Plant Molecular Biology 10: 105-116 (1987) ( Martinus Nijhoff Publishers, Dordrecht - Printed in the Netherlands

105

Transformation of cotton (Gossypium hirsutum L.) by Agrobacterium tumefaciens and regeneration of transgenic plants
Ebrahim Firoozabady, David L. DeBoer, Donald J. Merlo, Edward L. Halk, Lorraine N. Amerson, Kay E. Rashka and Elizabeth E. Murray Agrigenetics Advanced Science Company, 5649 E. Buckeye Road, Madison, 14"153716, USA
Received 3 August 1987; accepted in revised form 28 September 1987

Key words: Agrobacterium tumefaciens, cotton, Gossypium hirsutum L., regeneration, transformation
Abstract

Cotton (Gossypium hirsutum L.) cotyledon tissues have been efficiently transformed and plants have been regenerated. Cotyledon pieces from 12-day-old aseptically germinated seedlings were inoculated with Agrobacterium tumefaciens strains containing avirulent Ti (tumor-inducing) plasmids with a chimeric gene encoding kanamycin resistance. After three days cocultivation, the cotyledon pieces were placed on a callus initiation medium containing kanamycin for selection. High frequencies of transformed kanamycin-resistant calli were produced, more than 807oof which were induced to form somatic embryos. Somatic embryos were germinated, and plants were regenerated and transferred to soil. Transformation was confirmed by opine production, kanamycin resistance, immunoassay, and DNA blot hybridization. This process for producing transgenic cotton plants facilitates transfer of genes of economic importance to cotton.

Introduction

Agrobacterium tumefaciens provides a natural genetransfer mechanism that can be utilized to transfer a specific DNA sequence into the genomes of cells of many dicotyledonous plants [1]. With species such as tobacco and petunia, transformed plants can be produced in a simple process that involves a selectable marker [2]. More recently, transformed plants have also been regenerated in other crops such as tomato [3], potato [4], and alfalfa [5] using similar Agrobacterium-mediated transformation techniques. Cotton is attractive for genetic engineering because of its worldwide importance as a crop plant. Cotton transformation has been reported by Zhou et al. [6] through injection of DNA directly into embryos in immature cotton bolls. Since the injected

DNA was obtained from Sea Island cotton (Gossypium barbadense L.) plants with different morphological characteristics from the recipient Upland cotton (G. hirsutum L.) plants, the putative transformed plants were identified by phenotypes such as boll size and fiber length. The absence of any discrete foreign gene precluded analysis by DNA hybridization to genomic Southern blots, so these results must await molecular confirmation. Mutation or physiological changes might also account for the variations observed. Recently, Umbeck et al. [7] reported preliminary results on cotton (G. hirsutum L.) transformation via Agrobacterium using hypocotyls as explants. In that report, the technologies for transformation, selection, and regeneration were not thoroughly characterized. In our hands, similar hypocotyl inoculations [8] resulted in high levels of false-positive

106 (nontransformed) regenerated plants (unpublished results). A major obstacle to the application of Agrobacterium-mediated transformation to cotton has been the absence of a high-efficiency plant regeneration method [9-12]. A second barrier to cotton transformation has been the optimization of methods for using a selectable marker such as kanamycin resistance. In a system such as tobacco, transformed tissue at any stage of development can be selected on high levels of kanamycin (up to 1000 mg/1 [131), while in cotton such levels are toxic. Conversely, several factors can lead to breakthrough of nontransformed cotton calli, or to chimeric calli consisting of predominantly nontransformed tissues. These factors include low kanamycin levels, selection in later stages of callus proliferation, and use of explants such as hypocotyls that are only in partial contact with the selective media (our unpublished results). We present here a simple, reproducible, and efficient transformation/regeneration system which overcomes the above difficulties. We have used A. tumefaciens strains containing avirulent Ti (tumorinducing) plasmids to transform cotton cotyledon tissues, and we report significant modifications to the general transformation and selection regime which allows efficient regeneration of transgenic cotton plants with normal morphologies. E. coil selection were: ampiciUin, 50; kanamycin, 25; tetracycline, 10; gentamycin, 10. The latter three antibiotics were used for Agrobacterium selection at 25, 10, and 100 mg/l, respectively and streptomycin was used at 250 mg/l. Plasmids pRK292 [21] and pTJS75 were from the laboratory of D. Helinski (University of California, San Diego).

Binary vector system The details of the construction of the binary vector pH575 (Fig. 1)are available elsewhere [221. The cloning and recombinant DNA manipulations [19] of pH575 and its derivatives were all performed using E. coli MCI061 as the host strain. Because this strain is resistant to streptomycin, it is unsuitable for use as a donor strain for conjugations into LBA4404. Consequently, the binary vector was transformed into E. coli strain K802 for use in triparental matings [23]. Streptomycin- and kanamycin-resistant colonies were purified, and t ransconj ugants were identi fled by a miniprep procedure adopted from the alkaline lysis procedure [19]. The DNA between the border repeats is mobilized in trans for integration into plant cells by vir gene functions borne on the co-resident Ti plasmid pAL4404.

Cis vector system

Materials and methods

Bacterial strains and plasmids Escherichia coli strains MC1061 [14] or K802 [15] were used for recombinant DNA procedures. MM294(pRK2013) was employed as a plasmidmobilizing strain [16] in triparental matings and 2174 (pPHIJ1) as an excluding plasmid donor in marker exchange experiments [17]. A. tumefaciens strains employed were 15955 (American Type Culture Collection) and LBA4404 [18]. E. coli strains were grown in L medium [19] at 37 C and A. tumefaciens strains in YEP (17oyeast extract, 1070peptone, 0.570 NaC1) or Schilperoort's minimal (SM) medium [20] with 0.2070 sucrose, at 28 C. Antibiotics (mg/1) used for

A cis vector system based on A. tumefaciens 15955 was constructed by using the T-region from pH575 to replace the wild-type Ti sequences o f pTi15955. The 9530-bp HindlII-partial fragment of pH575 that contains the T-region was re-cloned into the unique HindllI site ofpRK292. The resulting plasmid, pH592, was then mated from K802 into a streptomycin-resistant spontaneous mutant of wildtype 15955, and transconjugants (I5955(pH592)) were selected for resistance to streptomycin, kanamycin, and tetracycline on SM medium. Single crossover events, by which a cointegrate plasmid containing pH592 and pTi15955 sequences was generated, were selected on SM by resistance to kanamycin, tetracycline and gentamycin following a

107 crossover event that eliminated both the pRK292 sequences and TL-DNA bp 1016 to 11 207 sequences [25], was verified by DNA blot analysis (not shown).

I~

Plant material
u .... p. 75 16 6kbp

Seeds of G. hirsutum cv. Coker 201 were surfacesterilized as described [26] except that seeds were exposed to bleach for only 8 - 1 0 min, germinated on MS0 medium (hormone-free MS medium [27] solidified with 0.2% Gel-rite (Kelko)) and incubated as
Fig. 1. Map of pH575, pH575 is a "micro Ti" binary vector based

described [261.

on the broad-host-range plasmid pTJS75 (T. Schmidhauser) and the T L region of the octopine-type plasmid pTi 15955. Resistance to kanamycin is conferred to transformed plant cells by the neomycinphosphotransferase II (NFI" 11) gene from bacterial transposon Tn5, under control of the cauliflower mosaic virus (CaMV) 19S promoter [39]. The 5' region of the NPT II coding region was modified by oligonucleotide site-directed mutagenesis to introduce a B a m H l recognition site between the "false" ATG at Tn5 base pair (bp) 1535 and the NPT II start codon at Tn5 bp 1 551 [40]. A unique Bglll recognition site, situated between the octopine synthase (OCS) and 19S/NPT II genes, is used for the introduction of foreign genes. This placement is desirable in that the gene of choice is integrated between genes used for selection (NPT 1I) and screening (OCS) of transformed cells. The various regions (I-IX) of the pH575 T-DNA are comprised of the following nucleotide sequences, oriented clockwise. T-DNA bases are numbered according to Barker et al. [25], CaMV nucleotides are according to Franck et al. [41], Tn903 nucleotides are according to Oka et al. [42], Tn5 nucleotides are according to Beck et al. [40] and Mazodier et al. [43], and pBR322 nucleotides are according to Sutcliffe [44]. I: T-DNA bp 603 to 1617, II: Tn903 bp 834 to 2 264, I|I: CaMV bp 7 667 to 7 018, IV: Tn5 bp 2 936 to 2399, V: T-DNA bp 22440 to 21727, VI: Tn5 bp 2517 to 1 540 (bp 1 544 "G" was mutated to "C"), VII: CaMV bp 5765 to 5376, VIII: T-DNA bp 11 208 to 14710, IX: pBR322 bp 376 to 652. Other abbreviations: A and B (circled), the 25-bp border repeats, which specify T-DNA ends; "AATAAA", RNA polyadenylation signals; 19S, CaMV 19S promoter; OCS, octopine synthase coding region; N I T I, bacterial kanamycin resistance; Tcr, tetracyclin resistance; B, BarnHl; E, EcoRl; H, HindllI. Additional details of the construction are in Materials and methods and in ref. [22].

Transformation and selection

The bacteria used for inoculation of cotyledon segments were scraped off the agar medium and suspended to a concentration of = 108 cells/ml in a cotton callus initiation liquid medium (G2) containing MS salts (Gibco), 100 mg/l myo-inositol, 0.4 mg/1 thiamine HCI, 5 mg/l 6-(%%dimethylallylamino)-purine (2iP), 0.1 mg/l anaphthaleneacetic acid (NAA) (all from Sigma), 3 %o (w/v) glucose, pH 5.9. Cotyledon pieces ( = 0.5 cm 2 surface area) from sterile 12-day-old seedlings were dipped in the A. tumefaciens suspension in petri dishes and gently shaken for a few seconds to ensure contact of all cotyledon edges with the bacterial cultures. The cotyledon pieces were then blotted dry and placed on Whatman No. 1 filter paper on callus initiation medium G 2 solidified with 0.2% Gel-rite. Inclusion of the filter paper was not necessary for transformation but greatly reduced bacterial overgrowth on plant tissues. Tissues were incubated at 25 C with a 16-h photoperiod (90 #E m -2 s-l). After three days cocultivation, cotyledon pieces were transferred to petri plates (without the filter paper) containing the same medium supplemented with 500 mg/1 carbenicillin (to control bacterial growth) and 15 - 3 5 mg/l kanamycin sulfate (US Biochemicals). Tissues were incubated at 30 C with a 16-h photoperiod (90 ~E m -2 s-l). After 3 - 4 weeks, calli were excised from the original explants, transferred to fresh kanamycin-containing medium and incubated under lower light intensity (10 ~E
m-2 s-l).

second triparental mating in which plasmid pPH1J1 was introduced into 15955(pH592). One of these colonies was then subjected to five rounds of cycloserine enrichment as described [24], and 2150 single colonies were screened for sensitivity to tetracycline. One colony, 1592, was found, and the expected structure of the T-DNA, resulting from a second

108

Regeneration of transgenic plants

After two to three weeks, calli were placed and maintained on embryogenic medium (G 2 with no hormones) under kanamycin selection (25 mg/l). Mature somatic embryos ( 5 - 1 0 m m in length with cotyledons, hypocotyl, and radicle structures) were transferred to G R M g n medium, which is a lower ionic strength medium [28] modified by addition of 0.1 m g / l gibberellic acid (Sigma, filter-sterilized), 0.01 rag/1 N A A (Sigma) and 0.5o7o glucose instead of sucrose (with no antibiotics), and incubated at 30C, 16-h photoperiod (90 #E m -2 s -1) to germinate and form plantlets. Plantlets were transferred to GA-7 Magenta cubes (Magenta Corp.) containing G R M g n medium and incubated at 25 C with a 16-h photoperiod (90 # E m -2 s-l). Plants with 3 to 5 leaves were transferred to a commercial peat moss/perlite mixture (Growing Mix No. 2; Fafard Ltd, Canada), maintained in a growth chamber at 25 C night, 30 C day, 16-h photoperiod (150 #E m -2 s -i) and watered and fertilized as needed. Plants were gradually hardened off, repotted in soil and moved to a greenhouse.

dogenous opines produced by feeding tissues on arginine [29] were not present on the TLC plates at the time of scoring (Fig. 2). This is due to the use of a TLC buffer in which arginine migrates much more slowly on the plate than opines. It is also important that phenanthrenequinone staining be very light, and that the plate be read immediately after staining under UV light. The intensity of octopine production varied, with some plants showing much stronger octopine production than others (Fig. 2). Similar results were obtained when the same plants were retested for octopine several weeks later. By this method, 3 ng octopine/mg tissue could be detected (Bookland and Paaren, in preparation).

N P T H assays: enzyme-linked immunosorbent assay (ELISA) and immunoblot

NPT II was detected and quantified in cotton ex-

TLC octopine assay

To detect octopine in callus or shoot material, 1 0 - 20 mg fresh weight of tissue was incubated overnight on MS0 medium containing 5 mM L-arginine to enhance octopine formation. Tissues were homogenized in microfuge tubes, centrifuged (15 000 g, 2 min), and 6 #1 of the supernatants were spotted (2/zl at a time) on W h a t m a n K-5 silica gel plates (20 20 cm) 1 cm apart. The plate was placed upright in a thin-layer chromatography (TLC) developing tank containing 20 ml of TLC buffer (methanol : 2-butanol : 0.1 M sodium acetate (pH 4.6), 15:1:4). After one hour, plates were removed, air-dried, sprayed faintly (under a fume hood) with a 1:I flesh mixture of 1070 N a O H in 607o ethanol : 0.04O7o phenanthrenequinone (Aldrich Chemical Co.) in 100O7o ethanol, and immediately visualized under UV light (254 nm). The octopine assay used here is highly reliable. With this method, background artifacts or en-

Fig. 2. TLC octopine detection in transformed tissues of cv.

Coker 201. Callus from uninoculated tissues (negative control). Lanes 2- 7 Transtbrmed calli from cotyledon sections inoculated with 1592 (lane 2) and LBA4404(pH575) (lanes 3, 4); and leaves (lanes 5-7) from transgenic plants (20, 07, 40) regenerated from these calli, respectively. Lane 8 Tissues transformed callus with virulent strain of 15955. Lane 9 Octopine and arginine (A) standards. (O) Ori, origin; -, octopine not detected; +, detectable levels and + +, high levels of octopine were produced. Lane 1

109 tracts by an ELISA assay (Halk et al., in preparation). The ELISA was constructed with rabbit antiNPT II immunoglobulin G (IgG), a biotin conjugate of this antibody [30] and streptavidin alkaline phosphatase conjugate (Bethesda Research Laboratories). Rabbit anti-NPT II antiserum was raised against NPT II purified from E. coli containing plasmid pKS4 [31]. Purified NPT II was used as the standard for calculating NPT II content in tissue extracts. Plant extracts for ELISA were made by grinding four 5-mm leaf discs in 200/xl of phosphate-buffered saline (127 mM NaC1, 2.6 mM KC1, 8.5 mM NaH2PO 4, 1.1 mM KH2PO4) containing 0.05% Tween-20, and 1% polyvinyl pyrollidone 40 000 (Sigma). Extracts were centrifuged (15 000 g, 5 rain) and three three-fold dilutions of the sample supernatants were loaded into ELISA plates. Soluble protein concentrations were determined by the Bradford [32] dye-binding assay (Biorad). NPT II levels are expressed as ng NPT II per mg of soluble protein in the extracts. Cotton leaf extracts for immunoblot analysis were prepared in 4% SDS, 5-/0 2-mercaptoethanol, 207o glycerol in 0.068 M Tris-HC1 (pH 6.8), electrophoresed in 13% polyacrylamide gels [33], and blotted to nitrocellulose. Blots were processed in rabbit anti-NPT II IgG (0.1 /~g/ml) followed by goat anti-rabbit IgG alkaline phosphatase conjugate (0.2/~g/ml, Kirkegarrd and Perry) by standard procedures [34]. Radioimmune precipitation assay buffer (RIPA) [35] was used as the wash buffer and RIPA containing 3% BSA and 1% goat serum was used as the antibody dilution buffer. Blots were developed in nitroblue tetrazolium/5-bromo-4-chloro3-idolyl phosphate (both from Sigma) substrate solution [36]. glycol, 10 mM orthophenanthroline, 10 mM NaHSO3). Triton X-100 was added to a final concentration of 1%0and the mixture was centrifuged at 300g, 4C for 5 min. The nuclear pellet was resuspended in nuclei buffer and lysed by the addition of an equal volume of 30 mM EDTA, 1.5 M NaCI, and 1%0cetyltrimethylammoniumbromide in the presence of proteinase K (30/zg/ml, Bethesda Research Laboratories) for 30 min at 65 C. DNA was precipitated in 2.5 M NHaOAc, 50070 isopropanol, followed by phenol extraction and isopropanol precipitation. Five #g of DNA was digested with BamHI, electrophoresed through 0.8% agarose, and transferred [38] onto nylon membrane (GeneScreen Plus, New England Nuclear). Hybridization was carried out with 32p-labeled synthetic OCS transcripts and nick-translated NPT II fragments from pH575. Filters were prehybridized overnight (in heat-sealed bags) at room temperature in 100 mM NaH2PO 4 (pH 7.8), 20 mM Na4P2OT, 5 mM EDTA, 1 mM orthophenanthroline, 0.1% SDS, 500 #g/ml heparin sulfate, 10% sodium dextran sulfate, and 50 #g/ml each herring sperm DNA and yeast RNA (Sigma). A portion of the prehybridization solution was removed from the bag, thoroughly mixed with the probe, and then returned to the bag containg the filter. The filter was hybridized for 6-12 h at 65C and washed (20 mM NaH2PO 4, 5 mM Na4P207, 1 mM EDTA and 0.1% SDS) at 65 C for 1 h with five buffer changes. Autoradiography was performed on Kodak XAR-5 film with an intensifier screen at -70C.

Results

Plant cell transformation and selection After 7-10 days on 25 mg/l kanamycin, cotyledon pieces inoculated withLBA4404(pH575) and 1592 initiated transformed kanamycin-resistant microcalli (0.5 mm) at wound sites (Fig. 3A), while no callus from control untreated tissues or from tissues treated with LBA4404 alone grew on kanamycin. Two to three weeks later, the number of calli which developed on sections were counted. Normally, 2 to 10 kanamycin-resistant calli were obtained per coty-

DNA isolation and blot hybridization DNA was prepared by isolation of nuclei from cotton leaf tissues using a modification of the method of Murray and Kennard [37]. Young leaf tissues were ground in a Bellco glass-glass homogenizer at 4 C in nuclei buffer (20 mM 1,4-piperazinediethane sulfonic acid (pH 7), 3 mM MgC12, 0.5 M hexylene

110 l e d o n s e c t i o n (Fig. 3B). S e c t i o n s p l a t e d o n m e d i u m w i t h n o k a n a m y c i n p r o d u c e d c a l l u s o n all w o u n d sites. A t t h i s stage, 2 - 4 - m m kanamycin-resistant calli w e r e excised f r o m t h e o r i g i n a l e x p l a n t s a n d transferred to fresh medium. selected calli that continued resistant in the subsequent The percentages of to be kanamycin-

s u b c u l t u r e were d e t e r -

m i n e d ( T a b l e 1). S i m i l a r r e s u l t s were o b t a i n e d w h e n t h e e x p e r i m e n t s were r e p e a t e d , e x c e p t t h a t t h e n u m -

Fig. 3. Regeneration of transgenic cotton plants. (A) A kanamycin-resistant microcallus (shown with arrow) developed at wound sites 10 days after coculture (bar = 1 mm). A cotyledon section showing several distinguished calli on 25 mg/l kanamycin three weeks after coculture (bar = 1 mm). Friable calli developed 4 weeks after transfer to hormone-free medium, G O (bar = 1 mm). Embryogenic calli developed 6 - 8 weeks after transfer to medium G O (bar = 1 mm). Somatic embryos with different morphologies (bar = 1 mm). Regenerated transgenic plants 10-12 days after tran sfer to soil.

(B)

(C) (D, E) (F) (G)

Table 1. Transformation frequencies of cotton (G. hirsutum cv. Coker 201) cotyledon sections using A. turnefaciens. Four weeks after inoculation, calli were excised and placed on fresh kanamycin-containing media, and 8 weeks after inoculation, calli were scored for kanamycin resistance and octopine production. Bacteria Number of sections inoculated Kanamycin (mg/1) Number of microcalli per section developed on kanamycin 1.9 4.1 b 1.3 1.6 1.4
0.1

Transformation frequencies (070)a Kanamycin resistance (number tested) 87 (46) 98 (361) 100 (49) 97 (64) 97 (56)
0 (1)

Octopine-positive (number tested)

LBA4404(pH575)

24 88 38 40 40
9

15 25 35 25 35
15

75 (8) 95 (130) 98 (18) 94 (33) 95 (20)

0 (4)

1592

LBA4404c

10 10

25 35

0.0 0.0

0 0

(0) (0)

0 0

(4) (4)

a Transformation frequencies were determined as the percentages of selected microcalli that were kanamycin-resistant in subsequent selection, or the percentages of calli that were octopine-positive. Total numbers of calli tested for each are shown in parentheses. All kanamycin-resistant calli tested had detectable levels of NPT II protein. b In later experiments, the number of microcalli per section was higher (6.8) than in early experiments, most likely due to improvements in transformation protocol. c In the case of LBA4404, calli that were cultured on medium with no kanamycin were assayed for octopine.

111 ber of microcalli per section obtained was higher ( 5 - 1 0 calli/section), presumably due to improvements in the inoculation protocols. A total of 221 putative transformed calli from LBA4404(pH575) and 1592 inoculations (Table 1) were tested for octopine; 75 to 987o were octopine-positive. On lower levels of kanamycin (15 mg/1), some nontransformed calli proliferated very slowly but eventually turned brown and died. The percentage of kanamycin-resistant, octopine-positive calli was increased when higher levels of kanamycin (25 or 35 mg/1) were used for selection (Table 1). There was no difference between 25 and 35 mg/1 kanamycin selection regimes for transformation parameters measured (Table 1). All the kanamycin-resistant calli tested were positive for NPT II using ELISA assays; the amount of protein ranged from 2.3 to 54.1 ng per mg total soluble protein (mean = 17.3 _+ 8.4). These results suggest that'25 mg/1 kanamycin is sufficient to inhibit the growth of nontransformed cotton cells under the conditions applied. Also, cotyledons are desirable explants for transformation and selection, since they provide callus proliferation only on the wound sites, remai'n in contact with the selective media, and consequently, produce predominantly kanamycin-resistant calli due to expression of NPT II protein. In the absence of kanamycin selection, barely detectable octopine-positive calli were obtained at frequencies of 10-207o of total calli tested, suggesting that transformation frequencies among the cell populations of some calli were reasonably high. Selection was applied to these calli one to two months after coculture. For example, 5 - 8-mm calli were transferred to kanamycin-containing media on a step-wise selection basis (initially 50, later 100 and eventually 200 mg/1). Some of these grew well, but none were positive when retested for octopine. Eight out of 12 plants regenerated from these calli were analyzed. None were transgenic based on octopine assay, kanamycin resistance, or DNA analyses (data not shown). These results suggest that transformed cells, even under kanamycin selection, may grow more slowly than normal cells; that it is difficult to select for transformed tissues in chimeric calli (tissues either die or grow on kanamycin and eventually consist of nontransformed tissues); or that kanamycin does not pass through cotton cells in the callus mass, as it does in tobacco calli.

Plant regeneration
The transformed calli were placed and maintained on embryogenic medium under selection. On this medium, more than 80% of the calli became embryogenic, i.e., friable cream-colored granular calli that produced somatic embryos (Fig. 3 C - E ) . Somatic embryos varied considerably in size and morphology (Fig. 3F), and some matured faster than others. Approximately 307o of mature somatic embryos germinated on GRMgn medium and formed plantlets. To date, 158 transformed (octopinepositive and/or kanamycin-resistant) plantlets have been produced using different transforming constructs, and 54 of these regenerated into normal plants. Plants were hardened off in a growth chamber and transferred later to greenhouse conditions (Fig. 3G) for further analyses. Regenerated plants are fertile and set seed. The whole process from infection until transgenic plants are transferred to soil takes about 6 - 8 months for Coker 201.

Analyses of transformed plants

To determine transformation frequencies, embryos or plants regenerated from kanamycin-resistant calli were analyzed for octopine production and kanamycin resistance (Table 2). For kanamycin resistance, leaf sections from regenerated plants were placed on callus initiation medium containing 25 mg/1 kanamycin and scored for callus formation two weeks later. Ten of these calli were tested for NPT II protein using ELISA assays and all were positive. This indicates that callus formation by leaf sections on kanamycin is due to production of NPT II protein. The percentages of octopine-positive plants regenerated from octopine-positive calli varied from 76 to 927o and kanamycin-resistant plants from 85 to 100% (Table 2). Interestingly, 100% of plants regenerated from octopine-negative, kanamycin-

112
Table 2, Transformation frequencies of embryos or plantlets regenerated from kanamycin-resistant Coker 201 calli.

Bacteria

Octopine production by calli + + -

Number of embryos (plantlets) tested 12 17 50 110

Transformation frequencies (%)* Kanamycin resistance 100 100 85 30 Octopine-positive 92 100 76 10

LBA4404(pH575) LBA4404(pH575) 1592 1592

* Transformation frequencies were determined as the percentages of embryos or plantlets that were kanamycin-resistant on the basis of leaf callus assay (see the text) or octopine-positive. In one experiment all calli (produced on 25 mg/l kanamycin from leaf disks) tested showed detectable levels of N P T II protein.

resistant calli transformed withLBA4404(pH575) were octopine-positive and kanamycin-resistant. However, in the population of plants regenerated from octopine-negative calli transformed with 1592, only 30% were kanamycin-resistant and 10% were octopine-positive (Table 2). All the octopinepositive plants tested were kanamycin-resistant, whereas not all kanamycin-resistant plants assayed were octopine-positive (Table 2). These results have several possible interpretations: some calli are chimeric and some nontransformed cells have been carried through selection; one, or both, of the OCS and NPT II genes are not expressed in some plants; the expression of these genes is below detection levels in the assays used; the OCS gene is not present in the kanamycin-resistant plant; or a combination of these phenomena exists. DNA was isolated from 15 plants. All were kanamycin-resistant and all but two were octopinepositive. DNA blot hybridizations showed the expected B a m H I fragment containing the NPT II gene in all the plants tested (data for seven of these are presented in Fig. 4A). This was predictable since tissues were maintained under kanamycin selection. Plants 22 and 41 were octopine-negative; predictably they did not show the expected 2.9-kb band when DNA was probed with OCS transcripts (Fig. 4B). This suggests that the OCS gene is not present in these plants. Plants 20, 21, and 42 showed the expected 2.9-kb B a m H I fragment containing the OCS gene. Plants 20 and 21, regenerated from the same original microcalli, showed identical additional bands with both probes, suggesting rearranged co-

pies of NPT II and OCS genes. These results suggest that plants 20 and 21 are clones and derived from a single transformed cell. Plants 07 and 40, regenerated from the same original calli, showed a higher molecular weight band (3.4 kb) when DNA was probed with the OCS transcripts. One possible explanation is that the B a m H I recognition site next to T-DNA B border is methylated and the enzyme cuts at an external B a m H I site in the plant genome, -~200 bp from the border. If this is the case, then plants 07 and 40 also appear to be derived from a single cell. Similar results were obtained by Sukhapinda et al. [45] where transgenic tomato plants derived from the same callus obtained via cotyledon inoculation by A. rhizogenes, showed identical banding patterns. When eight of the transgenic plants were tested for NPT II protein using the ELISA assay (Table 3), the amount of protein ranged from 16 to 50 ng per mg total soluble protein. The same plants also contained protein of the expected molecular weight in the immunoblot analysis (Fig. 5). All the kanamycinresistant, octopine-positive calli were positive for NPT II protein, and they contained protein of the expected molecular weight. The results of two of these calli is presented in Fig. 5.

Discussion

Transgenic cotton plants are readily obtained with the transformation-regeneration system described here. Keys to the transformation system were rapid

113
Table 3. The a m o u n t of N P T II protein in leaves of transgenic Coker 201 plants. All transgenic plants were kanamycinresistant in leaf callus assay. For description of octopine levels, see Fig. 2.
Plant no.* Octopine production + + + + + + + + + N P T II (ng)/mg of soluble protein 0.0 17.9 27.6 13.4 49.9 36.8 16.2 36.6 30.4

C201 (Control) 01 (575) 07 (575) 20 (1592) 21 (1592) 22 (1592) 40 (575) 41 (1592) ~2 (1592)

* The numbers in parentheses refer to the vector systems used for inoculation of cotyledon section and production of the transgenic plants.

Fig. 4. Southern hybridization analyses of D N A from transgenic cotton plants. D N A blot hybridized to 32p-labeled BamHI nicktranslated NPT II fragment (A, 20-h exposure) and synthetic OCS transcript (B, 20-h exposure). NC is nontransformed control plant. All the plants predictably showed the expected BamHI NPT I1 fragment (A). Plants 20 and 21 derived from the same callus clone showed identical banding patterns with both probes. The same is true for plants 07 and 40 which were also derived from the same callus clone. However, these two showed a higher molecular weight band (3.4 kb) probably due to methylation in BamHl site close to border B. Plant 01 (positive control) had additional 4.3-kb D N A sequences from Bacillus thuringiensis and showed the expected 7.2-kb BamHI fragment (B). Several plants including NC showed an endogenous hybridizing faint band ( -~ 4.5 kb) with the OCS probe (B).

Fig. 5. Immunoblot analyses of NPT II protein. NC is the extract from leaves of a regenerated plant from uninoculated tissue (control). C1, and C2 are extracts from calli transformed with LBA4404(pH575) and 1592, respectively. 5 - 4 0 ng purified NPT II was used as standard. Extracts from leaves of all transgenic plants and calli showed the expected protein band. Extract from plant 40 showed a very faint band not visible in the figure.

callus initiation at the cotyledon wound sites and the use of the selectable kanamycin resistance marker. Rapid callus initiation (on media with high 2iP levels) was important for the recovery of high numbers

of transformed microcalli at the periphery of the inoculated tissues. This could be due to better recovery of transformed cells on these media, high competency of dividing cells for transformation with A. tumefaciens [46, 47], or both. In early experiments, a range of callus initiation media were tested to op-

114 timize the method for cotton transformation. On slow callus initiation media, however, no callus was recovered even on 15 mg/l kanamycin (data not shown). Cotyledon sections are preferred for transformation and selection. Calli are produced only at the wound sites that are in contact with the selective media. In the experiments using hypocotyls as explants for inoculation [8], large masses of nontransformed calli rapidly proliferated. These tissues were highly chimeric due to a lack of complete contact of explants with kanamycin-containing media. Twentyfour plants regenerated from these calli were not transformed when analyzed by octopine assay, kanamycin resistance and DNA hybridization (data not shown). It is very important to apply the selection regime to cotyledon tissues immediately after cocultivation with the bacteria. The level of kanamycin (25 mg/1) used was sufficient to efficiently obtain transformed tissues containing at least one intact copy of the NPT II gene in the plant genome (Fig. 4). The frequencies of kanamycin-resistant microcalli which developed at the wound sites (Fig. 2A) were used as the quantitation criterion to optimize the method for cotton transformation. It was also important to use a low titer of bacteria ( = 108 cells/ml) for inoculation. High titers of bacteria (101 cells/ml) resulted in overgrowth on plant tissues. Excision of 2 - 4-mm calli from explants was essential to promote growth of these calli and to avoid Agrobacterium contamination which otherwise usually developed on top of cotyledon sections 3 - 4 weeks after inoculation. These calli, upon further culture, produced somatic embryos and transgenic plants within 6 months of inoculation. This is relatively rapid compared to other reports with regeneration times of at least one year for nontransformed normal plants [9-12]. The transformation/regeneration system presented here differs in many ways from the report by Umbeck et al. [7] and brings major improvements to transformation of cotton. About 85 to 907o of the cotyledon sections used as explants under welldefined conditions produced kanamycin-resistant microcalli, and 95 - 100070of the microcalli remained kanamycin-resistant upon subculture (Table 1). These calli were screened for octopine production using TLC, a highly reliable, simple, and less laborious assay than the more widely used published method [48]. The calli consisted mainly of transformed cells since they produced a high percentage of transgenic plants (Table 2). Glucose was used as the carbohydrate source all through tissue culture, since sucrose encourages production of phenolics and delays development (data not shown). The system is simple, efficient, reproducible, and rapid. The method could be used with either cis or binary disarmed vector systems containing kanamycin resistance as a selectable marker. We did not see significant differences in transformation frequencies between cis and binary vector systems. Also, it may not be essential to use a strong promoter to express the selectable marker gene, since CaMV 19S promoter is sufficiently active in cotton. Clearly, the efficiency of this transformation-regeneration system will permit the introduction of desirable genes such as insect resistance from B. thuringiensis, herbicide resistance and virus resistance to cotton.

Note added in proof

Northern blot analyses of total RNA isolated from transgenic cotton plants showed correlations between NPT II RNA and protein expression, and between OCS RNA expression and octopine production. Plants 22 and 41, which were OCS negative on Southern blots and in octopine assays lacked OCS RNA on Northern blots (data not shown).

Acknowledgements
We thank M. Maroney and G. Staffeld for their technical assistance, M. Murray for his advice on DNA isolation, H. Paaren for NPT II purification, J. Adang for the artwork, J. Ingle, J. Rowe, L. Hoffman, M. Adang, and C. Stock for their comments on the manuscript, and L. Bausch for typing the manuscript. This is Agrigenetics Advanced Science Company manuscript No. 76.

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Note added in proof

RI p r o g e n y p o p u l a t i o n f r o m s e l f - p o l l i n a t i o n o f p l a n t no. 21 s h o w e d a 4:1 p o s i t i v e : n e g a t i v e s e g r e g a t i o n for k a n a m y c i n resistance, o c t o p i n e , a n d N P T II p r o d u c t i o n , w h i c h is close to a n e x p e c t e d M e n d e l i a n p a t t e r n o f i n h e r i t a n c e for single d o m i n a n t g e n e s for O C S a n d N P T II in this plant. T h e r e was a g o o d c o r r e l a t i o n b e t w e e n o c t o p i n e , N P T II p r o d u c t i o n , a n d k a n a m y c i n resistance in t h e p r o g e n y p o p u l a t i o n ( d a t a n o t shown).