Optimization of Gamma Radiation Dose for Induction of Genetic Variation in Sugarcane (Saccharum spp) Callus and Regenerated Shoot Cultures

Callus cultures were established in three commercial sugarcane varieties viz., CoJ 64, CoJ 83 and CoJ 86 from spindle explants on MS + 2,4-D (4 mg l^?1) + BAP (0.5 mg l^?1) medium. Shoots were regenerated from two-month-old calli on MS + BAP (0.5 mg l^?1) medium. Callus and callus derived shoots were treated with gamma (γ) radiation at 20, 40, 60 and 80 Gray (Gy). Per cent shoot regeneration from y-irradiated calli in the three varieties ranged from 90 to 93.8 at 20 Gy, 83.3 to 87.5 at 40 Gy, 30 to 36.4 at 60 Gy and 0 at 80 Gy. Upon irradiating shoots, subsequent shoot proliferation in the three varieties ranged from 90.9 to 93.1% at 20 Gy, 82.6 to 84.0% at 40 Gy and 27 to 32.3% at 60 Gy, whereas 80 Gy dose was 100% lethal. Thus, 60 Gy dose of y-radiation was found to be optimum for carrying out mutagenesis of both callus and callus derived shoots. In the field, different irradiated clones of the same variety exhibited huge variability with respect to number of canes, cane girth, cane height and sucrose content.     

An efficient plant regeneration system through callus for Pseudarthria viscida (L.) Wright and Arn., a rare ethnomedicinal herb

The purpose of this study was to develop a protocol to induce high frequency of callus and subsequent plantlet regeneration for Pseudarthria viscida; an important medicinal plant. The cotyledonary node and young leaf pieces (1 × 0.5 cm, length × breadth) were used as explants for callus induction and subsequent shoot regeneration and adventitious roots induction from the shoots. The best results were achieved on the following media: (1) 96 % callus induction from cotyledonary node explants on MS medium supplemented with 1.5 mgl⁻¹ 2, 4 dichlorophenoxyacetic acid (2, 4-D) and 0.5 mgl⁻¹ 1-naphthalene acetic acid (NAA), (2) 97 % shoot regeneration from cotyledonary node derived calli with an average of 44.9 shoots per explant on MS medium fortified with 3.0 mgl⁻¹ N₆-benzylaminopurine (BA) and 1 mgl⁻¹ NAA,37 (3) 98 % rooting with an average number of 3.3 roots per shoot on MS medium containing indole-3-butyric acid (IBA) or NAA (0.5–4 mgl⁻¹) after 45 days. The plantlets were transferred to the field after acclimatization. Of the 40 plantlets transplanted to the soil, 29 survived (72.5 %).     

Efficient plant regeneration from shoot apices of sorghum

An efficient and rapid regeneration protocol was developed using shoot apices from germinating seedlings of two cultivars of sorghum, SPV-462 and M35-1, as explants. A vertical slit given from the base of each dissected apex enhanced the efficiency of callusing response by two fold. MS medium containing 0.5 mg dm⁻³ each of 2,4-D and kinetin was most effective in producing friable and embryogenic calli. Scanning electron microscopy of these calli detected somatic embryogenesis. Calli thus induced gave rise to approximately 42 green shoots per callus in both the genotypes when transferred to regeneration medium containing 1.5 mg dm⁻³ kinetin.     

Comparison of 2,4-D and picloram for selection of long-term totipotent green callus cultures of sugarcane

Long-term regeneration of sugarcane (Saccharum spp. hybrid and Saccharum spontaneum L.) callus cultures was achieved by selection of green callus on MS agar medium containing 0.5 mgl^-1 picloram or 2,4-D. Newly initiated sugarcane callus cultures were a complex mixture of different tissue types including white, nonregenerative and green, regenerative tissues. The proportion of the tissue types changed as a function of time in culture, genotype, and amount and kind of auxin. Green callus on picloram media always regenerated green plants. Nine hybrids and ten wild relatives of sugarcane produced green calli on picloram media whereas only three hybrids were grown as green calli on 2,4-D media in long-term culture. Green calli were inoculated into liquid MS medium with 0.5 mgl^-1 picloram for suspension culture. These cultures were totipotent after 19 months. For routine culture, we initiated callus cultures on modified MS medium with 3 mgl^-1 2,4-D, then in two to three weeks we subcultured callus on MS medium with 0.5 mgl^-1 picloram and selected for green callus. Green calli regenerated large numbers of green plants after more than four years.     

Plant regeneration from callus of Puccinellia distans (L.) Parl

Callus was induced from seeds of Puccinellia distans (L.) Parl. on MS medium supplemented with 2 mgl^-1 2,4-dichlorophenoxyacetic acid and 0.5 mgl^-1 kinetin. Morphogenesis initiation was achieved during subculture on medium containing 0.1 mgl^-1 2,4-D. From the point of morphogenetic capacity, 3 types of callus were selected. High frequency of plant regeneration was obtained by selection of embryogenic type of callus, and culture on N₆ medium and N₆ medium supplemented with kinetin (5–10 mgl^-1), or kinetin (2 mgl^-1) and IAA (0.5 mgl^-1). A high ratio of albinos among regenerants was observed.     

Plant regeneration of Chorispora bungeana via somatic embryogenesis

Summary A method was developed for in vitro regeneration of plants via somatic embryogenesis in Chorispora bungeana, an alpine plant with freeze-tolerance, using cell suspensions initiated from leaf-derived callus. Primary calli were induced from leaves of C. bungeana grown on Murashige and Skoog (MS) media supplemented with 4.0 mg l⁻¹ gibberellic acid (GA₃), 0.2 mgl⁻¹ α-naphthaleneacetic acid (NAA) and 0.2 mgl⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D). Suspension culture was initiated by incubating the callus particulates in liquid MS medium supplemented with 1.0 mgl⁻¹ kinetin (KT) and 0.2 mgl⁻¹ NAA. Individual early cotyledonary-stage somatic embryos isolated from cell suspension developed into whole plants on medium containing high levels of sucrose (60 and 90 gl⁻¹), whereas lower sucrose concentrations (0 and 30 gl⁻¹) were inhibitory to main root development. On the MS medium with 90 gl⁻¹ sucrose, one regenerated plant exhibited hetero-morphologic leaves, while other plants grown on different media showed a transformation from stem to root.     

Micropropagation of sugarcane (Saccharum spp.)

A method for callus induction, adventitious bud regeneration, shoot multiplication and rooting of in vitro formed shoots of Helianthus annuus L. var. Argentario is described. Hypocotyl and cotyledon explants formed callus on medium containing 2 mgl^–1 naphthalene acetic acid and 0.5 mgl^–1 benzyladenine. Adventitious buds were formed on hypocotyl segments on medium containing 0.5–2 mgl^–1 benzyladenine. The optimal level of sucrose concentration for shoot regeneration from hypocotyls was 1.5%. Multiplication from shoot apices was promoted by kinetin (2 mgl^–1) plus gibberellic acid (5 mgl^–1), benzyladenine (2 mgl^–1) plus gibberellic acid (10 mgl^–1) or at lower frequency by benzyladenine (1 mgl^–1). A general feature of the plantlets formed in vitro was the precocious flowering.     

Organogenesis and embryogenesis inHelianthus tuberosus and in the interspecific hybridHelianthus annuus ×Helianthus tuberosus

A method of plant regeneration from cotyledons ofHelianthus tuberosus, Helianthus annuus ×Helianthus tuberosus and for the backcross of the interspecific hybrids onH. annuus was developed. Induction of somatic embryogenesis and plantlet regeneration from anther culture of the interspecific hybridsH. annuus ×H. tuberosus is reported.Cotyledons were cultured on Murashige and Skoog basal medium (MS) supplemented with indole-3-acetic acid (IAA) and 6-furfurylaminopurine (kinetin) or N⁶-benzylaminopurine (BAP). Shoot regeneration occurred on most of the media tested, but the best results were obtained on media with a high concentration of cytokinins (BAP or kinetin: 4 mg l^–1) and lower concentration of auxin (IAA: 0.5–1 mg l^–1).Embryogenic callus and adventitious buds were initiated from only two anthers of the hybridH. annuus ×H. tuberosus cultured on the MS medium containing BAP (0.2 mg l^–1) and 1-naphtalenacetic acid (NAA: 0.1 mg l^–1). Prolonged culture of these embryogenic calli and buds on the original medium with successive subculture on MS basal medium without growth regulators resulted in embryo formation and shoot differentiation. The plantlets, after rooting, were established in soil.     

Somatic embryogenesis from leaf- and petiole-derived callus of Vitis rupestris

Somatic embryogenesis from leaf- and petiole-derived calli of Vitis rupestris was obtained with an efficiency of 3.2% and 4.2% of plated explants, respectively on two combinations of 6-benzyladenine and 2,4-dichlorophenoxyacetic acid (1/0.1 and 1/1 mgl^–1) added to MS medium. Embryogenic callus, embryo subcultures and somatic embryogenesis from somatic embryos were obtained either in the presence of 1 mgl^–1 indole-3-acetic acid or 0.1 mgl^–1 indole-3-butyric acid added to MS or NN media. Within a 4-month culture, embryo germination occurred at a frequency of 13% of explanted embryos when chilling at 4°C was provided for two weeks and a combination of 6-benzyladenine (1 mgl^–1) with indole-3-butyric acid (0.1 mgl^–1) was added to NN medium supplemented with casein hydrolysate (250 mgl^–1). A higher frequency (51%) was obtained in a longer culture time (9 months) when only indole-3-butyric acid was present in the medium and in absence of chilling.Abbreviations BA 6-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MS Murashige and Skoog (1962) - NN Nitsch and Nitsch (1969) - NOA 2-naphthoxyacetic acid     

Somatic embryogenesis and regeneration of Cenchrus ciliaris genotypes from immature inflorescence explants

An efficient, highly reproducible system for plant regeneration via somatic embryogenesis was developed for Cenchrus ciliaris genotypes IG-3108 and IG-74. Explants such as seeds, shoot tip segments and immature inflorescences were cultured on Murashige and Skoog (MS) medium supplemented with 2.0–5.0 mg dm^?3 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 mg dm^?3 N⁶-benzyladenine (BA) for induction of callus. Callus could be successfully induced from all the three explants of both the genotypes. But the high frequency of embryogenic callus could be induced only from immature inflorescence explants. Somatic embryos were formed from nodular, hard and compact embryogenic calli when 2,4-D concentration was gradually reduced and BA concentration increased. Histological studies of somatic embryos indicated the presence of shoot apical meristem with leaf primordia. Ultrastructural details of globular and scutellar somatic embryos further validated successful induction and progression of somatic embryogenesis. Shoots were differentiated upon germination of somatic embryos on MS medium containing 2,4-D (0.25 mg dm^?3) and BA or kinetin (1–5 mg dm^?3). Roots were induced on ½ MS medium containing charcoal (0.8 %), and the regenerated plants transferred to pots and established in the soil showed normal growth and fertility.     

Induction and comparison of different in vitro morphogenesis pathways using embryo of cumin (<Emphasis Type="Italic">Cuminum cyminum</Emphasis> L.) as a model material

In this study, using cumin embryo as explant and manipulating plant growth regulators (PGRs) in regeneration medium, the main in vitro morphogenesis pathways including direct shoot organogenesis, direct somatic embryogenesis, indirect somatic embryogenesis, and indirect shoot organogenesis were obtained. The effects of PGRs, subculture, and light on the induction and progression of different pathways were studied in detail. Direct shoot organogenesis occurred on the meristematic zone, while direct somatic embryogenesis was observed on hypocotyl part of cumin embryo (more differentiated part). Application of BAP (0.1 mgl⁻¹) was the sole triggering factor for induction of callus and indirect regeneration pathways. Exogenous IAA played the central role in the direct somatic embryogenesis pathway; however, the combined effects of IAA and NAA along with the high endogenous cytokinin level resulted in direct shoot organogenesis. Subculturing revealed accelerating effects on direct somatic embryogenesis pathway and callus formation. Conversely, subculturing had negative effect on direct shoot organogenesis pathway. In certain combinations of PGRs, like 0.4 mgl⁻¹ IAA + 0.4 mgl⁻¹ NAA, co-induction and co-regeneration of different pathways were observed. Investigation of genotype dependencies of different pathways showed that direct pathways are more genotype-dependent, stable, and faster than indirect pathways. This research presents the embryo of cumin as a convenient model material for induction and comparison of different morphogenesis pathways.     

Improvement of efficient in vitro regeneration potential of mature callus induced from Malaysian upland rice seed (Oryza sativa cv. Panderas)

A new and rapid protocol for optimum callus production and complete plant regeneration has been assessed in Malaysian upland rice (Oryza sativa) cv. Panderas. The effect of plant growth regulator (PGR) on the regeneration frequency of Malaysian upland rice (cv. Panderas) was investigated. Mature seeds were used as a starting material for callus induction experiment using various concentrations of 2,4-D and NAA. Optimal callus induction frequency at 90% was obtained on MS media containing 2,4-D (3 mg L⁻¹) and NAA (2 mg L⁻¹) after 6 weeks while no significant difference was seen on tryptophan and glutamine parameters. Embryogenic callus was recorded as compact, globular and light yellowish in color. The embryogenic callus morphology was further confirmed with scanning electron microscopy (SEM) analysis. For regeneration, induced calli were treated with various concentrations of Kin (0.5–1.5 mg L⁻¹), BAP, NAA and 0.5 mg L⁻¹ of TDZ. The result showed that the maximum regeneration frequency (100%) was achieved on MS medium containing BAP (0.5 mg L⁻¹), Kin (1.5 mg L⁻¹), NAA (0.5 mg L⁻¹) and TDZ (0.5 mg L⁻¹) within four weeks. Developed shoots were successfully rooted on half strength MS free hormone medium and later transferred into a pot containing soil for acclimatization. This cutting-edge finding is unique over the other existing publishable data due to the good regeneration response by producing a large number of shoots.Abbreviations: 2,4-D, 2,4-dichlorophenoxyacetic acid; NAA, naphthaleneacetic acid; Kin, kinetin; MS, Murashige and Skoog; BAP, benzylaminopurine; TDZ, thidiazuron     

Optimization of a mature embryo-based in vitro culture system for high-frequency somatic embryogenic callus induction and plant regeneration from japonica rice cultivars

Optimization of the conditions for an efficient induction of somatic embryogenic calli and regeneration of plants from mature seeds of japonica rice cultivars was attempted. The number, color, size, shape, and appearance time of the induced embryogenic calli varied among the rice cultivars depending on the type of basal medium (LS, MS, N6). Presence of adequate amount of sucrose in the medium was an absolute requirement for embryogenic callus formation and shoot induction. Induction of the embryogenic calli, whose overall rates ranged from 30 to 56%, was most efficient in N6 medium supplemented with 3.0 mg l^–1 of 2,4-D and 30 g l^–1 of sucrose. Agar concentration in the regeneration medium was also critical for the shoot induction. Kinetin was found to be more effective for shoot regeneration compared with BA, while the highest shoot regeneration frequencies were observed when either cytokinin was combined with high concentration (2.0 mg l^–1) of NAA. The optimal concentration of kinetin for the highest shoot regeneration frequency (6777%) was different among the cultivars tested. The embryogenic calli-derived shoots rooted on a plant growth regulator-free MS medium were successfully established in soil, producing fertile seeds.     

Regeneration of sorghum from shoot tip cultures and field performance of the progeny1

A system for rapid plant regeneration through somatic embryogenesis from shoot tip explants of sorghum [Sorghum bicolor (L.) Moench] is described. Somatic embryogenesis was observed after incubation of explants in dark for 6–7 weeks through a friable embryogenic callus phase. Linsmaier and Skoog medium supplemented with 2,4-dichlorophenoxyacetic acid (2 mg l⁻¹) and kinetin (0.1 mg l ⁻¹) was used for induction of friable embryogenic calli and somatic embryos. Germination of somatic embryos was achieved about 5 weeks after transfer onto Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (2 mg l⁻¹) and indole-3-acetic acid (0.5 mg l ⁻¹) under light. Seeds from in vitro-regenerated plants produced a normal crop in a field trial, and were comparable to the crop grown with the seeds of the mother plant used to initiate tissue culture. The simplicity of the protocol and possible advantages of the system for transformation over other protocols using different explants are discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Picolinic acid-induced direct somatic embryogenesis in sweet potato

Somatic embryos are being considered as an alternative material for in vitro germplasm conservation of sweet potato [(Ipomoea batatas (L.) Lam.)]. Picolinic acid was tested for somatic embryo production in sweet potato apical meristem tip cultures. Low level (0.2 mgl^-1) of picolinic acid combined with kinetin or 6-benzylamino purine (6-BAP) (1.0 and 2.0 mgl^-1) suppressed shoot growth and induced callus proliferation. Increased amount of picolinic acid (2 and 3 mgl^-1) in combination with kinetin (0.25 and 1.0 mgl^-1) induced direct somatic embryogenesis from apical meristem tips of variety Regal but not in Jewel. The primary embryos matured and germinated bipolarly yielding whole plantlets and unipolarly producing embryogenic hyperhydrated-fasciated shoots. The hyperhydrated-fasciated shoots, when cultured in picolinic and kinetin-enriched medium, produced secondary embryos. The secondary embryos also germinated bipolarly and unipolarly, resulting in subsequent cycles of embryogenesis. This recurrent embryogenesis ensures maintenance and proliferation of embryogenic tissues. Somatic embryos were also formed in mannitol-induced hyperhydrated shoots in response to picolinic acid and kinetin or 6-BAP treatment. Embryogenesis did not occur in non-hyperhydrated leaf, petiole, and internode sections.     

<Emphasis Type="Italic">In vitro</Emphasis> plant regeneration through somatic embryogenesis and direct shoot organogenesis in <Emphasis Type="Italic">Pennisetum glaucum</Emphasis> (L.) R. Br.

An efficient in vitro plant regeneration protocol through somatic embryogenesis and direct shoot organogenesis has been developed for pearl millet (Pennisetum glaucum). Efficient plant regeneration is a prerequisite for a complete genetic transformation protocol. Shoot tips, immature inflorescences, and seeds of two genotypes (843B and 7042-DMR) of pearl millet formed callus when cultured on Murashige and Skoog (MS) medium supplemented with varying levels of 2,4-dichlorophenoxyacetic acid (2,4-D; 4.5, 9, 13.5, and 18 μM). The level of 2,4-D, the type of explant, and the genotype significantly effected callus induction. Calli from each of the three explant types developed somatic embryos on MS medium containing 2.22 μM 6-benzyladenine (BA) and either 1.13, 2.25, or 4.5 μM of 2,4-D. Somatic embryos developed from all three explants and generated shoots on MS medium containing high levels of BA (4.4, 8.8, or 13.2 μM) combined with 0.56 μM 2,4-D. The calli from the immature inflorescences exhibited the highest percentage of somatic embryogenesis and shoot regeneration. Moreover, these calli yielded the maximum number of differentiated shoots per callus. An efficient and direct shoot organogenesis protocol, without a visible, intervening callus stage, was successfully developed from shoot tip explants of both genotypes of pearl millet. Multiple shoots were induced on MS medium containing either BA or kinetin (4.4, 8.8, 17.6, or 26.4 μM). The number of shoots formed per shoot tip was significantly influenced by the level of cytokinin (BA/kinetin) and genotype. Maximum rooting was induced in 1/2 strength MS with 0.8% activated charcoal. The regenerated plants were transferred to soil in pots, where they exhibited normal growth.     

Agrobacterium-mediated transformation of ginseng (Panax ginseng) and mitotic stability of the inserted β-glucuronidase gene in regenerants from isolated protoplasts

Cotyledonary expiants of ginseng zygotic embryos were cocultured with Agrobacterium tumefadens strain LBA4404 harboring the binary vector pBI121 for 48 h and transferred onto MS medium supplemented with 1 mgl^–1 2,4-dichlorophenoxyacetic acid (2,4-D), 0.1 mgl^–1 kinetin, and 100 mgl^–1 kanamycin. After 8 weeks of culture, kanamycin-resistant calli formed on the cut surfaces of cotyledonary expiants and subsequently they gave rise to numerous somatic embryos. Eight weeks after transfer onto medium containing 1 mgl^–1 each of 6-benzyladenine (BA) and gibberellic acid, most of them developed into plantlets. Southern analysis confirmed that the -glucuronidase (GUS) gene was incorporated into the genomic DNA of regenerants. Protoplasts were enzymatically isolated from transformed somatic embryo segments and cultured in liquid medium containing 60 gl^–1 myo-inositol, 1 mgl^–1 2,4-D, 0.5 mgl^–1 BA, and 0.5 mgl^–1 kinetin. Plants were regenerated from protoplasts via somatic embryogenesis. The polymerase chain reaction method revealed that 92% of the regenerants retained the GUS gene. When treated with X-glucuronide, 78% of the regenerants showed a GUS-positive response. The overall results indicate that the transgene is stably transmitted during somatic ontogeny and stably expressed in most the regenerants, whereas it may be deleted or impaired in some portion of them.Abbreviations BA 6-benzyladenine; 2,4-D,2,4-dichlorophenoxyacetic acid - DIG digoxigenine - GA₃ gibberellic acid - X-gluc X-glucuronide - GUS -glucuronidase - MS Murashige and Skoog (1962)     

Picloram induced somatic embryogenesis in Gasteria and Haworthia

Various leaf sections of Gasteria verrucosa Haw. and Haworthia fasciata Haw. were cultured on media to examine the effect of picloram (4-amino 3, 5, 6-trichloropicolinic acid) and 2, 4-D (2, 4-dichlorophenoxy acetic acid) on somatic embryogenesis. Picloram (0.5, 1.0, 2.0, 3.0 mgl^-1) outperformed 2, 4-D (0, 1.0, 2.0, 3.0 mgl^-1) as the auxin source of both earliness of callus and embryo induction and final yield of embryos produced at both kinetin levels examined (0.25, 1.0 mgl^-1). Embryos arose initially as a yellow, compact globular masses from the area just beneath the epidermis in linear pattern parallel with the main axis of the leaf and then developed a heartshaped appearance. Embryo formation was preceded by growth of callus almost crystalline in appearance on the cut surface. Subsequent shoot formation developed from green pigmented loci in crystalline callus derived from embryos. Shoot and root development in Gasteria was induced on a defined medium containing quarter strength MS or B5 salts with no hormonal supplementation.     

Somatic embryogenesis from mature zygotic embryos of Distylium chinense (Fr.) Diels and assessment of genetic fidelity of regenerated plants by SRAP markers

The objective was to establish an efficient regeneration protocol for Distylium chinense based on somatic embryogenesis and evaluate the genetic stability of plants regenerated in vitro. To induce callus mature zygotic embryos were cultured on Murashige and Skoog’s (MS) medium that was supplemented with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and N⁶-benzyladenine (BA). After 20 days, the highest rate of callus formation (88.9 %) occurred on MS medium supplemented with 0.5 mg l^?1 2,4-D and 0.1 mg l^?1 BA. It was observed that light-yellow, compact, dry, nodular embryogenic calli had formed. These calli were then subcultured on fresh MS medium supplemented with 0.1 mg l^?1 BA and 0.5 mg l^?1 α-naphthaleneacetic acid (NAA) for proliferation for an additional 30 days. To induce somatic embryos and plant regeneration, the embryogenic callus was transferred to fresh MS medium that was supplemented with different concentrations of BA and NAA. After 30 days, 0.5 mg l^?1 BA in combination with 0.5 mg l^?1 NAA produced the best result in terms of somatic embryogenesis (%), shoot differentiation (%), number of shoots per callus and shoot length. Next, the plantlets were transferred to the field for 5 weeks and a 95 % survival rate was observed. The sequence-related amplified polymorphism markers confirmed genetic stability of plants regenerated in vitro. To our knowledge, this is the first report that describes a plant regeneration protocol for D. chinense via somatic embryogenesis to be used for germplasm conservation and commercial cultivation.     

Spontaneous somatic embryogenesis on in vitro root segment cultures of Rauvolfia micrantha Hook. F.—A rare medicinal plant

Summary Plant regeneration through direct somatic embryogenesis was achieved from root segments derived from in vitro shoots of Rauvolfia micrantha Hook. f. (Apocynaceae) grown for 6 wk in half-strength Murashige and Skoog (MS) medium with 3% sucrose, 100 mgl⁻¹ myo-inositol, and 0.5 mgl⁻¹ α-naphthaleneacetic acid (NAA). The effects of photoperiod and plant growth regulators (PGRs) in half-strength MS medium were studied for the rapid and maximum induction of somatic embryos. The characteristic globular or heart-shaped stages of somatic embryogenesis were not found and cotyledonary stage embryos occasionally appeared without the intervention of callus in total darkness and 16-h photoperiod. Root segments cultured in the medium containing 0.1 mgl⁻¹ NAA and 0.2 mgl⁻¹ 6-benzyladenine (BA) under 16-h photoperiod showed the maximum frequency (39%) of embryogenesis. The frequency of embryo formation was increased to 63% when they were cultured in medium with 0.1 mgl⁻¹ NAA and 0.2 mgl⁻¹ BA in the dark for 4wk, then grown under the 16-h photoperiod. Explants with developing embryos developed into plants after transfer to half-strength MS medium supplemented with 0.1 mgl⁻¹ BA and 0.05 mgl⁻¹ NAA. The well-developed plants were hardened and most plants (80%) survived and were phenotypically similar to the mother plants.