Improvement of Plant Regeneration from Linum Protoplasts by the Induction of Somatic Embryogenesis

J. PlantPhysiol. Vol. 139. pp. 422-426(1992)

Improvement of Plant Regeneration from Linum Protoplasts by the Induction of Somatic Embryogenesis H. Q.

LING

and H.

BINDING

Botanical Institute, Christian-Albrechts-University, Am Botanischen Garten 1-9, W-2300 Kiel, F.R.G. Received July 24,1991 . Accepted September 16,1991

Summary Plant regeneration was obtained through the induction of somatic embryogenesis from protoplast-derived calli of the Linum species L. alpinum, L. amurense, L. hologynum, and L. perenne. Globular embryoids were formed on MS agar medium (Murashige and Skoog, 1962) with 5 pM NAA from calli that were previously cultivated for 1 month on MS medium containing 50 pM 2,4-D. This concentration was evaluated as optimum with L. perenne, revealing highest speed and highest frequency of embryoid formation. About 20 % differentiated embryoids could be germinated into complete plantlets on MS medium containing 0.25 ~M each of BAP, kinetin and zeatin, and 0.5 ~M IDA, while 70 % developed only shoots and 10% only roots, respectively. Differentiation of shoots from regenerated root tips was also observed. Plantlets transferred to the greenhouse grew vigorously. In the four species, regeneration frequencies were significantly improved by somatic embryogenesis as compared with adventitious shoot formation.

Key words: Somatic embryogenesis, Linum, protoplasts, regeneration. Abbreviations: BAP = benzylaminopurine; NAA rophenoxyacetic acid; IBA = indole-3-butyric acid.

Introduction The genus Linum is one of the largest genera of flowering plants, comprising more than 200 species (Gill and Yermanos, 1967). Linum usitatissimum L. is an important crop yielding useful oil and natural fibre. The wild species are supposed to be a rich natural resource for flax breeding. Most of those species which are already known to carry agronomically valuable genes are not cross-fertile with the crop (Seetharam, 1972); the sexual incompatibility may be overcome by the help of biotechnological methods. Regeneration of plants is a precondition for realization of gene transfer with in vitro techniques. In L. usitatissimum, diverse multicellular explants have already been grown to shoots in vitro since 1975 (Rybczynski, 1975; Gamborg and Shyluk, 1976; Mathews and Narayanaswamy, 1976; Murray et al., 1977; Nataraja and Ravi, 1984; Pretova and Williams, 1986; Zhan et al., 1989 a). Isolated pro© 1992 by Gustav Fischer Verlag, Stuttgart

=

naphthalene-1-acetic acid; 2,4-D

2,4-dichlo-

toplasts have been grown to callus (Gamborg and Shyluk, 1976) and to plants (from shoot tips: Binding et al., 1982; from cotyledons and roots: Barakat and Cocking, 1983). Different regeneration capacities from protoplasts among different genotypes were demonstrated by Ling and Binding (1987). There exist only three articles on cell and tissue cultures in Linum wild species. So far, nine species, namely L. lewissii, L. strictum (Barakat and Cocking, 1985), L. alpinum, L. amurense, L. hologynum, L. perenne, L. sufJruticosum ssp. salsoloides (Ling and Binding, 1987), L. marginale (Zhan et al., 1989b) and L. catharticum (Binding et al., 1991; in press) have been grown to plants from protoplasts via adventitious shoots. Apart from L. lewissii and L. sufJruticosum ssp. salsoloides, the regeneration frequencies of plants are unsatisfactory. This paper presents the improvement of plant regeneration via embryogenesis from protoplast-derived calli in four Linum species; a few preliminary data were presented previously (Ling and Binding, 1990).

Embryogenesis from Linum Protoplasts

120

Materials and Methods Plant materials The induction of somatic embryos was tried in eight species of the genus Linum: L. alpinum Ockendon, L. amurense Alef., L. catharticum L., L. hologynum Rchb., L. leonii F. W. Schultz, L. per· enne L., L. su./fruticosum L. ssp. salsoloides (Lam.) Rouy, and L. usita· tissimum L. Shoot cultures of these species have already been established (Ling and Binding, 1987). The cultures were grown at 25 ± 2 °C and 16-h day with white fluorescent light at about 20-40/Lmolm -2S-1.

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Induction ofsomatic embryos An embryogenesis procedure was followed that was modified from Pickardt et al. (1988). About 5 to 6-week-old calli from protoplasts of the eight Linum species were cultivated on MS agar containing 50 pM 2,4-D at 25 ± 2°C and 16-h day with white fluorescent light of about 60 - 80 /Lmol m - 2 S - 1 for 1 month. Subsequently, the calli were grown on MS agar with 5 pM NAA under the same culture conditions to differentiate embryoids. The calli were subcultured every 4-5 weeks using the NAA medium. Embryoids were separated from the calli with a sterile needle and placed on MS agar medium containing 0.25 ~ BAP, 0.25 pM kinetin, 0.251'M zeatin and 0.5 pM IBA. Well organized plantlets were transferred to greenhouse conditions. In order to evaluate the optimal 2,4-D concentration for the induction of somatic embryogenesis, each ten 4-week-old calli of L. perenne were cultured on agar media with 2,4-D in concentrations of 5~, 25 pM, 50 pM and 100 I'M for 1 month.

Results In order to find a suitable concentration of 2,4-D for the induction of somatic embryos, 4-week-old calli of L. perenne

Table 1: Numbers of embryoids at calli of L. perenne grown 4 weeks on 2,4-D media and then 12 weeks on medium containing 5 I'M NAA.

5 25 50 100

numbers of embryoids at each of calli 3 0 2 5 13 27 31 18 28 14 40 29 47 43 43 42 13 54 211 8 140 78 38 36 7 59 0 2 2 0 0 7 2 27 14 15

25

average numbers 12.5 31.9 64.4 6.9

60

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For protoplast isolation, shoot tips of about 5 mm in length were cut into small segments with a sterile razor blade. The pieces were incubated for 14-16h on a rotatory shaker (30 rpm} in a solution containing 3 % w/v Rohament PC enzyme (Roehm, Darmstadt, F.R.G.), 5 mM Ca(N0 3h and 0.5 M sorbitol. The suspensions were poured through a steel sieve of 40 pm pore size and centrifuged at about 100 x g for 5 min. The protoplasts were washed by an additional centrifugation in washing solution V 47.5/NaCI (Binding and Mordhorst, 1984) at 80 x g for 5 min. The protoplasts were grown to calli as described by Ling and Binding (1987).

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Fig. 1: Time, after the transfer from media with different concentrations of 2,4-D onto NAA medium, needed to differentiate somatic embryos in L. perenne. --: days up to the detection of the first globular embryoid; - - - - -: additional number of days up to the formation of embryoids from all of each the ten calli.

were grown on MS agar medium with different 2,4-D concentrations (see above) for 1 month. The growth rates of the calli decreased with increasing amounts of 2,4-D. At a concentration of 100 JLM, no proliferation was observed and recovery was retarded on NAA medium. After transfer to the NAA medium, all calli formed embryoids. The 2,4-D concentration of 50 JLM was most efficient with regard to the time of appearance of the first embryoid and to further development of embryoids (Table 1, Fig. 1). Shoot cultures were established from embryoids initiated with any of the 2,4-D concentrations. Microcalli from protoplasts of the eight Linum species tolerated 50 JLM 2,4-D, but growth of the calli was reduced. Chlorophyll was degraded within 7 to 10 days. After transfer onto the differentiation mediu~ with NAA, rapid growth was restored. The calli became soft and watery. Calli of L. hologynum and L. catharticum greened while most of the calli of the other species remained white-yellow or formed green spots. Somatic embryos were obtained in five species (Table 2). Subsequent growth of plants was obtained with four species, namely L. alpinum, L. amurense, L. hologynum and L. perenne. First Globular embryoids were observed in L. alpinum (Fig. 2 a) 3 weeks, in L. perenne 5 weeks, in L. amurense 8 weeks and in L. hologynum 12 weeks after the transfer from 2,4-D medium onto NAA medium, respectively. A mean number of 27.5 embryoids per gram callus was counted in L. perenne after 10 weeks on NAA medium. Embryogenesis continued over more than 1 year. Globular embryoids were separated and transferred onto the MS agar medium containing BAP, kinetin, zeatin, and IBA. Here, about 20 % of the differentiated embryoids developed into well organized plantlets (Fig. 2 b, c, d); the others formed either shoots (70%) or roots (10%). Multiple shoots were often observed (Fig. 2 e). Some of the embryoids left at calli on the NAA

424

H. Q. LING and H. BINDING

Table 2: Induction of somatic embryogenesis from protoplast-derived calli in the eight Linum species investigated. species

L. alpinum L amurense L catharticum L hologynum L leonii Lperenne L suffruticosum spp. salsoloides L usitatissimum

somatic embryogenesis after one year developmental stages

number of investigated calli

number of embryogenic calli

regeneration frequencies (%)

E P E P

96 63 60 83 30 107

88 49 0 50 0 97

91.7 77.8

50 100

0 0

n

E P n

E P E G

shoot formation frequencies (%)"

60.2

<1 20 1 50

90.6

<5 >90 0-8

.. = reported by Ling and Binding (1987) and for L catharticum, by Binding et al. (1991, in press) E = somatic embryos P = plants from somatic embryos n - no globular structures G = globular structures

medium developed to plantlets (Fig. 2 f), but appeared watery. Regeneration frequencies of plantlets from protoplast-derived calli were more than 90 % in L. alpinum and L. perenne, about 80 % in L. amurense and 60 % in L. hologynum. Plantlets from somatic embryos in the four species grew vigorously after transfer to the greenhouse (Fig. 2 g). The calli also produced roots besides somatic embryos on NAA medium. A few of the roots differentiated shoots at the tips on the NAA medium in L. alpinum, L. amurense and L. perenne (Fig. 2 h). Embryoids were also induced from calli of L. sufJruticosum ssp. salsoloides (Fig. 2 i). However, after transfer to the cytokinin-containing MS medium, they formed callus that then regenerated adventitious shoots. In L. usitatissimum, numerous globular structures were formed on the N AA medium (Fig. 2 j), which, however, did not develop to embryoids on the cytokinin-containing MS medium. In L. leonii and L. catharticum, even no globular structures were observed.

Discussion Regeneration from protoplasts through somatic embryogenesis was achieved in L. alpinum, L. amurense, L. hologynum and L. perenne. The efficiencies in plant regeneration from protoplasts were significantly improved in these four species, as compared with regeneration frequencies

through adventitious shoots (see Table 2); especially in L. aipinum and L. perenne, plant regeneration was raised by more than 2a-fold. However, the Linum species responded largely differently from failure in L. catharticum and L. leonii to high efficiencies in L. alpinum, L. amurence, L. hologynum and L. perenne. The optimum of 2,4-D concentration evaluated with L. perenne was the same as found for Vicia narbonensis (Pickardt et al., 1988); nevertheless, it might be argued that the differences in response of Linum species were due to other optima. Pretova and Williams (1986) reported on direct somatic embryogenesis from immature zygotic embryos of L. usitatissimum on Monnier's basal medium supplemented with O.OSmgL - I BAP, 1.0gL - I yeast extract and 0.4 g L -I glutamine. At these culture conditions, shoot-protoplast-derived calli and embryo-like structures of L. usitatissimum failed to form embryoids (data not shown). The fact that a similar sequence was efficient in Vicia narbonensis (Pickardt et al., 1988) and Actinidia chinensis (Mii et aI., 1988) suggested that this principle might be applicable to a wide range of taxa. With Linum, the process of embryogenesis was divided into three phases: induction phase (2,4-D phase), development phase of embryoids (NAA phase) and germination phase (cytokinin phase). Also in other taxa, auxins were the most important factors for induction and development of embryoids. The presence of 2,4-D is usually necessary in the initiation phase of embryogenesis. Apparently, 2,4-D allows reprogramming of somatic cells to embryogenic development and stimulation of mitosis.

Fig. 2 a-j: Regeneration through somatic embryogenesis from protoplast-derived Linum calli. a) Globular embryoids differentiated within 21 days after the transfer from 2,4-D medium onto NAA medium in L alpinum, bar - 0.3 mm; b) and c) embryoids developed on cytokinin medium, bar - 0.2 mm; d) complete plantlet formed on cytokinin medium, bar m 0.5 mm; e) multiple shoot formation from a somatic embryo of L perenne, bar = 0.5 mm; f) plantlet from somatic embryo at the callus on NAA medium, bar - 0.4 mm; g) young plant of L perenne in greenhouse, bar = 20 mm; h) shoot regeneration at a root tip of L alpinum, bar - 0.4 mm; i) somatic embryos of L suffruticosum ssp. salsoloides, bar - 0.2 mm; j) globular structures of L. usitatissimum, bar - 0.5 mm.

Embryogenesis from Linurn Protoplasts

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H. Q. LING and H. BINDING

Acknowledgements A grant of the Ernst und Berta Grimmke-Stiftung to Ling Hong Qing is greatfully acknowledged. We thank D. Brauer, N. P. Z., Holtsee, and Dr. H.-P. Piitz, G. F. P., Bonn, for their helpful interest.

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