Plant Cell Reports
Plant Cell Reports (1991) 10:296-299
9 Springer-Verlag1991
Long-term optimized embryogenie cultures in durum wheat ( Triticum durum Desf.) G . M . BorreUi 1, E. Lupotto 2, E Locatelli 2, and G. Wittmer 1 1 Isfituto Sperimentale per la Cerealicoltura, S.S. 16 Kin. 675 - 71100 Foggia, Italy 2 Istituto Sperimentale per la Cerealicoltura, Via Stezzano 24, 24100 Bergamo, Italy Received April 22, 1991/Revised version received June 13, 1991 Communicated by H. L6rz
Five varieties of durum wheat: Appulo, Ofanto, Latino, Creso, and Castello (Triticum durum Desf.) adapted to the semi-arid mediterranean environment have been tested for their in vitro response. Compact, embryogenic, highly regenerable calli originated from primary callus derived through proliferation of the scutellum of immature embryos explanted in the presence of 2,4 dichlorophenoxyacetic acid. Selective subculture of the white, compact, embryogenic sectors led to the establishment of long-term cultures. Regeneration occurred on hormone-free medium either via germination of somatic embryos, or via multiple-shoot formation probably due to precocious germination of somatic embryos. The three varieties, Ofanto, Creso and Appulo, were the best responding genotypes. Callus fragmentation and two subsequent transfers onto fresh medium at 7-day intervals yielded a frequency of plant regeneration of some 2540 plantlets per gram fresh weight callus in 21 days on Murashige and Skoog's hormone-free medium. Plantlets could be efficiently established in soil, thus confirming the possibility of biotechnological approaches with varieties of this crop species. ABSTRACT.
ABBREVIATIONS: E, embryogenic; NE, non embryogenic.
MS, Murashige and Skoog's (1962) medium; 2,4-D, 2,4 dichlorophenoxyacetic acid; DAA, days after anthesis; FWT, fresh weight tissue. INTRODUCTION
Although not yet a routine method, microprojectile bombardment (the biolistic approach, Sanford 1990) and protoplast direct gene transfer (Potrykus 1990), appear as the most promising methods for stable transformation of cereal cells. In either system, highly regenerable, longterm embryogenic cultures represent a major need for any approach to gene transfer in cereals. To date, optimized embryogenic cereal cultures for this purpose have been reproducibly obtained in maize (e.g. Armstrong and Green 1985; Duncan et al. 1985), rice (e.q. Yamada et al. 1986) and bread wheat (Redway et al. 1990; Vasil et al. 1990). Other cereals provide embryogenic and/or organogenic cultures not completely optimized, which need further refinement for proper use in gene transfer methodology. Plant regeneration from tissue cultures of various hexaploid wheat genotypes (Tritic~um aestivum L.) has been successfully achieved (Ozias-Akins and Vasil 1982; Lazar et al. 1983; Maddock et al. 1983). More recently, the identification of specific callus types for long-term cultures (Redway et al. 1990) and the establishment of Offprint requests to." G.M. Borrelli
embryogenic suspension cultures and protoplast systems in hexaploid wheat have been reported (Vasil et al. 1990; Chang et al. 1991; Redway et al. 1991). Conversely, very little information is available on tissue culture of the tetraploid durum wheat (Triticum durum Desf.) Which is the most recalcitrant species among wheats (Bennici 1986). Plant regeneration has been achieved in callus derived from mature (Eapen and Rao 1982) and immature embryos (Bennici et al. 1988), as well as from mesocotyl and leaf base tissues (Greco et al. 1984), and the regenerative pathway mainly described as organogenic. To our knowledge, no protoplast system in durum wheat has yet been established and no transformation techniques have been applied to optimized embryogenic cultures. In order to broaden the spectrum of genotypes to be used in tissue culture in search of optimized embryogenic cultures for genetic manipulation, we considered different durum wheat varieties, chosen among those adapted for cultivation in the dry mediterranean areas. In the present work we have assessed the possibility of establishing embryogenic cultures from immature embryos of five durum wheat varieties, and of selecting long-term embryogenic callus types characterized by high regenerative capability. MATERIALS AND METHODS
Seeds and plants were provided by and grown at the Section of the Experimental Institute for Cereal Research in Foggia. The following varieties, namely Appulo, Ofanto, Latino, Creso and Castello, suitable for cultivation in the dry mediterranean areas, were chosen. Plants were grown in a growth chamber in the following conditions, established for simulating the natural growing season in the field: 10 days at 6~ complete darkness, relative humidity 70%; 12 days at 10/14 hours light/dark regime, at 8 and 6~ respectively, relative humidity 70%; 10 days at 15/9 hours light/dark regime, at 12 and 8~ respectively, relative humidity 55%; I0 days at 15/9 hours light/dark regime, at 15 and 10~ respectively, relative humidity 50%; 10 days at 15/9 hours light/dark regime, at 20 and 10~ respectively, relative humidity 45%; and finally, until complete maturation, 16/8 hours light/dark regime, at 23 and 180C respectively, relative humidity 40%. Light was Provided by Philips TLD 58 W fluorescent lamps with an intensity of about 4000 lux. Immature embryos, taken at 15 DAA, were used as explants. The caryopses were removed from the spike and sterilized by rinsing in 70% (v/v) ethanol, washing in 20% NaOCI (v/v) with 0.1% (v/v) Tween 80 surfactant (Merck) for 20 minutes, and finally
297 rinsed four times in sterile distilled water. The immature embryos (no more than 1.5 mm long) were excised under a stereo dissecting microscope and explanted onto the culture medium with the scutellum exposed. Cultures were initiated, maintained and regenerated on 0.8% Difco Bacto Agar gelled medium autoclaved at 1.2 kg/cm2 at 121~ for 20 minutes, in 90 mm @ Petri dishes. Callus initiation and maintenance was at 24 + 2~ in complete darkness. For callus induction and propagation, three formulations derived from MS basic medium (Murashige and Skoog, 1962) were considered respectively indicated as MS (MS salts and vitamins, 30 g/1 sucrose), MSTDI (MS salts and vitamins, I g/l enzymatic hydrolysate of casein (Sigma), thiamineHCl raised up to 0.4 rag/l, 30 g/l sucrose) and MSTD2 (MS salts and vitamins, m-inositol raised up to 200 mg/1, 200 mg/l L-asparagine, thiamine HC1 raised up to 0.4 mg/l, 25 g/l sucrose). Media were all adjusted to pH 5.8 w i t h 0.i M KOH a n d autoclaved. Three concentrations, 1, 2 and 5 rag/1 2,4-D were tested for callus initiation. Primary callus induction frequency was evaluated 20 days after the explant in a total of 300 embryos/genotype/treatment in three replicates. Primary callus was subcultured every 3 weeks onto the same media supplemented with 1 mg/l 2,4-D in complete darkness at 24+2~ Callus cultures derived from each original zygotic embryo explanted, were considered different cell lines. Following the third subculture embryogenic, white, compact and nodular sectors (E callus) arose at the callus surface in some of the cultures. This E callus was separately subcultured in the presence of 1 rag/1 2,4D onto the same media solidified with 0.7% Sigma Agarose (type II - medium EEO). In three-four subcultures, the white nodular callus represented the majority of the callus structure. These were then indicated as "optimized cultures"
or "aged callus", according to Redway e_ttal__. (1990). Callus growth was recorded as growth index (GI) at day 21, calculated as follows: ](FWTt21 - FWTt0)/FWTt0 I. For regeneration, five-month old aged callus was transferred onto MS, hormone-free medium and incubated at 24+2~ with a 16/8 hours light/dark regime. The light source was Philips TLD 58 W fluorescent lamps. After 7 days, germinating somatic embryos were moved onto fresh medium twice at 7 day intervals; at each transfer, callus was fragmented and plated. Regenerated plantlets were separately transplanted. This procedure promoted extensive plant regeneration; 10-30 mm h i g h plantlets were moved to 200 ml glass flasks for complete development and rooting onto the same agar medium. When plantlets reached 15-18 cm in size, they were transferred into peat pots, after washing the roots free of agar medium. Roots were dipped a few seconds in a 0.05% (w/v) solution of antifungine (Zineb| and potted in autoclaved 3:1 (v/v) compost:washed sand. Plants were hardened in a growth chamber at 21~176 in a 16/8 hours day/night cycle, then transferred outdoors. RESULTS AND DISCUSSION
Callus induction and propagation. Scutellum of immature embryos of the five durum wheat varieties formed callus in about 20 days on the various media tested. Preliminary experiments were performed u s i n g three different concentrations of 2,4-D: 1, 2, and 5 mg/l (results not shown); the concentration of 2 mg/l 2,4-D was subsequently chosen as routine for callus induction, and i mg/l 2,4-D for callus propagation. The optimal response in culture was given by embryos taken at 15 DAA, which were 1.0-1.3 mm long. These results are consistent with those obtained by Bennici et al. (1988) and differ from the response reported
Table I. Genotypicresponse of five durum wheat varieties in callus induction frequency, secondary embryogenic callus establishment, and regeneration
Variety APPULO
Basic culture medium a MS MSTD1 MSTD2
106 109 115
14 8 16
900
94.5
330
38
300 300 300
71.4 67.3 73.5
31 26 25
0 0 1
900
70.7
82
1
MS MSTD1
300 300
84.1 82.0
36 39
16 9
MSTD2
300
82.9
41
12
900
83.0
116
37
300 300 300
73.2 67.1 74.5
55 25 34
0 12 8
900
71.6
114
20
300 300 300
96.7 96.4 92.8
86 119 136
69 14 19
900
95.3
341
102
MS MSTD1 MSTD2
Total
LATINO
MS MSTD1 MSTD2
Total OFANTO
Total
N u m b e of r secondary calli propagated
93.4 92.1 98.0
Total CRESO
Number of primary calli propagated c
300 300 300
Total CASTELLO
Number of Callus zygotic embryos induction explanted b frequency (%)
MS MSTD1 MSTD2
a media are supplemented with 2 mg/1 2,4-D b pool of three replicates per culture medium formulation c media are supplemented with 1 mg/l 2,4-D
Number of regenerable cultures
12
-
%
31.6
-
13
35.1
7
35.0
65
63.7
298
Figure I. Somatic embryogenesis and plant regeneration in the durum wheat variety Ofanto. a) compact, highly embryogenic secondary calli (24X) in propagation on MSTD2 medium in the dark; b) extensive regeneration of the E callus after 15 days transfer onto MS hormone-free medium; c) an isolated regenerated plantlet (24X). for bread wheat (T. aestivum) in which a younger developmental age (10-14 DAA) seemed to be the most responsive stage for explants (OziasAkins and Vasil 1982). Callus developed from the upper part of the scutellum in contact with the culture medium. The first callus produced could not be clearly classified as embryogenic tissue, being yellowish, rather wet, meristematic and nodular in appearance. Callus induction frequency was 95.3% and 94.5% for Ofanto and Appulo respectively, which were the best responding genotypes. Creso was slightly less responsive with a callus induction frequency of 83%, followed by Latino and Castello with values of 71.6% and 70.7%, respectively (Table 1). Within each genotype, no significant difference among values of callus induction frequency was detected depending on the basic culture medium used. For all genotypes, however, the lowest value was registered for the MSTD1 formulation. The presence of various organic compounds did not affect callus induction frequency and/or callus texture, and these results confirm the observations reported in hexaploid wheat (Redway e t al. 1990). The real performance in culture of each variety was better described by the percentage of primary calli propagated in culture. Primary calli were subcultured explantfree 20 days after embryo plating and each cell line derived from an individual zygotic embryo was independently propagated. Primary calli were subcultured twice (3 weeks each subculture) before the secondary white E callus started to appear in a few cultures per genotype. Variety Ofanto again was the best genotype, establishing 102 E callus cultures, followed by Appulo, Creso and Latino; only one E callus line could be established for Castello (Table I). From this time onwards, calli were selectively subcultured by choosing the most compact nodular tissues. Upon selective subculture of the E tissues, a gradual conversion of the callus phenotype toward the more E compact callus type was obtained (Fig. la). Medium solidified with agarose instead of agar was more suitable for propagation of the E calli. This procedure and the derived cultures are similar t o those recently described in hexaploid
wheat as "aged callus" (Redway et al. 1990). The obtainment of these optimized cultures, which were recently reported to be the most competent tissues for initiating embryogenic suspension cultures (Redway et al. 1991) and regenerable protoplasts (Vasil et al. 1990; Chang e t a l . 1991) represents, to our knowledge, a unique result in durum wheat studies. Values of the growth index (GI) calculated at the fifth subculture from primary callus establishment on five E cultures per genotype, were indicative of their performance (Table 2). E calli for each genotype were subcultured onto the same medium in which they originated. Growth rates were different in the various cases. It has to be noted that callus growth was totally due to the proliferation of the E callus. Subsequent experiments demonstrated that the formulation of MSTD2 was better suited for the long-term E culture propagation in the various genotypes. MSTD2 also was the most suitable culture medium for suspension cultures and for establishing friable cell lines in the variety Appulo (results will be reported elsewhere). These embryogenic cultures have been efficiently propagated for 10 months to date, without loosing or changing their embryogenic phenotype and regenerative capability. Plant regeneration. Regeneration was tested on the selected cultures, 5 subcultures after the E culture establishment. For each variety several cell lineages were considered and regeneration tested on basic MS hormonefree medium. For plant regeneration, calli were firstly transferred as 100 mg FWT pieces onto MS hormonefree medium. After a 7-days incubation in the light, calli were further fragmented and plated back onto fresh medium. Plantlets were removed throughout the culture period. Fragmentation and plating were repeated twice: this three-step regeneration procedure yielded on the average approximately 25-40 plantlets per gram FWT depending on the genotype. Consistent genotypic differences were detected in the culture ability to initiate shoots and plantlets, despite the culture medium used for callus
299 Table 2. Average value of growth index (GI) calculated from the fresh weight of 5 independent E callus cultures in three different varieties onto MS, MSTD1, and MSTD2 media, following a 21 day subculture Culture medium
GI
OFANTO
MS MSTD1 MSTD2
6.5 5.1 3.2
APPULO
MS MSTD1 MSTD2
5.1 4.6 4.1
CRESO
MS MSTDI MSTD2
2.7 5.7 6.1
Genotype
Growth index is measured as increment in the fresh weight of the tissue at the end of a 21 day subculture GI represents the ratio: FWTt21 - FWTt0 FWTt0
maintenance. The variety Ofanto was the most responsive genotype, in which 65 out of 102 cell lineages produced plantlets. Regenerative capabilities registered for Creso, Latino and Appulo were 35.1, 35.0 and 31.6% respectively, although the number of propagated cell lineages was far less (37, 20 and 38 respectively). Callus cultures of the variety Castello were the least responsive and could not be propagated past the second subculture as E callus. Each regenerable cell line gave rise to multiple shoots and seedlings and subsequently developed into complete plantlets (Fig. Ib, c). Depending on the genotype a high percentage of regenerated plantlets (in the range of 70-90%), could successfully be established in soil. Nearly 80% of the established plants were f e r t i l e a n d set seeds. With regard to t h e type of regeneration from the embryogenic cultures, we could recognize two pathways of development: one probably identifiable as multiple-shoot formation (Shimada and Yamada 1979; Maddock et al. 1983), the other via true somatic embryogenesis of single plantlets (Ozias-Akins and Vasil 1982). However, because of the highly embryogenic texture of the callus cultures, we are in general agreement with the studies performed on hexaploid wheat by OziasAkins and Vasil (1982), who concluded that regeneration occurs via somatic embryogenesis. Somatic embryos germinated "precociously" forming multiple shoots before their complete development. In all cases shoots rooted promptly in the same medium. The high number of regenerated complete plants and the establishment of longterm embryogenic cultures in several varieties of durum wheat as described in the present work, is of particular importance for the application of genetic manipulation to this crop species. Acknowledgements. The present work was promoted and strongly supported by the enthusiasm of Prof. G. Wittmer, director of the Section in Foggia, who died on September 12, 1990. The work was developed within the framework of the program of research "Advanced Biotechnologies applied to Plants" held by Ministero Agricoltura e Foreste, Roma. G.M.B. and F.L. hold fellowships within the program.
REFERENCES
Armstrong CL, Green CE (1985) Establishment and maintenance of friable, embryogenic maize callus and the involvement of L-proline. Planta 164: 207-214. Bennici A (1986) Durum wheat (Triticum durum Desf.). In: Bajaj Y.P.S. (ed) Biotechnology in Agriculture and Forestry, Vol. 2, Crops I. Springer Verlag, Berlin Heidelberg New York, pp 89-104. Bennici A, Caffaro L, Dameri R M, Gastaldo P, Profumo P (1988) Callus formation and plantlet regeneration from immature Triticum durum Desf. embryos. Euphytica 39: 255-263.. Chang Y-F, Wang WC, Warfield CY, Nguyen HT, Wong JR (1991) Plant regeneration from protoplasts isolated from long-term cell cultures of wheat (Triticum aestivum L.). Plant Cell Rep. 9: 611-614. Duncan DR, Williams ME, Zehr BE, Widhlolm JM (1985) The production of callus capable of plant regeneration from immature embryos of numerous Zea mays. genotypes. Planta 165: 322-332. Eapen S, Rao PS (1982) Plant regeneration from callus cultures of Durum and Emmer wheat. Plant Cell Rep. 1: 215-218. Greco B, Tanzarella OA, Blanco A (1984) Plant regeneration from leaf base callus in durum wheat (Triticum durum Desf.). Cereal Res. Comm. 12: 171- 177. Lazar MD, Collins GB, Vian WE (1983) Genetic and environmental effects on the growth and differentiation of wheat somatic cell cultures. J. of Heredity 74: 353357. Maddock SE, Lancaster VA, Risiott R, Franklin J (1983) Plant regeneration from cultured immature embryos and inflorescences of 25 cultivars of wheat (Triticum aestivum). J. Exp. Bot. 34: 915-926. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco culture. Physiol. Plant. 15: 473-497. Ozias-Akins P, Vasil IK (1982) Plant regeneration from cultured immature embryos and inflorescences of Triticum aestivum L. (wheat). Evidence for somatic embryogenesis. Protoplasma 110: 95-105. Potrykus I (1990) Gene transfer to plants: assessment and perspectives. Physiol. Plantarum 79: 125-134. Redway FA, Vasil V, Lu D, Vasil IK (1990) Identification of callus types for long-term maintenance and regeneration from commercial cultivars of wheat (Triticum aestivum L.). Theor. Appl. Genet. 79: 609617. Redway FA, Vasil V, Vasil IK (1991) Characterization and regeneration of wheat (Triticum aestivum L.) embryogenie cell suspension cultures. Plant Cell Rep. 9 (in press). Sanford JC (1990) Biolistie plant transformation. Physiol. Plantarum 79: 206-209. Shimada T, Yamada Y (1979) Wheat plants regenerated from embryo cell cultures. Jap. J. Genet. 54: 379-385. Vasil V, Redway F, Vasil IK (1990) Regeneration of plants from embryogenic suspension culture protoplasts of wheat (Triticum aestivum L.). Bio/Technology 8: 429434. Yamada Y, Yang ZQ, Tang DT (1986) Plant regeneration from protoplast-derived callus of rice (0ryza sativa L.). Plant Cell Rep. 5: 85-88.
Plant Cell Reports (1991) 10:296-299
9 Springer-Verlag1991
Long-term optimized embryogenie cultures in durum wheat ( Triticum durum Desf.) G . M . BorreUi 1, E. Lupotto 2, E Locatelli 2, and G. Wittmer 1 1 Isfituto Sperimentale per la Cerealicoltura, S.S. 16 Kin. 675 - 71100 Foggia, Italy 2 Istituto Sperimentale per la Cerealicoltura, Via Stezzano 24, 24100 Bergamo, Italy Received April 22, 1991/Revised version received June 13, 1991 Communicated by H. L6rz
Five varieties of durum wheat: Appulo, Ofanto, Latino, Creso, and Castello (Triticum durum Desf.) adapted to the semi-arid mediterranean environment have been tested for their in vitro response. Compact, embryogenic, highly regenerable calli originated from primary callus derived through proliferation of the scutellum of immature embryos explanted in the presence of 2,4 dichlorophenoxyacetic acid. Selective subculture of the white, compact, embryogenic sectors led to the establishment of long-term cultures. Regeneration occurred on hormone-free medium either via germination of somatic embryos, or via multiple-shoot formation probably due to precocious germination of somatic embryos. The three varieties, Ofanto, Creso and Appulo, were the best responding genotypes. Callus fragmentation and two subsequent transfers onto fresh medium at 7-day intervals yielded a frequency of plant regeneration of some 2540 plantlets per gram fresh weight callus in 21 days on Murashige and Skoog's hormone-free medium. Plantlets could be efficiently established in soil, thus confirming the possibility of biotechnological approaches with varieties of this crop species. ABSTRACT.
ABBREVIATIONS: E, embryogenic; NE, non embryogenic.
MS, Murashige and Skoog's (1962) medium; 2,4-D, 2,4 dichlorophenoxyacetic acid; DAA, days after anthesis; FWT, fresh weight tissue. INTRODUCTION
Although not yet a routine method, microprojectile bombardment (the biolistic approach, Sanford 1990) and protoplast direct gene transfer (Potrykus 1990), appear as the most promising methods for stable transformation of cereal cells. In either system, highly regenerable, longterm embryogenic cultures represent a major need for any approach to gene transfer in cereals. To date, optimized embryogenic cereal cultures for this purpose have been reproducibly obtained in maize (e.g. Armstrong and Green 1985; Duncan et al. 1985), rice (e.q. Yamada et al. 1986) and bread wheat (Redway et al. 1990; Vasil et al. 1990). Other cereals provide embryogenic and/or organogenic cultures not completely optimized, which need further refinement for proper use in gene transfer methodology. Plant regeneration from tissue cultures of various hexaploid wheat genotypes (Tritic~um aestivum L.) has been successfully achieved (Ozias-Akins and Vasil 1982; Lazar et al. 1983; Maddock et al. 1983). More recently, the identification of specific callus types for long-term cultures (Redway et al. 1990) and the establishment of Offprint requests to." G.M. Borrelli
embryogenic suspension cultures and protoplast systems in hexaploid wheat have been reported (Vasil et al. 1990; Chang et al. 1991; Redway et al. 1991). Conversely, very little information is available on tissue culture of the tetraploid durum wheat (Triticum durum Desf.) Which is the most recalcitrant species among wheats (Bennici 1986). Plant regeneration has been achieved in callus derived from mature (Eapen and Rao 1982) and immature embryos (Bennici et al. 1988), as well as from mesocotyl and leaf base tissues (Greco et al. 1984), and the regenerative pathway mainly described as organogenic. To our knowledge, no protoplast system in durum wheat has yet been established and no transformation techniques have been applied to optimized embryogenic cultures. In order to broaden the spectrum of genotypes to be used in tissue culture in search of optimized embryogenic cultures for genetic manipulation, we considered different durum wheat varieties, chosen among those adapted for cultivation in the dry mediterranean areas. In the present work we have assessed the possibility of establishing embryogenic cultures from immature embryos of five durum wheat varieties, and of selecting long-term embryogenic callus types characterized by high regenerative capability. MATERIALS AND METHODS
Seeds and plants were provided by and grown at the Section of the Experimental Institute for Cereal Research in Foggia. The following varieties, namely Appulo, Ofanto, Latino, Creso and Castello, suitable for cultivation in the dry mediterranean areas, were chosen. Plants were grown in a growth chamber in the following conditions, established for simulating the natural growing season in the field: 10 days at 6~ complete darkness, relative humidity 70%; 12 days at 10/14 hours light/dark regime, at 8 and 6~ respectively, relative humidity 70%; 10 days at 15/9 hours light/dark regime, at 12 and 8~ respectively, relative humidity 55%; I0 days at 15/9 hours light/dark regime, at 15 and 10~ respectively, relative humidity 50%; 10 days at 15/9 hours light/dark regime, at 20 and 10~ respectively, relative humidity 45%; and finally, until complete maturation, 16/8 hours light/dark regime, at 23 and 180C respectively, relative humidity 40%. Light was Provided by Philips TLD 58 W fluorescent lamps with an intensity of about 4000 lux. Immature embryos, taken at 15 DAA, were used as explants. The caryopses were removed from the spike and sterilized by rinsing in 70% (v/v) ethanol, washing in 20% NaOCI (v/v) with 0.1% (v/v) Tween 80 surfactant (Merck) for 20 minutes, and finally
297 rinsed four times in sterile distilled water. The immature embryos (no more than 1.5 mm long) were excised under a stereo dissecting microscope and explanted onto the culture medium with the scutellum exposed. Cultures were initiated, maintained and regenerated on 0.8% Difco Bacto Agar gelled medium autoclaved at 1.2 kg/cm2 at 121~ for 20 minutes, in 90 mm @ Petri dishes. Callus initiation and maintenance was at 24 + 2~ in complete darkness. For callus induction and propagation, three formulations derived from MS basic medium (Murashige and Skoog, 1962) were considered respectively indicated as MS (MS salts and vitamins, 30 g/1 sucrose), MSTDI (MS salts and vitamins, I g/l enzymatic hydrolysate of casein (Sigma), thiamineHCl raised up to 0.4 rag/l, 30 g/l sucrose) and MSTD2 (MS salts and vitamins, m-inositol raised up to 200 mg/1, 200 mg/l L-asparagine, thiamine HC1 raised up to 0.4 mg/l, 25 g/l sucrose). Media were all adjusted to pH 5.8 w i t h 0.i M KOH a n d autoclaved. Three concentrations, 1, 2 and 5 rag/1 2,4-D were tested for callus initiation. Primary callus induction frequency was evaluated 20 days after the explant in a total of 300 embryos/genotype/treatment in three replicates. Primary callus was subcultured every 3 weeks onto the same media supplemented with 1 mg/l 2,4-D in complete darkness at 24+2~ Callus cultures derived from each original zygotic embryo explanted, were considered different cell lines. Following the third subculture embryogenic, white, compact and nodular sectors (E callus) arose at the callus surface in some of the cultures. This E callus was separately subcultured in the presence of 1 rag/1 2,4D onto the same media solidified with 0.7% Sigma Agarose (type II - medium EEO). In three-four subcultures, the white nodular callus represented the majority of the callus structure. These were then indicated as "optimized cultures"
or "aged callus", according to Redway e_ttal__. (1990). Callus growth was recorded as growth index (GI) at day 21, calculated as follows: ](FWTt21 - FWTt0)/FWTt0 I. For regeneration, five-month old aged callus was transferred onto MS, hormone-free medium and incubated at 24+2~ with a 16/8 hours light/dark regime. The light source was Philips TLD 58 W fluorescent lamps. After 7 days, germinating somatic embryos were moved onto fresh medium twice at 7 day intervals; at each transfer, callus was fragmented and plated. Regenerated plantlets were separately transplanted. This procedure promoted extensive plant regeneration; 10-30 mm h i g h plantlets were moved to 200 ml glass flasks for complete development and rooting onto the same agar medium. When plantlets reached 15-18 cm in size, they were transferred into peat pots, after washing the roots free of agar medium. Roots were dipped a few seconds in a 0.05% (w/v) solution of antifungine (Zineb| and potted in autoclaved 3:1 (v/v) compost:washed sand. Plants were hardened in a growth chamber at 21~176 in a 16/8 hours day/night cycle, then transferred outdoors. RESULTS AND DISCUSSION
Callus induction and propagation. Scutellum of immature embryos of the five durum wheat varieties formed callus in about 20 days on the various media tested. Preliminary experiments were performed u s i n g three different concentrations of 2,4-D: 1, 2, and 5 mg/l (results not shown); the concentration of 2 mg/l 2,4-D was subsequently chosen as routine for callus induction, and i mg/l 2,4-D for callus propagation. The optimal response in culture was given by embryos taken at 15 DAA, which were 1.0-1.3 mm long. These results are consistent with those obtained by Bennici et al. (1988) and differ from the response reported
Table I. Genotypicresponse of five durum wheat varieties in callus induction frequency, secondary embryogenic callus establishment, and regeneration
Variety APPULO
Basic culture medium a MS MSTD1 MSTD2
106 109 115
14 8 16
900
94.5
330
38
300 300 300
71.4 67.3 73.5
31 26 25
0 0 1
900
70.7
82
1
MS MSTD1
300 300
84.1 82.0
36 39
16 9
MSTD2
300
82.9
41
12
900
83.0
116
37
300 300 300
73.2 67.1 74.5
55 25 34
0 12 8
900
71.6
114
20
300 300 300
96.7 96.4 92.8
86 119 136
69 14 19
900
95.3
341
102
MS MSTD1 MSTD2
Total
LATINO
MS MSTD1 MSTD2
Total OFANTO
Total
N u m b e of r secondary calli propagated
93.4 92.1 98.0
Total CRESO
Number of primary calli propagated c
300 300 300
Total CASTELLO
Number of Callus zygotic embryos induction explanted b frequency (%)
MS MSTD1 MSTD2
a media are supplemented with 2 mg/1 2,4-D b pool of three replicates per culture medium formulation c media are supplemented with 1 mg/l 2,4-D
Number of regenerable cultures
12
-
%
31.6
-
13
35.1
7
35.0
65
63.7
298
Figure I. Somatic embryogenesis and plant regeneration in the durum wheat variety Ofanto. a) compact, highly embryogenic secondary calli (24X) in propagation on MSTD2 medium in the dark; b) extensive regeneration of the E callus after 15 days transfer onto MS hormone-free medium; c) an isolated regenerated plantlet (24X). for bread wheat (T. aestivum) in which a younger developmental age (10-14 DAA) seemed to be the most responsive stage for explants (OziasAkins and Vasil 1982). Callus developed from the upper part of the scutellum in contact with the culture medium. The first callus produced could not be clearly classified as embryogenic tissue, being yellowish, rather wet, meristematic and nodular in appearance. Callus induction frequency was 95.3% and 94.5% for Ofanto and Appulo respectively, which were the best responding genotypes. Creso was slightly less responsive with a callus induction frequency of 83%, followed by Latino and Castello with values of 71.6% and 70.7%, respectively (Table 1). Within each genotype, no significant difference among values of callus induction frequency was detected depending on the basic culture medium used. For all genotypes, however, the lowest value was registered for the MSTD1 formulation. The presence of various organic compounds did not affect callus induction frequency and/or callus texture, and these results confirm the observations reported in hexaploid wheat (Redway e t al. 1990). The real performance in culture of each variety was better described by the percentage of primary calli propagated in culture. Primary calli were subcultured explantfree 20 days after embryo plating and each cell line derived from an individual zygotic embryo was independently propagated. Primary calli were subcultured twice (3 weeks each subculture) before the secondary white E callus started to appear in a few cultures per genotype. Variety Ofanto again was the best genotype, establishing 102 E callus cultures, followed by Appulo, Creso and Latino; only one E callus line could be established for Castello (Table I). From this time onwards, calli were selectively subcultured by choosing the most compact nodular tissues. Upon selective subculture of the E tissues, a gradual conversion of the callus phenotype toward the more E compact callus type was obtained (Fig. la). Medium solidified with agarose instead of agar was more suitable for propagation of the E calli. This procedure and the derived cultures are similar t o those recently described in hexaploid
wheat as "aged callus" (Redway et al. 1990). The obtainment of these optimized cultures, which were recently reported to be the most competent tissues for initiating embryogenic suspension cultures (Redway et al. 1991) and regenerable protoplasts (Vasil et al. 1990; Chang e t a l . 1991) represents, to our knowledge, a unique result in durum wheat studies. Values of the growth index (GI) calculated at the fifth subculture from primary callus establishment on five E cultures per genotype, were indicative of their performance (Table 2). E calli for each genotype were subcultured onto the same medium in which they originated. Growth rates were different in the various cases. It has to be noted that callus growth was totally due to the proliferation of the E callus. Subsequent experiments demonstrated that the formulation of MSTD2 was better suited for the long-term E culture propagation in the various genotypes. MSTD2 also was the most suitable culture medium for suspension cultures and for establishing friable cell lines in the variety Appulo (results will be reported elsewhere). These embryogenic cultures have been efficiently propagated for 10 months to date, without loosing or changing their embryogenic phenotype and regenerative capability. Plant regeneration. Regeneration was tested on the selected cultures, 5 subcultures after the E culture establishment. For each variety several cell lineages were considered and regeneration tested on basic MS hormonefree medium. For plant regeneration, calli were firstly transferred as 100 mg FWT pieces onto MS hormonefree medium. After a 7-days incubation in the light, calli were further fragmented and plated back onto fresh medium. Plantlets were removed throughout the culture period. Fragmentation and plating were repeated twice: this three-step regeneration procedure yielded on the average approximately 25-40 plantlets per gram FWT depending on the genotype. Consistent genotypic differences were detected in the culture ability to initiate shoots and plantlets, despite the culture medium used for callus
299 Table 2. Average value of growth index (GI) calculated from the fresh weight of 5 independent E callus cultures in three different varieties onto MS, MSTD1, and MSTD2 media, following a 21 day subculture Culture medium
GI
OFANTO
MS MSTD1 MSTD2
6.5 5.1 3.2
APPULO
MS MSTD1 MSTD2
5.1 4.6 4.1
CRESO
MS MSTDI MSTD2
2.7 5.7 6.1
Genotype
Growth index is measured as increment in the fresh weight of the tissue at the end of a 21 day subculture GI represents the ratio: FWTt21 - FWTt0 FWTt0
maintenance. The variety Ofanto was the most responsive genotype, in which 65 out of 102 cell lineages produced plantlets. Regenerative capabilities registered for Creso, Latino and Appulo were 35.1, 35.0 and 31.6% respectively, although the number of propagated cell lineages was far less (37, 20 and 38 respectively). Callus cultures of the variety Castello were the least responsive and could not be propagated past the second subculture as E callus. Each regenerable cell line gave rise to multiple shoots and seedlings and subsequently developed into complete plantlets (Fig. Ib, c). Depending on the genotype a high percentage of regenerated plantlets (in the range of 70-90%), could successfully be established in soil. Nearly 80% of the established plants were f e r t i l e a n d set seeds. With regard to t h e type of regeneration from the embryogenic cultures, we could recognize two pathways of development: one probably identifiable as multiple-shoot formation (Shimada and Yamada 1979; Maddock et al. 1983), the other via true somatic embryogenesis of single plantlets (Ozias-Akins and Vasil 1982). However, because of the highly embryogenic texture of the callus cultures, we are in general agreement with the studies performed on hexaploid wheat by OziasAkins and Vasil (1982), who concluded that regeneration occurs via somatic embryogenesis. Somatic embryos germinated "precociously" forming multiple shoots before their complete development. In all cases shoots rooted promptly in the same medium. The high number of regenerated complete plants and the establishment of longterm embryogenic cultures in several varieties of durum wheat as described in the present work, is of particular importance for the application of genetic manipulation to this crop species. Acknowledgements. The present work was promoted and strongly supported by the enthusiasm of Prof. G. Wittmer, director of the Section in Foggia, who died on September 12, 1990. The work was developed within the framework of the program of research "Advanced Biotechnologies applied to Plants" held by Ministero Agricoltura e Foreste, Roma. G.M.B. and F.L. hold fellowships within the program.
REFERENCES
Armstrong CL, Green CE (1985) Establishment and maintenance of friable, embryogenic maize callus and the involvement of L-proline. Planta 164: 207-214. Bennici A (1986) Durum wheat (Triticum durum Desf.). In: Bajaj Y.P.S. (ed) Biotechnology in Agriculture and Forestry, Vol. 2, Crops I. Springer Verlag, Berlin Heidelberg New York, pp 89-104. Bennici A, Caffaro L, Dameri R M, Gastaldo P, Profumo P (1988) Callus formation and plantlet regeneration from immature Triticum durum Desf. embryos. Euphytica 39: 255-263.. Chang Y-F, Wang WC, Warfield CY, Nguyen HT, Wong JR (1991) Plant regeneration from protoplasts isolated from long-term cell cultures of wheat (Triticum aestivum L.). Plant Cell Rep. 9: 611-614. Duncan DR, Williams ME, Zehr BE, Widhlolm JM (1985) The production of callus capable of plant regeneration from immature embryos of numerous Zea mays. genotypes. Planta 165: 322-332. Eapen S, Rao PS (1982) Plant regeneration from callus cultures of Durum and Emmer wheat. Plant Cell Rep. 1: 215-218. Greco B, Tanzarella OA, Blanco A (1984) Plant regeneration from leaf base callus in durum wheat (Triticum durum Desf.). Cereal Res. Comm. 12: 171- 177. Lazar MD, Collins GB, Vian WE (1983) Genetic and environmental effects on the growth and differentiation of wheat somatic cell cultures. J. of Heredity 74: 353357. Maddock SE, Lancaster VA, Risiott R, Franklin J (1983) Plant regeneration from cultured immature embryos and inflorescences of 25 cultivars of wheat (Triticum aestivum). J. Exp. Bot. 34: 915-926. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco culture. Physiol. Plant. 15: 473-497. Ozias-Akins P, Vasil IK (1982) Plant regeneration from cultured immature embryos and inflorescences of Triticum aestivum L. (wheat). Evidence for somatic embryogenesis. Protoplasma 110: 95-105. Potrykus I (1990) Gene transfer to plants: assessment and perspectives. Physiol. Plantarum 79: 125-134. Redway FA, Vasil V, Lu D, Vasil IK (1990) Identification of callus types for long-term maintenance and regeneration from commercial cultivars of wheat (Triticum aestivum L.). Theor. Appl. Genet. 79: 609617. Redway FA, Vasil V, Vasil IK (1991) Characterization and regeneration of wheat (Triticum aestivum L.) embryogenie cell suspension cultures. Plant Cell Rep. 9 (in press). Sanford JC (1990) Biolistie plant transformation. Physiol. Plantarum 79: 206-209. Shimada T, Yamada Y (1979) Wheat plants regenerated from embryo cell cultures. Jap. J. Genet. 54: 379-385. Vasil V, Redway F, Vasil IK (1990) Regeneration of plants from embryogenic suspension culture protoplasts of wheat (Triticum aestivum L.). Bio/Technology 8: 429434. Yamada Y, Yang ZQ, Tang DT (1986) Plant regeneration from protoplast-derived callus of rice (0ryza sativa L.). Plant Cell Rep. 5: 85-88.
