Plant Cell, Tissue and Organ Culture 34: 83-90, 1993. © 1993 Kluwer Academic Publishers. Printed in the Netherlands.

Efficient plant regeneration from protoplasts of Arachis paraguariensis Chod. et Hassl. using a nurse culture method

Zhijian Li 1, Robert L. Jarret 2, Roy N. Pittman z, Kerry B. Dunbar 2 & James W. Demski 1'* 1Department of Plant Pathology, Georgia Station, University of Georgia; ZUSDA-ARS Regional Plant Introduction Station, Georgia Station, 1109 Experiment Street, Griffin, Georgia 30223, USA (*requests for offprints)

Received 29 September 1992; accepted in revised form 21 January 1993

Key words: Arachis species, nurse culture, plant regeneration, protoplasts, tissue culture, wild peanut

Abstract

An efficient protocol has been developed for protoplast culture and plant regeneration from wild peanut (A. paraguariensis) using a nurse culture method. Protoplasts were isolated from suspension cultures initiated from leaf-derived callus, imbedded in agarose blocks and co-cultured with nurse cells of the same species. Up to 10% of the protoplasts divided and formed compact callus colonies. The protoplast plating efficiency was correlated with both the length of the nurse cell co-cultivation period and the protoplast plating density. The optimal nurse culture duration was 14 d. The optimal plating density was 2 x 104 protoplasts/ml plating medium. Multiple shoots (up to 10 shoots per colony) were readily regenerated from protoplast-derived callus after transfer of callus to semi-solid modified MS medium containing 0.5 mg 1-1 NAA and 1 mg 1-1 BA. Plantlets with normal leaflets were obtained by rooting shoots on porous rootcubes saturated with modified MS medium containing 1 mg 1-1 NAA.

Abbreviations: B A - N6-benzyladenine, N A A - 1-naphthaleneacetic acid, M S - Murashige and Skoog medium.

Introduction

Protoplasts provide an ideal experimental system for studies of genetic transformation, protoplast fusion, organelle transfer and somatic mutations (Davey & Kumar 1983). However, progress in the development of efficient protoplast regenera- tion schemes for Arachis species has been slow. Plant regeneration has been achieved from both wild and cultivated Arachis species (Bajaj et al. 1981; Pittman et al. 1983, 1984; Still et al. 1987; Ozias-Akins 1989; McKently et al. 1989; Sellars et al. 1990; Baker & Wetzstein 1992; Durham & Parrott 1992). However, regeneration of plants occurred via direct organogenesis or adventive embryogenesis from differentiated plant tissues.

In only a few instances have plants been regener- ated from callus of Arachis spp. (Narasimhulu & Reddy 1983; Still et al. 1987). Although cell division and subsequent callus formation were observed from cultured protoplasts isolated from immature and mature leaves, cotyledons and root tips of A. hypogaea (Oelck et al. 1982; Rugman & Cocking 1985), plant regeneration from protoplast-derived calli has not been re- ported.

While efforts continue to achieve successful plant regeneration of cultivated peanut from protoplasts, we believe that the development of a protoplast regeneration system, based upon the use of wild Arachis species, may provide a necessary insight into factors affecting regenera-

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tion from A. hypogaea and Arachis species in general. Previous reports demonstrated that the diploid wild peanut A. paraguariensis Chod. et Hassel. possessed a high capacity for plant re- generation from both cultured somatic tissues (Sellars et al. 1990) and long-term suspension cultures (Still et al. 1987). In this paper we report plant regeneration from protoplasts of A. paraguariensis using a nurse culture method.

Materials and methods

Plant material

Mature leaves of A. paraguariensis were col- lected from greenhouse-grown plants obtained from the S-9 Plant Germplasm Collection (Jarret et al. 1990). Leaves were rinsed in tap water, surface-sterilized by immersion in 70% (v/v) ethanol for 1.5min and 0.5% (w/v) sodium hypochlorite solution for 10 min with vigorous agitation, and then rinsed in sterile water with 3 to 4 changes. Five mm 2 blocks were cut from the surface-sterilized leaves and used as explants for callus induction. A nurse cell line (AP1) was developed from a long-term suspension culture (over 8 months) of A. paraguariensis that was also initiated from leaf-derived callus. Nurse cell lines of rice (Oryza sativa L. cv. Labelle, Li et al. 1992) and tobacco (Nicotiana tabacum L.) were kindly provided by Dr. N. Murai, Louisiana State University, Baton Rouge, LA. Nurse cells were maintained using the suspension culture conditions described below.

Culture media, callus induction and suspension culture initiation

The basal medium (MSB) used in this study contained MS basal salts (Murashige & Skoog 1962) (Sigma Chemical), B5 vitamins (Gamborg et al. 1968), 3% (w/v) sucrose, 1 mg 1-1 NAA and 1 mg 1-1 BA. The pH was adjusted to 5.8 with 0 .5M NaOH prior to autoclaving. To induce callus formation leaf explants were placed, abaxial side downwards, on 25ml of semi-solid MSB solidified with 0.8% (w/v) Sigma agar in 100 × 15 mm Petri dishes. Cultures were maintained at 26°C on a 16 h photoperiod with a

light intensity of 40 ixmol m -2 s -1. After 25 d callus was excised from the explant and trans- ferred to fresh semi-solid MSB. Subcultures were made at 25 d intervals thereafter.

Suspension cultures were initiated by inoculat- ing 125 ml Erlenmeyer flasks containing 25 ml of liquid MSB with about 2 g of two-month old leaf-derived callus. Cultures were rotated on a gyratory shaker (85 rpm) at 26-+ 2°C under dim light (approximately 20 p~mol m -2 s- l) . At week- ly intervals the culture medium was withdrawn using a sterile pasteur pipet attached to a vac- uum pump and replaced with an equal volume of fresh MSB. At each subculture cells in excess of 2 g were discarded in order to maintain a roughly equivalent culture inoculum density. Brown cal- lus dumps were also removed during the subcul- ture process.

Protoplast isolation

Two-month-old suspension cultures were used for protoplast isolation. About 5 g of suspension- cultured cells were harvested 4 d after the most recent subculture, rinsed once in fresh MSB and digested in 30 ml of filter-sterilized enzyme solu- tion at 28°C in the dark for 3 to 4 h. The enzyme solution consisted of 5% (w/v) Cellulase RS (Yakult), 1% (w/v) Macerozyme RS (Yakult), l m g 1-1 each of NAA and BA, CPW salts (Frearson et al. 1973), and 9% (w/v) mannitol. After digestion the protoplasts were filtered through a 40 p~m nylon mesh, collected in a 50 ml centrifuge tube by centrifugation of the filtrate at 100 x g for 8 min, and washed once with 40 ml KMC solution (Harms & Potrykus 1978). Proto- plasts were resuspended in protoplast culture medium (PCM) composed of MSB modified to contain 13.7% (w/v) sucrose.

To determine the effect of protoplast density on protoplast regeneration, a ran~ge of plating densities from 0.5 x 104 tO 4 × 10 protoplasts/ ml of plating medium was tested. Protoplast densities were adjusted using a hemato- cytometer.

Protoplast culture

Protoplast culture was performed using the aga- rose bead culture method as described by Shillito

et al. (1983) and the nurse cell co-cultivation method of Kyozuka et al. (1987). Four ml of protoplast solution with 2 x final protoplast den- sity were gently mixed with an equal volume of pre-warmed PCM (40°C) containing 2.5% (w/v) Seaplaque agarose (FMC). The mixture was plated in a 100 x 15 mm Petri dish, allowed to solidify for at least i h and then cut into I cm 2 blocks (30 blocks/plate). The protoplast-contain- ing agarose blocks were then separated into two 100 × 15 mm Petri dishes containing 15ml of PCM. After adding about 100 mg of 4-day-old nurse cells to each plate, protoplasts were cul- tured on a gyratory shaker at 35 rpm at 26°C in the dark. For experiments determining the effect of protoplast plating density, protoplast-contain- ing agarose blocks were co-cultured with nurse cells for a period of 14d. To determine the effects of the nurse co-cultivation period on cell division protoplast-containing agarose blocks were co-cultured with nurse cells for periods of from 6 to 16 d, in 2 day increments.

Following nurse cell co-cultivation the protop- last-containing agarose blocks were removed and washed with fresh PCM. They were then trans- ferred into 15 ml of modified PCM containing 8.3% (w/v) sucrose and cultured under the same conditions for 7 d. At this time protoplast plating efficiency, defined as the percentage of callus- forming protoplast colonies/the total number of protoplasts plated per unit volume, was esti- mated by counting at least 15 blocks for each treatment. To propagate protoplast-derived colonies for plant regeneration, agarose blocks were subcultured to semi-solid MSB sup- plemented with 0.2% (w/v) Type I agarose (Sigma) and maintained at 26°C on a 16 h photo- period (40 I~mol m -2 s -1) for 14 d.

All protoplast culture experiments were re- peated twice.

Plant regeneration

Protoplast-derived calli of 3 to 5 mm in diameter were used for plant regeneration. To determine the effect of NAA and BA on callus differentia- tion, protoplast-derived calli were transferred to modified MSB containing different concentra- tions of NAA and BA (Table 1) and 0.6% (w/v) Sigma agar. Cultures were kept at 26°C under a

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16-h photoperiod (40 I~mol m -2 s-l). After two rounds of 25d subcultures the number of colonies showing differentiation, and the number of shoots regenerated from each colony, were determined.

To achieve plant regeneration, protoplast-de- rived colonies were transferred to MSB contain- ing 0.5rag 1-1 NAA, l mg 1-1 BA and 0.6% (w/v) Sigma agar and cultured at 26°C under a 16h photoperiod (40~mol m -2 s- l) . Subcul- tures were made every 25 d. Regenerated shoots of about 5 mm were excised at their base and placed on the top of a sterile 3 cm long cylindri- cal Oasis rootcube (Smithers-Oasis, Kent, OH) in a 25 × 150 mm test tube containing 5 ml of hormone-free MSB. The culture medium was removed with a vacuum pump and replaced with an equal amount of fresh medium once every 14 d. When shoots were about 5 cm in length, and had produced true leaves, the hormone-free MSB medium was replaced with MSB sup- plemented with Img 1-1 NAA to induce root formation. Rooted plantlets were transferred to 6 cm pots containing a soil mixture of Promix BX (A.H. Hummert Seed Co., St. Luis, MO), clay and sand (volume ratio: 1:1:1.5) and grown on a covered mist bench. Surviving plants were transferred to 15 cm pots and maintained in the greenhouse.

Results

Suspension culture initiation

Callus was readily induced from leaf explants of A. paraguariensis cultured on MSB containing NAA and BA. Friable callus was evident at the edges of explants 3 to 4 weeks after culture initiation. Sporadic shoot primordia formation was frequently observed during subsequent sub- cultures. Compact callus clumps, transferred to MSB for initiation of suspension cultures, grew slowly during the first week of culture and sloughed off smaller clumps of callus and loose cells while the larger callus mass eventually turned brown. The presence of these brown callus masses appeared to inhibit the growth of the cell suspension and their removal was essen- tial in order to maintain viable cultures. The

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smaller callus clumps, produced from the initial inoculum, proliferated during subsequent subcul- tures and ultimately yielded, after one and a half months, a finely dispersed light-green cell sus- pension. Suspension-cultured cells proliferated rapidly (3-fold increase in fresh wt/7 d). Liquid A. paraguariensis callus cultures did not differen- tiate roots or shoots under our culture condi- tions. The callus remained light-green in color.

Protoplast isolation and culture

Four hours of enzymatic digestion yielded an average of 2 x 10 6 protoplasts/g fresh wt cells from one to two month-old suspension cultures. Protoplasts were easily separated from undi- gested cell clumps and debris after filtration and washing in KMC solution. The protoplasts were spherical and cytoplasmically rich (Fig. 1).

Fig. I. Protoplasts isolated from A. paraguariensis suspension. Scale bar = 30 ~m. Fig. 2. Protoplast-derived callus colonies after 8 d (a and b) and 20 d (c and d) following nurse culture. Scale bars = 30 I~m. Fig. 3. Protoplast-derived colonies in agarose blocks after 14 d in soft agarose MSB medium. Scale bar = 1.0 cm. Fig. 4. Multiple shoots developed from protoplast-derived callus after one month on semi-solid MSB medium containing 0.5 mg 1-1 N A A and 1 mg 1-1 BA. Fig. 5. Protoplast-derived plants of A. paraguariensis.

About 5% of the protoplasts fused to form giant cells with large vacuoles during the isolation. Fused protoplasts eventually burst during the protoplast culture.

Protoplasts were immobilized in agarose and co-cultured with nurse cells. Cell wall formation, as evidenced by the formation of elliptic cells, was observed 4 to 5 d after culture initiation. First cell divisions occurred about 8d after exposure to the nurse culture (Fig. 2a, b).

The period of exposure of the plated proto- plasts to the nurse culture had a dramatic effect on protoplast plating efficiency. Maximum plat- ing efficiency (up to 10.9%) was achieved when protoplasts were co-cultured with nurse cells for a period of 14d (Fig. 6). Protoplast plating efficiency dramatically decreased after a 16d exposure to the nurse culture. The reduction in protoplast plating efficiency may be accounted for by the depletion of growth nutrients in the culture media by the rapidly growing nurse cells.

Protoplast plating efficiency was also influ- enced by the protoplast plating density. The highest plating efficiency was achieved when protoplast cultures were plated at a density of 2 × 10 4 protoplasts/ml (Fig. 7). Higher or lower plating densities resulted in reduced protoplast plating efficiencies.

Transfer of small protoplast-derived callus colonies to PCM containing a reduced concen- tration of sucrose (8.3%) promoted cell division and resulted in colonies of about 20 cells (Fig.

~ 1 2

t -

"~3 8 I J J

O }

" 6

e~

a .

0 6 8 10 t2 14 16

Nurse Culture Duration (day)

Fig. 6. Effect of the length of nurse culture duration on protoplast plating efficiency. The values represent the means of 2 experiments. Standard errors are indicated.

87

16

:o LU

13..

"6. o

14

12

10

8

6

4

2

0

Protoplast Plating Density (10 4 0,5 1

m

2 3 4 per ml plating medium)

Fig. 7. Effect of protoplast plating density on protoplast plating efficiency. Data were averaged from 2 experiments. Standard errors are indicated.

2c, d) after 20 d. Callus colonies reached a size of about I mm in diameter 14 d after transfer to soft agarose MSB medium (Fig. 3).

Co-cultivation of protoplasts with A. para- guariensis nurse cells for a period of 14 d in PCM resulted in an average plating efficiency of 10.9%. However, no cell division was observed when protoplasts were cultured without nurse cells or with nurse cell lines derived from rice or tobacco. Under these conditions, most proto- plasts burst while others turned brown and eventually disintegrated.

Plant regeneration

Organ differentiation was readily induced from protoplast-derived callus colonies after transfer to semi-solid medium. Leaf-like structures and dark-purple shoot primordia were observed 2 to 3 weeks after subculture to semi-solid MSB containing NAA and BA. Different combina- tions of NAA and BA were tested for their efficacy in promoting shoot formation. Protop- last-derived callus colonies cultured on hormone- free medium did not produce shoots (Table 1). The addition of BA, at concentrations of 0.5 to 3 mg 1-1, resulted in the production of 4 to 6 shoots/colony, suggesting that BA is required for shoot formation. However, BA concentra- tions in excess of 0.5 mg 1-1 did not further enhance the frequency of shoot formation. The addition of 0.1 mg 1-1 NAA, in combination with

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Table I. Regeneration of shoots from protoplast-derived callus colonies of A. paraguariensis on MS medium supplemented with different concentrations of NAA and BA.

Mean no.

NAA BA Colonies Responsive Shoots Shoots / (mg 1-1) (mg 1-1) (total) ~ colonies (total) 2 colony

0 0 15 0 0 0 0 0.5 15 13 79 6.1 0 1.0 15 13 54 4.2 0 3.0 15 12 59 4.9 0.1 0.5 15 15 85 5.7 0.1 1.0 15 15 139 9.3 0.1 3.0 15 9 90 10.0 0.5 0.5 15 10 52 5.2 0.5 1.0 15 15 134 8.9 0.5 3.0 15 13 109 8.4

1Five protoplast-derived colonies were inoculated in one Petri plate. Each treatment contains 3 replicate plates. 2Data were collected 50 d after inoculation.

1 or 3 mg 1-1 BA, approximately doubled the number of shoots/colony. Increasing the NAA concentration from 0.1mg 1-1 to 0.5mg 1-1 slightly reduced the frequency of shoot forma- tion. However, shoots derived from callus cul- tured on modified MSB containing BA only, or BA in combination with 0 .1mg 1-1 NAA, became thickened and translucent after 2 to 3 rounds of subculture. These shoots eventually reverted to a soft callus. Shoots induced from, and maintained on, MSB containing 0.5 mg 1-1 N A A ~ n d 1 mg 1-1 BA continued to develop normally (Fig. 4). Thus, these culture conditions were used for subsequent plant regeneration experiments.

Although shoots were readily produced from protoplast-derived callus cultured on semi-solid mediu'tn, continuous subculture of shoots on semi-solid medium stimulated callus proliferation at the shoot base. When excised and cultured individually on semi-solid media shoots often produced phenolic compounds, resulting in browning of the shoot bases. This localized accumulation of phenolic compounds around the developing shoots inhibited shoot development and growth. These problems were avoided by culturing shoots on a porous support with liquid medium. The use of liquid media improved nutrient uptake by cultured shoots and effective- ly diluted the phytotoxic effects of the phenolic compounds. An enhancing effect of liquid media

on shoot regeneration and somatic embryo de- velopment has been reported for wild and culti- vated peanuts (Still et al. 1987; Durham & Parrott 1992). Shoots transferred to Oasis root- cubes grew rapidly reaching 10cm or more within 3 to 4 weeks. Callus growth at the shoot base was minimal. Leaf growth was also pro- moted. Leaves were dark green, elongated and heavily pubescent, similar to those of the donor plants. Shoot cultures occasionally flowered, a phenomena previously reported by Still et al. (1987).

Transfer of shoots to MSB containing 1 mg 1-1 NAA stimulated root initiation. Adventitious roots grew normally and were able to penetrate the rootcube reaching the liquid medium (Fig. 5). More than 20 rooted plants have been trans- ferred to soil. These plants are being grown in the greenhouse for observation of plant mor- phology and fecundity.

Discussion

Co-cultivation with nurse cells was essential for successful regeneration from A. paraguariensis protoplasts. This observation is in agreement with previous studies of rice (Kyozuka et al. 1989) and maize (Rhodes et al. 1988) proto- plasts. In previous studies utilizing rice, nurse

cells of Triticum monococcum were able to support division of rice protoplast-derived callus at a very low frequency, while nurse cells of carrot (Daucus carom) and tobacco (N. tabacum) failed to induce cell division (Kyozuka et al. 1989). We found that protoplasts of A. paraguariensis responded only to nurse cells derived from their donor cell lines and not to nurse cells of rice or tobacco. These results suggest that nurse cells either release substances into the culture medium that buffer toxic metabolites or produce substances that stimulate cell division. The effect of such putative stimulat- ory substances may be species-specific (Bellin- campi & Morpurgo 1987).

The use of a nurse culture provides a simple and reliable technique for enhancing callus pro- liferation from isolated protoplasts. When nurse cells are not employed, more sophisticated cul- ture medium such as that of Kao & Michayluk (1975), or the addition of complex organic ad- denda, are commonly required in order to in- duce cell division following protoplast plating. In addition, progressive dilution of the protoplast culture medium may be necessary (Dhir et al. 1991). In other instances regeneration from protoplasts, in the absence of a nurse culture, may depend upon the selection of rapidly grow- ing cell lines from long-term suspension cultures (Abdullah et al. 1986; Vasil et al. 1990). How- ever, plant regeneration from long-term cultures is frequently associated with low frequencies of plant regeneration and high incidences of somatic variation (Larkin & Scowcroft 1981). In this study co-culturing protoplasts isolated from relatively short-term suspension cultures (1 to 2 months of age), with nurse cells, promoted a high frequency of cell division under simplified culture conditions. This method permits the routine regeneration of a large number of plants from A. paraguariensis protoplasts. A similar stimulatory effect of nurse culture on plant regenerat ion from protoplasts has been reported for rice (Kyozuka et al. 1989; Li et al. 1990) and maize (Petersen et al. 1992).

The techniques reported here provide a powerful tool enabling successful plant regenera- tion from suspension culture-derived protoplasts of A. paraguariensis and may, with modification, be useful in efforts to regenerate plants from A.

89

hypogaea. The development of plants regenera- tion protocols from protocols of peanut will facilitate the application of transformation and fusion technology to this important crop plant.

Acknowledgements

We wish to thank Drs. Carol Robacker and Jerry T. Walker for critical reading of the manuscript and Dr. N. Murai for kindly providing rice and tobacco nurse cell lines. This work was sup- ported in part by Peanut CRSP, U.S. AID grant DAN-4048-G-00-0041-00; in part by the Georgia Commodity Commission for Peanuts and in part by State and Hatch Funds allocated to the University of Georgia.

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