the steps of
(d) isolating an explant from a first shootlet wherein said explant comprises a pre-existing meristem,
(e) inducing the proliferation of a second shootlet from the explant of the first shootlet by culturing said explant with a nutrient media supplemented with a concentration of cytokinin which promotes shootlet proliferation, and
(f) rooting the second shootlet(s)
12. A cotton plant produced by the method of claim 1
13. A cotton seed produced by the plant of claim 12
14. A cotton plant germinated from the seed of claim 13
15. A method for genetically engineering cotton plants comprising the steps of
(a) isolating an explant of a cotton plant wherein said explant comprises a pre-existing meristem, (b) transforming the explant,
(c) inducing the proliferation of a shootlet from the explant by culturing said explant with a nutrient media supplemented with a concentration of cytokinin which promotes shootlet proliferation and selecting for a transformant in the presence of a selecting agent; and (d) rooting the shootlet
16. The method of claim 15 wherein the explant is transformed by the introduction of foreign DNA via Agrobacterium tumefacians
17. The method of claim 16 wherein the explant is transformed by co- cultivating the explant with a culture of Agrobacterium tumefacians, said Agrobacterium tumefacians being transformation competent and harboring a plasmid comprising both a foreign gene and a selection agent resistance gene, both genes including appropriate regulatory sequences so as to be expressed in the cells of the cotton plant.
18. The method of claim 17 wherein the selection agent is an antibiotic and the resistance gene codes for antibiotic resistance.
19. The method of claim 18 wherein the antibiotic is kanamycin and the resistance gene is the NPTII gene.
20. The method of claim 19 wherein the pre-existing meristem is isolated from a seedling which is less than about 28 days old.
21. The method of claim 15 wherein the pre-existing meristem is isolated from a nodal or apical meristem of a seedling about 14 to about 28 days old.
22. The method of claim 15 wherein the pre-existing meristem is isolated from an apical meristem of a seedling about 1 or 2 days old.
23. The method of claim 21 wherein the nodal meristematic tissue comprises leaf nodes and cotyledonary nodes.
24. The method of claim 15 wherein the cytokinin is benzyladenine.
25. The method of claim 24 wherein the concentration of benzyladenine is no greater than about 1.0 μM.
26. The method of claim 25 wherein the concentration of benzyladenine ranges from about 0 15 μM to no greater than about 1 0 μM
27. The method of claim 26 wherein the concentration of benzyladenine is about 0.3 μM
28. The method of claim 15 further comprising the step of maturing the shootlet prior to rooting.
29. The method of claim 15 further comprising the steps of.
(d) isolating an explant from a first shootlet wherein said explant comprises a pre-existing meristem,
(e) inducing the proliferation of a second shootlet from the explant of the first shootlet by culturing said explant with a nutrient media supplemented with a concentration of cytokinin which promotes shootlet proliferation; and
(f) rooting the second shootlet.
30. A cotton plant produced by the method of claim 15
31 A cotton seed produced by the cotton plant of claim 30
32. A cotton plant germinated from the seed of claim 31.
33. A cotton plant produced by the method of claim 17.
34. The cotton plant of claim 33 comprising a genome in to which the foreign gene and the selection agent resistance gene have been inserted, wherein said plant produces a foreign cellular product encoded by the foreign gene.
35. A cotton seed produced by the cotton plant of claim 33.
36. A cotton plant germinated from the seed of claim 35.
37. A cotton plant which is the progeny of a cotton plant produced by the method of claim 15.
38. A cotton seed produced by the cotton plant of claim 38.
PCT/US1997/008242 1996-05-16 1997-05-15 A rapid in vitro regeneration scheme of cotton plants compatible with agrobacterium-mediated transformation WO1997043430A1 (en)

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