WO1995026628A1

WO1995026628A1 - Regeneration of green plants from protoplasts isolated from microspore cultures of barley

Info

Publication number: WO1995026628A1
Application number: PCT/FI1995/000186
Authority: WO
Inventors: Marjatta Salmenkallio-Marttila; Ulrika KURTÉN; Leena Mannonen; Anneli Ritala; Veli Kauppinen
Original assignee: Valtion Teknillinen Tutkimuskeskus
Priority date: 1994-04-05
Filing date: 1995-04-05
Publication date: 1995-10-12
Also published as: AU2216895A; FI941551A; FI941551A0

Abstract

The present invention relates to plant biotechnology and specifically to a method for regeneration of fertile plants from protoplasts isolated from cultured microspores of barley. Direct gene transfer methods can be used to transform the protoplasts with foreign gene(s), whereby regeneration of the transformed protoplasts provide transgenic green plants.

Description

Regeneration of green plants from protoplasts isolated from microspore cultures of barley

Field of the invention

The present invention relates to plant biotechnology and specifically to a method for regeneration of fertile plants from protoplasts isolated from cultured microspores of barley. Although the method has been developed for barley, it is applicable also for the other cereals for which microspore and anther culture methods have been developed, e.g. wheat, rye, oat, maize and rice. Direct gene transfer methods can be used to transform the protoplasts with foreign gene(s), whereby regeneration of the transformed protoplasts provide transgenic green plants.

Background of the invention

Eventhough representatives of most of the world's crop plants have been regenerated from protoplasts, the regeneration of a whole plant from single protoplasts still remains a problem (Lazzeri et al. 1990, Roest & Glissen 1993). Regeneration methods often apply only to particular varieties or even genotypes. Cells or tissues that in themselves are regenerable are most likely the ones that yield protoplast cultures capable of regenerating new plants. Therefore most of these methods are based on the production of embryogenic suspension cultures.

Methods for protoplast regeneration have been developed for the major cereals rice (Fujimura et al. 1985), maize (Rhodes et al, 1988a), wheat (Vasil et al. 1990), sorghum (Wei and Xu 1990) and barley (Yan et al. 1990, Jahne et al. 1991). Key steps in these methods are the production of suspension cultures that are a good source of protoplasts while they still retain their regeneration capacity. The establishment of regenerable cell suspension cultures of barley has proven difficult as is the case with other gramineous species. Friable embryogenic callus that is suitable for the initiation of suspension cultures is in the case of barley difficult to obtain; the establishment of suspension cultures usually takes at least six months and only a fraction of the suspension culture lines yield regenerable protoplasts. Moreover, the morphogenic capacity of suspension cultures decreases during long periods of culture (Lϋhrs and Lδrz 1988, Datta et al. 1992, Jahne et al. 1991).

Plant biotechnology provides new possibilities for the improvement of crop plants. However, cereals have proved to be very recalcitrant to genetic engineering. At present there is no satisfactory method available for routine transformation of barley or other cereals (Potrykus 1991, Karp & Lazzeri 1992). The failure to achieve Agrobacterium-ind ced transformation in cereals has led to increased interest in other methods for the production of transgenic plants, such as the use of particle bombardment (Mendel et al. 1989, Ritala et al. 1994) and direct DNA-uptake into cells or protoplasts stimulated either electrically or chemically (Lazzeri et al. 1991).

In addition to the delivery and integration of foreign DNA into the target cell, a successful transformation also implies the regeneration of the cell to a new plant and production of progeny with the transferred new trait. This means that one basic requirement for the use of any of these transformation methods is an efficient culture and regeneration system that guarantees regeneration of fertile plants from single transformed cells.

For the genetic modification of plants protoplasts are of great value. Protoplasts can be transformed by direct gene transfer, uptake of DNA through the plasma membrane stimulated either chemically or electrically. When using multicellular explant systems in transformation, as is the case with e.g. particle bombardment, chimaeric calli and plants may be produced as a result, while using protoplast systems for transformation, true transgenic clones can be obtained. The problem with the recovery of transgenic plants from protoplasts relates to plant regeneration, not to the transformation methods (Potrykus 1991).

Transient gene expression in protoplasts has been used to optimize gene transfer methods and to study the function of regulatory elements of plant genes. Transgenic rice (Toriyama et al. 1988, Zhang & Wu 1988, Zhang et al. 1988) and maize (Rhodes et al. 1988b) plants have been obtained following direct DNA transfer to protoplasts. Transgenic callus lines of barley but no plants have been obtained using PEG-induced direct DNA uptake into protoplasts (Lazzeri et al. 1991).

At the moment plant regeneration remains the major limitation to the application of genetic engineering of plant cells to crop improvement. The currently used gene transfer techniques require the use of embryogenic suspension cultures, which are rather difficult to establish and maintain. Barley plants have been regenerated from protoplasts derived from embryogenic suspension cultures (Yan et al. 1990, Jahne et al. 1991, Funatsuki et al. 1992). However, considering the use of the protoplast system for transformation, the relatively unstable plant regeneration reported in these studies remains a problem.

Summary of the invention

We have now developed a method for the production of microspore cultures, from which regenerable protoplasts can be isolated, thus avoiding the problems related to embryogenic suspension cultures. With our protocol good plant production from protoplasts was obtained repeatedly and plantlets regenerated from protoplasts were potted in soil within 4-5 months of collecting the spikes for microspore culture. The protoplasts obtained with this method have been successfully used for the production of transgenic barley plants.

Detailed description of the invention

The present inventors have now found that from a microspore mass cultured in an optimized medium regenerable protoplasts can be isolated with a high yield. From the isolated protoplasts green plants can be regenerated by cultivating in appropriate media. When the isolated protoplasts are transformed with foreign genes, transgenic barley plants are obtained after regeneration. The present invention thus provides a method for generating fertile barley plants, comprising cold treatment of barley spikes, isolating microspores from the cold treated spikes, culturing the isolated microspores as a suspension in a medium containing maltose, nitrogen sources and growth regulators, isolating protoplasts from the microspore mass obtained using conventional methods, cultivating the said protoplasts in a medium containing maltose, a nurse culture and growth regulators, and regenerating the protoplasts to generate fertile barley plants.

In another aspect the invention provides a method for generating transgenic barley plants, comprising culturing microspores isolated from cold treated barley spikes in a medium containing maltose, nitrogen sources and growth regulators, isolating protoplasts from the microspore mass obtained using conventional methods, transforming the isolated protoplasts with foreign gene(s), and cultivating the transformed protoplasts in a medium containing maltose, a nurse culture and growth regulators, and regenerating the protoplasts to generate transgenic barley plants.

The plant material used in the present invention is obtained by growing barley grains (Hordeum vulgare L.) e.g. in a growth room for a few weeks, preferably about 6 to 8 weeks, until the spikes of the tillers are developed to have microspores in the late uninucleate to early binucleate stage. The harvested tillers are cold treated by keeping them at a temperature under 10 °C, preferably at 7 °C for 1 to 4 weeks, preferably about 2 to 4 weeks. The optimal cold treatment time is 3 weeks.

The microspores are isolated from the cold treated spikes by methods known in the art. The spikes are e.g. cut into pieces and macerated with a teflon rod or, alternatively, by machine in a blendor. Different appropriate buffers and media can be used in the isolation. In this work the 108-medium, supplemented as described in the examples, was used. The crude preparation obtained is subsequently filtered to separate the microspores from the debris and the microspores are collected in an appropriate medium for cultivation. The isolated microspores are then cultured for 2 to 4 weeks to get an appropriate amount of microspore mass for further prosecution. In the culturing procedure of the microspores there are four factors which have importance in view of the subsequent protoplast isolation. These factors are the cultivating time, the maltose concentration of the culturing medium, the growth regulators used, and the amount and nature of the nitrogen source in the medium.

After 2 weeks of culture the amount of the microspore mass is still rather small, but it will increase tenfold within the third week. A preferable culturing time is from 3 to 4 weeks. According to the experiments in the present work protoplast yields obtained were on the average 12 x 10⁶ per g fresh weight when cultivating the microspores for 3 weeks and on the average 3 x 10⁶ per g fresh weight as the cultivation time was 4 weeks. As the microspore mass is further doubled within the fourth week, a most preferable cultivating time proved to be from 22 to 24 days.

Maltose is a preferable carbon source in the microspore cultivating medium. The amount of maltose in the medium is a critical factor when protoplasts are to be isolated from the microspore mass. An appropriate amount is from about 0.150 M to about 0.200 M. Concentrations higher than 0.200 M decrease the protoplast yield substantially. A preferable concentration according to the present invention is about 0.175 M.

The growth regulators are also important in the microspore culture medium affecting e.g. the differentiation as well as the division (callus formation) of the cells. In conventional methods 6-benzylaminopurine (BA) is often the only growth regulator used. A preferable medium used in this invention comprises 2,4-D (2,4-dichloro- phenoxyacetic acid), which increases the division of the cells, and kinetin (KIN), which affects the differentiation of the cells. Appropriate concentration of 2,4-D in the microspore culture medium is from about 4.5 μM to about 22.6 μM, a preferable concentration being about 7.9 μM. For kinetin an appropriate concentration is from about 0.5 μM to about 4.6 μM, a preferable concentration being about 1.2 μM. As inorganic nitrogen sources in the microspore culture medium e.g. NO₃ and NH₄ can be used. In the N6 basal medium described by Chu et al. (1975) the ratio and concentration of NO₃ and NH₄ are optimal for the purposes of the present invention. Thus said medium supplemented with the growth regulators discussed above, and further with glutamine as an organic nitrogen source, is preferable in the method of this invention.

Protoplasts are isolated from the microspore mass by methods analogous to the prior known protoplast isolation procedures. The isolation can be performed in a medium described e.g. by Lazzeri et al. (1991) with appropriate lytic enzymes, e.g. cellulase, macerozyme and pectolyase. After a few hours' incubation the suspension is filtrated and the protoplasts are washed with the same medium and centrifuged.

The protoplasts so obtained can be used for transformation with direct gene transfer methods, e.g. electroporation or PEG-transformation. These techniques are well known in the art. Subsequent regeneration of the transformed protoplasts produces plants carrying the foreign gene in all of their cells.

For culture the protoplasts are suspended at a density of 1-3 x 10⁶ per ml in an appropriate medium containing agarose as gelling agent. The suspension is plated on culture plate inserts or on Petri dishes. After the agarose has solidified, a nurse culture is added to the plates to support the division of the protoplasts. The nurse cultures are obtained by suspending an aliquot of the microspore mass or an appropriate suspension culture to the protoplast medium used (without the gelling agent). The importance of nurse cultures for the regeneration of barley protoplasts was evident, as the protoplasts failed to divide without the support of nurse cells.

In the protoplast culture and regeneration procedure same factors have importance as in the microspore culturing method, i.e. the maltose concentration of the culturing and regeneration media, the growth regulators used, the amount and nature of the nitrogen source(s), and the time of cultivation in the different media. Maltose is the preferable carbon source for the culture and regeneration of protoplasts. The appropriate amount in the protoplast medium is about 0.50 to about 0.65 M. For the division of protoplasts the optimal composition of growth regulators is 2.3 μM 2,4- D. The addition of coconut water into the protoplast medium is also important for the division and growth of the protoplasts.

Appropriate regenerating media for the protoplast cultures are e.g. the regeneration media II and III, which are the modified regeneration media II and III, respectively, described by Olsen (1987). However, preferable media according to this invention contain maltose instead of sucrose. For the regeneration of plants the essential factors are the sequential lowering of the sugar concentration of the medium and the addition of the plant growth regulator 6-benzyl-aminopurine, which promotes the differentiation of the proembryos. The plantlets regenerated on medium II are transferred to medium III for rooting and further development. The essential features of this medium are the low concentration of sucrose and the omission of plant growth regulators.

High concentrations of sugar are used to adjust the osmotic pressure of the protoplast culture medium. The type of sugar used is important also for other than osmotic reasons. For the culture of protoplasts isolated from barley microspores maltose was superior to glucose at same osmolality. In media containing glucose the protoplasts divided poorly, the plating efficiencies were only a few percent of the ones obtained with maltose and no plants were regenerated.

Brief description of the drawings

Fig. 1 The vector pHTT303 used in transformation of barley containing the marker gene nptll coding for neomycin phosphotransferase II under the control of cauliflower mosaic virus 35S transcript promoter.

Fig. 2 The vector pKAH36 used in transformation of barley containing the gene egll of Trichoderma reesei coding for endo-β-glucanase under the control of barley α-amylase promoter. Fig. 3 The vector pKAH21 used in Example 5 containing the gene egll of Trichoderma reesei coding for endo-β-glucanase under the control of cauliflower mosaic virus 35S transcript promoter

Fig. 4 NPTII activity in leaf extracts of transgenic barley plants regenerated from protoplasts. Lane NC represents a negative control from a non- transformed barley plant and lane PC a positive control from tobacco transgenic for 35S-nptII. Samples 1, 2 and 3 represent leaf extracts of three transgenic barley plants regenerated from protoplasts.

The following examples describe the procedures used in the present invention.

Experimental

Example 1. Isolation and culturing of microspores

Plant material. Barley grains (Hordeum vulgar e L. cv. Kymppi, an elite cultivar of spring barley) were obtained from Kesko Agronomic Station, Hauho. Plants were grown in a growth room (22/13°C day/night, 16 h light 4000 μmol πrY¹). The duration of the growth period was 6-8 weeks. The tillers were harvested when the sheath of the flag leaf had emerged 5-10 cm, which correlates with the late uninucleate to early binucleate stage of microspore development. The tillers were wrapped in aluminium foil and placed in beakers in a cold room at 7°C for 3-4 weeks.

Microspore isolation and culture. Microspores were isolated and cultured in 108- medium (Table 1), which is the N6 basal medium (Chu et al 1975) supplemented with 1.1 mM of glutamine, 2.8 mM of m-inositol, 0.175 M of maltose, 7.9 μM of 2,4-D and 1.2 μM of kinetin. For microspore isolation ten spikes were cut into 3-4 pieces and macerated with a teflon rod in the 108-medium. The crude microspore preparation was then filtered through an 80 μm nylon sieve and the microspores were collected by centrifugation for 5 min at 300 x g. The microspores were resuspended in 9 ml of the same medium and the total number of microspores and the number of embryogenic microspores were counted in a Fuchs-Rosenthal haemo- cytometer. The number of microspores isolated per spike varied between 0.7 and 1.6 x 10^s with a mean of 1.1 x 10⁵ per spike. The microspores were plated as crude preparations and their viability varied between 33 % and 53 % (mean 42 %). The microspores were cultured in 108-medium at a density of 0.8-1.0 x 10⁵ ml^"1 in 5 cm Petri dishes in the dark. The cultures were incubated stationary for the first 1.5 weeks and in a rotary shaker for another 1.5 weeks (65 rpm, stroke radius 2.5 cm).

Table 1. Composition of the medium 108 for microspore isolation and culture

Macroelements mg/1 Iron mg/1

KN0₃ 2830 FeS0₄.7H₂0 27,8

CaCl₂.2H₂0 166 Na₂EDTA.2H₂0 37,3

KH₂P0₄ 400

(NH₄)₂SO₄ 463

MgSO₄.7H₂O 185

Microelements mg/1 Vitamins mg/1

MnS0₄.4H₂0 4.4 Nicotinamide 0.5

H₃BO₃ 1.6 Thiamine-HCl 1.0

ZnSO₄.7H₂O 1.5 Pyridoxine-HCl 0.5

KI 0.8

Amino acids mg/1 Growth regulators mg/1

Glutamine 160 2,4-D 1.75

Glycine 2 Kinetin 0.25

Carbohydrates mg/1

- pH 5.8

Maltose 63000 - sterilized by filtration thr ough 0.22

/n-Inositol 500 μm membrane

Example 2. Isolation and culturing of protoplasts, and regeneration of plants

Protoplast isolation. Protoplasts were isolated from three to four weeks old cultures of isolated microspores according to the protocol of Lazzeri et al. (1991) for barley suspension cultures. 2-3 g of microspore mass were incubated in 20 ml of enzyme solution containing 1.0% cellulase Onozuka RS, 0.5% Macerozyme RIO and 0.05% pectolyase Y23 in washing solution (LW, Table 2). After 2-3 h incubation the suspension was diluted with equal volume of LW solution and filtrated through 160 μm, 55 μm and 20 μm nylon sieves. The protoplasts were washed twice with LW (100 x g, 5 min). The protoplasts were treated with heat shock by incubating the protoplast suspension in 45°C water bath for 5 min and then cooled on ice. After incubation in the cold room at 7 °C for approximately two hours the protoplasts were collected by centrifugation (100 g, 5 min) and either plated for regeneration or used for transformation experiments.

Protoplast culture. The protoplasts were suspended in Ll-protoplast medium (Table 2) containing 2.3 μM of 2,4-D, 0.5 M of maltose and 1.2% agarose (Sea- Plaque^®, FMC Corporation) at a density of 2-3 x 10° ml^-1. The suspension was plated on Millicell™-CM culture plate inserts, which were placed in 9 cm Petri dishes containing 8 ml of nurse culture. The nurse cultures were prepared by suspending approximately 0.5 g of the same microspore mass as was used for protoplast isolation in 8 ml of LI protoplast medium without agarose. Cultures were incubated on a rotary shaker (65 rpm, stroke radius 2.5 cm) at 23°C in the dark. After one week of culture the feeder cells were removed and 2 ml of fresh 108 medium (0.175 M maltose, 7.9 μM 2,4-D, 1.2 μM KIN) was added in the plates. After another week of culture the old medium was replaced with fresh 108 medium. After one more week the agarose pieces were transferred on L2 medium (Table 2) solidified with 0.3% gellan gum (Gelrite™, Scott Laboratories). The plating efficiencies were determined as the number of protoplasts producing microcalli in four weeks.

Table 2. Composition of the media used for protoplast isolation and culture.

LW mg/1 LI mg/1 L2 mg/1

Macroelements

NH₄N0₃ 750 750 1500

KH₂PO₄ 200 200 200

KN0₃ 1750 1750 1750

CaCl₂.2H₂O 450 450 450

MgS0₄.7H₂0 350 350 350

Iron

FeS0₄.7H₂0 _ 27.8 27.8

Na₂EDTA.2H₂0 - 37.3 37.3

Microelements

MnSO₄.H₂O 15 15 15

H₃BO₃ 5 5 5

ZnSO₄.7H₂O 13.35 13,35 13.35

KI 0.75 0.75 0.75

NaMoO₄.2H₂O 0.27 0.27 0.27

CuS0₄.5H₂0 0.025 0.025 0.025

CoCl₂.6H₂0 0.025 0.025 0.025

Vitamins

Nicotinic acid 1 1

Thiamine-HCl - 10 10

Pyridoxine-HCl - 1 1

Ascorbic acid - 1 1

Choline chloride - 0.5 -

Ca-pantothenate - 0.5 1

Folic acid - 0.2 - p -Aminobenzoic acid - 0.01 -

Biotin - 0.005 -

Vitamin A - 0.005 -

Vitamin D₃ - 0.005 -

Amino acids

Glutamine 750 750 750

Proline 150 150 150

Asparagine 100 100 100 Table 2. cont.

LW mg/1 LI mg/1 L2 mg/1

Growth regulators

2,4-D - 0.5 2.5

Carbohydrates

Maltose — 180 000 30 000 -Inositol - 100 200

Mannitol 110 000 125 -

Sucrose - 125 -

Mannose - 125 -

, Fructose - 125 -

Ribose - 125 -

Xylose - 125 -

Rhamnose - 125 -

Cellobiose - 125 -

Sorbitol - 125 -

Organic supplements

- 10 -

Coconut water (ml/1) pH 5.7

- LW-medium sterilized by autoclaving, LI- and L2-media sterilized by filtration through 0.22 μm membrane

Plant regeneration. The regeneration media II and III (Table 3) were the modified MS-media II and III, respectively, used by Olsen (1987) for barley anther culture. The medium II contained maltose instead of sucrose (Hunter 1987) and the media were solidified with 0.3% gellan gum. Embryogenic structures developing from the dividing protoplasts were transferred to regeneration medium II for further development. The plates were incubated at 23°C with illumination (50 μmol m^'V¹). Green plantlets were transferred to medium III for further growth and potted in soil when about 10 cm tall. Table 3. Composition of the plant regeneration media II and III.

II mg/1 III mg/1

Macroelements

NH₄N0₃ 165 165

KH₂P0₄ 170 170

KN0₃ 1900 1900

CaCl₂.2H₂O 440 440

MgSO₄.7H₂O 370 370

Iron

FeS0₄.7H₂O 27.8 27.8 Na₂EDTA.2H₂0 37.3 37.3

Microelements

MnS0₄.4H₂0 22.3 22.3

H₃BO₃ 6.2 6.2

ZnSO₄.7H₂O 8.6 8.6

KI 0.83 0.83

NaMoO₄.2H₂O 0.25 0.25

CuS0₄.5H₂O 0.025 0.025

CoCl₂.6H₂O 0.025 0.025

Vitamins

Thiamine-HCl 0.4 0.4

Amino acids

Glutamine 750 750

Growth regulators

6-benzyl-aminopurine 0.4

Carbohydrates -Inositol 100 100

Maltose 35000

Sucrose 20000

- pH 5.6 The microspores isolated from 20 spikes produced approximately one gram of mass in three weeks. Protoplast yield varied between 5 x 10⁶ and 15 x 10⁶ per g fresh weight. Therefore one spike corresponds to approximately 0.5 x 10⁶ protoplasts.

Protoplasts failed to divide without the support of nurse cells. When cultured with nurse cells, the protoplasts started to divide after one week of culture and colonies were visible after 2-3 weeks. Protoplast derived calli resembled very much in appearance the embryogenic calli derived from microspore cultures.

The results from the protoplast regeneration experiments are presented in Table 4. Plating efficiencies obtained in our experiments, between 0.002 % and 0.015 %, are relatively low. However, we obtained good plant production repeatedly. The production of green plants varied from one to 20 per 1 x 10⁶ protoplasts with a mean of 8.3. This is approximately one tenth of the regeneration capacity of the microspore cultures. The plants transferred to soil are morphologically similar to the plants regenerated from microspore cultures and they are fertile.

Table 4. Regeneration of barley protoplasts, summary of seven separate experi¬ ments.

Total number of spikes 100

Total number of protoplasts 55.5 x 10⁶

Total number of microcalli (plating efficiency) 5116 (0.01%)

Number of green and albino plants 459 584

Green plants per 1x10° protoplasts 8.3

Green plants per spike 4.6 Example 3. Effect of the age of the microspore culture on the regeneration of protoplasts

Microspores were isolated and cultured as described in Example 1. 3.0 g of callus mass from microspore cultured for 23 days and 4.5 g of callus mass from microspores cultured for 30 days were used for protoplast isolation. Protoplasts were isolated and cultured as in Example 2. The results of the experiment are presented in Table 5.

Table 5. Effect of the age of the microspore culture on regeneration of protoplasts

Age of culture

23 d 30 d

Yield of protoplasts/g fw 8.0 x 10⁶ 2.7 x 10⁶

Green plants/lxlO⁶ protoplasts 2.5 0.8

Isolation of protoplasts from microspores cultured for three weeks appears to be optimal. At three weeks the yield of protoplasts is high and the protoplasts are highly regenerable. At two weeks there is not yet enough cell mass for protoplast isolation and over five weeks old cultures yield only very low numbers of proto¬ plasts.

Example 4. Transformation of barley protoplasts by electroporation

Protoplasts were suspended in electroporation buffer (0.55 M mannitol, 130 mM KC1, 10 mM NaCl, 4 mM CaCl₂ and 10 mM HEPES^ pH 7.0 or 35 mM aspartic acid monopotassium salt, 35 mM glutamic acid monopotassium salt, 5 mM calcium gluconate, 5 mM MES and 0.55 M mannitol, pH 7.0) at a density of 3-5 x 10⁶/ml. 300 μl samples were mixed with 30 μg of plasmid DNA (pKAH36 or pHTT303) in multi-well dishes (Sterilin 33F24L) and chilled on ice for 10 min before electropo¬ ration. An electrical field of 400-900 v/cm was applied at 0°C by discharge of a 200 μF capacitor that had been charged with an electrophoresis power supply. The protoplasts were kept on ice for 10 min after which the protoplast suspension was mixed with the protoplast culture medium and cultured as described in Example 2. Control protoplasts were not treated with DNA and electroporation. The protoplasts were regenerated without any selective agents in the medium and the leaves of the green plantlets obtained were screened for the transferred trait as described in Example 6.

Table 6. Regeneration of control and transformed protoplast cultures after treatment by electroporation

Treatment No. of pro¬ No. of No. of Transgenic toplasts microcalli green green plants plants pKAH36 Control 5 x 10⁶ 56 113 Sample 1 5 x 10⁶ 36 18 Sample 2 5 x 10⁶ 238 40 6 Sample 3 5 x l0⁶ 143 17 pHTT303 Control 3.25 x 10⁶ 18 Sample 3.25 x 10⁶ 20 3 3

Example 5. Transformation of barley protoplasts by treatment with polyethylene glycol (PEG).

The protoplasts were suspended in F medium (140 mM NaCl, 5 mM KC1, 5 mM HEPES, 5 mM glucose, 125 mM CaCl₂, pH 7.0) at a density of 5 x 10⁶ per ml. The suspension was divided in 0.5 ml aliquots in centrifuge tubes and 50 μg of pKAH36 DNA was added. After 5 min 1 ml of 40% w/v PEG 1500 or PEG 4000 in F solution was added slowly to the protoplast suspension. The mixture was incubated for 15 min with gentle shaking at intervals. 2 ml aliquots of F solution were added four times at 5 min intervals. The protoplasts were then pelleted by centrifugation at 100 g for 5 min and cultured as described in Example 2. Control protoplasts were treated with PEG without addition of plasmid DNA. The transformed protoplasts were cultured without any selective agents in the medium and the green plantlets obtained were screened for the transferred trait as described in Example 6. Table 7. Regeneration of protoplast cultures after treatment with PEG, 4.5 x 10⁶ protoplasts treated per each sample

Treatment Number of Number of Green plants microcalli green plants per 1 x 10⁶ protoplasts

PEG 1500 Control 75 32 7.1 Sample 1 63 63 14.0 Sample 2 22 62 13.8

PEG 4000 Control 34 3 0.7 Sample 1 88 6 1.3 Sample 2 89 1 0.2

Example 6. Screening for the transformed plants.

Assay for NPTII activity. Neomycin phosphotransferase II activity in the plants was assayed with the NPTII gel assay (Reiss et al. 1984, modified by Van den Broeck et al. 1985) (Fig. 4).

Screening of plants by PCR technique. The plant material was screened by the polymerase chain reaction (PCR) carried out in a Perkin Elmer cetus 9600 thermocycler. The complete PCR mixture contained 100-500 ng of genomic or 0.5 pg of pKAH21 DNA, 12.5 pmol of each oligonucleotide primer, 200 μM dNTPs, 0.5 U Dynazyme and buffer supplied by the enzyme manufacturer (Finnzymes Oy) in a total volume of 100 μl. Thirty cycles were performed under following conditions: 75 s at 95°C, 2 min at 55°C, and 3 min at 72°C. Primers were designed to amplify a 557 bp fragment of the cDNA for EGI. The forward primer was 5 -AGGA- CACCTCGGTGGTCCTT-3' and the reverse primer 5'-AGAGTGAGGGGT- CAAGGCATT-3'. The PCR performance was controlled by including a primer pair amplifying the promoter fragment of one of the α-amylase genes. Total DNA isolated from a transgenic barley cell line (K96/3a) was used as a positive control. The amplified samples were analyzed by electrophoresis in a 2% agarose gel. References

Chu, C.C., Wang, C.C., Sun, C.S, Hsu, C, Yin, K.C., Chu, C.Y. & Bi, F.Y. (1975) Establishment of an efficient medium for anther culture of rice through comparative experiments on the nitrogen sources. Sci. Sin. 18:659-668

Datta, K., Potrykus, I., Datta, S.K. (1992) Efficient fertile plant regeneration from protoplasts of the Indica rice breeding line IR72 (Oryza sativa L.) Plant cell reports 11:229-233

Fujimura, T., Sakurai, M., Agaki, H., Negishi, T. & Hirose, A. (1985) Regeneration of rice plants from protoplasts. Plant Tissue Culture Letters 2:74-75. Funatsuki, H., Lorz, H. & Lazzeri, P.A. (1992) Use of feeder cells to improve barley protoplast culture and regeneration. Plant Sci. 85:179-187

Hunter, C.P. (1987) Plant generation method. European patent application No. 245 898

Jahne, A., Lazzeri, P.A. & Lorz, H. (1991) Regeneration of fertile plants from protoplasts derived from embryogenic cell suspensions of barley (Hordeum vulgare L.). Plant Cell Reports 10:1-6 Karp, A. & Lazzeri, P.A. (1992) Regeneration, stability and transformation of barley. In: Shewry, P.R. (Ed) Barley: genetics, biochemistry, molecular biology and biotechnology, (pp 549-571). C.A.B. International, Wallingford

Lazzeri, P.A., Brettschneider, R., Lϋhrs, R. & Lorz, H. (1991) Stable transformation of barley via PEG-induced direct DNA uptake into protoplasts. Theor. Appl. Genet. 81:437-444

Lazzeri, P.A. & Lorz, H. (1990) Regenerable suspension and protoplast cultures of barley and stable transformation via DNA uptake into protoplasts. In: Lycett, G.W. & Grierson, D. (Eds.) Genetic engineering of crop plants. Butterworths, London, pp. 231-237.

Lϋhrs, R. & Lorz, H. (1988) Initiation of morphogenic cell suspension and protoplast cultures of barley (Hordeum vulgare L.). Planta 175:71-81

Mendel, R.R., Mϋller, B., Schulze, J., Kolesnikov, V. & Zelenin, A. (1989) Delivery of foreign genes to intact barley cells by high-velocity microprojectiles. Theor. Appl. Genet. 78:31-34 Olsen, F.L. (1987) Induction of microspore embryogenesis in cultured anthers of Hordeum vulgare. The effects of ammonium nitrate, glutamine and asparagine as nitrogen sources. Carlsberg Res. Commun. 52:393-404 Potrykus, I. 1991 Gene transfer to plants: Assessment of published approaches and results. Ann. Rev. Plant Physiol. Plant Mol. Biol. 42:205-225

Reiss, B., Sprengel, R., Will, H. & Schaller, H. (1984) A new sensitive method for qualitative and quantitative assay of neomycin phosphotransferase in crude cell extracts. Gene 30:211-218

Rhodes, C.A., Lowe, K.S. & Ruby, K.L. (1988a) Plant regeneration from protoplasts isolated from embryogenic maize cell cultures. Bio/Technology 6:56-60

Rhodes, C.A., Pierce, D.A., Mettler, I.J., Mascarenhas, D. & Detmer, J.J. (1988b) Genetically transformed maize plants from protoplasts. Science 240:204-207

Ritala, A., Aspegren, K., Kurten, U., Salmenkallio-Marttila, M., Mannonen, L., Hannus, R., Kauppinen, V., Teeri, T.H. & Enari, T.-M. (1994) Fertile transgenic barley by particle bombardment of immature embryos. Plant Mol. Biol. (In press)

Roest, S. & Glissen, L.I.W. (1993) Regeneration from protoplasts - a supplementary literature review. Acta Bot. Neerl 42:1-23

Toriyama, K., Arimoto, Y., Uchimiya, H. & Hinata, K. (1988) Transgenic rice plants after direct gene transfer into protoplasts. Bio/Technology 6:1072-1074.

Van den Broeck, G., Timko, M.P., Kausch, A.P., Cashmore, A.R., Van Montagu, M. & Herrera-Estrella, L. (1985) Targeting of a foreign protein to chloroplasts by fusion to the transit peptide from the small subunit of ribulose 1,5-phosphate carboxylase. nature 313:358-363

Vasil, V., Redway, F. & Vasil, I.K. (1990) Regeneration of plants from embryogenic suspension culture protoplasts of wheat (Triticum aestivum L.). Bio/Technology 8:429-434

Wei, Z. & Xu, Z. (1990) Regeneration of fertile plants from embryogenic suspension culture protoplasts of Sorghum vulgare. Plant Cell Reports 9:51-53

Yan, Q., Zhang, X., Shi, J. & Li, J. (1990) Green plant regeneration from protoplasts of barley (Hordeum vulgare L.). Kexue Tongbao 35:1581-1583.

Zhang, W. & Wu, R. (1988) Efficient regeneration of transgenic plants from rice protoplasts and correctrly regulated expression of the foreign gene in the plants. Theoretical and Applied Genetics 76:835-840.

Zhang, H.M., Yang, H., Rech, E ., Golds, T.J., Davis, A.S., Mulligan, B.J., Cocking, E.C. & Davey, M.R. (1988) Transgenic rice plants produced by electropo- ration-mediated plasmid uptake into protoplasts. Plant Cell Reports 7:379-84.

Claims

We claim:

1. A method of generating fertile barley plants comprising (a) cold treatment of barley spikes at a temperature under 10 °C for 1 to 4 weeks,

(b) isolating microspores from the cold treated barley spikes,

(c) culturing the isolated microspores as a suspension for up to 4 weeks in a medium containing maltose, the concentration of maltose being from about 0.150 M to about 0.200 M, nitrogen sources and growth regulators, (d) isolating protoplasts from the microspore mass obtained using conventional methods, (e) cultivating said protoplasts in a medium containing maltose, the concentration of maltose being from about 0.500 M to about 0.650 M, a nurse culture and growth regulators, and (f) regenerating the protoplasts in protoplast regeneration medium to generate fertile barley plants.

2. The method according to claim 1, wherein the growth regulators used are 2,4-D and kinetin.

3. The method according to claim 1, wherein as an organic nitrogen source used is glutamin.

4. The method according to claim 1 wherein the nurse culture comprises a suspension of the microspore mass obtained at stage (c).

5. Use of the protoplasts obtained at stage (d) of claim 1 for transformation with foreign gene(s).

6. A method of generating transgenic barley plants comprising

(a) cold treatment of barley spikes at a temperature under 10 °C for 1 to 4 weeks,

(b) isolating microspores from the cold treated spikes,

(c) culturing the isolated microspores for up to 4 weeks in a medium containing maltose, the concentration of maltose being from about 0.150 M to about 0.200

M, nitrogen sources and growth regulators,

(d) isolating protoplasts from the microspore mass obtained using conventional methods,

(e) transforming the isolated protoplasts with foreign gene(s), (f) cultivating the transformed protoplasts in a medium containing maltose, the concentration of maltose being from about 0.500 M to about 0.650 M, a nurse culture and growth regulators, and (g) regenerating the protoplasts in protoplast regeneration medium to generate trans genie barley plants.

7. A method according to claim 6 wherein the nurse culture comprises a suspension of the microspore mass obtained at stage (c).

8. The method according to claim 6, wherein the growth regulators used are 2,4-D and kinetin.