High frequency generation of fertile transgenic rice plants after PEG-mediated protoplast...

Plant Molecular Biology Report pages 276-291 Volume 8(4)

Protocol

High Frequency Generation of Fertile Transgenic Rice Plants after PEG-Mediated

Protoplast Transformation

Zhijian Li, Mark D. Burow, and Norimoto Murai

Department of Plant Pathology and Crop Physiology, College of Agriculture, Louisiana State University, Baton Rouge, LA 70803-

1702 USA

Keywords: Oryza sativa L., hygromycin, transformation, regeneration, polyethylene glycol

Abstract: An efficient method for the transformation and regeneration of fertile transgenic rice (Oryza sativa L.) plants is presented. In this protocol, seed calli from the varieties Nipponbare and Taipei309 were used to produce rice suspension cultures in General Medium. Protoplasts were isolated from suspension cells (8 x 10 + protoplasts per g fresh weight), then were incubated with sterile DNA in the presence of MaMg solution, followed by addition of PEG to a final concentration of 25%. A hygromycin phosphotransferase (hph) gene under the plant tran- scriptional regulatory signals was used as a selectable marker gene. Hygromy- cin-resistant colonies were selected in the presence of 95 bd~r hygromycin B with apparent frequencies of 2xl0aand 5x10 ~ for Nipponbare and Taipei 309, respectively. Plantlets were regenerated from resistant colonies in Murashige and Skoog plant regeneration medium. Among 628 transgenic plants grown to maturity in the greenhouse, two-thirds bore viable seeds.

T r HE molecular biology of rice was, until recently, limited in part by the difficulty of obtaining sufficient numbers of fertile transgenic plants for gene expression studies. Rice transformation has

Abbreviations and gene names: 2,4-D, 2,4-dichlorophenoxyacetic acid; BAP, 6- benzylaminopurine; C.aMV 35S, cauliflower mosaic virus 35S promoter; IAA, indole-3-acetic acid; MES, 2-[N-morpholino]ethanesulfonic acid; PEG, polyethylene glycol 8000; hph, gene coding for hygromycin B phosphotransferase; nos, gene coding for nopaline synthetase; tml, gene for tumor morphology large.

276

Fertile Transgenic Rice from Pro toplasts 277

been achieved through infection with Agrobacterium tumefaciens (Raineri et al., 1990), DNA application to pollen tubes (Luo and Wu, 1989), electroporation (Toriyama et al., 1988; Shimamoto et al., 1989), and PEG treatment of protoplasts (Zhang and Wu, 1988; Hayashimoto et al., 1990). Of these methods, the PEG-mediated procedure appears to be the most efficient and reliable, possessing several other advantages. This method is simple and inexpensive. Rice protoplasts are capable of regenerating into whole plants when provided appropriate in-vitro conditions. Large populations of protoplasts canbe obtained readily for the transformation, selection and subsequent regeneration of transgenic plants. Finally, the delivery of foreign DNA into plant protoplasts by PEG is through membrane interactions (MacDonald, 1985), resulting in less severe physical damage and thus higher protoplast viability.

We are interested in studying gene expression in a homologous rice transforma tion system. To establish an efficient gene transfer procedure, we selected the General medium of Yang et al. (1980; cf. also Chen, 1986), a mixed nurse culture of Kyozuka et al. (1987) and a PEG-mediated protoplast transformation of Negrutiu et al. (1987). The improved procedu re resulted in protoplast plating efficiencies of up to 17%, a plan t regeneration frequency of 67%, an apparent transformation efficiency of 5 x 10 ~ and generation of large numbers of fertile transgenic rice plants (Li and Mural, 1990; Hayashimoto et al., 1990).

In this report, we describe in detail a protocol for the transformation and regeneration of rice protoplasts into fertile transgenic plants. This method should facilitate genetic study and applications in crop improve- ment.

Materials and Methods

Special chemicals, solutions and equipment required Cellulase RS and Macerozyme R-101 were purchased from Yakult Honsha Co. (Tokyo, Japan), and SeaPlaque agarose was acquired from FMC Co. (Rockland, ME, USA). Chemicals for media preparation were obtained from J. T. Baker Chemicals, Biorad Laboratories, Calbiochem, Fisher Scientific, Mallinckrodt, and Sigma Chemical Co.

All the solutions and media for tissue culture were autoclaved for 20 rain at 121 ~ C. Enzymes were dissolved in deionized water7 stirred for at least one hour, centrifuged for 10 min at 1500 xg to remove undissolved sediments, and finally filter-sterilized through a 0.22-/~m diameter Mil- lipore filter.

278 Li et al.

MS2 medium (per liter) 4.4 g MS salts (Sigma #M 5524), 30 g sucrose ~, 2.0 mg 2,4-D, and 10 g

Difco Bacto-agar a. Adjust pH to 5.8 with 1.0 M NaOH, and make up to 1 L final volume.

General liquid medium (per liter) (Yang et al., 1980) 3000 mg KNO~, 400 mg NH4t-I2PO4, 166 mg CaCL2H~O, 185 mg

MgSO~.7H20, 42.1 mg NaFeEDTA, 4.0 mg MnS(~4-4H20 , 1.5 mg ZnSOJH~O, 1.6 mg H3BO 3, 0.80 mg KI, 0.025 mg CuSO~5H20, 0.25 mg NaMoO -2H20, 0.025 mg CoCl .6H20, 2.0 mg glycine, 1.0mg thiamine-H~l, 0.5 mg pyridoxine-~Cl, 0.5 mg nicotinic acid, 1.0 mg 2,4-D, and 30 g sucrose. Adjust pH to 5.8 with 1.0 M NaOH.

General protoplast medium (per liter) Same ingredients as liquid medium except 136.9 g sucrose was used.

Agarose protoplast medium (approx 100 mL) 2.5 g Seaplaque agarose plus 100 mL General protoplast medium.

Soft agarose medium (per liter) Same ingredients as General liquid medium except 2.5 g agarose

(Sigma type I) was added.

Enzyme solution (per 100 mL) 4.0 g cellulase RS, 1.0 g macerozyme R-10, and 7.29 g mannitol. Adjust

pH to 5.6 with 1.0 M NaOH.

KMC solution (per 900 mL) (Harms and Potrykus, 1987) 26.1 g KCI, 49.9 g MgC12"6H20, and 37.3 g CaC122H20. Adjust pH to

6.0 with 1.0 M NaOH.

MS plant regeneration medium (per liter) (Cao et al., 1990) 4.4 g MS salts (Sigma #M5524), 30 g sucrose, 100 mg myo-inositol, 500

mg casein hydrolysate, 0.50 mg IAA, 0.80 mg BAP, and 8.0 g agarose (Sigma type I). Adjust pH to 5.8 with 1.0 M NaOH.

MaMg solution (per 100 mL)(Negrutiu et al., 1987) 0.31 g MgC122H20, 0.10 g MES, and 7.29 g mannitol. Adjust pH to 5.6

with 1.0 M NaOH.

40% (w/v) PEG solution (per 100 mL) 40 g polyethylene glycol-8000 (Sigma # P5413 ), 0.31 g MgC122I--I20, 0.10

g MES, and 7.29 g mannitol. Titrate pH to 5.6 with 1.0 M NaOH.

Fertile Transgenic Rice from Protoplasts 279

TE buffer 1.21 g Trizma base and 0.37 g Na2EDTA.

concentrated HCI. Adjust pH to 8.0 with

Other solutions 2.5% (v/v) sodium hypochlorite (1 part commercial bleach + 1 part

water) 70% (v/v) EtOH sterile H20.

Equipment Shaker incubator, laminar flow hood, Beckman TJ-6 centrifuge, Spotlite

hemocytometer (Scientific Products, McGaw Park, IL, USA), light microscope.

Supplies Sterile 50-mL capped conical-bottom centrifuge tubes (Onieda Con-

tainer Co., Inc., New York, USA) 125-mL erlenmeyer flasks, funnels lO0-mm diameter sterile petri dishes (Scientific Products, McGaw

Park, IL,USA) 400- and 40-~m pore diameter nylon macroporous filters (Medical

Industries, Inc., Los Angeles, CA, USA) 22-U~n pore diameter nitrocellulose filters (Gelman Sciences, Ann

Arbor, MI, USA) sterile transfer pipettes (Beral Enterprises, Inc., Chatsworth, CA, USA)

Plant materials Mature seeds of the japonica rice cultivars Nipponbare and Taipei 309

were harvested from plants grown at the LSU Rice Research Station, Crowley, LA, USA, and stored at-20~ Seeds are available upon request.

Notes 1. Cellulase R-10 (Cat. No. 68485) and Macerozyme R-10 (Cat. No. 91827) are

available from Gallard-Schlesinger Industries, Inc., 584 Mimeola Avenue, Carle Place, NY 11514,USA.

2. Tap water was deionized through ion-exchange resins to a conductance of 3 megaohm-cm. Deionized water was purified further by the MilliQ Type I reagent grade water system, or glass-distilled in the Corning Mega-Pure system.

3. Purified sucrose (free of RNase and DNase) from Biorad or Amresco was used for protoplast media. For other media, refined cane sugar from local super- markets was found to be adequate.

280 Li et al.

4. Use of highly-purified Seaplaque agarose and Sigma type I agarose was essential for protoplast and plant regeneration medium, respectively. For other purposes, Difco Bacto-agar or agar (AX0410-1) from EM Science (Gibbstown, NJ,USA) was found to be adequate.

Plant expression vectors The transformation vectors used in this experiment were pTRA131 and pTRA132, which were constructed to confer hygromycin resistance to rice (Hayashimoto et al., 1990). The vector pTRA131 contained the coding region for hph from Escherichia coli under control of the nos promoter of Agrobacterium tumefaciens, and the termination and polyadenylation sites of tml from A. tumefaciens. The remainder of the vector consisted of the pUC12 plasmid. The vector pTRA132 was similar, except that hph was under control of the CaMV 35S promoter instead of the nos promoter.

Suspension culture initiation Callus was initiated from mature rice seeds. Dehulled mature seeds were surface-sterilized in 70% EtOH and bleach, and plated on MS2 medium. Friable secondary calli were propagated from primary scutellum-derived calli after one subculturing, and were transferred into liquid General medium for the initiation of suspension culture. Careful selection of small, yellow secondary calli is critical for establishing rapidly growing suspension cultures. Fresh calli should be induced regularly for producing young suspension cultures, since cultures grown beyond six months after the initiation of primary callus were found to be unsatisfactory as a source of protoplasts with high plant regeneration capability. Old suspension cultures, however, can be used as nurse cells (see section on transformation).

P r o c e d u r e

�9 Grind mature seeds with a mortar and pestle. Select dehulled seeds with an intact embryo. Eliminate seeds with fungal or bacterial contamination.

�9 Transfer about 200 dehulled mature seeds into a sterile 125-mL erlenmeyer flask containing 50 mL of 70% EtOH. Incubate for 1.5 rain with occasional agitation, then wi thdraw the EtOH from the flask.

�9 Add 50 mL of 2.5% sodium hylx~hlorite and incubate on a shaker at 135 rpm for 45 min.

�9 Remove the bleach completely. Rinse seeds with 100 mL of sterile water three times.


�9 Use sterile forceps to transfer 20 seeds to petri dishes each containing 25 mL of solidified MS2 medium. Seal plates with Parafilm.

�9 Incubate in the dark at 27~ for 14 days. �9 Remove the endosperm, elongated shoots, and roots from the

germinated seeds. Transfer those embryos containing compact primary callus onto fresh MS2 plates and continue culturing for another 14 days under the same conditions. I

�9 By the end of this second subculture, small, yellow callus clumps should grow out from the primary calli. Excise these secondary calli and transfer calli from each dish to a separate sterile 125-mL erlenmeyer flask containing 30 mL of liquid General medium and cover with sterile foil, then seal with Parafilm?

�9 Incubate the new suspension culture on a shaker (85 rpm) at 27~ under dim light (8.4 ~E-m<s -1) for 7 days.

�9 At the end of the first week of suspension culture, allow cell clumps to settle at the bot tom of the flasks, withdraw 20 mL of the upper liquid and supplement with an equal volume of freshly-prepared General liquid medium.

�9 Subculture weekly. Protoplast isolation may be performed after between one and three months of suspension culture.

Notes 1. Calli can be obtained from tissues other than scutellum. However, scutellum-

derived calli are superior because they grow quickly in liquid media and because the resulting suspension is not contamina ted with the elongated cells that occur otherwise in suspension cultures.

2. Primary scu tellum-derived calli released toxic phenolic substances and turned brown soon after inoculation. These brown calli spoil the suspension culture rapidly, and therefore should be eliminated as soon as they are visible. Secondary calli grew actively and resulted in healthy cell aggregates (Fig. 1).

Protoplast isolation Protoplasts with dense cytoplasm are obtained from one- to five-month- old suspension cultures. Suspension cultures older than five months should not be used, for they produce highly vacuolated protoplasts which are unsuited for plant regeneration. The procedure used for protoplast isolation was that of Kyozuka et al. (1987) with minor modi- fications.

Procedure �9 Transfer 3 g of three to four day-old suspension cell aggregates

using a sterile spatula to a sterile petri dish. Add 20 mL of filter- sterilized enzyme solution, suspending the cell aggregates evenly. Seal the Petri dish with two layers of Parafilm.

282 Li et al.

�9 Incubate the digestion mixture in the dark at 30~ for 3.5 h without agitation2

�9 Transfer the digestion mixture into a sterile 125-mL flask. Add 20 mL of KMC solution to the flask and swirl the digested cell clumps gently to release protoplasts. Prepare two layers of sterile nylon membranes by placing a 400-/am pore diameter membrane on top of a 40-/am membrane inside a small sterile funnel. After allowing the protoplast solution to settle, gravity-filter the supernatant through the nylon membranes into another sterile flask. Rinse the undigested cell clumps in the flask twice, each time with 25 mL of KMC solution, collecting the new filtrate into the same flask as the original filtrate.

�9 Transfer the filtrate to two sterile 50-mL centrifuge tubes, balance, cap, and spin at 130 xg for 8 min.

�9 Withdraw the enzyme solution with a sterile pasture pipet connected through a trap to a small vacuum p u m p 2.

�9 Add 10 mL of KMC solu t-ion to each centrifuge tube to resu spend the protoplast pellet, then add 35 mL more KMC solution. Centrifuge at 130 xg for 4 min.

Notes 1. The time required for digestion depends on the condition of digested cell

aggregates. If the cell clumps are small (about I mm in diameter) and light yellow, 3.5 hours of digestion should be adequate for the release of protoplasts in high yield (up to 8 x 10 6 protoplasts per g fresh weight). Digestion for more than 4 hours usually reduces protoplast quality and regenerability.

2. Complete removal of enzyme solution from the protoplast pellet can cause bursting of protoplasts. A small amount of solution should be retained with the protoplasts during the entire isolation procedure.

PEG-mediated transformation PEG-mediated DNA transformation into plant protoplasts was developed initially by Krens et al. (1982), and was modified subsequently by several investigators (Paszkowski et al., 1984; Negrutiu et al., 1987). These studies indicated that protoplasts from different plant species or explant sources responded to PEG treatments differently. We found that the PEG concentration was the most significant factor affecting the protoplast viability and apparent transformation frequency of rice protoplasts (Hayashimoto et al., 1990).

Nipponbare protoplasts were resuspended in MaMg solution, then incubated briefly with plasmid DNA for 10 min to allow sufficient contact between the DNA and the plasma membrane. A 40% (w/v ) PEG solution was added slowly to bring the final PEG concentration to 25%


(w/v). Protoplast solutions were swirled gently during the addition of PEG, because the rapid addition of concentrated PEG can cause a sharp increase in osmotic potential and result in fusion or breakage of protoplasts. After a 15-min incubation, PEG was washed away from protoplasts with KMC solution, the protoplasts were embedded in agarose sheets, and nurse cells were added. No heat shock or cold treatment was included in this procedure.

Procedure �9 Resuspend the protoplast pellet in 1 to 2 mL of MaMg solution.

Determine the protoplast density with a hemocytometer. Add 5 ~tl of protoplast solution to a hemocytometer with cover glass, then under a light microscope count the total number of intact protoplasts in 10 randomly-chosen grid fields of 1/16 mm 2. Calculate the total number of protoplasts per mL by multiplying the average number of protoplasts per grid field by 1.6 x 104.

�9 Adjust the protoplast density to 4 xl06 protoplasts per mL using an appropriate volume of MaMg solution.

�9 Prepare sterile intact plasmid DNA in 0.1xTE 1-2. Add 80 uL of DNA solution containing 40 ug of plasmid DNA to a 50-mL sterile plastic tube.

�9 Slowly transfer I mL of protoplast solution to the DNA-containing tube. Incubate at room temperature (24~ for 10 min.

�9 Apply 1.75 mL of 40% (w/v) PEG stock solution dropwise with gentle swirling to the protoplast-DNA mixture using a sterile pipette. Allow to incubate at room temperature for 15 min.

�9 Stop the PEG treatment by adding 40 mL of KMC solution to the mixture. Gently invert the tube several times to wash PEG from the protoplasts thoroughly.

�9 Centrifuge at 130 xg and remove the supernatant. Resuspend the protoplasts in 4 mL of protoplast medium.

�9 Prepare warm (40~ agarose-containing protoplast med ium and add 4 mL of this dropwise and with gentle swirling to the protoplast suspension using a sterile pipette. Quickly transfer the mixture into a 100-mm diameter petri dish and allow to solidify for at least one hour in a laminar flow hood.

�9 Cut the agarose-containing sheet into square blocks 10 mm on a side, and float the blocks by adding 15 mL of protoplast medium, loosening blocks by scooping with a sterile spatula where needed. An agarose block (10xl0x0.7mm) prepared as above should contain 7.0 x 104 protoplasts.

�9 Add 400 mg (fresh weight) of actively growing nurse cells 3 to the petri dish. Seal the plate with two layers of Parafilm.

284 Li et al.

�9 Culture the plates on a shaker (30 rpm) in the dark for 10 days at 27~

�9 Wash agarose blocks from each petri dish in 25 mL of fresh protoplast medium to remove nurse cells. Then transfer the blocks to another petri dish containing 20 mL of fresh protoplast medium.

�9 Wrap and incubate the plates as in the previous step, except for only 4 days. Protoplast-derived colonies should be visible at the end of this culture period.

�9 Transfer agarose blocks from each plate to 20 mL of protoplast medium containing 95 ~tM (50 mg/L) of hygromycin B in a petri dish.

�9 Maintain protoplasts under selection for 12 days on a shaker (30 rpm) in the dark at 27~

�9 Use a flamed spatula to remove the agarose blocks from selection medium, briefly wash them in fresh protoplast medium and transfer them into a petri dish containing 25 mL of soft agarose medium.

�9 Incubate in the dark for 14 days at 27~ Most of the resistant colonies should grow to from 1 to 2 mm in diameter at the end of incubation period.

Notes 1. Plasmid DNA for protoplast transformation was isolated by the modified

method of Hansen and Olsen (1978). Plasmid DNA was further purified by CsC1 density gradient centrifugation.

2. To sterilize DNA for transformation, resuspend 50 fig of intact plasmid DNA in 100 ~tL of TE buffer containing 0.1 M NaOAc (pH 6.0) in a sterile Eppendorf tube. Add 250/2.1of cold (-20~ absolute EtOH and allow DNA to precipitate at -80~ for 60 min. After centrifugation for 10 min at 10,000 x g, remove the supernatant and rinse the DNA pellet with 500 ~tl of 70% EtOH. Air-dry the pellet in the laminar-flow hood to remove EtOH. Resuspend the DNA in 100 gl of sterile 0.1x TE and allow 2 h for the DNA to dissolve.

3. Nurse cells are required for rice protoplast regeneration (Kyozuka et al., 1987; Li and Murai, 1990). Unlike the suspension culture for protoplast propagation and plant regeneration, nurse cell lines have been maintained beyond 6 months of culture duration. Cultures containing dispersed, fast-growing clumps of cells (the majority <0.5 mm in diameter) are selected for further subculturing and use as nurse cells.

Plant regeneration and propagation We used a method developed by Cao et al. (1989) for the regeneration of plantsfromprotoplastsofNipponbareandTaipei309. Plant regeneration starts with small calli (1 to 2 mm in diameter) in MS regeneration medium. A large number of plantlets can be regenerated from calli after two to three subcultures of 20 days each. Regenerating plantlets were


transferred to eflenmeyer flasks and grown until they were 5 cm tall. These planflets were transferred to the potted soil in the greenhouse and grown in a water-flooded bench until seed set.

Procedure �9 Transfer 30 protoplast-derived calli of from I to 2 mm in diameter

onto each plate containing 25 mL of MS regeneration medium. �9 Incubate the plates on a shelf under daily cycles of 16 hours of light

(56 ~_E-m-2-s -1) and 8 hours of darkness for 20 days at 27~ �9 For the first subculturing, pick up groups of actively-growing

clumps of calli from 10 initial inoculants and place separately onto fresh medium in a petri dish. Let grow under the same conditions as the previous step for another 20 days.

�9 Calli producing green spots and plantlets should appear after the first subculturing. Transfer such calli to fresh medium, not more than five calli per plate. Culture for 20 days.

�9 Transfer regenerating calli derived from a single protoplast colony to a single 125-mL erlenmeyer flask containing 25 mL of regeneration medium. Culture for 10 days to promote the growth of shoots and roots of the regenerating plantlets.

�9 Plantlets from 5 to 10 cm tall with at least three true leaves are ready for transplanting to the greenhouse. Before transferring, grow the plantlets in a liquid solution containing 0.1x MS salts for 2 days in an uncapped Erlenmeyer flask to allow plantlets to adapt to reduced humidity.

�9 Wash plantlets in tap water, then transplant to 20-cm-diameter plastic pots containing a sterilized 2:1:1 mixture of Crowley silt loam, sand, and peat moss. Plants were grown in the greenhouse in a water-flooded bench with water maintained from 10 to 15 cm deep. Apply Osmocote time-release fertilizer (13:13:13 N:P:K) at a rate of 5 g per pot, once in the first week of transplanting, and a second time at the beginning of flowering.

�9 Maintain the temperature of the greenhouse between 20 ~ and 35~ Rice pollen is inactivated by temperatures higher than 35~ and active pollen will not be produced when the temperature is below 23~ two weeks prior to or during the flowering stage.

Resul ts and D i s c u s s i o n

In order to develop a reliable suspension culture for protoplast isolation, mature seeds of the varieties Nipponbare and Taipei 309 were used to initiate callus tissues which were developed subsequently into suspen-

286 Li et al.

Table 1. Rate of regeneration of transformed Nipponbare protoplasts'.

Plasmid pTRA132

Hygromycin concn, in selection 95 }dVI 190 ~M Treated protoplasts 4 x 106 4 x 106 Resistant colonies 788 704 Calli for plant regeneration 73 148 Regenerating calli with shoots 18 51 Regenerated plants 60 179

Plasmid pTRA131

Hygromycin concn, in selection 95 p.M 190 p.M Treated protoplasts 4 x 10 ~ 4 x 106 Resistant colonies 304 236 Calli for plant regeneration 49 57 Regenerating calli with shoots 14 25 Regenerated plants 47 98

* Data were obtained on 18 June 1989.

sion cultures. Primary callus was induced readily from the scutellum of rice embryos after 14 days of incubation in MS medium containing 9 uM 2,4-D. However, when transferred into liquid medium, these compact calli were frequently arrested in growth, and released brown phenolic compounds which spoiled the suspension culture. To avoid this, primary calli were propagated further by a subculture in callus induction medium to allow the generation of secondary callus. Secondary calli, produced along the edges of primary callus clumps, were yellow, small, round, uniformly-sized callus aggregates. These callus tissues grew continuously without any lag in growth. Fine, friable callus aggregates were produced after three or four subsequent weekly subcultures. Comparison of callus growth rates in General medium and four other media indicated that General medium supported the highest increase of callus fresh weight after three weekly subcultures (Li and Murai,1990).

Microscopic observation revealed that cell aggregates in General medium were composed of round-shaped, actively-dividing cells containing dense cytoplasm. When subjected to enzymatic digestion with 4% cellulase and 1% macerozyme, three- to four- day-old cell aggregates released viable protoplasts in high yield (up to 8 x 106 protoplasts per g


Fig. 1. Two-month old Nipponbare suspension cultures ready for protoplast isolation.

fresh weight). General medium was employed, because the plating efficiency in General medium (10.4%) was higher than in other four media tested (Li and Murai, 1990). Fewer protoplasts derived from General medium contained large vacuoles.

Suspension cultures more than five months old yielded highly vacuolated protoplasts and were not adequate as a source of protoplasts for plant regeneration. Somatic variations, including albino and other phenotypically-abnormal plants, occurred at higher frequency in the older cultures.

Direct gene transfer by PEG treatment has been investigated in other plant species (Krens et al., 1982; Paszkowski et al., 1984; Negrutiu etal., 1987). For transformation of rice protoplasts, PEG concentration was found to be the most important factor influenc- ing the efficiency of stable transformation (Hayashimoto et al., 1990). The MaMg solution followed by PEG treatment provided the most effective condition for

Fig. 2. Regenerating shootsand roots gene u p t a k e by p r o t o p l a s t s from protoplast-derived hygromycin- (Negrutiu et al., 1987; Zhang and resistant calli. Wu, 1988). The optimal concen-

288 Li et al.

Fig. 3. Fertile transgenic rice plants grown and matured in the greenhouse.

tration of PEG for transformation was 30%, but solubility considerations limited the practical concentration to 25%. The overall apparent transformation rates for Nipponbare and Taipei 309 were 2x10 -4 and 5x10 ~, respectively, which are from 200 to 500 hygromycin-resistant colonies per million protoplasts (Hayashimoto et al., 1990). These transformation frequencies were approximately ten times the rate obtained after electroporation (Shimamoto et al., 1989).

The addition of nurse (callus suspension) cells was essential for efficient rice protoplast regeneration (Kyozuka et al., 1987). The survival rates of Nipponbare and Taipei 309 protoplasts were 0% and 0.7% when nurse cells were omitted, far lower than the rate of over 10% when nurse cells were present (Li and Murai, 1990.)

Selection of transformed cells was accomplished by the addition of hygromycin B to a concentration of 95/3.M 14 days after transformation. More stringent selection with 190 uM hygromycin B relsulted in a slightly reduced number of resistant colonies (Table 1). Addition sooner or later than 14 days had a detrimental effect on either the survival rate or tightness of selection. The hph gene was used as a selectable marker,


Table 2. Time required to regenerate mature rice plants from protoplast transformation of Nipponbare.

Stage Time

Suspension Culture Primary callus initiation 2 weeks Secondary callus induction 2 weeks Suspension development 4-6 weeks

Protoplast Culture Nurse culture 10 days Calli propagation in liquid medium 4 days Selection for hygromycin resistance 12 days Calli propagation in soft agarose medium 14 days

Plant Regeneration 5-8 weeks Greenhouse Growth

Time to emergence of first panicle 5-7 weeks Additional time to flowering 1 week Time for maturation of seeds 3-4 weeks

Total time required 28-36 weeks

for methotrexate was not as effective as desired for selection in tobacco, and there was endogenous neomycin phospho-transferase activity in rice (Hauptmann et al., 1988). It also appears that kanamycin may cause sterility in cereal plants.

Small calli (1 to 2 mm in diameter) were transferred onto MS regeneration medium. Starting from the second week, green spots and then shoots appeared in regenerating calli (Fig. 2). Whole plants were developed within 5 to 8 weeks following 20-day periodic subcultures. A total of 628 transgenic rice plants were regenerated from hygromycin- resistant colonies in 5 experiments. As the data from one of the experiments indicated (Table 1), the overall plant regeneration frequency from hygromycin-resistant colonies was approximately 30%.

Transgenic plants were grown to maturity in the greenhouse. Most of the transgenic plants were phenotypically normal, and two-thirds of the transgenic plants grown in the greenhouse set viable seeds. A small number of plants showed typical somaclonal mutations. For example, fewer than 10 albino plants were found among the regenerated plants. Southern blot hybridization of genomic DNA from five independently transformed plants indicated that I to 10 copies of the hygromycin gene were present in the rice genome (Hayashimoto et al., 1990). Genetic analysis of hygromycin resistance in the selfed progenies of 29 indepen- dent transgenic plants showed the hygromycin resistance gene was

290 Li et al.

loca ted at one o r t w o loci in the rice g e n o m e ( m a n u s c r i p t in p repa ra t ion ) . These resu l t s d e m o n s t r a t e d tha t the t r ansgen ic rice p l an t s o b t a i n e d w e r e t rue t r a n s f o r m a n t s , a n d the t r ans fe r r ed hph g e n e w a s loca ted in the c h r o m o s o m e s .

The h i g h p l a n t r e g e n e r a t i o n ra te o f the a b o v e - d e s c r i b e d p r o c e d u r e m a k e s it poss ib le to g e n e r a t e a la rge n u m b e r o f t r ansgen i c rice p l an t s for g e n e express ion s tudies . The s i x -mon th total t ime p e r i o d for r e g e n e r a t i o n (Table 2) is a r e d u c t i o n f r o m s o m e p r e v i o u s m e t h o d s ( T o r i y a m a et al., 1986; Y a m a d a et al., 1986), a n d m a k e s it p rac t ica l to p e r f o r m the exper i - m e n t in a r e a s o n a b l e t ime.

Acknowledgments. The authors would like to express their gratitude to Dr. Akio Hayashimoto for constructing the plant expression vectors and Drs. M.C. Rush and Qingjung Xie for providing Taipei 309 seeds and advice in plant regeneration. This work was supported in part by grants from the Louisiana Educational Quality Support Fund (1987-90)-RD-A-6.

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Harms, Ch. T., and I. Potrykus. 1978. Fractionation of plant protoplast types by iso- osmotic density gradient centrifugation. Theor. Appl. Genet. 53:57-63.

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High frequency generation of fertile transgenic rice plants after PEG-mediated protoplast...

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