Planta

Planta (1984) 162:1-7

9 Springer-Verlag 1984

Plant regeneration from long-term suspension cultures of white clover Derek W.R. White Grasslands Division, Department of Scientific and Industrial Research, Private Bag, Palmerston North, New Zealand

Abstract. A genotype of Trifolium repens L. capa-

ble of sustaining high-frequency plant regeneration from long-term (24-month old) cell cultures has been selected. Numerous densely cytoplasmic meristemoids were formed in suspension cultures following the coordinate removal of 2,4-dichlorophenoxyacetic acid (2,4-D) and trichloropicolinic acid (picloram) from the medium and an increase in the NH2 concentration. Some meristemoids arose from single cells in culture. Increasing the NH + concentration in the medium resulted in increased meristemoid formation and decreased the growth rate. Ammonium stimulated meristemoid formation when it was the sole source of nitrogen only if a lethal shift in the pH of the medium was prevented. Meristemoids plated on hormone-free agar medium developed directly into shoots which spontaneously formed roots. Key words: A m m o n i u m - Cell culture (plant regeneration) - Meristemoid - Trifolium (regeneration).

Introduction

A low efficiency or lack of plant regeneration from cultured cells is still a limitation to the use of invitro genetic manipulation techniques, such as variant selection and transformation, for the improvement of many plant species, including several legumes. Factors contributing to low efficiency include the decline in regeneration capacity that often occurs with increasing time in culture, and the presence of a high proportion of non-morphogenic cells in the cultures (e.g. McCoy and Bingham 1977; Syono 1965; Wochok and Wetherell 1972; Abbreviations: 2,4-D = dichlorophenoxyacetic acid; MS = Murashige-Skoog (1962) medium; N A A - c~-naphthaleneacetic acid; SH = Schenk-Hildebrandt (1972) medium

Murashige and Nakano 1965). Amongst the legumes, genetic variation in plant regeneration capacity has been reported for alfalfa (Saunders and Bingham 1975; McCoy and Bingham 1977; Kao and Michayluk 1980), peas (Malmberg 1979), red clover (Keyes et al. 1980) and white clover (Mohapatra and Gresshoff 1982). In alfalfa, improvement in regeneration efficiency has been obtained by selecting exceptional clones from material bred for the ability to regenerate from culture. These clones have been used to obtain plant regeneration from suspension cultures (McCoy and Bingham 1977) and mesophyll protoplasts (Johnson et al. 1981), and to identify factors affecting the regeneration efficiency of alfalfa (Walker and Sato 1981). In white clover, variation between ecotypes in the capacity for plant regeneration from shortterm (three-month old) cultures has been reported (Mohapatra and Gresshoff 1982). However, since white clover is a outbreeding species, ecotypes or varieties are highly heterogeneous populations. Therefore, there are marked differences in the morphogenic response of different genotypes from a variety (Bhojwani and White 1982). In this paper, plant regeneration at high efficiency from a longterm (24-month old) culture of a selected white clover genotype is reported. Some of the factors affecting morphogenesis are also investigated. Materials and methods Plant material. Trifolium repens L. seeds from four geographic origins: (1) Regal Ladino - USA, (2) Kent Wild white clover England, (3) C.P.I. 48907 - Spain, and (4) Louisiana white clover - Kenya (Grasslands Division, DSIR, seed batch. Nos. C2014(1), C2126(2), C2634(3) and C2734(4)) were used. Chemicals. Chemicals used were of " A n a l a r " grade. Kinetin (6-furfurylaminopurine) and 2,4-dichlorophenoxyacetic acid (2,4-D) were obtained from Sigma Chemical Company, St. Louis, Mo., USA, N6-benzylaminopurine, c~-naphthaleneacetic

2

D.W.R. White : Plant regeneration from white clover cells

acid (NAA) and indole-3-acetic acid from Calbiochem, San Diego, Calif., USA. Picloram (4-amino 3,5,6-trichlorpicolinic acid) was a gift from Prof. I.K. Vasil, University of Florida, Gainesville, USA.

lated (2 ml/plate) onto agar medium of the same composition. Cell aggregates (0.5-2.0 mm in diameter) producing shoot meristems were subcultured onto half-strength MS medium for further shoot development and root formation.

Callus induction, maintenance, and selection of a high-regeneration genotype. Fifty seeds from each population were surfacesterilized for 30 s in 95% ethanol followed by 6 min in 0.2% HgC12 acidified with 5 ml/1 concentrated HC1, rinsed five times in sterile distilled water, allowed to imbibe for 6 h in sterile water, and germinated for 4 d on 0.8% aqueous agar in standard plastic Petri dishes at 27~ in continuous light of 10 W m -2 from 40-W "cool-white" fluorescent lamps (Thorn, Auckland, New Zealand). For callus induction, four hypocotyl explants 2-3 mm in length were taken from each seedling and placed on B 5 medium (Gamborg et al. 1968) containing 1 mg/1 2,4-D, 0.2 rag/1 kinetin, 0.1% casein hydrolysate (" trypticase ;" BBL, Cockeysville, Md., USA), and 0.8% agar. Three weeks later, the calli were transferred to B s medium containing 0.5 rag/1 2,4-D, 0.5 rag/1 kinetin, 0.05 rag/1 picloram and 0.8 % agar, and were thereafter subcultured at three-weekly intervals onto the same medium. At each subculture, 1-cm diameter pieces of calli were also subcultured onto a regeneration medium (B s medium with 2 mg/1 kinetin, 0.2 mg/1 indole-3-acetic acid), to detect genotypes capable of a high level of plant regeneration. Genotypes that gave no plant regeneration after two subcultures were discarded. A genotype, WR8, that gave plant regeneration from all callus pieces at each subculture over a 12-month period was selected for further study.

Chromosome counts. Root tips from regenerated plants were treated in saturated 8-hydroxyquinoline + 0.05% Triton X-100 (Rohm and Haas, Philadelphia, Pa., USA) for 3-3.5 h at 4 ~ C, fixed in 1 : 3 (v/v) acetic acid: ethanol, and stained with Feulgen stain.

Suspension culture. Friable calli (approx. 1 g fresh weight) of the WR8 genotype were inoculated into 30 ml of B s medium containing 0.5 rag/1 2,4-D, 0.5 mg/1 kinetin and 0.05 rag/1 picloram, in 250-ml Erlenmeyer flasks, and incubated on a gyratory shaker at 100 rpm. The resulting fine suspension was maintained by subculturing to fresh medium every 4 d (twofold dilution). Organogenesis was initially studied by washing the suspension culture twice in B 5 medium lacking hormones and inoculating 2 ml of the culture, containing about 200 mg fresh weight of cells, onto agar medium. Media tested were B 5 medium lacking hormones, and Murashige and Skoog's (1962) medium (MS) containing combinations of N A A (0.25, 0.5, 1.0 rag/l) + benzylaminopurine (0.25, 0.5 rag/l) or N A A (1.0, 2.0 rag/l) + kinetin (1.0rag/i). Organogenesis of suspension-cultured cells in liquid medium was obtained by washing cultures twice in MS medium containing 1 rag/1 N A A + 1 rag/1 kinetin (MSH1), inoculating 500 mg fresh weight of cells into 30 ml of liquid MS-H1 medium in a 250-ml Erlenmeyer flask, and incubating for 10 d on a gyratory shaker at 100 rpm. All growth and organogenesis studies were replicated at least four times. For fresh-weight and dry-weight determinations cells were collected by filtration on a Millipore M F support pad (Millipore Corp., Bedford, Mass., USA). Fresh-weights were determined immediately and dry-weights were measured after 20 h at 80 ~ C. To determine meristemoid numbers, suspensions were subcultured after 10 d (twofold dilution), cultured for a further 7 d, and then sieved through a i-ram-pore-diameter screen to remove large cell aggregates. Samples of 10 mg fresh weight were diluted, and the number of meristemoids counted under a stereomicroscope, Plant regeneration from meristemoids. Meristemoids formed in suspension culture were maintained in the same medium for four subcultures, each of 4 d, washed twice in Schenk and Hildebrandt's (1972) medium (SH) with 1% sucrose, and inocu-

Electron microscopy. Suspension-cultured cells were treated for 2 h with Karnovsky fixative in 0.1 M sodium-phosphate buffer, pH 7.2, washed, mixed with 20% bovine serum albumen (BSA) (Sigma Chemical Co., St. Louis, Mo., USA) and pelleted by centrifugation (5 rain at 100 g). The BSA was cross-linked with glutaraldehyde (25% solution) and 1- to 2-ram slices of the embedded cell aggregates were fixed in 1% osmium tetroxide for 1 h. Segments were embedded in Epoxy resin (DurcupanA C M ; Fluka, Buchs, Switzerland) and thin sections examined using an EM 200 (Philips, Eindhoven, The Netherlands) electron microscope.

Results

Selection of a genotype capable of sustaining high levels of plant regeneration. Callus from only one of the 200 genotypes examined sustained high levels of plant regeneration. This genotype, designated WR8, came from Grasslands seed batch No. C2126. The WR8 cell line was distinguishable at the first subculture by the spontaneous development of shoot buds towards the end of the threeweek passage on callus-maintenance medium. Although transfer to regeneration medium accentuated shoot formation, shoot development also occurred when callus was transferred directly to B 5 or half-strength MS media lacking hormones. Shoots spontaneously formed roots on halfstrength MS medium whereas attempts to induce root formation with low concentrations (0.1-1.0 mg/1) of the auxins N A A or indole-3acetic acid led only to shoot proliferation. Plantlets transferred to soil developed into plants with small leaves and an intensely branching prostrate habit. Calli initiated from stolon segments taken from these plants retained the shoot-forming characteristics of the original WR8 cell line. The chromosome numbers and fertility of six plants regenerated after 12 months of callus culture were examined. Five of these plants had the normal chromosome number for white clover (2 n = 32) and produced viable seed, and one sterile plant had 33 chromosomes.

Organogenesis in suspension culture. Suspension cultures of WR8 consisted of a mixture of spherical, cytoplasm-rich ceils, the majority of which were in small cell aggregates, and single, elongate,

D.W.R. White: Plant regeneration from white clover cells

3

Fig. I A - G . Direct shoot formation from suspension-cultured cells of white clover, line WR8. A WR8 suspension-cultured cells growing in maintenance medium, x 140. B Meristemoids formed 7 d after transfer of WR8 cells to MS-HI medium, x 140. C Individual meristemoids, x 1600. D A thick section through cell aggregates grown in maintenance medium, x 500. E A thick section through cell aggregates 7 d after transfer to MS-H1 medium, x 315. F Developing shoots two weeks after plating meristemoids on SH with 1% sucrose. G Proliferating shoots derived from WR8 suspension cultured cells three months after meristemoid induction

4

D.W.R. White: Plant regeneration from white clover cells

Fig. 2 A - D . An ultrastructnral comparison of white-clover (WR8) cells grown in maintenance medium and MS-H1 medium. A, B Cells grown in maintenance medium. A Cells grown in maintenance medium had a large central vacuole. B Plastids were undeveloped and contained starch granules (arrowhead). C, D Cells grown in MS-HI medium for 7 d. C Meristemoid cells were densely cytoplasmic with small vacuoles (arrowheads). D Chloroplasts had begun to develop thylakoids (arrowhead) 7 d after transfer to induction medium. A, C bars = 5 gm, x 4 300; B, D bars = 2 gm; x 8 500

vacuolate cells (Fig. 1 A, D). Suspension cultures plated on a variety of solid media, both without hormones and with combinations of N A A and benzylaminopurine or N A A and kinetin (see Materials and methods), formed callus which formed shoots. For a further examination of the factors controlling organogenesis, attempts were made to in-

duce formation of either somatic embryos or shoots directly from WR8 cells in suspension culture. A high proportion of cells washed with and inoculated into MS medium containing 1 mg/1 N A A and i mg/1 kinetin (MS-H1) became dense and non-vacuolate after 3 4 d of culture and developed into dense, globular, green meristematic cell masses (meristemoids) 7 d after inoculation

D.W.R. White: Plant regenerationfrom white clover cells

5

Table I. The effect of ammonium source on growth, final medium pH, and meristemoidformation of white-clover (WR8) cells after 10 d of culture. Hi = 1 rag/1NAA + 1 rag/1kinetin. The mediumwas initiallyadjusted to pH 5.8. Data are means+ SE Medium

FW increase (g)

DW increase (g)

Final pH

No. of meristemoids

SH-HI SH SH-H1 with 20.6 mM NH+ a s ( N H 4 ) 2 S O 4 SH with 20.6 mM NH+ as (NH4)2SO4 SH with 20.6 mM NH+ as (NH4)C1

5.00+ 0.06 4.46 • 0.18

0.56_+0.03 0.45 _+0.03

5.42 5.59

0 52• 7

0.34_+0.09

0.05 _+0.01

4.79

593 _+48

0.77 + 0.12

0.09 + 0.01

4.79

689_+29

0.66 + 0.21

0.087- 0.01

4.70

695 _+51

(Fig. 1 B). Although some of the meristemoids were observed as discrete spherical cell masses in the medium (Fig. 1 C), most were initially part of larger cell aggregates. Sections through such cell aggregates (Fig. 1 E) showed individual cells and small groups of cells that were more densely staining. Also, some of the elongate single cells in the suspension culture underwent repeated divisions and formed meristemoids. In some, only one of the two cells arising from the first division continued dividing to form the meristemoid while the other elongate cell remained attached. Meristemoids maintained in MS-H1 medium proliferated but did not develop further. However, when plated on solid SH medium (1% sucrose) they developed numerous shoot meristems after two weeks of culture (Fig. 1 F). Subculture onto half-strength MS medium led to shoot development (Fig. 1 G). Direct regeneration of shoots from suspension-cultured WR8 cells still occurred at the same high frequency 24 months after the cells were placed in culture (i.e. 12months as callus followed by 12 months in suspension culture). A comparison of the ultrastructure of cells growing in maintenance medium and cells which had formed meristemoids after 7 d growth in MSH1 medium (Fig. 2) showed that the former cells had a large central vacuole (Fig. 2A), proplastids containing starch granules (Fig. 2 B) and numerous osmophilic globules. Meristemoid cells were characterised by a dense cytoplasm with only few, small vacuoles (Fig. 2C) which contained both large globular and small particulate osmophilic deposits and in some cells folded membrane-like material. Chloroplasts in meristemoid cells had partially differentiated and begun to form thylakoids (Fig. 2 D).

Effect of medium composition on meristemoid formation. To determine some of the factors influenc-

ing meristemoid formation in suspension culture, a series of media were tested (Table 1). When SH medium was substituted for MS medium, either no (SH-HI) or only a relatively few (SH) meristemoids were formed. Since one of the major differences between SH medium and MS medium is in the concentration of ammonium ions (2.6 mM and 20.6 mM, respectively), the influence of the NH~ concentration on meristemoid formation was determined. When the N H + concentration of either SH-HI or SH medium was raised to 20.6 mM by adding either ( N H 4 ) 2 S O 4 or NH4C1 , high frequency meristemoid formation was obtained. The addition of either 2,4-D (0.5 rag/l) or picloram (0.05 mg/1) to SH medium containing 20.6 mM N H f resulted in the complete inhibition of meristemoid formation (data not shown).

Effect of ammonium concentration on growth, final medium pH, and meristemoid formation. The formation of meristemoids in SH or SH-HI media containing 20.6 mM N H + was accompanied by a reduction in final medium pH, and growth measured as fresh-weight or dry-weight increase (Table 1). Therefore, the effect of N H ~ concentration on growth (dry-weight increase), final medium pH and meristemoid formation was examined (Fig. 3). Cells grown in SH medium formed meristemoids in the presence of lower concentrations (2.6 raM, 5.0 raM) of N H ~ than cells grown in SH-H1 medium, indicating that the presence of hormones influenced the sensitivity of the response to N H 2 . Nevertheless high-frequency meristemoid formation required at least 5 mM N H 2 and was inversely correlated with growth and final medium pH (Fig. 3). To determine whether meristemoids would form when NH4~ was the sole source of nitrogen, cells were inoculated into medium containing KC1 in place of KNO3, and either 10 mM or 20.6 mM

6

D.W.R. White: Plant regeneration from white clover cells 800

600

700 500

O

600 400 500 400

300

m

-4

O

300 200

"11 m o) m

200 100 100

v

0

0 2.6

5.6

5.0

10.0

15.0

AMMONIUM

(raM)

5.0

10.0

20.6

0

5.5 5.4 5.3

pH

5.2 5.1 5.0 4.9 4.8 4.7

B 2.6

15.0

20.6

AMMONIUM (mM)

Fig. 3A, B. Influence of NH~ concentration on growth and meristemoid formation in white-clover (WR8) cell cultures, and final medium pH. A Dry-weight increase after 10 d of culture (mg): m - - m , SH-HI ; n - - D , SH; and number of meristemoids per 10 mg FW 17 d after transfer to induction media: e - - e , SH-H1 ; o - - o , SH. B Final medium pH after 10 d of culture, e--e, SH-H1 ; o - - o , SH

NH~. In both cases, severe growth inhibition and a final medium pH of about 3.6 resulted. However, when the media were buffered to pH 5.4 with 50 mM N-morpholinoethanesulfonic acid (Mes), both growth and meristemoid formation were obtained. The final pH of the medium was 4.1. Discussion

In most species, plant regeneration capacity, if present, declines with increasing time in cell culture. The results presented in this report indicate that this problem may be overcome, at least in white clover, by selecting genotypes with long-term morphogenic capacity. In this case the selected

white-clover genotype (WR8) retained its capacity for plant regeneration throughout 24 months of culture. Once identified, such a genotype can be vegetatively propagated and becomes a reliable source of explants for initiating new morphogenic cell cultures. Although the reason(s) for the sustained plant regeneration capacity of WR8 cells is at present unknown, a number of characteristics indicate that it may be a consequence of high levels of endogenous cytokinins. These characteristics include: spontaneous shoot-bud formation on callusing medium and shoot proliferation in the presence of low concentrations of indole-3-acetic acid or NAA. Normally to obtain plant regeneration from whiteclover callus a regeneration medium containing a high cytokinin to auxin balance is required and regenerated shoots placed on media containing low concentrations of indole-3-acetic acid or NAA form roots (Gresshoff 1980). The mode of plant regeneration from cell culture usually follows one of two alternative pathways, organogenesis or somatic embryogenesis. In the organogenic pathway, separate shoot and root meristems form within the callus in response to a balance of auxin and cytokinin. Somatic embryos are thought to arise from single cells which undergo a pattern of development similar to zygotic embryos. Where this occurs, it is frequently in response to the withdrawal or reduction of high auxin levels, usually 2,4-D (for a review, see Kohlenbach 1977). The direct regeneration of shoots from WR8 suspension cultures has features in common with both of these developmental pathways. Both individual cells within cell clumps and separate single cells developed into globular meristematic cell masses, similar in morphology and ultrastructure to the proembryos formed during the early stages of somatic embryogenesis. However, the early maturation of chloroplasts, 7 d after cells were transferred to regeneration medium (Fig. 2 D), and the development of shoot meristems are both features more consistent with organogenesis. Formation of large numbers of meristemoids in WR8 suspension cultures required the removal of 2,4-D and picloram, and addition of at least 5 mM NH~. Meristemoid formation was also obtained in buffered medium containing 10 mM NH,~ as sole nitrogen source, indicating that it was the presence of NH~- per se and not an interaction between NH 2 and NO~ that was critical for this development. Similar effects of NH + in inducing morphogenesis via somatic embryogenesis in carrot (Halperin 1966; Wetherell and Dougall 1976;

D.W.R. White: Plant regeneration from white clover cells

Dougall and Verma 1978; Kamada and Harada 1979) and alfalfa (Walker and Sato 1981) have been reported. The extent to which WR8 cultures responded to increased NH + levels was influenced by the presence of phytohormones in the medium. Addition of 1 rag/1 NAA and 1 rag/1 kinetin increased growth and decreased meristemoid formation at 1Qwer NH2 concentrations (Fig. 3 A). This correlation between increased meristemoid formation, NH4~ concentration and decreased growth rate, raises the possibility that NH~- may be stimulating morphogenesis in an indirect manner by reducing the growth rate. The WR8 genotype described in this report should be a suitable material for studies on the regulation of organogenesis from suspension cultures. It can be used to obtain plant regeneration from white-clover mesophyll protoplasts (for a preliminary report, see White 1983), and may be useful for in-vitro genetic manipulation procedures such as transformation where a high regeneration efficiency is desirable. The excellent technical help of Ms. F. Olliver, and the assistance of Mr. D. Hopcroft with electron microscopy are acknowledged.

References Bhojwani, S.S., White, D.W.R. (1982) Mesophyll protoplasts of white clover: isolation, culture and organogenesis. Plant Sci. Lett. 26, 265-271 Dougall, D., Verma, D.C. (1978) Growth and embryo formation in wild-carrot suspension cultures with ammonium as a sole nitrogen source. In Vitro 14, 180-182 Gamborg, O.L., Miller, R.A., Ojima, K. (1968) Nutrient requirements of suspension cultures of soybean root cells. Exp. Cell Res. 50, 151 158 Gresshoff, P.M. (1980) In vitro culture of white clover: callus, suspension, protoplast culture and plant regeneration. Bot. Gaz. (Chicago) 141,157-164 Halperin, W. (1966) Alternative morphogenetic events in cell suspensions. Am. J. Bot. 53, 443 453 Johnson, L.B., Stuteville, D.L., Higgins, R.K., Skinner, D.Z. (1981) Regeneration of alfalfa plants from protoplasts of selected Regen S clones. Plant Sci. Lett. 20, 297-304 Kamada, H., Harada, H. (1979) Studies on the organogenesis in carrot tissue cultures. II. Effects of amino acids and inorganic nitrogenous compounds on somatic embryogenesis. Z. Pflanzenphysiol. 91, 453-463

7 Kao, K.N., Michayluk, M.R. (1980) Plant regeneration from mesophyll protoplasts of alfalfa. Z. Pflanzenphysiol. 96, 135-141 Keyes, G.J., Collins, G.B., Taylor, N.L. (1980) Genetic variation in tissue cultures of red clover. Theor. Appl. Genet. 58, 265 271 Kohlenbach, H.W. (1977) Basic aspects of differentiation and plant regeneration from cell and tissue cultures. In: Plant tissue culture and its bio-technological application, pp. 354366, Barz, W., Reinhard, E., Zenk, M.H., eds. Springer, Berlin Heidelberg New York Malmberg, R.L. (1979) Regeneration of whole plants from callus culture of diverse genetic lines of Pisum sativum L. Planta 146, 243-244 McCoy, T.J., Bingham, E.T. (1977) Regeneration of diploid alfalfa plants from cells grown in suspension culture. Plant Sci. Lett. 10, 59-66 Mohapatra, S.S, Gresshoff, P.M. (1982) Ecotypic variation of in vitro plantlet formation in white clover (Trifolium repens). Plant Cell Rep. 1, 189-192 Murashige, T., Nakano, R. (1965) Morphogenetic behaviour of tobacco tissue cultures and implication of plant senescence. Am. J. Bot. 52, 819-827 Murashige, T., Skoog, F. (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15, 473-497 Saunders, J.W., Bingham, E.T. (1975) Growth regulator effects on bud initiation in callus cultures of Medicago sativa. Am. J. Bot. 62, 850-855 Schenk, R.U., Hildebrandt, A.C. (1972) Medium and techniques for induction and growth of monocotyledonous and dicotyledonous plant cell cultures. Can. J. Bot. 50, 199-204 Syono, K. (1965) Changes in organ forming capacity of carrot root calluses during subculture. Plant Cell Physiol. 6, 403-419 Walker, K.A., Sato, S.J. (1981) Morphogenesis in callus tissue of Medicago sativa: the role of ammonium ion in somatic embryogenesis. Plant Cell Tissue Organ Cult. 1, 109-121 Wetherell, D.F., Dougall, D.K. (1976) Sources of nitrogen supporting growth and embryogenesis in cultured wild carrot tissue. Physiol. Plant. 37, 97-103 White, D.W.R. (1983) Plant regeneration from mesophyll protoplasts of white clover (TrifoIium repens L.) In: Protoplasts 1983, Poster Proc., pp. 60-61, Potrykus, I., Harms, C.T., Hinnen, A., Hutter, R., King, P.J., Shillito, R.D., eds. Birkhfiuser, Basel Boston Stuttgart Wochok, Z.S., Wetherell, D.F. (1972) Restoration of declining morphogenetic capacity in long-term tissue cultures of Daucus carota by kinetin. Experientia 28, 104~105

Received 25 October 1983; accepted 16 April 1984