Plant Cell Reports
Plant Cell Reports (1989) 7:644-647
© Springer-Verlag 1989
Somatic embryogenesis and plant regeneration in inflorescence and seed derived callus cultures of Poa pratensis L. (Kentucky bluegrass) P. van der Valk 1, M. A. C. M. Zaal 1, and J. Creemers-Molenaar 2 i Foundation for AgriculturalPlant Breeding, P.O. Box 117, 6700 AC, Wageningen, The Netherlands 2 Barenbrug Holland BV, Oosterhout, The Netherlands
Received April 5, 1988/Revised version received January 30, 1988 - Communicated by F. Constabel
ABSTRACT Callus induction and plant regeneration were studied in 15 cultivars of the facultative apomictic species Po___aapratensis L. (Kentucky bluegrass). The tissue culture responses of mature seeds and immature inflorescences were compared. Murashige and Skoog's (MS) medium, supplemented with 2 mg/l 2,4-dichlorophenoxyaeetic acid (2,4-D) was used for callus induction and maintenance. Plants could be re'generated ~from compact and friable callus on MS medium devoid of 2,4-D. Plants were recovered from 14 cultivars at a high frequency (up to 79% of the callus cultures) when young inflorescences were used as the explant material and from only 3 cultivars, at a low frequency (up to 3%), with seeds. Somatic embryos were obseryed in callus cultures of many cultivars. Fully developed germinating somatic i embryos were occasionally observed. Plant regeneration appeared to take place both via somatic embryogenesis and organogenesis. Plants were generally green but albino shoots developed at a low frequency from friable callus. Abbreviations 2,4-D: 2,4-dichlorophenoxyaoetic acid; MS: Murashige and Skoog's (1962) medium; IAA: indole-3-acetic acid; N6: medium of Chu et al. (1975). INTRODUCTION The type of explant used to initiate in vitro cultures appears to be a critical factor determining the capacity of cells and tissues for plant regeneration (Maddock 1985; Vasil 1987). Consistent shoot formation in gramineous species has been obtained mainly from callus induced from very immature material, in particular immature zygotic embryos and young inflorescences (Maddock 1985; Vasil and Vasil 1984; Vasil 1985, 1987). Plant regeneration from callus cultures of the common forage and turf grass species Poa pratensis L. (Kentucky bluegrass) has been studied using mature seeds (Krans 1981) and mature embryos (McDonnell and Conger 1984; Boyd and Dale 1986) as the explant material. In the latter studies plants could be recovered at low frequencies from callus of a small number of cultivars (McDonnell and Conger 1984; Boyd and Dale 1986). Some information about the tissue culture response of young inflorescences (Manton e__%t a_!l. 1982) and shoot tips (Wu and Liu 1985; Wu and Jampates 1986) of this grass species is available. In the present report we compare plant regeneration from
Offprint requests to: P. van der Valk
callus cultures induced from mature seeds and immature infloreseences using 15 cultivars of the highly apomietic grass species P. pratensis. MATERIALS AND METHODS Seeds Seeds of 15 Poa pratensis L. cultivars (Table !), all in ~ common use in the Netherlands, were obtained from the National Institute of Cultivar Research (RIVRO), Wageningen, The Netherlands. Seeds of genotype VB 577 were kindly provided by Barenbrug Holland BV, Oosterhout, The Netherlands. Explant materials Seeds were dehusked by agitation in 50% (v/v) H~SO 4 in H20 for 2 5 m i n and rinsed in running tap water (i min) (Torello et al. 1983). They w e r e then surfacesterilized in 70% (v/v) ethanol (30 s), foliowed by a 2.5% (w/v) sodium hypochlorite solution, containing 2 drops of Tween-20 per I00 ml (20 min). They were rinsed in sterile tap water ( 6 times; 2 min each) and placed in 9 cm diam. Petri dishes containing 25 ml of callus-induction medium (see below) (I0 seeds per dish). Petri dishes were sealed with parafilm. Plants with inflorescences at the appropriate developmental stage were taken from field plots or outdoor pots. Tillers were trimmed 3 cm below and 6 cm above the highest node. They were rinsed in 70% (v/v) ethanol (I min), followed by surface-sterilization in a 5% (w/v) sodium hypochlorite solution, containing 2 drops of Tween-20 per I00 ml (I0 min) and rinsed in sterile tap water (6 times; 2 min each). Immature inflorescences, 4-8 mm long, were excised aseptically using a stereo microscope and cut into segments 2 mm long. Only the basal 2 inflorescence segments were cultured. Six segments were placed in a 9 cm diam. Petri dish containing 25 ml of callus induction medium. Inflorescence segments were cultured within 3 h after collection of plant material. Culture media The basal growth medium used in this study consisted of Murashige and Skoog's (MS) medium (Murashige and Skoog 1962) (without sucrose, IAA and kinetin) (Flow laboratories, Irvine, Scotland), supplemented with 0.4 mg/l thiamine-HCl, 30 g/l sucrose, 2 mg/l 2,4-D, and 0.8% (w/v) agar, pH 5.8. This medium has proven successful for tissue culture of many grass species (Vasil and Vasil 1984; Ahloowalia 1984). In some callus induction experiments N6-medium (Chu et al. 1975), supplemented with 30 g/l sucrose, 2 mg/l 2,4-D, and 0.8% (w/v) agar (pH 5.6) was used. Media were
645 filter sterilized before use. Agar was autoclaved separately, as a concentrated solution for use as required. To compare the tissue culture response of seeds and inflorescence segments, explants and ealli were subjected to the same treatment (cf. Ahloowalia 1984, Vasil and Vasil 1984), as follows: I. Callus induction: MS (or N6) + 2 mg/l 2,4-D (MS2 or N62 resp.) (6-7 weeks) 2. Callus maintenance and growth: MS2 (two transfers, 2-3 weeks each, depending on growth rate) 3. Callus organization: MS + 0,2 mg/l 2,4-D (MS0.2) (3 weeks) 4. Plant regeneration: MS without 2,4-D (MSO) Treatments 1-3 were in the dark (25 °C); treatment 4 was 16 h light (ca 2000 lux white fluorescent light) and 8 h dark (25 °C). After growth for 6 weeks on MSO, green plantlets were transferred to culture tubes containing the same medium. Two to three months later plants were transferred to soil. Scanning electron microscopy Tissues were fixed in 2.5% (v/v) glutaraldehyde in 0.i M Na-cacodylate (pH 7.2) for 2 h, washed in eacodylate buffer and distilled water, consecutively. Material was dehydrated in an ethanol series, critical point dried, using CO 2 as the drying liquid. The material was then sputter coated with gold and viewed in a Jeol JSM-T300 scanning electron microscope. RESULTS General Preliminary callus induction experiments with seeds as the explant material had shown that for cv___ss.Baron and Geronimo 2,4-D at 2 mg/l was optimal for the development of morphogenic callus. Thus in further experiments 2,4-D at 2 mg/l was used to induce callus from both seeds and inflorescences. Three types of callus were distinguished: I. a watery, translucent, smooth type of callus. 2. a slow-growing compact callus, which was either white and rough or slightly green-yellow and nodular. 3. a rapidly growing friable (F) callus, containing compact sectors. Seeds Seed germination frequencies varied considerably between cultivars on both MS2 and N62 medium, but for a particular cultivar germination frequencies were generally similar on both media (not shown). Callus induction from seeds was strongly associated with seed germination: callus was produced almost exclusively from germinating seeds. Germinating seeds developed watery callus within 3 weeks after the start of callus induction. The production of this callus was often associated with the formation of root-like structures. Three to five weeks after eallus initiation a slowgrowing compact callus was formed in varying amounts depending on the cultivar. In some cultivars (e_~g.. Baron, Barblue, Geronimo) such callus was rough and white, whereas in others (Entopper, Fylking, Monopoly) compact callus was slightly green-yellow and rather nodular. The fraction of germinating seeds developing compact callus varied considerably between cultivars (I-30%) on both MS2 and N82, although induction frequencies of such callus for a particular cultivar were generally similar on both media (Table i). In some cultivars a rapidly growing friable (F) type of callus developed in close association with compact callus, 4-5 weeks after callus induction (Table i). Many compact calli turned brown only a few weeks after their appearance. Browning of callus was also frequently observed after transfer onto fresh medium, particularly if calli were small. While callus induction on both MS2 and N62 was studied
and found to yield similar results, plant regeneration was studied on MS only (Table i). Structures resembling somatic embryos at different stages of development were observed on and below the callus surface of some cultivars, notably of those producing friable callus (Figs. 1-4; Table i). Transfer of callus to low 2,4-D medium (MSO.2 or MSO) increased the number of somatic embryos and was also followed by the appearance of shoot primordia on the callus surface (Fig. I). Fully developed germinating embryos were occasionally observed. Scanning electron microscopy showed the atypical morphology of many somatic embryos: multiple embryos with fused scutella (Fig. 5) and leafy scutella (Figs. 6, 7). Culture of isolated somatic embryos on MSO-medium failed to induce plant regeneration. Frequently leaves, apparently breaking through the outer layers of the friable callus, were observed (Fig. 8). Plant regeneration appeared to take place both through the outgrowth of shoot primordia and via the germination of somatic embryos (Fig. 7). Plants could be regenerated from compact and friable callus of 3 cultivars (i.e. Baron, Geronimo and Kimono) (Table i). Up to 40 plants were regenerated from callus of a single explant. The vast majority of the shoots were green but some albino shoots were produced. Green plantlets were transferred to soil without difficulty. Callus cultures of many cultivars only produced roots and/or shoot primordia (green spots), that failed to grow out into plants (Table i). Inflorescences The fraction of inflorescence segments producing compact callus was notably higher as compared to seeds (compare Tables i and 2) and varied between 43 and 95%. Browning of compact callus was common in some cultivars (e_~g.. Ampellia, Aquila, Barblue, Parade, and VB577). In these cultivars compact callus was white, rough, dry and slow-growing. Browning was generally confined to the older parts of this callus. Nodular callus, produced by cultivars such as Entopper, Fylking and Monopoly, had a lesser tendency towards browning. Friable callus was produced by the same cultivars as with seeds (i.e. Baron, Geronimo and Kimono), and also by others (e~g.. Aquila, Cynthia, Julia) (Table 2). Embryo-like structures appeared on the callus surface in several cultivars at various frequencies (Table 2). Highest frequencies (up to 42 %) were found in cvs. Baron, Geronimo, Kimono and VB577, i.e. in the only cultivars that produced somatic embryos on seed-derived callus at a low frequency (compare Table i). Green plantlets were regenerated from all cultivars but one, at frequencies of up to 79% (Table 2). Shoots and somatic embryos developed exclusively from compact or friable callus, stressing the morphogenic capacity of these callus types. Albino shoots were formed along with green shoots in 6 cultivars. Highest frequencies of albino production were observed in evs. Baron and Geronimo, i.e. in the same eultivars that produced albinos from seed-derived callus. Albino shoots were produced almost exclusively from friable callus, while green shoots were formed from both compact and friable callus. Inflorescence-derived calli that failed to produce shoots formed non-developing primordia and/or roots at much higher frequencies than seed-derived calli (compare Tables 1 and 2). DISCUSSION This study has shown that in Pea pratensis the morphogenic response of immature inflorescence segments is much higher than that of germinating mature seeds. The strong tissue-culture response of inflorescence segments as compared to seeds was
646 Table I Callus induction and plant regeneration from germinating mature seeds of Poa pratensis L. on MS-medium morphogenic response (%)e Cultivar
germinating seeds (N)
germinating seeds producing compact callus (%)a
somatlc b embryos
green shoots
Ampellia Aquila Barblue Baron Cynthia Delft Entopper Fylking Geronimo Kimono Monopoly Obelisk Parade VB 577
90 170 125 166 136 189 152 70 150 176 165 70 113 79
27 ii 18 13 I0 2 3 9 15 i 13 i 19 i0
~ + + + +
0 0 0 3 0 0 0 0 1.5 0.6 0 0 0 0
(F) d (F)
(F) (F)
green/ albino shoots c
+ + + -
nondeveloping primordia
0 0 2 i 0 0.5 0 0 7 0.6 0 0 4 5
~ercentages represent fraction of germinating seeds bthe fraction of germinating seeds producing compact callus (%) was scored after 7 weeks of culture cpresent at a low frequency (1-3%)(+), absent (-) dCallus cultures producing both green and albino shoots; present at low frequency (+), absent (-) cultures producing friable callus in addition to compact callus efractlon of cultures (%) showing morphogenic response noted, was scored after culture for 6 weeks on MSO Table 2 Callus induetlon and plant regeneration from immature inflorescences of Poa pratensls L. on MS-medium morphogenic response (%)a cultivar
explants cultured (N)
explants developing compact . callus (%)b
somatic embryos (%)
green shoots
green/ albino shoots c
nondeveloping primordia
roots only
Ampellla Aquila Barblue Baron Cynthia Delft Entopper Fylking Geronimo Julia Kimono Monopoly Obelisk Parade VB 577
94 89 64 77 69 82 56 70 20 55 58 65 72 66 47
60 43 83 69 54 72 59 94 90 78 69 44 71 52 95
0 5 9 36 14 7 0 0 40 0 26 ii 19 0 42
18 21 17 56 43 54 50 79 65 13 66 32 54 0 75
0 2 0 15 3 0 0 0 15 0 3 0 3 0 0
64 6 70 6 7 37 11 3 25 33 9 20 8 94 4
0 67 2 31 7 9 14 17 0 49 22 45 33 0 15
(F) d (F) (F) (F) (F) (F) (F)
~fraction of cultures (%) showing morphogenic response noted, was scored after culture for 6 weeks on MSO fraction of explants producing compact callus (%) was scored after Ii weeks ~callus cultures producing both green and albino shoots cultures producing friable callus in addition to compact callus expressed in the proportion of explants producing I) compact (morphogenic) callus, 2) somatic embryos, and 3) green plants. Immature inflorescences have been successfully used for the induction of shoot-forming callus cultures in cereals and grasses other than Poa pratensis (Dale 1983; Ahloowahlia 1984; Maddock 1985; Tomes 1985; Vasil 1985). The fact that we could regenerate plants from virtually all ~. pratensis cultivars tested, using a simple and generally applicable callus induction and
plant regeneration system, may encourag~ efforts for cultivar improvement in this species through the isolation of somaclonal variants. This is of particular interest since the generation of genetic variation is difficult to obtain by conventional breeding procedures in this highly apomictic grass species (Pepin and Funk 1971). Although plant regeneration from inflorescence-derived callus was established in 14 out of 15 ~. pratensis cultivars, clear differences in tissue culture response were observed between the cultivars used.
647
Figs. I and 2. Light micrographs showing morphogenic callus cultures, Fig. I. Friable callus with compact sectors (c) and shoot primordium (arrow). Bar = I mm. Fig. 2. Embryo-like structures at d i f f e r e n t stages of development. Note cup-shaped embryo (arrow). Bar = 0.5 mm. Figs. 3-8. Scanning electron micrographs of embryo-like structures at d i f f e r e n t stages of development. Fig. 3. Globular pro-embryos. Bar = 0.2 mm. Fig. 4. Somatic embryo with globular c o l e o p t i l e (S=scuteilum). Bar 0.2 mm. Fig. 5. Atypical somatic embryos. Note fused scuteiIa (large arrow) and s c u t e l l a r notch (small arrow). Bar = 0.2 mm. Fig. 6. Leafy s c u t e l l a (LS) with m u l t i p l e shoot primordia. Bar = 0.2 mm. Fig. 7 . F u l l y developed somatic embryo. Note leaf (arrow) emerging from c o l e o p t i l a r pore. Bar = 0.5 mm. Fig. 8. Leaf (L), apparently breaking through outer layers of the c a l l u s . Bar = 0.5 mm. REFERENCES Such differences became apparent in various ways, i.e. in i) the texture of compact callus, 2) the tendency of callus towards browning, 3) the frequency of production of friable callus, 4) the frequency of formation of embryo-like structures, 5) the frequency of formation of green shoots, and 6) the incidence of albino shoots. The genetic basis of these differences in tissue-culture response between cultivars still has to be established. The observed low morphogenie capacity of seed-derived callus in P. pratensis is similar to observations made by others regarding the tissue culture response of mature embryos in this species (McDonnell and Conger 1984; Boyd and Dale 1986). Plant regeneration in the Gramineae has been reported to occur by shoot morphogenesis (organogenesis) and through the germination of somatic embryos (Dale 1983; Maddock 1985; Vasil 1987). McDonnell and Conger (1984) and Boyd and Dale (1986) presented evidence to suggest that in P. pratensis plant regeneration from callus occurs via organogenesis. Our observations suggest that in this species plant regeneration on MS medium takes place via both organogenesis and somatic embryogenesis.
Acknowledgements This work was supported by INPLA grant number G 85-01. The authors wish to express their appreciation to Karin Grondhuis for her contribution to this research. We thank Tineke Oostendorp (Department of Botany, University of Nijmegen) for technical assistance with scanning electron microscopy.
Ahloowalia BS (1984) In: Ammirato PV, DA Evans, WR Sharp, Y Yamada (eds) Handbook of plant cell culture, Vol 3, MacMillan, New York, pp 91-125 Boyd LA, Dale PJ (1986) Plant Breeding 97:246-254 Chu CC, Wang CC, Sun CS, Hsu C, Yin KC, Chu CY, Bi FY (1975) Scientia siniea 18:659-668 Dale PJ (1983) Experientia Supp, 46:31-41 Krans JV (1981) In: Sheard RW ( e d ) Proe 4th Int Turfgrass Res Conference, Univ of Guelph, Ontario, Canada, July 19-23, 1981, Univ of Guelph Press, Guelph, pp 27-33 Maddock SE (1985) In: Bright SWJ and Jones MGK (eds) Cereal tissue and cell culture, Martinus Nijhoff, Dr W Junk, Dordreoht, pp 131-174 Manton M, Riordan TP, Shearman RC (1982) Proc Nebr Acad Science 92:80 (Abstr) McDonnell RE, Conger BV (1984) Crop Science 24:573-578 Murashige T, Skoog F (1962) Physiol Plant 15:473-497 Pepin GW, Funk CR (1971) Crop Science Ii:445-448 Tomes DT (1985) In: Bright SWJ and Jones MGK (eds) Cereal tissue and cell culture, Martinus Nijhoff, Dr W Junk, Dordrecht, pp 175-203 Torello WA, Mancino L, Troll J (1983) Rasen-Turf-Gazon 1:4-7 Vasil IK (1985) In: Henke RR, Hughes KW, Constantin MJ and Hollaender A (eds) Tissue culture in forestry and agriculture, Plenum, New York, pp 31-47 Vasil IK (1987) J Plant Physiol 128:193-218 Vasil V, Vasil IK (1984) In: Vasil IK ( e d ) Cell culture and somatic cell genetics of plants, Academic Press, Orlando, pp 36-42 Wu L , Jampates R (1986) Cytologia 51:125-132 Wu L, Liu L (1985) In: Lemaire F (ed) Proc 5th Int Turfgrass Res Conference, Inst Natl de la Recherche Agronomique, Avignon, July 1-5, 1985, pp 175-183
Plant Cell Reports (1989) 7:644-647
© Springer-Verlag 1989
Somatic embryogenesis and plant regeneration in inflorescence and seed derived callus cultures of Poa pratensis L. (Kentucky bluegrass) P. van der Valk 1, M. A. C. M. Zaal 1, and J. Creemers-Molenaar 2 i Foundation for AgriculturalPlant Breeding, P.O. Box 117, 6700 AC, Wageningen, The Netherlands 2 Barenbrug Holland BV, Oosterhout, The Netherlands
Received April 5, 1988/Revised version received January 30, 1988 - Communicated by F. Constabel
ABSTRACT Callus induction and plant regeneration were studied in 15 cultivars of the facultative apomictic species Po___aapratensis L. (Kentucky bluegrass). The tissue culture responses of mature seeds and immature inflorescences were compared. Murashige and Skoog's (MS) medium, supplemented with 2 mg/l 2,4-dichlorophenoxyaeetic acid (2,4-D) was used for callus induction and maintenance. Plants could be re'generated ~from compact and friable callus on MS medium devoid of 2,4-D. Plants were recovered from 14 cultivars at a high frequency (up to 79% of the callus cultures) when young inflorescences were used as the explant material and from only 3 cultivars, at a low frequency (up to 3%), with seeds. Somatic embryos were obseryed in callus cultures of many cultivars. Fully developed germinating somatic i embryos were occasionally observed. Plant regeneration appeared to take place both via somatic embryogenesis and organogenesis. Plants were generally green but albino shoots developed at a low frequency from friable callus. Abbreviations 2,4-D: 2,4-dichlorophenoxyaoetic acid; MS: Murashige and Skoog's (1962) medium; IAA: indole-3-acetic acid; N6: medium of Chu et al. (1975). INTRODUCTION The type of explant used to initiate in vitro cultures appears to be a critical factor determining the capacity of cells and tissues for plant regeneration (Maddock 1985; Vasil 1987). Consistent shoot formation in gramineous species has been obtained mainly from callus induced from very immature material, in particular immature zygotic embryos and young inflorescences (Maddock 1985; Vasil and Vasil 1984; Vasil 1985, 1987). Plant regeneration from callus cultures of the common forage and turf grass species Poa pratensis L. (Kentucky bluegrass) has been studied using mature seeds (Krans 1981) and mature embryos (McDonnell and Conger 1984; Boyd and Dale 1986) as the explant material. In the latter studies plants could be recovered at low frequencies from callus of a small number of cultivars (McDonnell and Conger 1984; Boyd and Dale 1986). Some information about the tissue culture response of young inflorescences (Manton e__%t a_!l. 1982) and shoot tips (Wu and Liu 1985; Wu and Jampates 1986) of this grass species is available. In the present report we compare plant regeneration from
Offprint requests to: P. van der Valk
callus cultures induced from mature seeds and immature infloreseences using 15 cultivars of the highly apomietic grass species P. pratensis. MATERIALS AND METHODS Seeds Seeds of 15 Poa pratensis L. cultivars (Table !), all in ~ common use in the Netherlands, were obtained from the National Institute of Cultivar Research (RIVRO), Wageningen, The Netherlands. Seeds of genotype VB 577 were kindly provided by Barenbrug Holland BV, Oosterhout, The Netherlands. Explant materials Seeds were dehusked by agitation in 50% (v/v) H~SO 4 in H20 for 2 5 m i n and rinsed in running tap water (i min) (Torello et al. 1983). They w e r e then surfacesterilized in 70% (v/v) ethanol (30 s), foliowed by a 2.5% (w/v) sodium hypochlorite solution, containing 2 drops of Tween-20 per I00 ml (20 min). They were rinsed in sterile tap water ( 6 times; 2 min each) and placed in 9 cm diam. Petri dishes containing 25 ml of callus-induction medium (see below) (I0 seeds per dish). Petri dishes were sealed with parafilm. Plants with inflorescences at the appropriate developmental stage were taken from field plots or outdoor pots. Tillers were trimmed 3 cm below and 6 cm above the highest node. They were rinsed in 70% (v/v) ethanol (I min), followed by surface-sterilization in a 5% (w/v) sodium hypochlorite solution, containing 2 drops of Tween-20 per I00 ml (I0 min) and rinsed in sterile tap water (6 times; 2 min each). Immature inflorescences, 4-8 mm long, were excised aseptically using a stereo microscope and cut into segments 2 mm long. Only the basal 2 inflorescence segments were cultured. Six segments were placed in a 9 cm diam. Petri dish containing 25 ml of callus induction medium. Inflorescence segments were cultured within 3 h after collection of plant material. Culture media The basal growth medium used in this study consisted of Murashige and Skoog's (MS) medium (Murashige and Skoog 1962) (without sucrose, IAA and kinetin) (Flow laboratories, Irvine, Scotland), supplemented with 0.4 mg/l thiamine-HCl, 30 g/l sucrose, 2 mg/l 2,4-D, and 0.8% (w/v) agar, pH 5.8. This medium has proven successful for tissue culture of many grass species (Vasil and Vasil 1984; Ahloowalia 1984). In some callus induction experiments N6-medium (Chu et al. 1975), supplemented with 30 g/l sucrose, 2 mg/l 2,4-D, and 0.8% (w/v) agar (pH 5.6) was used. Media were
645 filter sterilized before use. Agar was autoclaved separately, as a concentrated solution for use as required. To compare the tissue culture response of seeds and inflorescence segments, explants and ealli were subjected to the same treatment (cf. Ahloowalia 1984, Vasil and Vasil 1984), as follows: I. Callus induction: MS (or N6) + 2 mg/l 2,4-D (MS2 or N62 resp.) (6-7 weeks) 2. Callus maintenance and growth: MS2 (two transfers, 2-3 weeks each, depending on growth rate) 3. Callus organization: MS + 0,2 mg/l 2,4-D (MS0.2) (3 weeks) 4. Plant regeneration: MS without 2,4-D (MSO) Treatments 1-3 were in the dark (25 °C); treatment 4 was 16 h light (ca 2000 lux white fluorescent light) and 8 h dark (25 °C). After growth for 6 weeks on MSO, green plantlets were transferred to culture tubes containing the same medium. Two to three months later plants were transferred to soil. Scanning electron microscopy Tissues were fixed in 2.5% (v/v) glutaraldehyde in 0.i M Na-cacodylate (pH 7.2) for 2 h, washed in eacodylate buffer and distilled water, consecutively. Material was dehydrated in an ethanol series, critical point dried, using CO 2 as the drying liquid. The material was then sputter coated with gold and viewed in a Jeol JSM-T300 scanning electron microscope. RESULTS General Preliminary callus induction experiments with seeds as the explant material had shown that for cv___ss.Baron and Geronimo 2,4-D at 2 mg/l was optimal for the development of morphogenic callus. Thus in further experiments 2,4-D at 2 mg/l was used to induce callus from both seeds and inflorescences. Three types of callus were distinguished: I. a watery, translucent, smooth type of callus. 2. a slow-growing compact callus, which was either white and rough or slightly green-yellow and nodular. 3. a rapidly growing friable (F) callus, containing compact sectors. Seeds Seed germination frequencies varied considerably between cultivars on both MS2 and N62 medium, but for a particular cultivar germination frequencies were generally similar on both media (not shown). Callus induction from seeds was strongly associated with seed germination: callus was produced almost exclusively from germinating seeds. Germinating seeds developed watery callus within 3 weeks after the start of callus induction. The production of this callus was often associated with the formation of root-like structures. Three to five weeks after eallus initiation a slowgrowing compact callus was formed in varying amounts depending on the cultivar. In some cultivars (e_~g.. Baron, Barblue, Geronimo) such callus was rough and white, whereas in others (Entopper, Fylking, Monopoly) compact callus was slightly green-yellow and rather nodular. The fraction of germinating seeds developing compact callus varied considerably between cultivars (I-30%) on both MS2 and N82, although induction frequencies of such callus for a particular cultivar were generally similar on both media (Table i). In some cultivars a rapidly growing friable (F) type of callus developed in close association with compact callus, 4-5 weeks after callus induction (Table i). Many compact calli turned brown only a few weeks after their appearance. Browning of callus was also frequently observed after transfer onto fresh medium, particularly if calli were small. While callus induction on both MS2 and N62 was studied
and found to yield similar results, plant regeneration was studied on MS only (Table i). Structures resembling somatic embryos at different stages of development were observed on and below the callus surface of some cultivars, notably of those producing friable callus (Figs. 1-4; Table i). Transfer of callus to low 2,4-D medium (MSO.2 or MSO) increased the number of somatic embryos and was also followed by the appearance of shoot primordia on the callus surface (Fig. I). Fully developed germinating embryos were occasionally observed. Scanning electron microscopy showed the atypical morphology of many somatic embryos: multiple embryos with fused scutella (Fig. 5) and leafy scutella (Figs. 6, 7). Culture of isolated somatic embryos on MSO-medium failed to induce plant regeneration. Frequently leaves, apparently breaking through the outer layers of the friable callus, were observed (Fig. 8). Plant regeneration appeared to take place both through the outgrowth of shoot primordia and via the germination of somatic embryos (Fig. 7). Plants could be regenerated from compact and friable callus of 3 cultivars (i.e. Baron, Geronimo and Kimono) (Table i). Up to 40 plants were regenerated from callus of a single explant. The vast majority of the shoots were green but some albino shoots were produced. Green plantlets were transferred to soil without difficulty. Callus cultures of many cultivars only produced roots and/or shoot primordia (green spots), that failed to grow out into plants (Table i). Inflorescences The fraction of inflorescence segments producing compact callus was notably higher as compared to seeds (compare Tables i and 2) and varied between 43 and 95%. Browning of compact callus was common in some cultivars (e_~g.. Ampellia, Aquila, Barblue, Parade, and VB577). In these cultivars compact callus was white, rough, dry and slow-growing. Browning was generally confined to the older parts of this callus. Nodular callus, produced by cultivars such as Entopper, Fylking and Monopoly, had a lesser tendency towards browning. Friable callus was produced by the same cultivars as with seeds (i.e. Baron, Geronimo and Kimono), and also by others (e~g.. Aquila, Cynthia, Julia) (Table 2). Embryo-like structures appeared on the callus surface in several cultivars at various frequencies (Table 2). Highest frequencies (up to 42 %) were found in cvs. Baron, Geronimo, Kimono and VB577, i.e. in the only cultivars that produced somatic embryos on seed-derived callus at a low frequency (compare Table i). Green plantlets were regenerated from all cultivars but one, at frequencies of up to 79% (Table 2). Shoots and somatic embryos developed exclusively from compact or friable callus, stressing the morphogenic capacity of these callus types. Albino shoots were formed along with green shoots in 6 cultivars. Highest frequencies of albino production were observed in evs. Baron and Geronimo, i.e. in the same eultivars that produced albinos from seed-derived callus. Albino shoots were produced almost exclusively from friable callus, while green shoots were formed from both compact and friable callus. Inflorescence-derived calli that failed to produce shoots formed non-developing primordia and/or roots at much higher frequencies than seed-derived calli (compare Tables 1 and 2). DISCUSSION This study has shown that in Pea pratensis the morphogenic response of immature inflorescence segments is much higher than that of germinating mature seeds. The strong tissue-culture response of inflorescence segments as compared to seeds was
646 Table I Callus induction and plant regeneration from germinating mature seeds of Poa pratensis L. on MS-medium morphogenic response (%)e Cultivar
germinating seeds (N)
germinating seeds producing compact callus (%)a
somatlc b embryos
green shoots
Ampellia Aquila Barblue Baron Cynthia Delft Entopper Fylking Geronimo Kimono Monopoly Obelisk Parade VB 577
90 170 125 166 136 189 152 70 150 176 165 70 113 79
27 ii 18 13 I0 2 3 9 15 i 13 i 19 i0
~ + + + +
0 0 0 3 0 0 0 0 1.5 0.6 0 0 0 0
(F) d (F)
(F) (F)
green/ albino shoots c
+ + + -
nondeveloping primordia
0 0 2 i 0 0.5 0 0 7 0.6 0 0 4 5
~ercentages represent fraction of germinating seeds bthe fraction of germinating seeds producing compact callus (%) was scored after 7 weeks of culture cpresent at a low frequency (1-3%)(+), absent (-) dCallus cultures producing both green and albino shoots; present at low frequency (+), absent (-) cultures producing friable callus in addition to compact callus efractlon of cultures (%) showing morphogenic response noted, was scored after culture for 6 weeks on MSO Table 2 Callus induetlon and plant regeneration from immature inflorescences of Poa pratensls L. on MS-medium morphogenic response (%)a cultivar
explants cultured (N)
explants developing compact . callus (%)b
somatic embryos (%)
green shoots
green/ albino shoots c
nondeveloping primordia
roots only
Ampellla Aquila Barblue Baron Cynthia Delft Entopper Fylking Geronimo Julia Kimono Monopoly Obelisk Parade VB 577
94 89 64 77 69 82 56 70 20 55 58 65 72 66 47
60 43 83 69 54 72 59 94 90 78 69 44 71 52 95
0 5 9 36 14 7 0 0 40 0 26 ii 19 0 42
18 21 17 56 43 54 50 79 65 13 66 32 54 0 75
0 2 0 15 3 0 0 0 15 0 3 0 3 0 0
64 6 70 6 7 37 11 3 25 33 9 20 8 94 4
0 67 2 31 7 9 14 17 0 49 22 45 33 0 15
(F) d (F) (F) (F) (F) (F) (F)
~fraction of cultures (%) showing morphogenic response noted, was scored after culture for 6 weeks on MSO fraction of explants producing compact callus (%) was scored after Ii weeks ~callus cultures producing both green and albino shoots cultures producing friable callus in addition to compact callus expressed in the proportion of explants producing I) compact (morphogenic) callus, 2) somatic embryos, and 3) green plants. Immature inflorescences have been successfully used for the induction of shoot-forming callus cultures in cereals and grasses other than Poa pratensis (Dale 1983; Ahloowahlia 1984; Maddock 1985; Tomes 1985; Vasil 1985). The fact that we could regenerate plants from virtually all ~. pratensis cultivars tested, using a simple and generally applicable callus induction and
plant regeneration system, may encourag~ efforts for cultivar improvement in this species through the isolation of somaclonal variants. This is of particular interest since the generation of genetic variation is difficult to obtain by conventional breeding procedures in this highly apomictic grass species (Pepin and Funk 1971). Although plant regeneration from inflorescence-derived callus was established in 14 out of 15 ~. pratensis cultivars, clear differences in tissue culture response were observed between the cultivars used.
647
Figs. I and 2. Light micrographs showing morphogenic callus cultures, Fig. I. Friable callus with compact sectors (c) and shoot primordium (arrow). Bar = I mm. Fig. 2. Embryo-like structures at d i f f e r e n t stages of development. Note cup-shaped embryo (arrow). Bar = 0.5 mm. Figs. 3-8. Scanning electron micrographs of embryo-like structures at d i f f e r e n t stages of development. Fig. 3. Globular pro-embryos. Bar = 0.2 mm. Fig. 4. Somatic embryo with globular c o l e o p t i l e (S=scuteilum). Bar 0.2 mm. Fig. 5. Atypical somatic embryos. Note fused scuteiIa (large arrow) and s c u t e l l a r notch (small arrow). Bar = 0.2 mm. Fig. 6. Leafy s c u t e l l a (LS) with m u l t i p l e shoot primordia. Bar = 0.2 mm. Fig. 7 . F u l l y developed somatic embryo. Note leaf (arrow) emerging from c o l e o p t i l a r pore. Bar = 0.5 mm. Fig. 8. Leaf (L), apparently breaking through outer layers of the c a l l u s . Bar = 0.5 mm. REFERENCES Such differences became apparent in various ways, i.e. in i) the texture of compact callus, 2) the tendency of callus towards browning, 3) the frequency of production of friable callus, 4) the frequency of formation of embryo-like structures, 5) the frequency of formation of green shoots, and 6) the incidence of albino shoots. The genetic basis of these differences in tissue-culture response between cultivars still has to be established. The observed low morphogenie capacity of seed-derived callus in P. pratensis is similar to observations made by others regarding the tissue culture response of mature embryos in this species (McDonnell and Conger 1984; Boyd and Dale 1986). Plant regeneration in the Gramineae has been reported to occur by shoot morphogenesis (organogenesis) and through the germination of somatic embryos (Dale 1983; Maddock 1985; Vasil 1987). McDonnell and Conger (1984) and Boyd and Dale (1986) presented evidence to suggest that in P. pratensis plant regeneration from callus occurs via organogenesis. Our observations suggest that in this species plant regeneration on MS medium takes place via both organogenesis and somatic embryogenesis.
Acknowledgements This work was supported by INPLA grant number G 85-01. The authors wish to express their appreciation to Karin Grondhuis for her contribution to this research. We thank Tineke Oostendorp (Department of Botany, University of Nijmegen) for technical assistance with scanning electron microscopy.
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