Perfluorochemicals and plant biotechnology: an improved protocol for protoplast culture and plant regeneration in rice (Oryza sativa L.)

Biotednolog ELSEVIER

Journal

of Biotechnology 50 (1996) 47 54

Perfluorochemicals and plant biotechnology: an improved protocol for protoplast culture and plant regeneration in rice (Oryza sativa L.) J. Wardrop, Plant

Genetic

Manipulation

Group,

K.C. Lowe *, J.B. Power, Department

of Lifti

Nottingham

Science,

M.R. Davey

Unicersit),

NG7 2RD,

of Nottingham.

University

Park,

CK

Received 23 January 1996; revised 3 May 1996; accepted 5 May 1996

Abstract A novel approach is described for the culture of protoplasts isolated from cell suspensions of the Japonica rice (Oryza sativa L.) cultivar Taipei 309 involving supplementation with an oxygenated pertluorochemical (PFC) liquid, pertluorodecalin. Protoplasts cultured in the presence of oxygenated perfluorodecalin exhibited a 5-fold increase in mean plating efficiency (P < 0.01) compared to controls. This beneficial effect was sustained throughout culture with a 12% increase (P < 0.01) in shoot regeneration efficiency, leading to intact plants with morphometric characteristics similar to those of seed-derived individuals. Further refinement of the system, involving 24-well culture plates, similarly revealed a 7.5-fold increase in plating efficiency in response to PFC supplementation (P < 0.01). The enhanced growth of protoplast-derived colonies was confirmed by image analysis assessments of the cultures. Overall, these results demonstrate a beneficial effect of supplementing rice protoplast culture systems with oxygenated perfluorochemical liquid which will be of broader value in plant biotechnology. KQWW~S: Perfluorochemical;

Protoplasts;

Plant regeneration;

Orzya

L.

sativa

1. Introduction Optimisation Abbreviations: BAP, 6-bezylaminopurine ; 2,4-D, 2,4dichlorophenoxyacetic acid ; FDA, fluorescein diacetate ; Kin, kin∈ MS, Murashige and Skoog (1962); NAA, cc-naphthaleneacetic acid : PFC., nerfluorochemicat. . * Corresponding author.

0168-16561’961S15.000 1996 Elsevier Science B.V. All rights reserved PII

SO168-1656(96)01548-9

been several past

of protoplast-to-plant

a prime aspects

decade

Utomo global

and crucial of rice

(Jain

et al.,

object&e biotechnology 1995; Lynch

et al., 1995; Zhang, importance of rice

systems

has

underpinning during et al.,

the 1995;

1995). This reflects the as a major food crop.

48

J. Wardrop et al. / Journal of Biotechnology

The protoplast plating efficiency of this, and other economically-important cereals, is still low when compared to the generally more efficient protoplast systems of their dicotyledonous counterparts. Abdullah et al. (1986) reported that while initial division occurred in up to 57% of cells derived from cultured protoplasts isolated from several varieties of Japonica rice, only ca. 1% of such protoplasts underwent sustained mitotic division leading to visible cell colonies. The results obtained using rice protoplasts are dependant on a number of variables, primarily the source material and plant genotype. At present, embryogenic cell suspensions are the prime source material for the isolation of totipotent rice protoplasts. However, as well as the choice of plant variety, source material and culture medium used, additional innovative approaches are also required to elevate rice protoplast systems, in terms of their plating efficiency, to levels more comparable to those of dicotyledons. One explanation for poor mitotic division of isolated protoplasts is inadequate oxygen availability during the early and vital days of culture. Indeed, oxygen deprivation can adversely influence plant cell growth through inhibitory effects on respiratory adenosine triphosphate (ATP) production (van der Plas and Wagner, 1986). However, culture of protoplasts from several species in oxygen-enriched atmospheres can enhance the plating efficiency and mitotic division of protoplast-derived cells (Carman, 1987; d’Utra Vaz et al., 1992). A novel approach for supplying oxygen utilising perfluorochemical to such cultures, (PFC) liquids, has emerged with promising results using Petunia hybrids as a convenient model (Anthony et al., 1994a). In this system, the culture of cell suspension-derived protoplasts at an interface between liquid medium, supplemented with the co-polymer surfactant, Pluronic& F-68, and oxygenated PFC, enhanced the mean plating efficiency of protoplast-derived cells by up to 52”/0. However, it was not possible to assess the longerterm effects of culture in the presence of PFC on plant phenotype or fertility because the suspension cultured albino cells of P. hybrida from which protoplasts were derived had lost their shoot regeneration capability (Power et al., 1976).

50 (1996) 47 -54

PFC liquids are inert, organic compounds that have attracted considerable interest in medicine and biotechnology, primarily because of their ability to dissolve substantial amounts of respiratory gases (Lowe, 1994). PFCs have, for example, been widely employed for regulating gas supply in prokaryotic and eukaryotic cell cultures (King et al., 1989; Ju and Armiger, 1992; Elibol and Mavituna, 1995; Lowe et al., 1995). Such use of PFCs to facilitate gas supply to cultured cells is potentially relevant for less efficient protoplast systems, including those of rice, where the ultimate efficacy of conversion of protoplasts to tissues capable of shoot regeneration into plants is paramount, particularly for successful somatic hybridisation and transgenic plant production (Lynch et al., 1995). The present study has assessed the growth of cell suspension-derived protoplasts of the Japonica rice cultivar Taipei 309. A key objective was to determine whether protoplast-to-plant regeneration in rice could be promoted by the use of oxygenated PFCs during the initial stages of in vitro growth. A further, crucial objective was to demonstrate that PFCs had no long-term detrimental effects upon the morphological and fertility characteristics of regenerated plants. The use of image analysis, as a tool to evaluate cell and tissue growth in assessments of this kind, was also incorporated into this study, since previous work has demonstrated that such technology provides a convenient, non-invasive and accurate quantification of cell growth responses in PFC-supplemented cultures (Anthony et al., 1994b).

2. Materials

and methods

2.1. Plant materials, protoplast isolation, viability and culture Callus

derived

from

mature seed scutella of 309 was initiated on LS medium (Linsmaier and Skoog, 1965) semi-solidified with 0.4% (w/v) Sea Kern agarose (Flowgen Ltd., Shenstone, UK) and supplemented with 2.5 mg 1~ ’ 2,4-dichlorophenoxyacetic acid (2,4-D). This callus was used to initiate cell suspensions which were maintained by sub-culture every 7 d in Oryza sativa L. cv. Taipei

J. Wardrop et al.

I Journal of Biotechnology SO (1996) 47-54

liquid amino acid-based (AA2) medium supplemented with 2.0 mg l- ’ 2,4-D (Abdullah et al., 1986). The cell suspension cultures had been established for approximately 30 months at the time of protoplast isolation and, consequently, regeneration potential had declined, their thereby providing an ideal system to monitor and to authenticate any protocol designed specifically to improve plant throughput from protoplast-derived tissues. Protoplasts were isolated enzymatically from cell suspensions 4 d post-subculture and their viability determined by the uptake of fluorescein diacetate (FDA) (Widholm, 1972). In initial experiments, protoplasts were suspended at a final density of 2 x 10” ml-’ in 3 ml aliquots of KPR medium made semi-solid with 0.4% (w/v) Sea Plaque agarose (Thompson et al., 1986). Protoplasts were transferred, prior to gelling of the medium, to 30 ml glass screw-capped bottles (Beatson Clark and Co. Ltd., Rotherham, UK) either alone (control) or over 6 ml aliquots of sterile perfluorodecalin (Flutec” PP.5; BNFL Fluorochemicals Ltd., Preston, UK). The latter was saturated with oxygen (10 mbar; 15 min) prior to being overlaid with protoplasts in the agarose medium. Cultures were incubated in the dark at 28 + 1°C. After 21 d, the aliquots of agarose-medium containing the embedded protoplasts were divided into 4 equal segments. All 4 segments were transferred to a 5 cm Petri dish containing 3 ml of liquid KPR medium and the dish sealed with Nescofilm (Bando Chemical Industries Ltd., Kobe. Japan). Cultures were incubated as before for a further 14 d, at which time protoplast-derived colonies were visible. All experiments were replicated 3 times. In a variation of this protocol, which was developed primarily to improve and to simplify the handling procedure during culture, the 30 ml glass bottles were replaced with tissue culture plates each with 24 circular wells (Costar Corporation, Cambridge, USA). Each well contained either 1 ml of 0.4% (w/v) Sea Plaque agarose-solidified KPR medium with embedded protoplasts (control) or 1 ml of the same protoplast suspension over 2 ml of oxygenated PFC. The dishes were sealed with Nescofilm. After 7

49

d culture in the dark, the agarose layers containing dividing protoplasts were transferred to 3.5 cm diameter Petri dishes (1 layer per dish), the dishes each containing 2 ml of liquid KPR medium. Cultures were incubated as in the initial assessments. 2.2. Assessment of protoplast division frequency and shoot regeneration ef$ciency Protoplast plating efficiency was determined as the percentage of viable protoplasts which had undergone sustained division giving rise to visible colonies after 35 d of culture. This approach was used, since the nature. of the twoa phase culture regime did not permit non-destructive determination of initial plating efficiency. Shoot regeneration efficiency was also assessed in relation to the number of such colonies producing one or more shoots and the number of shoots produced per protoplastderived callus. 2.3. Image analysis assessments of protoplastderived colony growth Non-invasive image analysis was used to record the morphometric characteristics of protoplast-derived colonies, as described previously (Anthony et al., 1994b). Petri dishes, each containing an agarose layer with dividing protoplasts, were photographed with an Ilford FP4 film using a Nikon 601 35 mm camera with a 55 mm micro lens (1 s exposure at f16). Black and white prints (4 per treatment) were scanned using an Apple Document Scanner connected to an Apple Macintosh IIci computer. The images were imported into the image-analysis package (NIH Image, Version 1.44) at a resolution of 150 dpi. Protoplast-derived colonies were selected by thresholding which identified two populations of regenerated protoplasts, those giving discrete colonies, and those which had exhibited non-sustained division with insignificant colony formation; the latter were subsequently eliminated from the final thresholded image.

50

J. Wardrop et al. ! Journal of’ Biotechnology

2.4. Plunt regenerution Fifty protoplast-derived colonies were removed from each agarose disc and transferred to 9 cm Petri dishes (10 colonies per dish), each containing 15 ml MS based medium supplemented with 2.0 mg 1~ ’ kinetin (Kin) and 0.5 mg 1~ ’ a-naphthaleneacetic acid (NAA). The medium contained both 1.5% (w/v) maltose and 1.5% (w/v) sucrose as carbohydrate sources with 0.4% (w/v) Sea Kern agarose, pH 5.8. Protoplast-derived calli were cultured for 28 d in the dark at 28 +_ 1°C until regenerated shoots were well established; shoots were detached and transferred to MS-based medium with 0.5 mg l- ’ NAA alone, semi-solidified with 0.2% (w/v) Phytagel (Sigma, Poole, UK) at pH 5.8 to induce adventive rooting. Shoots were transferred individually to 40 ml aliquots of medium in 175 ml capacity screwcapped glass jars. Shoots were maintained with a 16 h photoperiod (27 PEm ~ ’ s _ ‘; Thorn Cool White Fluorescent Tubes, Thorn EMI Ltd., UK) at 26 f 1°C. Following rooting (21 d), 45 plants from the control group and the same number derived from protoplasts cultured with oxygenated PFC were transferred to the glasshouse and raised to flowering for phenotypic analyses and ploidy assessments. Protoplast-derived plants were established ex vitro in 7.5 cm diameter plastic pots, each containing a 6:6:1 (v/v/v) mixture of Levington M3 compost (Fisons plc., Ipswich, UK), John Innes No. 3 compost (J. Bentley, Barton-on-Humber, UK) and Perlite (Silvaperl Ltd., Gainesborough, UK), in covered propagator trays at 28 f 2°C for 42 d prior to their transfer to 15 cm diameter pots. The photoperiod and light intensity were the same as used previously. 2.5. Morphometric plun ts

unulyses

of protoplast-derived

Protoplast-derived plants in the glasshouse were scored for phenotypic parameters, including plant height, tiller number, days to flowering, length and width of flag leaves and leaf 1ength:width ratio. Panicle length and number of primary branches, the number of spikelets per panicle and

50 (1996) 47-54

pollen viability were also recorded. The number of tillers was determined 6 months after transfer of plants to compost; the vegetative growth period was taken as the period between potting and the time when the first flower opened. 2.6. Assessment qf’ pollen viability protoplust-derived plunts

oj

Pollen viability was assessed by gentle squeezing of pollen grains from harvested anthers into a drop of 4% (w/v) sucrose solution on a glass slide. One drop of a 0.005% (w/v) aqueous solution of FDA was added. The preparation was examined after 5 min by fluorescence microscopy. Fully rounded, strongly fluorescent pollen grains were considered viable, whereas lightly fluorescent, dimorphic grains were considered non-viable. A minimum of 500 pollen grains per plant and JO plants per treatment were assessed and the percentage viability determined.

2.7. Flow> cytometric analysis of ploidy levels in protoplast-derived plants Nuclei were isolated from young leaves of glasshouse-grown plants (30 plants per treatment) by chopping tissue with a scalpel blade in 2.5 ml aliquots of staining buffer (15 mM Tris, 2 mM ethylenediamine tetraaacetic acid (EDTA), 0.5 mM spermine, 80 mM KCI, 20 mM NaCl, 0.1% (v/v) Triton X-l 00 and 50 pg 1~ ’ ethidium bromide), pH 7.5 (Dolezel et al., 1989). The resulting suspension was passed through a nylon sieve of 30 pm pore size. A 50 ~1 aliquot of Immuno-Checka polystyrene beads (Coulter Electronics Ltd., Luton, UK) was used as an internal standard and fluorescence evaluations were made with a Coulter EPICS 541 flow cytometer. The output of the argon-ion laser was 100 mW at a wavelength of 488 nm. Ten thousand particles per extract were assessed and resulting histograms were transferred to an IBM PC computer. Relative mean linear fluorescence values of the G,/G, nuclei were evaluated using computer programmes developed by N.W. Blackhall (Department of Life Science, University of Nottingham).

J. Wardrop et 01.

Fig. I. Percentage

oxygen

saturation

51

‘Journal of Biotechnology 50 (1996) 47-54

of perfluorodecalin

with time (100% oxygen.

2.8. Optimisation of oxygenation-charging of per@4orodecalin Optimum oxygenation parameters for the perfluorodecalin media supplements were determined prior to the initiation of these experiments. Percentage oxygen concentrations in perfluorodecalin were measured using a Jenway 9015 dissolved oxygen meter with a POM102 probe (Scientific Laboratory Supplies Ltd., Nottingham, UK). The instrument was calibrated to measure percentage oxygen saturation for 100 ml perfluorodecalin which had been autoclaved for 20 min at 121°C (118 kPa nominal steam pressure). All measurements were taken in a sterile laminar flow cabinet. One hundred percent medical grade oxygen (B.O.C. Ltd., Guildford, UK) at 10mbar was bubbled into 100 ml perfluorodecalin held in a sterile Duran vessel for 1 min. followed by a short (10s)interval to allow a steady reading to be made; this was repeated at 1 min intervals for 20 min. Changes in oxygen saturation (n = 3) were plotted and the standard curve (Fig. 1) used to determine the optimal oxygenation time (cu. 15 min) for perfluorodecalin used in all experiments.

10 mbar).

Values are mean

t

S.E.M.

(n = 3).

2.9. Statistical methods Statistical analyses were performed according to methods of Campbell (1990). Means and standard errors (SEM) were used throughout. The non-parametric, Mann-Whitney U test was employed to compare treatments with non-normal distributions and where variances among treatments were highly unequal. Otherwise, a conventional, parametric Student’s t-test was used. A probability of P < 0.05 was considered significant.

3. Results Culture of rice protoplasts with oxygenated perfluorodecalin in glass bottles resulted in a 5-fold increase in mean plating efficiency, as assessed optically after 35 d, compared to untreated controls (P < 0.01) coupled with a 12”/0 increase (P < 0.01) in plant regeneration (Table 1). The mean number of shoots regenerating from each protoplast-derived colony after 63 d in response to culture with oxygenated perfluorodecalin (n = 40) was also increased from 4 + 1 to 8 _t 1 (P < 0.05: Table 1). A 7.5-fold increase (P < 0.01)

52

J. Wardrop et al. /Journal

Table I Mean protoplast colony following Treatment

Control Oxygenated

plating efficiency, number culture of rice protoplasts Plating

PFC

0.02 0.10

efficiency

& 0.01 * 0.01**

(‘Xl)

of Biotechnology

50 (1996) 47-54

of protoplast-derived colonies regenerating for 21 d with oxygenated PFC Protoplast-derived colonies atmg shoots (‘XI)” 25 f 37 *

shoots and number

regener-

I 1**

“Assessed at 35 d (n = 9); “Assessed at 63 d (n = 450); ‘Assessed Values are mean k S.E.M. *P < 0.05. **P c: 0.01

in the mean plating efficiency of protoplasts cultured with oxygenated perfluorodecalin in the 24-well culture dishes was also observed (Table 2) showing that this novel cultural approach could be readily extended to small-volume systems. Image analysis assessments revealed a significant increase (P < 0.01) in protoplast-derived colony growth following culture with PFC. For example, the mean area of the culture plates covered by colonies after 35 d (n = 4) was increased from 11.6% + 5.8% in controls to 49.8% ? 5.4% (P < 0.01) in response to media supplementation with oxygenated perfluorodecalin (Table 2). In contrast, visual assessments revealed a 7.5-fold increase in plating efficiency over the same time period. The ploidy of the protoplast-derived plants transferred to the glasshouse, as assessed by flow cytometry, was unaffected by culture of protoplasts with oxygenated perfluorodecalin. Thirty plants derived from protoplasts cultured in the presence of perfluorodecalin and the same number of plants regenerated from protoplasts cultured in the absence of this compound were all diploid. Plants regenerated from protoplasts exposed to oxygenated perfluorodecalin flowered 1 lo&200 d after their transfer to the glasshouse and showed no marked differences in morphology or pollen viability compared to plants derived from protoplasts cultured in the absence of perlluorodecalin (Table 3) or seed-derived individuals (Davey et al., 1991).

No. of shoots colony’ 4*1 8 *

of shoots

regenerated

per

per regenerating

I*

at 63 d (n = 40).

4. Discussion These results demonstrate the important, possibly crucial, role of oxygen in plant protoplast growth. Moreover, PFCs facilitate effective oxygen transfer to protoplasts, as well as providing a convenient, inert, potentially recyclable, medium support. All experiments were deliberately undertaken using protoplasts of a rice cell suspension which was sub-cultured routinely every 7 d for 30 months after its initiation. By this age, the suspension exhibited a marked decline in plant regeneration efficiency directly or indirectly from its protoplasts. The present study has shown that culture in the presence of oxygenated perfluorodecalin significantly rejuvenated protoplast division and shoot regeneration from protoplast-derived colonies. These results support previous findings, using P. hybrida as a model, that oxygenated perfluorodecalin can improve protoplast plating efficiency (Anthony et al., 1994a). However, the present results significantly extend earlier observations with the finding that there was a positive effect of co-culture with PFC, sustained up to and including the expression of totipotency, as judged by the proportion of protoplast-derived colonies exhibiting regeneration through somatic embryogenesis. The present results demonstrate, for the first time, that plants regenerated from protoplasts cultured in the presence of oxygenated perfluorodecalin were phenotypically and genotypitally normal. This reinforces the inertness of PFCs, specifically perfluorodecalin, in biological

53

J. Wardrop et al. //Journal qf Biotechnolog_t 50 (1996) 47-54 Table 2 Assessment

of PFC-enhanced

Treatment

Optically efficiency

Control Oxygenated PFC

0.02 0.15

rice protoplast assessed (‘/;I)”

growth

plating

f 0.01 i_ 0.02**

in 24 well culture

plates

using optical

and image analysis

techniques

Image analysis derived plate cover (“4,)’

Protoplast-derived colonies regenerating shoots (‘:;t)’

Number of shoots per regenerating colonyd

11.6 i 49.8 f

I9 * 2 38 T 2**

2*1 7 *

5.8 5.4**

I**

“Assessed at 35 d (n = 72); ‘Assessed at 35 d (n = 4); ‘Assessed at 63 d (n = 720); ‘Assessed at 63 d (n = 50). Values are mean k S.E.M. *P < 0.05, **P < 0.01.

systems (King et al., 1989; Ju and Armiger, 1992; Elibol and Mavituna, 1995; Lowe et al., 1995). Additionally, this provides a crucial baseline for the future broader exploitation of PFCs as respiratory gas-carrying media supplements in somatic cell systems, where plant regeneration is an essential component of the experimental system. The improved culture of rice protoplasts with oxygenated PFC supplements, as described here, will also be valuable in protoplast-based genetic transformation studies where it is essential to maximise the throughput of transgenic plants. Additionally, it has been noted previously (Anthony et al., 1994a; Elibol and Mavituna, 1995; Table 3 Comparison of the phenotypic characteristics of protoplastderived plants of the rice cultivar Taipei 309 following culture of protoplasts in the absence (control) or presence of oxygenated PFC Trait

Control

Plant height (cm) Number of tillers/plant Flag leaf length (cm) Flag leaf width (cm) Flag leaf length:width ratio Panicle length (cm) Number of primary branches per panicle Number of secondary branches per panicle Number of spikelets per panicle Pollen viability (‘XI)

90.6 I2 24.9 I.1 26.1

Oxygenated

k * i * +

2.5 1 1.4 0.1 3.1

90.8 I2 24.2 I.1 23.0

19.3 * 6kl

I.1

18.1 * 7+1

4*1 49 )

PFC

k 2.4 * I k I .4 + 0.1 + I.3 I.1

4+1 3

54 f

Lowe et al., 1995) that the physical properties of PFC liquids make them attractive as culture media supplements since they are inert and thermally stable. Such unique features permit sterilisation and recycling, overcoming the initial high cost of such compounds. The present cultural approach refined for Japonica rice is currently being extended to other protoplast and tissue culture systems, primarily those of woody species, which are considered relatively recalcitrant in culture. One further refinement of the culture regime described here will be to assess the potential benefits of ‘reloading’ the PFC liquid with oxygen during culture without disturbance to the actively dividing protoplasts. Such assessments would facilitate the scale-up of this approach and its extension, ultimately, to use in batch cultures of plant cells and transformed roots, to improve biomass production, leading, perhaps, to increased yields of economically-important secondary products. Acknowledgements

The authors thank BNFL Fluorochemicals Ltd, Preston, UK, for financial support and for perfluorodecalin, Dr. C. Washington for assistance with image analysis and Mr. N.W. Blackhall for assistance with flow cytometry.

3

References 87.1

Values are mean and S.E.M. (n = IO).

+

1.5

85.2

+

I.0

(n = 45), except pollen viability

Abdullah, R., Cocking, E.C. and Thompson, J.A. (1986) Efficient plant regeneration from rice protoplasts through somatic embryogenesis. BioiTechnol. 4, 1087~1090.

54

J. Cz’urdrop ct ai.

: Journui

Anthony, P., Davey, M.R., Power, J.B., Washington, C. and Lowe, K.C. (1994a) Synergistic enhancement of protoplast growth by oxygenated perfluorocarbon and Pluronic F-68 Plant Cell Rep. 13, 251-255. Anthony, P., Davey, M.R., Power, J.B., Washington, C. and Lowe, K.C. (1994b) Image analysis assessments of perfluorocarbon and surfactant-enhanced protoplast division. Plant Cell. Tiss. Org. Cult. 38, 39943. Campbell, R.C. (1990) Statistics For Biologists (3rd ed.). Cambridge University Press, Cambridge. Carman, J.G. (1987) Improved somatic embryogenesis in wheat by partial stimulation of the in-OGU/O oxygen, growth-regulator and desiccation environments. Planta 175. 417-424. Davey. M.R., Kothari, S.L., Zhang, H., Rech, E.L., Cocking, E.C. and Lynch, P.T. (1991) Transgenic rice: characterisation of protoplast derived plants and their seed progeny. J. Exp. Bot. 42. II 59- 1169. Dolezel, J., Binarova, P. and Lucretti, S. (1989) Analysis of nuclear DNA content in plant cells by flow cytometry. Biol. Plantarum 31, 1133120. d’Utra Vaz, F.B., Slamet, I.H., Khatun, A., Cocking, E.C. and Power, J.B. (1992) Protoplast culture in high molecular oxygen atmospheres, Plant Cell. Rep. I I, 416-418. Elibol, M. and Mavituna, F. (1995) Effect of perfluorodecalin as an oxygen carrier on actinorhodin production by Strrptornyes uwlicolor. Appl. Microbial. Biotechnol. 43. 206210. Jam, R.K., Khehra, G.S., Lee, S.-H., Blackhall, N.W.. Marchant, R., Davep, M.R., Power. J.B., Cocking, E.C. and Gosal, S.S. (1995) An improved procedure for plant regeneration from indica and japonica rice protoplasts. Plant Cell. Rep. 14, 515-519. Ju, L.-K. and Armiger, W.B. (1992) Use of perfluorocarbon emulsions in cell culture. BioTechniques 12, 258-263. King, A.T., Mulligan, B.J. and Lowe, K.C. (1989) Perfluorochemicals and cell culture. BioiTechnol. 7. lO37- 1042.

of Biotechnology

50 (1996) 47

54

Linsmaier, E.M. and Skoog, F. (1965) Organic growth factor requirements for tobacco tissue cultures. Physiol. Plant. IS, 100-127. Lowe. K.C. (1994) Perhuorochemicals Vast. Med. Rev. 4. 3 I-48.

in vascular

medicine.

Lowe, K.C., Davey. M.R. and Power, J.B. (1995). Perfluorochemicals and plant biotechnology. In: Banks, R.E. (Ed.), Fluorine in Agriculture, Rapra, Shrewsbury, pp. 157-164. Lynch, P.T., Jones, J.. Blackhall, N.W., Davey. M.R., Power, J.B., Cocking. E.C.. Nelson, M.R., Bigelow. D.M.. Orum, T.V., Orth. C.E. and Schuh, W. (1995) The phenotypic characterisation of R2 generation transgenic rice plants under held and glasshouse conditions. Euphytica 85. 395401. Murashige, T. and Skoog, F. (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant 15, 4733497. Power. J.B., Frearson, E.M., Hayward, C., George, D., Evans, P.K., Berry, S.F. and Cocking, E.C. (1976) Somatic hybridisation of Petlrnia hybrida and Petunia parodii. Nature 263, 500-502. Thompson, J.A.. Abdullah, R. and Cocking, E.C. (1986) Protoplast culture of rice (Ory-_a satica L.) using media solidified with agarose. Plant Sci. 47, 1733133. Utomo, MS., Croughan. S.S. and Croughan, T.P. (1995) Suspension and protoplast culture of U.S. rice cultivars. Plant Cell. Rep. 15, 34437. van der Plas. L.H.W. and Wagner. M.J. (1986) Effect of oxygen on growth and respiration of potato tuber callus. Plant Cell Tiss. Org. Cult. 7, 217-225. Widholm, J. (1972) The use of FDA and phenosafranine for determining viability of cultured plant cells. Stain Technol. 47, 1866194. Zhang. S. (1995) Efficient plant regeneration from lndica (group 1) rice protoplasts of one advanced breeding and three varieties. Plant Cell. Rep. 15, 6871.

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