Maturation of black spruce somatic embryos. Part I. Effect of l-glutamine on the number and germinability of somatic embryos

Different concentrations of l-glutamine and different nitrogen sources in the medium were compared during maturation of black spruce (Picea mariana (Mill.) B.S.P.) somatic embryos. l-glutamine can be used as the sole nitrogen source for the maturation of Picea mariana somatic embryos at 2 to 3 gl^-1. A significantly lower number of somatic embryos was obtained on a medium prepared with only inorganic nitrogen. Compared with a medium supplement to inorganic nitrogen resulted in a twofold increase in the number of embryos for six genotypes. The nitrogen source and concentration in the maturation medium significantly affected the germination sensus stricto of somatic embryos (radicle appearance), but not their development into plantlets; at the time of epicotyl appearance, an effect of the nitrogen source was no longer found. A comparison of the development of somatic embryos into plantlets from seven genotypes showed that the genotype had more effect in terms of epicotyl appearance and in conversion rate than the nitrogen source present in the maturation medium.Abbreviations HLM-1 half-Litvays's medium with 10 M 2,4-D and 5 M BA - i only inorganic nitrogen in the medium - i+1 gG inorganic nitrogen plus 1 g l^-1 glutamine in the medium - SMM standard maturation medium - 2.5gG only 2.5 g l^-1 glutamine in the medium     

An improved method for somatic plantlet production in hybrid larch (Larix × leptoeuropaea): Part 2. Control of germination and plantlet development

Germination and plantlet development in somatic embryos of Larix x leptoeuropaea were affected by the duration of the maturation treatment and the concentrations of sucrose and abscisic acid in the maturation media. Extension of the maturation period from 3 weeks to 4 weeks resulted in a significant decrease in germination and plantlet development frequencies. There was no significant effect of abscisic acid concentration on either the number of somatic embryos germinated or the number of plantlets obtained, but it affected the rapidity of the epicotyl development. Sucrose at 0.2 M, applied during maturation, was significantly more beneficial in attaining high germination rates than at 0.1 M. High germination rates (92 and 93%) and plantlet development rates (74 and 80%) were achieved when somatic embryos were matured for a 3-week period on media with either 40 or 60 M abscisic acid, respectively, and 0.2 M sucrose prior to transfer to the growth regulator-free germination medium. Two acclimatization methods were applied: the first required 10 to 12 weeks and ensured 97% plantlet survival under greenhouse conditions; the second required 2–3 weeks and ensured 86% plantlet survival. This represents the first detailed study of the effects of maturation regimes on the recovery of somatic embryo-derived plants of Larix.Abbreviations ABA abscisic acid - IBA indolebutyric acid - 2,4-d 2,4-dichlorophenoxyacetic acid - EM embryonal mass     

Somatic embryogenesis and plantlet regeneration in American ginseng (Panax quinquefolium L.)

Summary Somatic embryogenesis in American ginseng (Panax quinquefolium L.) was investigated from three explant sources (root, leaf and epicotyl) with Murashige and Skoog (MS) medium containing different growth regulators. Mature roots and leaves obtained from 3- to 5-yr-old field-grown plants, and seedling leaves and epicotyls from plantlets grownin vitro, were evaluated. From root and epicotyl explants, callus development was optimal with 3,6-dichloro-o-anisic acid (dicamba) (9.0 μM) and kinetin (KN) (5.0 μM) as the growth regulators. When these calluses were transferred after 3 mo. to dicamba alone (9.0 μM), somatic embryo formation was observed at an average frequency of 15.6% in root explants after an additional 3 mo., and 2% in epicotyl explants after an additional 6 mo. No plantlets were recovered because the embryos germinated to form shoots with no roots. From leaf explants, callus growth was optimal with α-naphthaleneacetic acid (NAA) at 10.0 μM and 2,4-dichlorophenoxyacetic acid (2,4-D) at 9.0 μM. Somatic embryos developed on this medium, with the highest frequency (40%) obtained after 3 mo. from seedling-leaf explants. Calluses on mature leaves formed somatic embryos after 7 mo. with NAA/2,4-D at an average frequency of 30%. Transfer of these somatic embryos to 6-benzyladenine/gibberellic acid (4.4/2.9 μM) promoted shoot development but no roots were observed. Up to 100% of germination was observed within 6 wk on half-strength MS salts containing activated charcoal (1%) and on NAA/2,4-D (5.0/4.5 μM) with charcoal (1%). On the latter medium, somatic embryos enlarged and frequently gave rise to new somatic embryos after a brief callusing phase. The embryos germinated through a two-stage process, involving the elongation of the root followed by the formation of a shoot. The highest recovery of ginseng plantlets from germinated embryos was 61.0%. Following transfer to potting medium and maintenance under conditions of high humidity and low light intensity, the plantlets elongated and developed new leaves. A high percentage (50%) of these plants have been acclimatized to soil.     

Diversity of epicotyl dormancy among tropical montane forest species in Sri Lanka

• Fruiting season of many Sri Lankan tropical montane species is not synchronised and may not occur when conditions are favourable for seedling establishment. We hypothesised that species with different fruiting seasons have different seed dormancy mechanisms to synchronise timing of germination with a favourable season for establishment. Using six species with different fruiting seasons, we tested this hypothesis.
• Germination and imbibition of intact and manually scarified seeds were studied. Effect of GA₃ on germination was examined. Embryo length:seed length (E:S) ratio of freshly matured seeds and of those with a split seed coat was determined. Time taken for radicle and plumule emergence and morphological changes of the embryos were recorded.
• The radicle emerged from Ardisia missionis, Bheza nitidissima and Gaetnera walkeri seeds within 30 days, whereas it took >30 days in other species. Embryos grew in seeds of B. nitidissima and G. walkeri prior to radicle emergence but not in Microtropis wallichiana, Nothapodytes nimmoniana and Symplocos cochinchinensis. A considerable delay was observed between radicle and plumule emergence in all six species. Warm stratification and/or GA₃ promoted germination of all species.
• All the tested species have epicotyl dormancy. Seeds of B. nitidissima and G. walkeri have non‐deep simple morphophysiological epicotyl dormancy, and the other four species have non‐deep physiological epicotyl dormancy. Differences in radicle and epicotyl dormancy promote synchronisation of germination to a favourable time for seedling development. Therefore, information on dormancy‐breaking and germination requirements of both radicle and epicotyl are needed to determine the kind of dormancy of a particular species.

     

Shoot organogenesis from cultured seed explants of peanut (Arachis hypogaea L.) using thidiazuron

Summary Thidiazuron (TDZ) was utilized to induce adventitious shoot formation from the hypocotyl region of cultured seed explants of peanut (Arachis hypogaea L.). Excision of the radicle from seed explants was more stimulatory to shoot initiation than removal of the epicotyl alone. Removal of both the radicle and the epicotyl from seeds resulted in a 37-fold increase in the frequency of shoot production when compared to intact seeds. Half seed explants with epicotyl and radicle removed produced the greatest number of shoots per explant. Explants from mature seeds were more responsive to TDZ than immature seed-derived explants. A 1-wk exposure to 10 μM TDZ was sufficient to stimulate the initiation of adventitious shoots that subsequently developed into plants. High frequency of shoot initiation was readily induced in a variety of genotypes ofA. hypogaea and a wild peanut (A. glabrata). Plants regenerated from shoots induced by TDZ were phenotypically normal and fertile.     

Repetitive somatic embryogenesis and plant regeneration in Zizyphus jujuba Mill

Summary A procedure for regenerating Zizyphus jujuba Mill. (Chinese date) plants through repetitive somatic embryogenesis (RSE) was developed. Primary somatic embryos were produced from cotyledon-derived cultures of germinated plants in vitro. The highest induction frequency of primary somatic embryogenesis (PSE) was obtained with a combination of 4.52 μM 2,4-dichlorophenoxyacetic acid (2,4-D), 0.49 μM indole-3-butyric acid (IBA), and 0.44 μM benzyladenine (BA) (17.4%). These primary somatic embryos were multiplied by RSE on media with different plant growth regulator (PGR) combinations. The highest RSE frequency (51.3%), was obtained with 0.58 μM gibberellic acid (GA₃). However, the highest number (4.4 per primary somatic embryo) of repetitive somatic embryos was obtained with 0.98 μM 6-r-r-dimethylallylaminopurine (2-iP). For germination of somatic embryos, different PGRs, cold and desiccation treatment were tested. Desiccation of somatic embryos at 25±1°C for 2 wk was the best treatment for germination with epicotyl elongation and root development. Of over 256 plants regenerated, 237 (92.6%) survived.     

High-frequency conversion of abnormal peanut somatic embryos

Peanuts (Arachis hypogaea L.) are widely cultivated as a rich source of protein and oil. Although protocols for the regeneration of peanut via somatic embryogenesis and organogenesis have been developed, most of them have resulted in low frequencies of plant recovery. In this report, we describe a protocol for plantlet formation at high frequency from somatic embryos. Morphologically abnormal somatic embryos germinated and produced roots only in medium devoid of growth regulators. Shoots emerged from the undeveloped plumule of these rooted embryos in medium containing both 6-benzyladenine (BA) and kinetin (KN), or in medium with thidiazuron (TDZ) alone. In Murashige and Skoog basal medium supplemented with 8.9 μm BA and 14 μm KN, 86% of the embryos developed shoots. Substitution of BA and KN with 22.7 μm TDZ increased plant recovery from 86% to 92%. Plants grown on TDZ had multiple shoots. Eighty-four percent of these plants survived in sandy soil and were grown to maturity. Received: 12 February 1996 / Revision received: 11 July 1996 / Accepted 30 April 1997     

In vitro regeneration of commercial cultivars of subterranean clover

Regeneration of subterranean clover (Trifolium subterraneum L.) was achieved by both shoot organogenesis and somatic embryogenesis. Shoots derived via organogenesis were initiated from the hypocotyls of mature imbibed seed. The hypocotyl, including the emerging radicle, was sliced longitudinally into two halves and cultured on shoot induction medium. After 30 days, adventitious shoots were formed from the hypocotyl region while the radicle showed no development. Shoots were then subcultured onto shoot multiplication medium and finally onto a root initiation medium. Histological studies revealed that shoots arose de novo and did not originate from pre-existing meristems. In the second regeneration protocol, shoot apical meristems from young seedlings were induced to form callus. Following four to six weeks culture in the dark, somatic embryos appeared spontaneously on the calli. A majority of embryos had a well-defined root pole, two cotyledonary lobes, and were capable of germination, albeit at a low frequency. Regenerated plants obtained from both protocols appeared phenotypically normal.     

Somatic embryogenesis, plant regeneration and cyanogenesis in Manihot glaziovii Muell. Arg. (ceara rubber)

 The report describes a system for somatic embryogenesis and direct plant regeneration from the embryos of Manihot glaziovii. Somatic embryos were obtained by culturing young leaf lobes (3–6 mm long) adjacent to the apex in Murashige and Skoog medium containing 18 μm 2,4-dichlorophenoxy acetic acid for 20 days and then transferring them to a maturation medium with 0.5 μm 6-benzylaminopurine. Secondary embryogenesis was induced from cotyledonary segments of somatic embryos by using the same protocol as that for primary embryogenesis. For regeneration, somatic embryos were cultured in medium supplemented with 10⁻⁴ m kinetin and 53.4% of them developed into plantlets. Linamarin and linamarase were not detected in calli or in somatic embryos. Linamarin content was found to be highest in leaves of regenerated plantlets, followed by stem and root tissues. Levels of linamarase activity were almost the same in leaves and stem tissues and very low in roots. Received: 19 April 1999 / Revision received: 11 August 1999 / Accepted: 17 August 1999     

Effect of partial drying and desiccation on somatic seedling quality in Norway and Serbian spruce

Somatic embryo quality is still a problem for many researchers. To improve the efficiency of germination, special procedures are used, such as partial drying of somatic embryos at high relative humidity or desiccation in the presence of supersaturated solutions of salt. In this work, cotyledonary somatic embryos of Norway spruce (Picea abies) and Serbian spruce (P. omorika) were placed on culture media (ME or BM-5) to germinate. We found that after 4 weeks of incubation on these media, hypocotyl and radicle growth of control (non-dried) somatic embryos of both species was not adequate to yield seedlings able to acclimatize to greenhouse conditions. Therefore, somatic embryos were partly dried at relative humidity of 97 % or desiccated at relative humidity of 79 %, for 2 or 3 weeks, and then placed on the Margara (ME) medium. Partial drying of somatic embryos at the higher relative humidity (97 %) enabled an improvement of radicle growth of germinating somatic embryos in both species. The highest conversion rate (45 %) was obtained for embryos of Norway spruce maintained for 2 weeks at relative humidity of 97 %. This treatment contributed to the improvement of germination and conversion efficiency of somatic embryos of Norway spruce, regardless of the drying period. Improved radicle growth facilitated development of better quality seedlings of this spruce species. In Serbian spruce, we did not obtain seedlings of sufficient quality, due to poor hypocotyl growth. Desiccation at humidity of 79 % for 3 weeks proved to be lethal to somatic embryos of both species.     

Genotypic control of peanut somatic embryogenesis

The protocol for obtaining a high frequency of plant development via somatic embryogenesis from mature zygotic embryo-derived leaflets of peanut (Arachis hypogaea L.) involves multiple stages; these include the induction of embryogenic masses, development of embryos, radicle emergence/conversion of embryos and the development of plants from rooted abnormal embryos. Sixteen genotypes were subjected to this protocol by exposing mature zygotic embryo-derived leaflets to the common media sequence and comparing responses. Although the protocol was effective for all the genotypes, variation in frequency of response at each stage of development indicated that, with the exception of root meristem differentiation and subsequent radicle emergence, the whole process of somatic embryogenesis depended on the genotypic constitution of the original plant. The failure of somatic embryos to undergo conversion to plantlets could be a genotype-dependent characteristic. Received: 5 June 1997 / Revision received: 2 December 1997 / Accepted: 12 December 1997     

Somatic embryogenesis and plant regeneration in Quercus acutissima

Immature embryos of Quercus acutissima were collected weekly beginning 5 weeks post-fertilization and cultured on modified MS(Murashige and Skoog) medium containing 1,000 mg/l glutamine and 5 mM proline with different combinations of IBA(0.5–10.0 mg/l) and BA(0 or 1.0 mg/l) in light. The highest percentage of embryogenic cultures occurred on the medium containing 0.5 mg/l IBA or 1.0 mg/l BA and 0.5 mg/l IBA. Four weeks after initiation, the embryogenic cultures were transferred to MS medium without plant growth regulators and cultured for 4 weeks. The somatic embryos were then transferred to germination medium. The best germination results were achieved from WPM(Woody Plant Medium) containing 0.1 mg/l BA. Plantlets from somatic embryos were incubated on WPM supplemented with 0.2 mg/l BA for 4 weeks and plantlets with well developed shoots and roots were transplanted to perlite and peat moss(11, v/v) mixtures and placed in a culture room. After being hardened off for 8 weeks, they were transferred outdoors where they grew.Abbreviation BA N⁶-benzyladenine - IBA indole-3-butyric acid - GA₃ gibberellic acid - ABA abscisic acid - MS Murashige & Skoog Medium - WPM Woody Plant medium     

Somatic embryo productions by liquid shake culture of embryogenic calluses in <Emphasis Type="Italic">Vigna mungo</Emphasis> (L.) Hepper

The regeneration of plants via somatic embryogenesis liquid shake culture of embryogenic calluses was achieved in Vigna mungo (L.) Hepper (blackgram). The production of embryogenic callus was induced by seeding primary leaf explants of V. mungo onto Murashige and Skoog (MS) (Physiol Plant 15:473–497, 1962) medium supplemented (optimally) with 1.5 mg/l 2,4-dichloro-phenoxyacetic acid. The embryogenic callus was then transferred to liquid MS medium supplemented (optimally) with 0.25 mg/l 2,4-dichloro-phenoxyacetic acid. Globular, heart-shaped, and torpedo-shaped embryos developed in liquid culture. The optimal carbohydrate source for production of somatic embryos was 3% sucrose (compared to glucose, fructose, and maltose). l-Glutamine (20 mg/l) stimulated the production of all somatic embryo stages significantly. Torpedo-shaped embryos were transferred to MS (Physiol Plant 15:473–497, 1962) liquid medium containing 0.5 mg/l abscisic acid to induce the maturation of cotyledonary-stage embryos. Cotyledonary-stage embryos were transferred to 1/2-MS semi-solid basal medium for embryo conversion. Approximately 1–1.5% of the embryos developed into plants.     

Induction of somatic embryogenesis in cultured cells of <Emphasis Type="Italic">Chenopodium quinoa</Emphasis>

A major limiting factor for quinoa cultivation as a grain crop on a large scale are virus diseases, in particularly seed borne diseases. Therefore, a somatic embryogenesis protocol is a necessary tool to produce virus free plants. Somatic embryogenesis offers the possibility of mass production of transgenic plants and therefore can be used easily to study the effect on plants resulting from breeding processes. An in vitro protocol has been developed for somatic embryogensis from calluses and cell cultures of Chenopodium quinoa. Callus was induced from hypocotyl explants within 2 weeks of culture on a modified Murashige and Skoog (MS) medium supplemented with 0.45 M 2,4-D. Calluses were cultured on solid or liquid MS medium and later the development of somatic embryos was observed on both employing the same MS medium without 2,4-D. To our knowledge this is the first report of somatic embryogenesis in Chenopodium quinoa.     

Somatic embryogenesis and in vitro rosmarinic acid accumulation in Salvia officinalis and S. fruticosa leaf callus cultures

The effect of explant age, plant growth regulators and culture conditions on somatic embryogenesis and rosmarinic acid production from leaf explants of Salvia officinalis and S. fruticosa plants collected in Greece was investigated. Embryogenic callus with numerous spherical somatic embryos could be induced on explants derived from both species and cultured for 3 weeks on a Murashige and Skoog (MS) medium supplemented with 1.8–18 μm 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin (Kin) or 10.5–21 μm 1-naphthalenacetic acid and 6-benzyladenine. Only explants from young plants (with six to eight leaves) responded to the culture treatments and, in general, low light intensities (50 μmol m^–2 s^–1) favoured callus formation and induction of somatic embryos. Somatic embryos were further developed on the same medium. Heart- and torpedo-shaped embryos (1–2 mm long) were subcultured on a growth-regulator-free MS medium for maturation. Maximum rosmarinic acid accumulation in S. officinalis and S. fruticosa callus cultured on 4.5 μm 2,4-D and 4.5 μm Kin was 25.9 and 29.0 g/l, respectively. Received: 17 January 1997 / Revision received: 26 May 1997 / Accepted: 30 June 1997     

High-efficiency plant production via direct somatic single embryogenesis from preplasmolysed cotyledons of Panax ginseng and possible dormancy of somatic embryos

Cotyledon explants of immature ginseng zygotic embryos cultured on Murashige and Skoog medium lacking growth regulators formed somatic embryos directly, most in a multiple state, fused together and to the parent cotyledon explants. When the cotyledon explants of ginseng were pretreated with 1.0 m sucrose for 24–72 h, all the somatic embryos developed individually from all surfaces of the cotyledons and the number of somatic embryos per explant was enhanced fourfold. Histological observation revealed that all the single somatic embryos from preplasmolysed cotyledons originated from epidermal single cells, whereas all the multiple embryos from cotyledons without pretreatment originated from epidermal and subepidermal cell masses. When the somatic embryos matured to the cotyledonary stage, further growth ceased and they remained white, probably indicating dormancy. Gibberellic acid (GA₃) (over 1.0 mg/l) or chilling treatment (–2°C for over 8 weeks) were prerequisites for the germination of somatic embryos. Ultrastructural observation revealed that the cotyledon cells of somatic embryos without chilling or GA₃ treatment contained numerous lipid reserves, dense cytoplasm, proplastids and non-activated mitochondria, whereas the cotyledon cells of somatic embryos after chilling or GA₃ treatment were highly vacuolated and contained well-developed chloroplasts and active-state mitochondria enclosing numerous cristae, indicating that in-vitro-developed somatic embryos of P. ginseng may be dormant after maturing in a manner similar to zygotic embryos. Received: 8 July 1998 / Revision received: 31 August 1998 / Accepted: 23 September 1998     

Evaluation of haemoglobin (erythrogen): for improved somatic embryogenesis and plant regeneration in cotton (Gossypium hirsutum L. cv. SVPR 2)

Somatic embryogenesis in cotton (Gossypium hirsutum L.) is accelerated when the plant regeneration medium is supplemented with haemoglobin (erythrogen). In cotton SVPR 2 lines, a higher frequency of embryoid formation was observed when the medium contained 400 mg/l haemoglobin. Fresh weight of the callus, rate of embryoid induction, number of embryoids formed and the percentage of plant regeneration from somatic embryos were increased. Among the two different cultivars tested, MCU 11 showed no response to the presence of haemoglobin when compared to SVPR 2, and embryogenic callus formation was completely absent in the former. Medium containing MS salts, 100 mg/l myo-inositol , 0.3 mg/l thiamine-HCL, 0.3 mg/l Picloram (PIC), 0.1 mg/l kinetin and 400 mg/l haemoglobin effected a better response with respect to embryogenic callus induction. After 8 weeks of culture, a high frequency of embryoid induction was observed on medium containing MS basal salts, 100 mg/l myo-inositol, 0.3 mg/l PIC , 0.1 mg/l isopentenyl adenine, 1.0 g/l NH₄NO₃ and 400 mg/l haemoglobin. Plant regeneration was observed in 75.8% of the mature somatic embryos, and whole plant regeneration was achieved within 6–7 months of culture. The regenerated plantlets were fertile and similar to in vivo-grown, seed-derived plants except that they were phenotypically smaller. A positive influence of haemoglobin was observed at concentrations up to 400 mg/l at all stages of somatic embryogenesis. The increase in the levels of antioxidant enzyme activities, for example superoxide dismutase and peroxidase, indicated the presence of excess oxygen uptake and the stressed condition of the plant tissues that arose from haemoglobin supplementation. This increased oxygen uptake and haemoglobin-mediated stress appeared to accelerate somatic embryogenesis in cotton.Abbreviations BAP Benzylaminopurine - 2,4-D 2,4-Dichlorophenoxyacetic acid - GA₃ Gibberellic acid - GR Glutathione reductase - 2iP Isopentenyl adenine - KT Kinetin - NAA Naphthaleneacetic acid - PFC Perfluorocarbon - PIC Picloram - PO Peroxidase - ROS Reactive oxygen species - SOD Superoxide dismutase - T.HCl Thiamine hydrochloride     

Micropropagation of <Emphasis Type="Italic">Kalopanax pictus</Emphasis> tree via somatic embryogenesis

Summary Kalopanax pictus (Thunb.) Nakai is a tall tree, and its wood has been used in making furniture, while its stem bark is used for medicinal purposes. Here, we report on the micropropagation of Kalopanax pictus via somatic embryogenesis. Embryogenic callus was induced from immature zygotic embryos. The frequency embryogenic callus induction is influenced by days of seed harvest. Callus formation was primarily observed along the radicle tips of zygotic embryos incubated on Murashige and Skoog (MS) medium with 4.4 μM 2,4-dichlorophenoxyacctic acid (2,4-D). Somatic embryogenesis was observed following transfer of embryogenic callus to MS medium lacking 2,4-D. Somatic embryos at the cotyledonary stage were obtained after 6 wk following culture. Frequency of conversion of somatic embryos into plantlets was low (35%) on a hormone-free MS basal medium, but it increased to 61% when the medium was supplemented with 0.05% charcoal. Gibberellic acid (GA₃) treatment markedly enhanced the germination frequency of embryos up to 83%. All plantlets obtained showed 98% survival on moist peat soil (TKS2) artificial soil matrix. About 30 000 Kalopanax pictus plants were propagated via somatic embryogenesis and grown to 3-yr-old plants. These results indicate that production of woody medicinal Kalopanax pictus plantlets through somatic embryogenesis can be practically applicable for propagation.     

Plant regeneration via adventitious bud formation from cotyledon explants of Panax ginseng C. A. Meyer

Cotyledon explants of ginseng (Panax ginseng C. A. Meyer) produced somatic embryos directly on medium without growth regulators, with 89% of the explants forming somatic embryos. Cytokinin treatment greatly suppressed somatic embryo formation but stimulated the direct formation of adventitious buds. BAP treatment was more effective than the kinetin treatment for adventitious bud formation. Auxin (0.05 mg/l IBA) in combination with cytokinin enhanced adventitious bud formation, with the highest frequency, 40%, at 0.05 mg/l IBA and 5 mg/l BAP. Adventitious buds were mainly formed near the distal portion of the cotyledons, while somatic embryos were formed near the proximal excised margins. Shoots were developed from adventitious buds after transfer to MS medium with 10 mg/l GA₃. Root formation from the shoots was obtained after the shoots were transferred to half-strength MS medium with auxin (IAA). When the plants derived from adventitious buds were transferred to greenhouse soil, 36% were successfully acclimatized. Received: 7 November 1997 / Revision received: 12 January 1998 / Accepted: 7 February 1998     

Epicotyl morphophysiological dormancy in seeds of Lilium polyphyllum (Liliaceae)

Dormancy-breaking and seed germination studies in genus Lilium reveal that the majority of Lilium spp. studied have an underdeveloped embryo at maturity, which grows inside the seed before the radicle emerges. Additionally, the embryo, radicle or cotyledon has a physiological component of dormancy; thus, Lilium seeds have morphophysiological dormancy (MPD). A previous study suggested that seeds of Lilium polyphyllum have MPD but the study did not investigate the development of the embryo, which is one of the main criteria to determine MPD in seeds. To test this hypothesis, we investigated embryo growth and emergence of radicles and epicotyls in seeds over a range of temperatures. At maturity, seeds had underdeveloped embryos which developed fully at warm temperature within 6 weeks. Immediately after embryo growth, radicles also emerged at warm temperatures. However, epicotyls failed to emerge soon after radicle emergence. Epicotyls emerged from >90% seeds with an emerged radicle only after they were subjected to 2 weeks of cold moist stratification. The overall temperature requirements for dormancy-breaking and seed germination indicate a non-deep simple epicotyl MPD in L. polyphyllum.