Plant Cell Reports

Plant Cell Reports (1995) 15:26-29

© Springer-Verlag1995

Thidiazuron-induced high frequency of shoot induction and plant regeneration in protoplast derived pea callus Petra Biihmer, Beate Meyer, and Hans-J6rg Jacobsen Lehrgebiet Molekulargenetik, Universitfit Hannover, Herrenh/iuserstr. 2, D-30419 Hannover, Germany Received 11 September 1994/Revised version received 20 February 1995 -Communicated by H. Ltrz

Abstract. Protoplasts isolated from lateral shoot buds of cotyledon-free pea embryo axes were regenerated to callus. Protoplast derived calluses with a diameter of about lcm were transferred to shoot induction media, containing different concentrations (1-50pM) of thidiazuron. Shoot formation was observed after 16 weeks up to 12% efficiency. Thidiazuron (10pM) was the most effective concentralJon in all experiments. Shoot buds elongated in medium supplemented with N-isopentenyl adenine and indole-3-butyric acid. Since rooting was almost impossible in these thidiazuron-induced shoots, shoots were grafted onto young pea seedlings and regenerated to fertile plants. Abbreviations 2ip, N-isopentenyl adenine; IAA, indole3-acetic acid; IBA, indole-3-butyric acid; MES, 2-(Nmorpholino)ethane acid; NAA, naphthaleneacetic acid; TDZ, thidiazuron; BAP, 6-benzylaminopurine; PEG, polyethylene glycol Introduction

In vitro-regeneration and transformation of economically important grain legumes like pea (Pisum safivum L.) are still far from being routine operations, although significant progress has been reported in recent years. Regeneration via organogenesis and somatic embryogenesis was achieved using different complex tissues (Gamborg et al. 1974, Malmberg 1979, Mroginski & Kartha 1981, Rubluo et al. 1984, Hussey & Gunn 1984, Kysely et al. 1987, Natali & Cava]lini Correspondence to: H.-J. Jacobsen

1987, Jackson & Hobbs 1990, Tetu et al. 1990, Nauerby et al. 1991, Ozcan et al. 1992, (3zcan et al. 1993) as well as from protoplasts (Puonti-Kaerlas & Eriksson 1988, Lehminger-Mertens & Jacobsen 1989a,b). For crop plants, regeneration protocols are generally being developed to be used in genetic transformation experiments and they must fulfill certain criteria. In this respect, most of the regeneration systems described for grain legumes up to now were not applicable to transformation, since (a) in Agrobactefium tumefaciens mediated gene transfer or particle bombardment, the transformation competence of regenerative tissue is not very efficient, and (b) the use of multicellular explants for transformation often resulted in chimeric callus and plants (Puonti-Kaerlas et al. 1992). The availability of an efficient protoplast regeneration system would help overcome these problems since transgenic clones are obtained by transformation of single cells. Both protoplast based regeneration systems published for pea so far, however, were found not to be suitable for transformation (Puonti-Kaerlas et al. 1992, own experience). In the present paper we report a protocol for pea protoplast regeneration via organogenesis which combines a high efficiency of regeneration with a high transformation competence. Material and Methods Lateral shoot buds of pea (Pisum sativum L.) cv. Finale and cv. Solara were used as source material for protoplast isolation. Seed material was kindly provided by Cebeco-Zaden (Lelystad, The Netherlands). Isolation and culture of protoplasts as well as regeneration up to the

2"7 microcallus stage were done as described by Lehminger-Mertens & Jacobsen (1989a), except that all media were prepared without casein hydrolysate. Shoot Induction. Protoplast derived-calluses (diameter lcm) were transferred to shoot induction media (MSI), supplemented with different concentrations of thidiazuron (1.5, 10, 15, 20, 25 or 50pM, respectively).MSI-medium consisted of MS-salts (Murashige & Skoog 1962), enriched with Bs-vitamins (Gamborg et al. 1968), 2% sucrose, 3% mannitol and 0.05% MES. The medium was solidified with 0.7% plant-agar (Duchefa, The Netherlands) and the pH was adjusted with 1N KOH to 5.7 prior to autoclaving. All hormones were added after sterilisation. Cultures were grown at 22+2°C with a 16 h photoperiod. Subculturing was done every 14 days until shoot buds were regenerated. Shoot Culture. For the multiplication of shoots and shoot elongation,

Fig. 1 .: Shoot bud formation after TDZ treatment (16 weeks)

explants with regenerated shoot buds were transferred to elongation media (MSE) with MS-salts, Bswitamins, 2% sucrose, 0.05% MES, lpM IBA, 12, 25 or 50pM 2iP and 0.45% Gelrite (Roth), pH 5.7. Shoots were subcultured every three weeks until they reached the length of lcm. Rooting and Grafting. Shoots > 1cm were carefully removed from the calluses and placed on various root induction media containing MS/2-salts, 3% sucrose and 0.7% plant agar, pH 5.7. Several procedures described for the rooting of pea shoots were tested (Malmberg 1979, Mroginski & Kartha 1981, Kublakova et al. 1988, Cardi et al. 1991). In addition, media with 2pM NAA, 5.3pM NAA, 0.5pM IAA+ 5tJM IBA+ 21JM NAA, 0.51JM IAA+ 5tiM IBA or media without any hormones were tested as putative media for inducing rooting in TDZ-induced shoots. All media were also used with 0.2% graphite added. In another experiment, shoots were grafted onto hypocotyls or epicotyis of sterile-grown6-10 day old pea-seedlings (same cultivar as the regenerated shoots) according to the procedure of Pickardt et al. (1992). Grafted shoots were cultured on medium consisting of 1/10 MS-salts, 2% sucrose, 0.05% MES and 0.7% plant-agar, pH 5.7 for 2-3 weeks. Developing plantlets were transferred to small pots with sterile soil, sand and perlite (2:1:1) and were acclimatized in the growth room before transfer into the greenhouse.

Results and Discussion

Regeneration of Plants from Protoplast Culture We developed a regeneration system for pea protoplast-derived callus via organogenesis, by using the strong cytokinin analogue thidiazuron (TDZ) as an effective shoot-inducing growth regulator (Fig. 1). Protoplast-derived pea calluses, however, survived the transfer to the regeneration medium (MSI) only after they had reached a diameter of lcm. Smaller calluses failed to develop further, turned brown and died. So it is likely that both the vitality and the size of the callus are important factors for successful regeneration.

TDZ has been shown to exhibit strong cytokinin activity, similar to that of N6-substituted adenine derivatives in various cytokinin bioassays (Mok et al. 1982; Capelle et al. 1983; Thomas & Katterman 1986). It is hypothesized that TDZ mimics the effects of cytokinins on organ formation (Leshem et al. 1994). TDZ induced shoot formation occurred in almost all concentrations tested (Tab.l), but 10pM was found to be optimal for inducing organogenesis in both cultivars. TDZ (10pM) was also shown to be the optimal level in some other legumes: induction of direct shoot formation in

Phaseolus vulgaris L. (Malik & Saxena 1992) and somatic embryogenesis in intact seedlings of Arachis hypogaea L. (Gill & Saxena 1992). We determined the shoot induction frequency of cv. Finale at 12% and cv. Solara at 8% (Tab.l).

Shoot Elongation and Multipfication Unfortunately, the use of TDZ results in extremely short shoots in many species, which elongate at much slower rates (this study, Preece & Imel 1991). This drawback was overcome by transferring the shoot-bud bearing calluses to MSE-medium. The application of 25pM 2iP resulted in a well-balanced relationship between shoot elongation and multiplication (Tab.2).

28 Table 1 : Effect of TDZ-concentration on frequency of shoot formation n=190 calluses for each treatment, % 2 n/100 calluses with 1 regenerated shoot; means for 101JMTDZ based on two replicate experiments; nd, not determined)





















Table 2: Influence of 2iP on shoot elongation and multiplication

Shoot cultures on MSE-medium continued to produce elongating shoots for more than one year.

(+: hardly any elongation/ multiplication,++: good elongation/ multiplication,+++:very good elongation/multiplication)

2ip conc.













The shoots elongated within 4-10 weeks to a size of ~lcm (Fig.2). When the calluses were Fig. 2: Regenerated

transferred to media containing only auxin or no

and elongated shoot

hormones, the shoot buds became stunted. Contrary

on MSE-medium

to the results published by Sriskandarajah et al. (1990) for apple (13.95pM 2iP), we obtained elongation of pea-shoots with 12pM 2iP. Good elongation was observed when we used 25pM 2iP. 50pM was also tested, but in contrast to Preece and Imel (1991, Rhododendron hybrids) elongation was not as good as

TDZ seems to be more effective in pea than BAP or other cytokinins when we compare our data with the results of protoplast-regeneration previously Puonti-Kaerlas

Shoots longer than 1cm (Fig.2) were excised from the calli and transferred to different rooting media. In all experiments no rooting of TDZ-induced, elongated shoots was achieved. Sometimes root primordia-like

with 25pM 2iP.


Rooting of Shoots




determined a shoot frequency of 1-1.4% for two cultivars. Lehminger-Mertens & Jacobsen (1989a) had a shoot regeneration which was relatively high, about

structures at the cut surface of the shoots were observed but no further development occurred. These results are consistent with the data published by Lu (1993) and Leshem et al. (1994). They reported that long exposure to TDZ results in difficulties to obtain shoots with regenerated roots. Grafting was an alternative way to overcome these problems (Fig .3).

10% for the cultivar Belman, but further development of normal shoots was not obtained (Jacobsen & Lehminger-Mertens, unpublished). First regeneration of shoots was obtained after 16 weeks on 10pM TDZ (Fig.l) and the main proliferation of shoot buds occurred between 16-20 weeks. Shoot buds appeared as small clusters at basal regions of the callus. These regions turned green before organized structures became visible.

Fig. 3: Shoot grafted onto hypocotyl (left) or epicotyl (right) of sterile grown decapititated pea seedlings

29 The grafting rate was found to be 100%. Plantlets and plants produced in this way were much more vital and showed only the typical symptoms of test tube derived plants detectable usually. More than onehundred regenerated pea plants have been successfully transferred into the greenhouse, where they subsequently flowered and produced seeds (Fig .4).

Fig. 4: Regenerated pea plants with flowers and pods The duration of the entire procedure reported here, starting with the isolation of protoplasts to the seed-bearing primary regenerants requires twelve months. The regeneration-system includes grafting. Applying this technique enabled us to regenerate whole plants, which is a major achievement compared to results reported earlier (Lehminger-Mertens & Jacobsen 1989a, Puonti-Kaerlas & Eriksson 1988), where rooting of regenerated shoots was unsuccessful and thus no plants could be obtained. Experiments with transgenic calluses derived from PEG-transformed pea protoplasts are on the way. First results indicate that the present system is also applicable for the development of transgenic peas through direct gene transfer.

Acknowledgements Parts of this work have been financed through a GFP/BML-grant to HJJ (OE 67/90NR).

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Thidiazuron-induced high frequency of shoot induction and plant regeneration in protoplast derived pea callus.

Protoplasts isolated from lateral shoot buds of cotyledon-free pea embryo axes were regenerated to callus. Protoplast derived calluses with a diameter...
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