Hereditas 120: 211-216 (1994)

Origin of somatic embryogenesis is proliferating root primordia in seed derived oat callus Z . CHEN, R. KLOCKARE and C. SUNDQVIST

Department of Plant Physiology, Botanical Institute, University of Goteborg, Goteborg, Sweden

CHEN, Z., KLOCKARE, R. and SUNDQWST, C. 1994. Origin of somatic embryogenesis is proliferating root primordia in seed derived oat callus ~ Hereditas 120: 21 1-216. Lund, Sweden. ISSN 0018-0661. Received December 8, 1993. Accepted March 2, 1994

The ontogeny of somatic embryos and proliferating root primordia in tissue cultures of mature seeds of oats is described. Callus formation was initiated on developing embryos of mature oat seeds maintained on Murashige and Skoog's medium containing 2 mg I- 2,4-dichlorophenoxyacetic acid. The root primordia on subculture medium did not elongate but multiplied by forming new primordia directly from root meristem, after callusing or by cleaving pre-existing root primordia. In the root meristem region somatic embryogenesis could also occur. The first somatic embryos were observed at the end of the first subculture. Different developmental stages of somatic embryos were histologically identified in the culture. Occasionally shoot buds were formed directly from root primordia. Among 5 tested cultivars Risto had the best response on callus culture medium. 200 sboots/plantlets per gram of a 10-month culture were obtained on MS germination medium containing 0.01 mg I- ' ABA and 6% sucrose.

Christer Sundqvist, Department of Plant Physiology, Botanical Institute, University of Goteborg, Carl Skottsbergs Gala 22, S-413 19 Goteborg, Sweden

Cultured tissues of oat and many other cereals tend to produce roots in excess on callus induction media (MADDOCK 1985). This type of outgrowth was found to consist of proliferating root meris- tems embedded in terminally differentiated cells (KING et al. 1978). CURE and MOTT (1978) con- sidered that shoot regeneration in oat callus culture might be directly related to adventitious bud for- mation from roots rather than from undifferenti- ated callus. Similarly, NABORS et al. (1982) showed that green spots, which anatomically resembled root apices, had a positive correlation with shoot formation. Improvement of the methods for tissue cultures of different crop species led to successful regeneration of plants via somatic embryogenesis (VASIL and VASIL 1986). HEYSER and NABORS (1982) were first to report green spots associated with somatic embryogenesis in oat tissue culture. However, they doubted the positive correlation between green spots and embryogenic callus be- cause isolated green spots could not produce em- bryogenic callus. In a later study friable embryo- genic oat callus cultures originating from immature embryos were developed (NASSUTH et al. 1987; BREGITZER et al. 1989). Callus in these cultures had a low capacity for root meristem/primordia formation. The differences obtained might depend on varying tissues being the source for the prolifer-

ating cells. A further study of the early stage of callus formation and development of somatic em- bryos seems motivated.

In this paper we show for the first time in oat that callus induced from oat seed developed prolif- erating root primordia and the meristems of these root primordia were sites of somatic embryo for- mation. Attention is focused on anatomical obser- vation on the site of proliferating root primordia and somatic embryogenesis.

Materials and methods Plant material

Dehusked oat seeds (Auena sativa L. cv. Risto, Victory, Sang, Sanna, Vital, and Sol, Svalof AB, Svalov, Sweden) were placed on moist filter paper in Petri dishes for 3 h. The seeds were then surface sterilized with 70 % ethanol for 0.5 min, 100 YO Klorin (4.4 % sodium-hypochlorite, pH 12.8, Col- gate-Palmolive AB) for 20 min, 0.1 % HgCl, for 7 min, and rinsed in sterile distilled water 5 times.

Callus induction and subculturing

Seeds were placed in jars (baby food jars, Sweden) containing 20 ml autoclaved MS (MURASHIGE and

212 Z. CHEN ET AL Hereditas 120 (1994)

SKOOG 1962) callus culture medium supplemen- ted with sucrose (30 g I-'), thiamine-HC1 ( I mg I-'), glutamine (200 mg 1-I), casein hydrolysate (500 mg 1- I), and 2,4-dichlorophenoxyacetic acid (2,4-D) (1, 2, or 4mg1-') and solidified with 0.13% Gellan gum after pH adjustment to 5.8. Six seeds were placed in each jar and kept in darkness at 25°C.

After one month of culture, callus with under- lying root or shoot pieces were transferred to a fresh supply of the same medium. The fresh weight from one-month-old callus was measured by weighing callus excised from explants. In the first subculture, outer callus tissue was excised from inner callus and both transferred to the culture medium for compar~son of growth rate, least 2 repeats were performed using 50 seeds for each experiment.

Fig. 1. Five-month-old compact and nodular embryo- genic callus. Bar = 2 mm.

Plant regeneration Results Callus induction and root primordium formation About 1 g embryogenic tissue (Fig. 1) was trans-

ferred to 20ml MS or N, (CHU et al. 1975) medium supplemented with 2,4-D (0.1 mg I-'), abscisic acid (0.1 or 0.01 mgl-') and sucrose (3 to 6%). After one month, germinated plantlets were transferred to a medium without hormones or to media containing gibberellic acid (GA) (0.01 mg I-'), zeatin (1 mg 1 I-') or kinetin (0.1 mgl-I). Cultures were kept at 25°C with 12 h daily illumination provided by 20 W fluores- cent tubes at a photon fluence rate of ca 22 pmol m-'s-'.

Regenerated plants were planted in soil, covered with beakers for about one week, and kept in a growth chamber at 25°C with 18 h daylight pro- vided by 40 W fluorescent tubes at a photon fluency rate of ca 300 pmol m-* s- ' until harvest.

Histological examination

Samples were collected one month after culture initiation and after the first subculture, fixed in 3 YO glutaraldehyde buffered with 0.05 M Na- cacodylate (pH 6.9) at room temperature for 3 h, dehydrated first in methoxyethanol and then in ethanol, and embedded in Technovit 7100 (Her- aeus, Kulzer). Sections were cut 3 pm thick with a Ralph-type glass knife, mounted on glass slides and stained with 0.025 'YO toluidine blue. Observa- tion with light microscope was carried out on a series sections of tissue specimens.

Oat seeds initiated translucent friable calli at the junction between the primary root and coleoptile after three days of incubation on callus induction medium (2,4-D 2 mg 1-I). Within one month, each responding embryo developed a shoot and translu- cent friable callus, although roots were not always present. Callus could also be produced from the apical meristem of the roots and along the root axis in samples where roots were present. Among the 5 cultivars tested, culture of Risto showed a callus induction frequency of 93 %, while Victory, Sang, Sanna, and Vital showed a frequency of 76 %, 63 %, 24% and 18 %, respectively. The fresh weight of the callus produced ranged from 30-200 mg per seed.

During callus induction, seedling roots became swollen from proliferating callus cells. Epidermal and cortical cells of the roots had a tendency to be expelled and fall off. Differentiated pericycle cells became meristematic. When these roots were sec- tioned longitudinally or transversely, numerous lat- eral root primordia were seen originating from the pericycle cells along the vascular strand. Callus induction occurred also in the basal portions of the shoot. Parenchymatic cells developed a mixture of meristematic cells and single xylem cells. The meristematic cells had prominent nuclei and could directly form root primordia. Groups of root pri- mordia were also formed indirectly after callusing in the vicinity of the shoot bases.

Herediras 120 (1994) SOMATIC EMBRYOS FROM ROOT PRIMORDIA OF OATS 21 3

Fig. 2A and B. Multiplication of root primordia. A Two root primordia (R) formed by direct division of a root meristem. Bar = 200 pm. B Three root primordia (R) formed from callus mass (C). Bar = 200 pm. Samples of A and B were randomly picked up from culture.

The callus tissue surrounding the explant could be divided into an outer part and an inner part. Growth of the outer callus was compared with inner callus after the first subculture. Callus stem- ming from the inner part produced about twice as much callus in fresh weight as that derived from the outer part. The outer part contained only nonembryogenic cells with slow growth rate, while the inner callus had a higher growth rate because of its content of root meristems.

Multiplication of root primordia

Root primordia embedded in the callus did not elongate during subculture in a medium containing 2 mg I-' 2,4-D. The root primordia remained be- tween 300 and 600 pm long.

The root meristems produced root cap cells which were highly vacuolated and loosely attached. Root meristems contained cytoplasmically dense cells with numerous, small vacuoles and nuclei with two or three nucleoli. The cells divided in various directions, so the organisation of root pri- mordia in vitro was not as tight as that in an intact plant.

The formation and multiplication of root pri- mordia took place in three ways. New root primor- dia were formed directly from the meristem region of existing root primordia (Fig. 2A), from callus

mass (Fig. 2B), or from cleavage of a pre-existing root primordium.

The callus growth rate was about the same when 1, 2, or 4 mg I-' of 2,4-D was used in the medium. However, when the level of 2,4-D was decreased from 2 to 1 mg I-' the root formation was in- creased, which indicated that the depression of root elongation was released. Thus 2mg I-' of 2,4-D was regarded as a suitable concentration for maintenance of this culture.

Embryogenesis in root primordia during the first subculture

The ontogenetic development of somatic embryos was examined in the basal regions of root meris- tems (Fig. 3A). The formation of cell complexes, representative of early proembryos, was apparent by a localised asymmetrical cell division activity, an increase in cell wall thickness, and a sequence of internal segmenting divisions in a number of cells (Fig. 3B). This type of proembryo was similar to that in the Pennisetum americanum culture ( BOTTI and VASIL 1984). The cell complexes had a ten- dency to separate from the surrounding cells, thus establishing an isolated environment for embryoge- nesis. Thus there is a strong connection between the root meristems and the formation of somatic embryos. Undetermined cells in the basal part

214 z. CHEN ET AL. Herediias 120 (1994)

Fig. 3A-F. Somatic embryogenesis. A A longitudinal section of a root initial showing formation of cell complexes (arrowhead) at the basal end of the root meristem. Bar = 100 pm. B Complexes (proembryos) formed by internal segmenting divisions. Bar = 50 pm. C A mid-proembryo. P, protoderm; E, embryonic cortex; S, suspensor. Bar = 50 pm. D A transition embryo. S, scutellum; C, coleoptile. Bar = 100 pm. E An embryo in the coleoptilar stage. S, scutellum; C, coleoptile; P, procambium; R, root meristem. Bar = 200 pm. F The same embryo as in Fig. 2E, but still attached to a root primordium. S, scutellum; R, root primordium. Bar = 200 pm.

Hereditus I20 (1994) SOMATIC EMBRYOS FROM ROOT PRIMORDIA OF OATS 215

Fig. 4. Adventitious shoot primordia (S) formed from a root initial (R). Bar = 100 pm.

of the root meristem might form the somatic embryos.

Different stages of somatic embryo development could be found in the same culture. Proembryos in young, mid, or late stages of development were easy to recognize. In the mid-proembryos (Fig. 3C), cell divisions seemed synchronized in each tier. Cells and vacuoles from the protoderm and suspensor were much smaller than those in the proper cortex, suggesting that cells in the former tissues had a higher division rate than those in the latter. Division of cortex cells of somatic embryos was irregular and no precise cell lines could be established. However, the protoderm was well de- lineated. During the transition from late proem- bryo to early differentiating embryo, an increase of cell division could clearly be seen in regions where scutellum and coleoptile were to be formed (Fig. 3D). By the end of the first subculture an incipient differentiation of the coleoptilar stage occurred where scutellum, coleoptile, procambium, and root meristem had formed (Fig. 3E). The generated embryo was occasionally found still to remain attached to the root primordium (Fig. 3F).

In addition, cell complexes giving rise to shoot primordia could be formed from the root meris- tems during the first subculture (Fig. 4). However these structures could not be found in long-term cultures. In this case the auxin (2 mg 1F' 2,4-D) in

the subculture medium might be inhibitory to bud initiation. Although various stages of development of somatic embryos occurred in the first few sub- cultures, most subsequently formed embryos in long-term cultures remained in the globular stage.

Plant regeneration through somatic embryos

Somatic embryos associated with underlying callus cells were germinated on different media (see Ma- terial and methods). MS medium containing 0.01 mg I - ' of ABA and 6 % sucrose was most suitable for germination. After 30-40 days on this medium single plantlets or clumps with 2-5 mm green leaves, with or without roots were obtained. Per gram a 10-month-old embryogenic tissue pro- duced up to 200 shoots or plantlets. When shoots/ plantlets were transferred to a medium containing cytokinin the growth was improved after 20-30 days. More robust plantlets with well developed roots were achieved upon transfer to a hormone- free medium. More than 90 % of the green plantlets transferred to soil survived and reached maturity. Embryogenic tissue has been maintained for more than 30 months to date.

Discussion Somatic embryogenesis from root explants has been reported from the pericycle/endodermis re- gion in rice (STICKLEN 1991), epidermal and corti- cal cells of Cichorium (DUBOIS et al. 1990) and Camellia japonica (VIEITEZ et al. 1991), and from root meristem cells of Vanilla planifolia (PHILIP and NAINAR 1986). However, our result is the first demonstration of somatic embryogenesis direct- ly from the meristem of root primordia in oat culture.

The observation of the different root types, such as lateral roots arising from proliferation of the root pericycle, basal roots developing in the basal region of the plant, adventitious roots arising from callus cells in the primary culture, and new root primordia formed during the subsequent subcultur- ing, supported the previous report ( MADDOCK 1985) that oat tissue cultured in vitro has a high root-forming capacity.

Proliferation of oat root primordia during long- term cultures has been reported by BRENNEMAN and GALSTON (1975) as well as HEYSER and NABORS (1982). However, the origin of these new root primordia was not clear. According to our

216 z. CHEN ET AL Hereditas 120 (1994)

anatomical observations, it seems that new root primordia are formed from the meristem region of the root primordium, from callus mass, or by cleavage of an already existing root primordium. Thus a histological evidence for new root pri- mordium formation directly from the root meri- stem in oat.

Generally the root meristem is considered to be an intrinsic stable structure and the pathway of development is determined for root elongation ( WAREING and PHILLIPS 1981). Nevertheless, basal end cells of the root primordia in the culture seem undetermined. They divided actively so that the normal regular pattern of differentiation was disturbed. Compared with barley seedlings, differ- entiation usually takes place in 70-80 pm long root primordia (LuxovA 1975). The further differ- entiation at the base end root primordia in this culture led to initiation of proembryos, shoots or roots. It is of interest to note that although shoot and root meristems are generally considered to be multicellular in origin and somatic embryos origi- nate from single cells, in our culture these three types of differentiation were observed in the same region of the tissue. This diversity of morpho- genetic pathways was similar to that observed in suspension cultures of Atropa belladonna L. (KONAR et al. 1972).

Somatic embryogenesis during long term cultiva- tion of callus was described by HEYSER and NABORS (1982). Both embryogenic and non-em- bryogenic callus was formed. Embryogenic callus could form plants after more than 36 weeks in cultivation. BREGITZER et al. (1989) differed be- tween nonfriable and friable embryogenic callus. Both types could form somatic embryos and plants. Even if root meristems were observed, the close connection to the formation of somatic em- bryos was not established.

In conclusion, even though various pathways of differentiation could occur in the same culture, formation of somatic embryos became the main activity during subculturing. This result fits with the explanation by KING et al. (1978) and HAL- PERIN (1986) that high concentration of 2,4-D in the medium suppresses root elongation, promotes secondary root initiation and favours proliferation of embryogenic cells.

Acknowledgements. -We thank Prof. Chris H. Bornman and Dr Ke-Cheng Hsiao for helpful discussions and Ms Nancy Ilenius for technical assistance. The financial support from the Swedish

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