Optimization of conditions for DNA uptake and transient GUS expression in protoplasts for different tissues of wheat and barley

plan;cience ELSEVIER SCIENTIFIC PUBI.ISHERS IR[ LAND

Plant Science 96 (1994) 179-187

Optimization of conditions for DNA uptake and transient GUS expression in protoplasts for different tissues of wheat and barley Isabel Diaz Centro Nacional de Biotecnoh>gia-CSlC-Madrid, Spain

(Received 26 August 1993: revision received 30 September 1993; accepted 18 October 1993)

Abstract

Conditions have been optimized for the routine isolation and transfection of protoplasts from developing endosperm, aleurone, leaves and roots from barley (Hordeum vulgare L.) and wheat (Triticum aestivum L.). Transient expression of the/3-glucuronidase (GUS) reporter gene under the control of the cauliflower mosaic virus 35S promoter was used to evaluate the best procedure for each tissue. Electroporation was -300% more efficient than polyethyleneglycol (PEG) mediating 35S-GUS transient expression in leaf and root protoplasts while endosperm protoplasts had to be PEG transfected since even the mildest electroporation parameters tested (250 V/cm, 200 #F, 10 ms) were lethal for them. Protoplasts isolated from dry aleurone showed the highest viability (approximately 90%), but GUS activity after PEG transfection was 50% of that obtained with endosperm protoplasts. Conditions optimized for a given tissue of a barley cultivar such as Bomi, were readily applied to other barley cultivars and to other cereal species. Key words." Barley; Wheat; Electroporation; PEG-mediated DNA uptake; Transient expression; Protoplasts

I. Introduction

Genetic transformation has become an important tool in the study of plant gene expression. Cereals are traditionally considered recalcitrant to in vitro manipulation, including transformation [1,2]. Although recently some of the important cereal crops, such as maize, rice and wheat, have been stably transformed after direct D N A uptake into protoplasts, or by particle bombardment of Present address: Bioquimica y Biologia Molecular, Escuela T~cnica Superior de Ingenieros Agr6nomos, UPM, Ciudad Universitaria, 28040 Madrid, Spain

embryogenic cells derived from cell suspensions, microspores or immature embryos [3-9], transformation of barley is still not routinely achieved. Transient gene expression in plant protoplasts has been used in the analysis of gene expression and regulation, as the activity of chimaeric constructs of promoters fused to reporter genes, unintegrated in the cell nucleus, can be quantitated within a few hours after D N A uptake [10]. These techniques have been successfully applied to many dicot and some monocot protoplasts and have been very useful in the characterization of cisacting motives in gene promoters [1 1-13]. Different procedures have been developed for

0168-9452/94/$06.00 © 1994 Elsevier Scientific Publishers Ireland Ltd. All rights reserved. SSDI 0168-9452(93)03756-L

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the DNA uptake into various types of plant protoplasts, and a number of reporter genes suitable for transient expression are available [14-21]. However, data on transient expression in cereal protoplasts is scarce, because metabolically active protoplasts from certain tissues, such as endosperm and roots, are difficult to obtain and transfect. Reproducible procedures for protoplast isolation from developing endosperm, aleurone, leaves and roots of barley and wheat as well as optimized conditions for transient gene expression are described here. These protocols with minor modifications could be extended to other small grain cereals. 2. Materials and methods

2.1. Plant material Barley (Hordeum vulgare L. cv Bomi and Riso 1508) and wheat (Triticum aestivum cv Chinese Spring) were used throughout this study. 2.2. Plasmids The plasmid p35S-GUS is a pUC19 based vector containing the Amp ® gene for selection in bacteria and the /3-glucuronidase (GUS) coding region under the control of the 35S cauliflower mosaic virus promoter (CaMV35S), followed by the 3' non-coding region of the nopaline synthase gene (NOS) [14]. A promoterless GUS construct was used as negative control. Plasmid DNA was purified with a maxi-column (Qiagen, lzasa, Spain) and resuspended in sterile water at a concentration of 1 mg/ml. 2.3. Isolation of protoplasts Endosperm. Wheat and barley endosperms from early developmental stages, up to 15 days after pollination (dap), were the starting material for the isolation of metabolically active protoplasts which were prepared essentially as described by Diaz and Carbonero [14]. After surface sterilization of the kernels by immersion in 10% (v/v) 'Domestos' Lever, Spain) for 30 min, endosperms were aseptically obtained and plasmolysed for 1 h in CPW salts [22] with 11% (w/v) mannitol (CPW 11 M). Plasmolysis was followed by incubation in the dark at 27°C for 14 h in the same medium contain-

L Diaz/Plant Sci. 96 (1994) 179-187

ing 0.4'¼, (w/v) Cellulase Onozuka RS. After washing off the enzyme solution with CPW 11 M medium and filtering through a nylon sieve (250 /~m pore size), the filtrate was allowed to sediment for 14 min and the supernatant gently centrifuged (40 x g, 3 min). Both protoplast pellets were combined and purified by centrifugation (60 x g, 5 min) onto a 50°/,, Percoll cushion and the protoplast band carefully collected at the Percoll interface. After transfection, protoplasts were cultured in the medium previously described [14]. Aleurone. Aleurone protoplasts were prepared by a method adapted from Jacobsen et al. [23]. The mature de-embryonated kernels were hand dissected after 24 h imbibition in water, to separate the aleurone layer from the starchy endosperm. Aleurones were digested with 4.5% (w/v) Cellulase Onozuka R10 for 35 h at 27°C. After washing, the protoplast suspension was layered onto a 50% Percoll cushion and centrifuged (80 x g, 5 min). The protoplast band collected from the Percoll interface was washed and after transfection, cultured in the medium previously described [23]. Leaves. Seeds were surface sterilised with 25% (v/v) ~Domestos' (Lever, Spain) and axenically germinated for 6 days at 27°C in the dark. Etiolated leaves (6-8 cm long) were cut into small pieces and plasmolysed for 1 h in CPW salts with 13% (w/v) mannitol (CPW 13 M) as osmoticum [22]. Digestion with an enzyme solution containing 1% (w/v) Cellulase Onozuka RS and 0.37% (w/v) Macerozyme R10 was carried out for 14 h at 27°C. After filtration (sieves of 500 and 64 #m pore size) and centrifugation (80 x g, 5 rain) the protoplasts were resuspended in CPW 21S medium (CPW salts with 21% (w/v) sucrose) and centrifuged (100 x g, l0 min). The floating band of viable protoplasts was diluted in the electroporation buffer. After transfection, protoplasts were cultured in the medium described by Gupta and Pattanayak [24]. Roots. Seeds were surface sterilised as previously described for the isolation of leaf protoplasts. After axenic germination for 2 days at 27°C in the dark, roots of about 1-2 cm of length, were cut into very small pieces (0.1-0.2 mm), plasmolysed for 1 h in CPW 13 M medium and then statically incubated (14 h, dark, 27°C) in an enzyme mixture of 4% (w/v) Cellulase Onozuka RS and 0.3% (w/v)

I. Diaz / Plant Sci. 96 (1994) 179-187

Pectolyase Y23 (Sheishin Pharmaceuticals), followed by 2 h incubation in the same solution under gentle agitation (40 rev./min). After filtering through a nylon sieve (64 ~m pore size) and washing, the protoplasts were pelleted by centrifugation (80 x g, 5 min) and freed of cell debris by resuspension in CPW 21S medium and centrifugation (110 x g, 10 min). The floating protoplast band was diluted in the electroporation buffer. The transfected protoplasts were cultured in the same medium previously described for leaf protoplasts [23].

2.4. Protoplast transfection. PEG-mediated DNA uptake. The transfection of protoplasts mediated by polyethyleneglycol (PEG) was adapted from Negrutiu et al. [17]. Aliquots of 500 #1, containing 0.5-1 x 10 6 protoplasts per ml in the appropriate culture medium (depending on the original tissue from which they were derived) were incubated with variable amounts of the p35SGUS plasmid DNA (10-40/zg), using as a carrier 50 t~g of herring sperm DNA. After 5 min, a PEG solution (40% PEG 3400, 0.4 mannitol, 0.1 M Ca(NO3)2, pH 8) was added to a final concentration of 15% PEG. Incubation was at 25°C for 20 min. This transformation mixture was gradually diluted in the solution described by Krens et al. [25] and centrifuged. Finally, protoplasts were washed with the corresponding medium and cultured for 12-48h in the dark at 27°C until sampiing for GUS determination. Electroporation-mediated DNA uptake. Electroporation was performed with the Electro Cell Manipulator 600 (BTX electroporation system). Aliquots of 400 ~1, containing 0.5-2 x 106 protoplasts per ml in 0.65 M mannitol, were mixed with 20-30 /~g of the p35S-GUS plasmid DNA and 50/~g of herring sperm DNA as a carrier, in prechilled cuvettes with a 2 mm gap between electrodes and incubated on ice for 10 min. Optimization of electroporation conditions for transfecting protoplasts from different tissues, was performed applying fields ranging from 250 up to 750 V/cm by discharge of 200 or 500/~F capacitors. The pulse length varied from 10 to 25 ms. The electroporated samples were diluted 20 times with the corresponding culture medium, centrifuged, and

181

incubated for 20-36 h in the dark at 27°C and harvested.

2.5. Protoplast viabili O' The number of surviving protoplasts were counted with a Neubauer chamber. Dead protoplasts were clearly distinguishable by their condensed appearance and dark color. Viability was confirmed by staining with the Evans blue dye [261. 2.6. GUS expression and protein assays Transfected protoplasts were collected from the incubation medium by centrifugation and lysed in the extraction buffer reported by Jefferson [19]. The extract was centrifuged and GUS determined in the supernatant, using 4-methyl umbelliferyl flglucuronide (MUG) as substrate [19]. Activity was expressed as pmol of 4-methyl umbelliferone (MU) per min per mg of total protein or per 106 protoplasts. Protein concentrations were determined with the BioRad Kit using bovine serum albumin as standard. 3. Results and discussion

3.1. Protoplasts isolation from d~[~rent cereal tissues The procedures described here allow the routine isolation of metabolically active protoplasts from developing endosperm, aleurone, leaves and roots of barley and wheat. Each tissue required a specific combination and concentration of enzymes and other parameters for maximum yield of isolated cells. The age and growth conditions of the tissues has a dramatic effect on the final yield of viable protoplasts and on their transient expression activity. The easiest to obtain were protoplasts from leaves and the more delicate to handle were those derived from developing endosperm. No viable protoplasts from endosperm could be obtained after 20 dap [14]. The aleurone protoplasts, after isolation of the aleurone layers, were not difficult to prepare and manipulate. In the case of leaf tissues, the protoplast yield was higher from seedlings grown in the dark for 6 days than from green leaves with well developed chloroplasts. Root pro-

182

toplasts could be obtained only from the apical portion ( - l0 ram) of the main roots. The number of viable protoplasts sharply decreased when roots were older than 2 days. The distinctive phenotypes of these four types of barley protoplasts are illustrated in Fig. 1. Protoplasts isolated from developing endosperms were big and vacuolated with an average size of 35 #m in diameter and contained large amounts of starch granules (Fig. la), which can easily damage cell membranes during isolation and transfection [14,15]. Some steps are crucial for -

L Dia--/Plant Sci. 96 (1994) 179-187

obtaining a good endosperm protoplast preparation: (i) selection of endosperms early after pollination (8-15 days) in order to keep the starch content as low as possible; (ii) preplasmolysis of the intact endosperms in CPW salts [22] with 11% (w/v) mannitol (CPW 11 M medium) to allow the separation of the cell wall from the cytoplasmic membrane before digestion with 0.4"/,, (w/v) Cellulase Onozuka RS for 14 h; (iii) a gravity sedimentation step after the enzymatic treatment; (iv) purification of the viable protoplasts from the starch granules and other cell debris by a gentle

Fig. I. Protoplasts from different tissues of barley (Hordeum vulgare L. cv Bomi). (a) Developing starchy endosperm protoplasts (I 3 dap), (b) aleurone protoplasts, (c) etiolated leaf protoplasts, (d) root protoplasts (bar = 10 /~m except in b where bar = 15 #m).

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L Diaz/Plant Sci. 96 (1994) 179-187

centrifugation (60 × g, 5 min) onto a 50% Percoll cushion. With this procedure a high yield of viable endosperm protoplast is obtained, about 3 x 10 6 protoplasts/100 developing kernels, both from barley (cvs Bomi and Riso 1508) and from wheat (cv Chinese Spring) (Table 1). Protoplasts (pp) derived from the aleurone layer, after 24 h water imbibition, were - 30/~m in diameter, showed a condensed cytoplasm (Fig. lb) and their cell membrane was quite resistant to manipulation. Compared to endosperm, aleurone cells tolerated higher concentrations of hydrolytic enzymes (4.5% vs 0.4% cellulase) and higher centrifugal forces. In the aleurone protoplasts cyclosis was frequently observed an indication of high metabolic activity. The average yields of aleurone protoplasts from barley cvs Bomi and Riso 1508 and from wheat cv Chinese Spring (Table 1) varied between 2.7 and 1.7 × 10 6 pp/100 kernels, a value which was only 2 - 3 times lower than that reported by Salmenkallio et al. [12] for barley cv Himalaya, a cultivar in which the aleurone layer is large and easy to separate from the starchy endosperm. The highest viability figure, about 90%, was obtained for barley aleurone protoplasts. Protoplasts isolated from etiolated leaves averaged 20/~m in diameter and had preplastids and small vacuoles in their cytoplasm (Fig. lc).

Yields of 3.5 x 10 6 pp per g of fresh tissue were consistently obtained (Table 1). The optimum cellulase concentration to be used with this tissue is 1%, an intermediate value of those needed to prepare endosperm and aleurone protoplasts. Low concentrations of Macerozyme R10 (0.37°/,, w/v) increased the yield by about 20%. Wheat and barley leaf protoplasts tolerated higher centrifugation speeds than those derived from endosperm and aleurone. Similar yields and viability figures were obtained with rice leaf protoplasts (data not shown). Root protoplasts were small, averaging 16/~m in diameter (Fig. ld), showed a dense cytoplasm and were quite resistant to external manipulations, tolerating centrifugal forces of 110 x g for 10 min. Binary combinations of three enzymatic preparations were tested: Rhozyme HP150, Cellulase Onozuka RS and Pectolyase Y23. The highest yield of metabolically active root protoplasts was obtained with a mixture of 4% (w/v) Cellulase Onozuka RS and 0.3% (w/v) Pectolyase Y23 (She±shin Pharmaceuticals). Only the most apical part ( = 10 mm) of the main roots produced good protoplast preparations but yield was low: about 0.8 x 10 6 per g of fresh tissue in barley, about 0.6 × 106 in wheat and 0.4 x 106 in rice (data not shown).

Table 1 Yield and viability of protoplasts isolated from different tissues of barley and wheat Tissue

Endosperm Aleurone Leaves Roots

Species

14. vulgare cv Bomi H. vulgare cv Riso 1508 72 aestivum cv Chinese Spring H. vulgare cv Bomi H. vulgare cv Riso 1508 7". aestivum cv Chinese Spring H. vulgate cv Bomi H. vulgare cv Riso 1508 T. aestivum cv Chinese Spring H. vulgare cv Bomi 72 aestivum cv Chinese Spring

Protoplasta.b Yield ( x 10 6)

Viability(%)

2.9 + 3.2 + 3.1 + 2.3 ± 2.7 + 1.7 + 3.6 + 3.1 + 3.5 ± 0.7 + 0.5 ±

75 81 70 92 90 83 86 78 83 87 81

0.6 0.3 0.7 0.9 0.8 0.8 0.3 0.4 0.3 0.5 0.6

aprotoplast yield resulted from either 100 developing kernels (endosperm), 100 dry kernels (aleurone), or 1 g fresh tissue (leavesand roots). Values are means +S.E.M. of at least three experiments. bprotoplast viability is expressed as percentage of total protoplasts which excluded the Evans blue dye [26].

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Fig. 2. Transient GUS expression after PEG-mediated DNA transfection of endosperm protoplasts. (A) Effect of plasmid concentration on GUS expression from starchy endosperm protoplasts (pmol MU/mg protein/min). (B) Time course of transient GUS activity in protoplasts from starchy endosperm (circles) and aleurone (squares). Thirty/~g of plasmid DNA/106 protoplasts together with 50 #g of carrier DNA was always used. Barley cv Bomi (O, II), wheat cv Chinese Spring (O rl). Data are means of three experiments; bars indicate standard error of the mean (S.E.M.).

3.2. T r a n s i e n t g e n e e x p r e s s i o n Conditions were optimized for DNA uptake a n d t r a n s i e n t e x p r e s s i o n in p r o t o p l a s t s u s i n g t h e p35S-GUS reporter gene. The same plasmid d e v o i d o f t h e 35S p r o m o t e r w a s u s e d as t h e n e g a tive c o n t r o l a n d its e x p r e s s i o n v a l u e w a s s u b -

t r a c t e d in all cases. P E G - m e d i a t e d u p t a k e a n d electroporation were the two transfection methods used. T h e e l e c t r o p o r a t i o n m e t h o d c o m p a r e d w i t h the PEG procedure was approximately three times as e f f i c i e n t in t e r m s o f f i n a l G U S a c t i v i t y p e r / ~ g o f plasmid DNA for leaf and root protoplasts, while

Table 2 Transient GUS expression in protoplasts isolated from different tissues of barley and wheat a Tissue

Endosperm (PEG) Aleurone (PEG) Leaves (E) Roots (E)

Barley

Wheat

cv Bomi

cv Riso 1508

cv Chinese Sring

4365 2415 5320 3720

5224 + 23 2126 + 30 4716 + 17 --

4250 1849 4650 3014

+ + + +

16 19 20 23

+ 4+ +

31 27 14 31

DNA uptake was PEG mediated in endosperm and aleurone protoplasts. Electroporation was used in leaves (625 V/cm, 200 #F, 10 ms) and roots (625 V/cm, 500 ~F, 25 ms). Thirty/zg and 20 #g of plasmid DNA were used in the PEG and in the electroporation mediated transfections, respectively. aGUS activity 24 h after transfection with p35S-GUS is expressed as pM methylumbelliferone/106 protoplasts/min + S.E.M. (average of at least 3 experiments).

1. Diaz / Plant Sci. 96 (1994) 179-187

185

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Fig. 3. Transient GUS expression after electroporation mediated DNA transfection of leaf protoplasts. Effect of input voltage (V/cm) on (A) GUS gene expression determined 24 h after discharging a 200 #F capacitor onto 106 protoplasts. Values are means of three experiments; bars indicate S.E.M. (B) Viability expressed as % of total protoplasts that excluded the Evans blue dye. Twenty t~g of plasmid DNA/106 protoplasts plus 50 ttg of carrier DNA was always used. Barley cv Bomi (O), wheat cv Chinese Spring (O).

the PEG-mediated DNA uptake [14,15] was the only possibility for endosperm transfection since even the mildest electroporation conditions assayed (250 V/cm, 200 /~F, 10 ms) produced > 90% cell mortality (data not shown). GUS activity in transfected endosperm protoplasts was directly proportional to DNA concentration in the transfection mixture up to 20/xg of plasmid DNA, reaching a plateau between 20 and 40 t~g (Fig. 2A). Thus 20 or 30/~g of plasmid (for electroporation or PEG-mediated transfection, respectively) and 50/~g of herring-sperm DNA were always used routinely (10 6 p r o toplasts). Maximum GUS activity in PEG-transfected endosperm and aleurone protoplasts from barley and wheat was detected 24 h after incubation (Fig. 2B). This incubation time had been reported as the optimum for oat mesophyll protoplasts [27] and for rapeseed hypocotyl protoplasts, although protoplasts derived from cell suspensions of either carrots or soybean showed a

maximum activity after 66 to 72 h after transformation [10]. In aleurone protoplasts the GUS activity per mg protein after PEG transfection with 30/zg of plasmid DNA was approximately 1/3 of that measured using the same conditions for endosperm protoplasts (Fig. 2B) while it was about 50% of the endosperm value when referred to 106 protoplasts (Table 2). To optimize the electroporation procedure for transient expression in leaf protoplasts of barley and wheat, we varied the input voltages between 375 and 750 V/cm, keeping the capacitor at 200 /~F, which automatically fixed the pulse length at 10 ms (Fig. 3A). The last two parameters (200/~F and 10 ms) had been described as the optimal for barley mesophyll protoplasts [28]. In our hands, maximal GUS activity with barley and wheat protoplasts was at 625 V/cm (Fig. 3A). At lower than 500 V/cm GUS gene expression diminished and at higher voltages (750 V/cm), very few protoplasts

186

I. Diaz/Plant Sci. 96 (1994) 179-187

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500/,tF

800

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200 #F

T E

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o

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i

i

i

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375

500

625

750

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i

i

625

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Fig, 4. Transient GUS expression after electroporation mediated DNA uptake by root protoplasts. Effect of input voltage (V/cm) on (A) activity determined 24h after discharging 200/~F (10 ms) or 500 #F (25 ms) capacitors. Values are means of three experiments; bars indicate S.E.M. (B) Viability expressed as % of protoplasts that excluded the Evans blue dye. Twenty t~g of plasmid DNA/106 pp plus 50/~g of carrier DNA was always used. Barley cv Bomi (O), wheat cv Chinese Spring (O).

(25%) remained alive 24 h after electroporation (Fig. 3B). It appeared that optimum GUS activity occurred when approximately 50% of the protoplasts were killed by the electrical treatment. Similar results have been reported for protoplasts isolated from different tissues of potato [29]. Consequently, wheat and barley leaf protoplasts were electroporated at 625 V/cm, 200/zF, 10 ms, using 20/~g of plasmid DNA per 106 protoplasts. No previous reports have been published on isolation and transient expression of protoplasts derived from root tissues of barley and wheat. To test the conditions that resulted in high GUS activity, electrical fields ranging from 375 to 750 V/cm were applied to protoplasts from young roots, by discharging 200 and 500 /~F capacitors which determined pulse durations of 10 and 25 ms, respectively. The highest GUS expression was achieved when using 20/~g of plasmid DNA and electroporating at 625 V/cm, 500 #F, 25 ms: 630 pmol MU/mg protein x minute (Fig. 4A), although the protoplast viability measured under

these conditions was about 40% lower than that obtained after a similar discharge from a 200/~F capacitor (Fig. 4B). Since these parameters (625 V/cm, 500/zF, 25 ms) have been also successfully applied to protoplasts isolated from roots and leaves of rice (data not shown), it can be concluded that root protoplasts from these three small grain cereals are more resistant to electrical discharge than leaf protoplasts. These methods are being used for the functional analysis of tissue specific cereal promoters, it seems unlikely that these procedures are useful for stable transformation in barley and wheat since the protoplasts of the different tissues have not been routinely regenerated into plants.

4. Acknowledgements Acknowledgements are gratefully due to Professor Pilar Carbonero for advice and useful discussions throughout this work and L. Lamoneda and J. Garcla for technical assistance in

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the preparation of the manuscript. Financial support by the Spanish Commission Interministerial de Ciencia y Tecnologia (grant Bio91-0782) and Ministerio de Educaci6n y Ciencia (grant PB89-

14

0190) a r e a l s o a c k n o w l e d g e d . 15

5. References 1

2 3

4

5

6

7

8

9

10

11

12

13

I.K. Vasil, Progress in the regeneration and genetic manipulation of cereal crops. Biotechnology, ~, (1988) 397-402. I. Potrykus, Gene transfer to cereals: an assessment. Biotechnology, 8 (1990) 535-542. C.A. Rhodes, D.A. Pierce, l.J. Mettler, D. Mascarenhas and J.J. Detmer, Genetically transformed maize plants from protoplasts. Science, 240 (1988) 204-207. K. Shimamoto, R. Terada, T. lzawa and H. Fujimoto, Fertile transgenic rice plants regenerated from transformed protoplasts. Nature, 338 (1989) 274-276. S.K. Dana, A. Peterhans, K. Datta and I. Potrykus, Genetic engineered fertile indica-rice recovered from protoplasts. Biotechnology, 8 (1990) 736-740. V. Vasil, A.H. Castillo, M.E. Fromm and I.K. Vasil, Herbicide resistant fertile transgenic wheat plants obtained by microprojectile bombardment of regenerable embryogenic callus. Biotechnology, 10 (1992) 667-674. D.A. Somers, H.W~ Rines, W. Gu, H.F. Kaeppler and W.R. Bushnell, Fertile, transgenic oat plants. Biotechnology, 10 (1992) 1589-1594. M.G. Koziel, G.L. Beland, C. Bowman, N.B. Carozzi, R. Crenshaw, L. Crossland, J. Dawson, N. Desai, M. Hill, S. Kadnell, K. Launis, K. Lewis, D. Maddox, M. McPherson, M,R. Heghji, E. Merlin, R. Rhodes, G.W. Warren, M. Nright and S.V. Evola, Field performance of elite transgenic maize plants expressing an insecticidal protein derived from Bacillus thuringiensis. Biotechnology, 11 (1993) 194-200. A. Ritala, L. Mannonen, K. Aspegren, M. SalmenkallioMarttila, U. Kurten, R. Hannus, J. Mendez-Lozano, T.H. Teeri and U. Kauppinen, Stable transformation of barley tissue culture by particle bombardment. Plant Cell Rep., 12 (1993) 435-440. J.O. Rasmussen and O.S. Rasmussen, PEG mediated DNA uptake and transient GUS expression in carrot, rapeseed and soybean protoplasts. Plant Sci., 89 (1993) 199-207. W.R. Marcotte Jr, C.C. Bayley and R.S. Quatrano, Regulation of a wheat promoter by abscisic acid in rice protoplasts. Nature, 335 (1988) 454-457. M. Salmenkallio, R. Hannus, T.H. Teeri and V. Kauppinen, Regulation of u-amylase promoter by gibberellic acid and abscisic acid in barley protoplasts transformed by electroporation. Plant Cell Rep., 9 (1990) 352-355. R.H. Hamptmann, P. Ozias-Akins, V. Vasil, Z. Tabaeizadeh, S.C. Rogers, R.B. Horsch, I.K. Vasil and

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17

18

19

20

21

22

23

24

25

26

27

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R.T. Traley, Transient expression of electroporated DNA in monocotyledonous and dicotyledonous species. Plant Cell Rep., 6 (1987) 265-270. 1. Diaz and P. Carbonero, Isolation of protoplasts from developing barley endosperm: A tool for transient expression studies. Plant Cell Rep., 10 (1992) 595-598. 1. Diaz, J. Royo and P. Carbonero, The promoter of barley trypsin-inhibitor BTI-CMe, discriminates between wheat and barley endosperm protoplasts in transient expression assays. Plant Cell Rep., 12 (1993) 698-701. J. Denecke, V. Gossele, J. Botterman and M. Cornelissen, Quantitative analysis of transiently expressed genes in plant cells. Methods Mol. Cell. Biol., 1 (1989) 19-27. 1. Negrutiu, R. Shillito, 1. Potrykus, G. Biosini and F. Sala, Hybrid genes in the analysis of transformation conditions 1. Setting up a simple method for direct gene transfer in plant protoplasts. Plant Mol. Biol., 8 (1987) 363-373. M.R. Davey, E.L. Rech and B.J. Mulligan, Direct DNA transfer to plant cells. Plant Mol. Biol., 13 (1989) 273-285. R.A. Jefferson, Assaying chimeric genes in plants: The GUS gene fusion system. Plant Mol. Biol. Rep., 5 (1987) 387-405. B. Reiss, R. Sprengel, H. Will and H. Schaller, Protein fusions with the kanamycin resistance gene from Tn5. Gene, 30 (1984) 211-218. D.W. Ow, K.V. Wood, M. DeLuca, J.R. de Wet, D.R. Helinski and S.H. Howell, Transient and stable expression of the firefly luciferase gene in plant cells and transgenic plants. Science, 234 (1986) 856-859. J.B. Power and J.V. Chapman, Isolation, culture and genetic manipulation of plant protoplasts, in: R.A. Dixon (Ed.), Plant Cell Culture, IRL Press, 1985, pp. 37-66. J.V. Jacobsen, J.A. Zwar and P.H. Chandler, Gibberellic acid responsive protoplasts from mature aleurone of Himalaya barley. Planta, 163 (1985) 430-438. H.S. Gupta and A. Pattanayak, Plant regeneration from mesophyll protoplasts of rice (Ory:a sativa L.). Biotechnology, 11 (1993) 90-94. F.A. Krens, L. Molendijk, G.J. Wullerus and R.A. Schilperoot, In vitro transformation of plant protoplasts with Ti-plasmid DNA. Nature, 296 (1982) 72-74. D.F. Gaff and O. Okong'O-ogola, The use of nonpermeating pigments for testing the survival of cells. J. Exp. Bot., 22 (1971) 756-758. D.C. Higgins and J.T. Colbert,/3-glucuronidase gene expression and mRNA stability in oat protoplasts. Plant Cell Rep., 12 (1993) 445-452. T.H. Teeri, G.K. Patel, K. Aspegren and V. Kauppinen, Chloroplast targeting of neomycin phosphotransferase I1 with a pea transit peptide in electroporated barley protoplasts. Plant Cell Rep., 8 (1989) 187-190. H. Jones, G. Ooms and M.G.K. Jones, Transient gene expression in electroporated Solanum protoplasts. Plant Mol. Biol., 13 (1989) 503-511.