PlantCell Reports

Plant Cell Reports (1996) 15:963-968

9 Springer-Verlag1996

Optimization of biolistic method for transient gene expression and production of agronomically useful transgenic Basmati rice plants Rajinder K. Jain 1, Sunita Jain 2, Baiyang Wang 2, and Ray Wu 2 1 Department of Genetics, Haryana Agricultural University, Hisar, India 2 Section of Biochemistry, Molecular and Cell Biology, Biotechnology Building, Cornell University, Ithaca, NY 14853-2703, USA Received 12 December 1995/Revised version received 23 February 1996 - Communicated by I. K. Vasil

ABSTRACT We have developed a reproducible biolistic procedure for the efficient transformation of embryogenic suspension cells of an improved aromatic Indica rice variety, Pusa Basmati 1. The 13glucuronidase gene was used to assay transient transformation; other plasmids carrying either a potato protease inhibitor 2 (Pin2) gene, or a late embryogenesis-abundant protein (LEA3) gene from barley, were used for the optimization of biolistic process and transgenic plant production. After optimization of the procedure, over 600 transient transformants and at least five fertile plants showing integrative transformation were obtained per bombarded filter. At least 30% of the plants were derived from independent transformation events. The new improved procedure involves the use of a reporter gene or other useful genes driven by the strong rice actin 1 gene (Actl) promoter, osmotic pre-conditioning of cells for 24 h on medium supplemented with 0.25 M mannitol prior to bombardment, use of gold particles for DNA delivery, and use of plant regeneration medium with high (1.0%) agarose concentration.

KEY W O R D S : Basmati rice, Group 1 Indica, biolistics, transformation, protease inhibitor gene, late embryogenesis-abundant protein gene. INTRODUCTION The biolistic method has been successfully used to produce fertile transgenic plants such as maize (Fromm et al. 1990, Gordon-Kamm et al. 1990), oat (Somerset al. 1992), rice (Christou et al. 1991, Cao et al. 1992), wheat (Vasil et al. 1992), barley (Wan and Lemaux 1994) and rye (Castillo et al. 1994). For rice, either immature embryos (Christou et al. 1991) or suspension cells (Cao et al. 1992) were used as targets for bombardments. However, the reported transformation efficiency is still relatively low. There could be several reasons to account for low efficiency; optimal bombardment conditions vary depending on the type and quality of target cells and tissues, and media requirements for inducing somatic embryogenesis and plant Correspondence to: R. Wu

formation may not be well understood. In rice, most success in producing transgenic plants via biolistics has been limited to tissue culture responsive Japonica rice varieties. However, economically important Indica rice varieties, especially those belonging to Varietal Group 1, remain difficult to transform and regenerate fertile plants. Improvement in current biolistic transformation procedures is required to increase the efficiency of integrative transformation. For practical plant regeneration, at least a few hundred potentially regenerable cells should transiently express the introduced DNA per bombardment. A number of factors that can potentially affect transformation frequency have been reviewed by Birch and Bower (1994). Russell et al. (1992a, b) reported a manifold increase in transformation frequencies depending upon the promoter strength, nature of the metallic particles used for DNA delivery, and osmotic pre-conditioning of tobacco suspension cells. Optimizing the concentration and duration of osmotic treatment resulted in a 6.8-fold increase in recovery of transformed maize clones following particle bombardment (Vain et al. 1993). There are reports that suggest the stimulatory effect of desiccation treatments on plant regeneration from rice calli (Tsukahara and Hirosawa 1992; Ranc6 et al. 1994; Jain et al. 1996). We have been working on transformation of Basmati rice (var. Pusa Basmati 1) using gene constructs that can potentially improve the defense against insect attack and drought tolerance. Pusa Basmati 1 is an improved, fine grain, aromatic, and semi-dwarf Indica rice variety which essentially belongs to Varietal Group 1 (Jain et al. 1995). In this paper, we report the results of biolistic experiments that have allowed us to dramatically improve the transformation of embryogenic suspension cells of Pusa Basmati 1. Effects of the microcarriers used for DNA delivery, target cell distance (distance between the target cells and launch point of the microprojectiles), and osmotic preconditioning of the target cells on transient gene expression and transgenic plant production have been described. ActlF-GUS plasmid (McElroy et al. 1990) was used for transient gene expression studies;

964 other plasmids carrying potato protease inhibitor 2 (Pin2) gene, or a late embryogenesis-abundant protein (LEA3) gene from barley, were used for optimization of the biolistic process and transgenic plant production. The importance of a protease inhibitor gene in developing resistance to insect pests is well documented in transgenic tobacco (Johnson et el. 1989). Lea protein genes have been suggested to act as protectants during seed desiccation and in water-stressed seedlings. A barley LEA3 gene was recently transferred to Japonica rice variety Nipponbare using the biolistic method; the transgenic plants showed increased tolerance to both water stress and salinity (Xu et al. 1995). MATERIALS

AND METHODS

Plant materials Seeds of Pusa Basmati 1 were provided by the International Rice Research Institute, Manila, Philippines. Cell suspension cultures of Pusa Basmati 1 were established using the mature seed scutellum-derived calli, as described by Jain et al. (1995). For the initiation and maintenance of cell suspensions, a modified R2 medium (Ohira et al. 1973), which contained 3.0% (w/v) maltose instead of sucrose and 560 mg/l of L-proline, was used. Cell suspensions of Pusa Basmati 1 were 5-8 months old at the time when these transformation experiments were conducted.

Plasmid DNA The structure of various plasmids is illustrated in Figure 1. The useful gene in plasmids is driven by either the strong Act1 promoter or the woundinducible Pin2 promoter. The plasmids also contained a phosphinothricin acetyl transferase (bar) gene, driven by 35S promoter, as the selectable marker gene. Xhol

Sstl

BJmaHl

I o,/1 I a,a,'

SJcd

Xb*l

I-j_

I nva, I r~aJ" I,~s, ,

pActlF-GLIS

ua"

,'Y--

BlemH1 BB~I P/a25" Aall~lP/n2. P/s2J' ~ br ..__~3" pTWa

Figure 1. Structure of pActlF-GUS, pBY520 and pTWa plasmids. Act1 5', rice actin 1 gene promoter; Act1 In, intron from Act1 5' promoter region; bar, phosphinothricin acetyl transferase gene; HVA1, late embryogenesis abundant (LEA3) protein gene; Nos 3', nopaline synthase gene 3' region; Pin2, potato protease inhibitor 2 gene; Pin2 3', Pin2 3' region; Pin2 5', Pin2 promoter region; 35S 5', cauliflower mosaic virus 35S promoter. Only important restriction endonuclease sites are indicated.

Microprojectile bombardment Plasmid DNAs were adsorbed to gold particles or tungsten particles (mean diameter of 1 ~tm) by

CaC12 and spermidine precipitation and delivered to the target cells using the DuPont Helium PDS1000 biolistic device (Cao et al. 1992). For the preparation of filters with overlaying cells for bombardment, about 0.5 ml settled cell volume (scv) of sieved (1,000 l.tm nylon mesh) cells obtained after 4 days of subculture, was spread on a 5.5-cm diameter Whatman #1 filter disc and washed two times with liquid Murashige and Skoog (1962) medium supplemented with 2.5 mg/1 2,4-D (MS 2.5) by vacuum filtration. The filter discs were placed on the surface of 9 cm Petri dishes that contain 25 ml of agarose-solidified (0.5% w/v, Agarose Ultra Pure, GIBCO BRL, USA) MS 2.5 medium. The dishes were incubated at 26~ in the dark. For osmotic preconditioning, the filter discs were placed on the surface of 9-cm Petri dishes that contain 25 ml of 0.5% w/v agarose-solidified MS 2.5 medium supplemented with 0.25 M mannitol. These dishes were used for the biolistic experiments the following day. Each filter disc was bombarded twice with the metallic particles coated with 2.5 ~tg of plasmid DNA, keeping the target-cell distance of 9 or 12 cm. After 24 h, filters were transferred to the new dishes with mannitol-free MS 2.5 medium. G U S assay Histochemical GUS assay was carried out 2 days after bombardments, essentially as described by McElroy et al. (1990). Blue loci, indicative of transient GUS expression, were counted 2 days after the addition of the X-Gluc substrate solution.

Selection of transformants Three days after bombardment, filter discs with overlaying cells were transferred to the Petri dishes containing 25 ml MS 2.5 medium supplemented with 8 mg/1 ammonium glufosinate. These filters were transferred every 10 days onto fresh selection medium. The dishes were incubated at 26~ in the dark. After 30 days, data were recorded on the number of putative transformed calli showing fresh growth on the selection medium.

Plant regeneration The putative transformed calli, as well as nonselected calli, were transferred onto the Petri dishes containing 25 ml of modified MS-based regeneration medium (Jain et al. 1995) containing maltose (30 g/l), kinetin (2.0 mg/1), u-naphthalene-acetic acid (0.5 mg/1), and ammonium glufosinate (5.0 mg/l). The regeneration medium was solidified with either 0.5% (w/v) or 1.0% of agarose. For partial desiccation, the calli were kept on an empty sterile Petri dish containing two sterile Whatman filter paper discs for 25 h in the dark before transferring them onto regeneration medium. The cultures were incubated in the dark at 26~ for 2 weeks and then transferred under light (55 ~tmol m-2 sec-1, daylight fluorescent tubes, 16 h photoperiod) for another 2 weeks. These calli were then transferred to the 0.5% agarose-solidified regeneration m e d i u m without ammonium glufosinate and kept under light. After 2-3 weeks, the calli with differentiated shoots were transferred to the 0.5% agarose-solidified regeneration medium containing 3 mg/1 of ammonium glufosinate and incubated under light. After 2 weeks, the surviving and normal growing shoots were

965 individually transferred to the rooting medium (0.25% w/v phytagel-solidified MS medium with 1.5 mg/1 NAA) and 3 weeks later, they were transferred to pots in the greenhouse.

BastaTM-resistanee test For detecting the presence and expression of bar gene in transgenic plants, a 0.25% (v/v) solution of commercial herbicide BastaTM (containing 162 g/1 ammonium glufosinate, Hoechst-Roussel Agri-Vet Company, Sommerville, NJ) and 0.1% (v/v) Tween20 was painted on both sides of a leaf. After one week, the damage caused by the herbicide was assessed and resistant/sensitive phenotype scored.

Molecular analysis PCR amplification was carried out using DNA samples isolated from leaf tissues of putative transgenic rice plants. A suitable pair of genespecific oligonucleotides were synthesized for the amplification of a DNA sequence between the promoter region (Act1 5' intron or CaMV35S promoter) and a marker or a useful gene (bar or Pin2) based on the published gene sequences. As an internal control, a set of primers which amplify a 304 bp product by PCR from Act1 promoter region, was used. PCR assays were performed in 25 pl reaction mixture using 0.15 gl AmpliTaq DNA polymerase, according to the manufacturer's instructions (Perkin Elmer, Roche Molecular Systems, Inc., Branchburg, NJ) with the following cycle parameters: 30 sec at 94~ 45 sec at 62~ and 45 sec at 72~ After 40 cycles, the PCR products were separated on a 1.0% agarose gel and stained with ethidium bromide. For DNA blot hybridization, 15 I.tg of rice genomic DNA, which had been isolated from each sample as described by Zhao et al. (1988), was digested by suitable restriction endonuclease(s), separated on a 1.0% agarose gel, transferred onto a nylon membrane, and hybridized with the appropriate 3zP-labeled probe. RESULTS AND DISCUSSION In the beginning, we used the biolistic procedure developed in this laboratory for transgenic plant production in Japonica rice varieties (Cao et ai. 1992) for DNA delivery into Pusa Basmati 1 suspension cells. The procedure involved bombardment of tungsten particles coated with plasmid DNA using the DuPont Helium PDS-1000 device keeping the bombardment pressure at 1,500 psi, gap distance at 1.0 cm, and target cell distance (distance between target cells and launch point of the microprojectiles) at 12 cm. Each filter carrying 0.5 ml settled cell volume (scv) of suspension cells was bombarded twice with 2.5 gg of plasmid DNA per bombardment. This procedure produced an average of 24 blue spots or 2.0-6.8 ammonium glufosinateresistant (AG R) calli per filter in Pusa Basmati 1 (Tables 1 and 2). To improve transformation frequencies, we investigated the effects of several factors on the transformation of Pusa Basmati 1 cells; the optimal conditions for each parameter were then combined to form a new, improved protocol.

Target cell distance (TCD) Reduction in TCD from 12 cm to 9 cm increased the average number of blue spots and AGR calli (Tables

1 and 2). The filters bombarded sequentially with TCD of 9 and 12 cm produced AGR calli at frequencies similar to those obtained using 9 cm TCD for both the bombardments (Table 2), but microscopic observations showed that sequential use of TCD of 9 and 12 cm allowed a more even distribution of coated particles on the surface of the filter disc or cells, with minimum damage to the cells.

Microcarriers There were 2.5- to 5.4-fold more GUS-expressing cells when gold particles were used for the biolistic process compared to the tungsten particles (Table 1). The number of transformed AGR calli was also several fold higher with the gold particles (Table 2). Compared to tungsten particles, gold particles have been preferred as microcarriers for biolistics because of their size uniformity, spherical shape, and inert nature. There is evidence that tungsten toxicity can reduce the recovery of stable transformants in some plant species (Russell et al. 1992).

Osmotic pre-conditioning of cells We compared the effect of osmotic-preconditioning of cells using 0.25 M mannitol on transient as well as stable transformation of Pusa Basmati 1 cells (Tables 1 and 2). A one-day treatment of Pusa Basmati 1 cells in medium with 0.25 M mannitol increased the number of GUS expressing cells by 3.9- to 8.8-fold (Table 1) and the number of AG k calli by 2.2- to 4.9-fold (Table 2), depending on the other biolistic conditions. The best values for transient GUS expression and stable transformation were obtained when cells were osmotically pre-conditioned with 0.25 M mannitol, and gold particles were used as microcarriers with TCD of 9 cm for both the bombardments or sequentially keeping the TCD of 9 and 12 cm for the two bombardments. In the case of pTWa, bombardments made using these conditions produced an average of 103 AG~ calli per filter disc. To obtain a higher degree of stable transformation, post bombardment handling of cells was also found to be critical. Since high concentrations of mannitol were inhibitory to cell growth, the filters with overlaying cells were transferred to the mannitol-free medium a day after the bombardment, and then after another day the filters were transferred to the selection medium containing 8 mg/1 ammonium glufosinate. Direct transfer of bombarded filters from mannitolsupplemented medium to the selection medium gave less satisfactory results (data not shown). Another beneficial effect of osmotic treatment of cells was that the fresh growth and identification of AGR calli were relatively clear and readily distinguished.

Plant regeneration transformed calli

from

putatively-

After 30 days, the new calli that showed active cell proliferation on the selection medium, and grew up to the size of about 1.0 cm diameter (referred to as resistant calli), were transferred to the regeneration medium containing 5.0 mg/1 ammonium glufosinate. This medium further helped in the selection of transformed AGR calli. The calli showed extensive embryogenesis, but formation of shoots was limited due to the presence of ammonium glufosinate in the

966 Table 1. Effect of metallic particles employed for DNA delivery~ osmotic pre-conditioning of cells, and target cell distance on GUS gene expression, as measured by development of blue loci per filter in Pusa Basmati cells. Particle type

Osmotic pre-conditioninga

Target cell distance (cm)

Number (+s.e.) of blue spots per filter

Tungsten

No

12, 12

24+5

Tungsten

No

9, 9

37+8

Tungsten

Yes

12, 12

207+27

Tungsten

Yes

9, 9

215_+41

Gold

No

12, 12

132+25

Gold

No

9, 9

158+16

Gold

Yes

12, 12

510+142

Gold

Yes

9, 9

684+91

m

D

Data represent the mean + standard error of three independentexperiments. For each experiment, three filters were used and the values averaged. Each filter carried 0.5 scv cells and was bombarded twice. aFilters carrying suspensions cells were kept on MS 2.5 medium supplemented with 0.25 M mannitol for 24 h before bombardment.

Table 2. Effect of metallic particles employed for DNA delivery, osmotic pre-conditioning of cells, and target cell distance on stable transformation in Pusa Basmati 1 cells. Plasmid

(A) pTWa

pLEA3

Osmotic preconditioninga

Particle type

Target cell distance (cm) (D)

Number of resistant calli per filter

(E)

(B)

(c)

No

Tungsten

12, 12

7+1

No

Tungsten

9,9

14+4

No

Tungsten

9, 12

13+2

No

Gold

9, 12

27+4

Yes

Tungsten

9, 12

43+1

Yes

Gold

9, 12

102+2 5

No

Tungsten

12,12

4+4

No

Tungsten

9,9

11+1

No

Tungsten

9, 12

11+1

No

Gold

9, 12

25+3

Yes

Tungsten

9, 12

24+4

Yes

Gold

9, 12

52+8

m

m

m

Data represent the mean + standarderror of three independentexperiments. For each experiment, three filters were used and the values averaged. Each filter carried 0.5 scv suspension cells and was bombarded twice with DNA-coated metallic particles (Column C) sequentially keeping the target cell distance shown in Column D. aFilters carrying target cells were kept on MS 2.5 medium supplementedwith 0.25 M mannitol for 24 h before bombardment.

967 medium even at this low concentration (data not shown). To induce high-frequency shoot formation, these calli were transferred to the ammonium glufosinate-free regeneration medium. After the calli showed regenerated shoots, the calli were again grown in medium with ammonium glufosinate to stop the growth of the non-transformed cells. In our experiments, osmotic stress created by using a high agarose concentration (1.0% w/v) for the solidification of regeneration medium was found to be better compared to the partial desiccation treatment for high-frequency plant regeneration from transformed AGR calli (Table 3). In different experiments, shoot regeneration frequencies on 1.0% agarose medium ranged between 80-91%, compared to 11-18% in medium with 0.5% agarose, and 3134% following 24 h partial-desiccation of calli. The calli transferred onto regeneration medium solidified with high (1.0%) agarose concentration experiences a milder osmotic stress in the beginning, which gradually increases due to constant water evaporation. The cells/tissues on this medium were relatively drier; a condition that may help the shoot regeneration process. After 4 weeks of growth, the percent water content in non-transformed calli on 0.5% agarose medium was 91% compared to 84% in calli grown on 1.0% agarose medium. The water content in 24 h desiccated calli was 80%. Partialdesiccation treatment, which resulted in greater water loss from calli in just 24 h compared to that lost by calli on 1.0% agarose medium in four weeks, was perhaps too harsh for the calli already growing under herbicide-stressed condition. The use of 1.0% agarose regeneration medium with 5 mg/1 ammonium glufosinate also maintained the stringent selection conditions, allowing only the transformed calli to grow.

PCR analysis A total of 235 plants (2-4 leaf stage, 5-10 cm plant heigh0 obtained after transformation with pTWa and pLEA3 plasmids were analyzed by PCR using a suitable set of primers derived from the promoter region, bar or Pin2 gene sequences. Of these, 188 plants showed amplification of the expected sized DNA fragment, indicating that 80% of the selected AG R plants could be transgenic (for an example see Figure 2).

Herbicide-resistance test Phosphinothricin acetyltransferase encoded by the bar gene can detoxify phosphinothricin-based herbicides. A total of 55 putative-transgenic plants harboring one of the two plasmids transferred to greenhouse, were tested for herbicide resistance. When painted with 0.25% commercial herbicide BastaTM, the leaves of 23 plants showed complete resistance; the leaves of the remaining 32 plants either showed partial resistance or were equally sensitive as the control non-transformed Pusa Basmati 1 plants. The leaves of seed-grown nontransformed control Pusa Basmati 1 plants were relatively more sensitive than those of Japonica rice variety Taipei 309 to Basta application; the leaves of Pusa Basmati 1 plants turned completely yellow and died even at 0.1% Basta concentration (data not shown).

DNA blot hybridization A random sample of 15 putative transgenic plants harboring LEA3 gene were analyzed by DNA blot hybridization using the HVA1 cDNA fragment as the probe (Figure 3). Digestion of pBY520 plasmid or genomic DNA from transgenic rice plants releases the 1.0-kb fragment containing the HVA1 coding region. Among 15 plants analyzed, 6 showed the

Table 3. Effect of agarose concentration and partial desiccation on plant regeneration from putativetransformed calli of Pusa Basmati 1. Shoot regeneration Plasmid pTWa

pt A

Agarose concentration/ desiccation treatment

Total no. of calli

No. of calli regenerating shoots

Percent

65

7

11

No desiccation, agarose 1.0%, 4 weeks and 0.5%, 2 weeks

210

167

80

Partial desiccation 24 h, agarose 0.5%

80

27

34

No desiccation, agarose 0.5%

72

13

18

No desiccation, agarose 1.0%, 4 weeks and 0.5%, 2 weeks

166

151

91

Partial desiccation 24 h, agarose 0.5%

75

23

31

No desiccation, agarose 0.5%

968 expected 1.0-kb hybridization band (lanes 3, 4, 8, 9, 14 and 18). Results of DNA blot hybridization were also consistent with those of the herbicide resistance test, which suggest that both the selectable marker gene and HVA1 gene on the same plasmid were efficiently co-integrated into the rice genome.

In summary, the biolistic transformation procedure described herein results in the production of over 600 transient transformants and at least five fertile plants showing integrative transformation per bombarded filter in an improved aromatic Indica rive variety Pusa Basmati 1. This procedure may accelerate the transfer of value-added genes to Basmati rice or other Indica rice varieties.

ACKNOWLEDGMENTS Support is acknowledged from the Rockefeller Foundation for a fellowship grant to R.K.J. and a research grant to R.W.

REFERENCES Birch RG, Bower R (1994) In: Particle Bombardment Technology for Gene Transfer, Yang N-S, Christou P (eds), Oxford UniversityPress, NY, p 3-37 Bower R, Birch RG (1992) The Plant J 2" 409-416 Cao J, Duan X, McElroy D, Wu R (1992) Plant Cell Rep

11:586-591 Figure 2. PCR-amplified DNA fragments from Act1 In (304 bp) and Act1 In-Pin2 (490 bp) regions from putativetransgenic plants. Lane 1 is 2 kb ladder;, lanes 2 to 8 represent the DNA amplified using pTWa plasmid DNA (lane 2), DNA from a non-trausgenic plant (lane 3), water control (lane 4), and DNA from putative transgenic plants obtained after transformation with pTWa plasmid containing the PIN2 gene. Absence of bands in lane 6 indicates a problem with DNA amplification because there was no amplification of the endogenousAct1 In (304 bp).

Figure 3. DNA blot hybridization analysis to show integration of the HVA1 gene into the genome of some of the transgenic Pusa Basmati 1 rice plants. Fifteen gg genomic DNA from each plant was digested with a combination of EcoRI and BamHI, separated on a 1.0% agarose gel, blotted onto a nylon membrane, and hybridized with a 32P-labeled HVA1 eDNA fragment. Digestion with EcoRI and BamHI releases the 1.0 kb fragment containing the HVA1 eDNA. Lanes 1 and 20, 1.0 kb DNA ladder, lane 2, pTWa plasmid DNA, lanes 3-18, putative transgenie plants; lane 19, non-transformedcontrol plant DNA.

Plant fertility Many workers have reported the problem of sterility or reduced fertility in plants regenerated from selected-transformed calli of Indica rice varieties. However, in our experiments, 90% of the transformed plants transferred to the greenhouse were fertile and showed good seed setting within 110-145 days of transplantation.

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Optimization of biolistic method for transient gene expression and production of agronomically useful transgenic Basmati rice plants.

We have developed a reproducible biolistic procedure for the efficient transformation of embryogenic suspension cells of an improved aromatic Indica r...
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