<Emphasis Type="Italic">In vitro</Emphasis> regeneration of witloof chicory (<Emphasis Type="Italic">Cichorium intybus</Emphasis> L.) from leaf explants and accumulation of esculin

Summary An efficient protocol has been developed for the regeneration of plantlets from leaf explants of witloof chicory (Cichorium intybus L.). Regeneration via callus was obtained on modified Murashige and Skoog semisolid medium (MS) containing 2.0 μM indole-3-acetic acid +5.0 μM 6-furfurylaminopurine (kinetin), and 1000 mgl⁻¹ casein hydrolyzate. At least five or more shoots regenerated from each callus. The shoots were rooted on MS +0.2 μM indole-3-butyric acid. The plantlets thus obtained were successfully established in soil after bardening. Esculin accumulation was recorded in plant tissues at different stages of differentiation in in vitro cultures and compared with in vivo-grown, plants. The esculin accumulation was higher in in vitro plants.     

<Emphasis Type="Italic">Cupressus</Emphasis> callus and cell suspension cultures: Effect of seiridins on their growth and sensitivity

Summary Cupressus macrocarpa and C. arizonica were examined for callus and cell culture production in vitro. Both species produced callus on agar-solidified MSCY medium supplemented with vitamins, antioxidants, 0.14 μM kinetin (KIN), and 10 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Suspension cultures of both species were established in liquid MSCY medium. Seiridin (SE) and iso-seiridin (ISE), two phytotoxic butenolides produced by Seiridium cardinale, S. cupressi, and S. unicorne, the causal agents of many canker diseases of cypress, were tested on callus or cell suspension cultures. In the medium without other plant growth regulators (PGR), SE promoted cell proliferation of cypress better than ISE, for callus initiation, callus maintenance, and cell suspension cultures. The growth rates of cypress callus tissues and suspension cultures of both cypress species on media containing 50–150 μM SE or ISE were measured. At concentrations of 50 μM and higher, growth rates increased exponentially with the SE concentration. A comparison with KIN and 2,4-D indicated that 50 μM SE promoted growth of callus tissues and cell suspension cultures more than 100 μM ISE. SE can also interact with, or counteract, KIN and 2,4-D. It was demonstrated that SE could replace KIN in the medium for C. arizonica. SE could be involved in cell enlargement and proliferation processes. The less susceptible cypress species (C. arizonica) had a high content of terpenoids than that of the more susceptible species (C. macrocarpa). SE could be a useful tool as a phytohormonal-like regulator to manipulate physiological changes at the cellular level and as an elicitor of sensitivity or tolerance of cypress germplasm to the phytotoxin.     

Induction of somatic embryogenesis in cashewnut (Anacardium occidentale L.)

Summary Somatic embryogenesis was induced in callus cultures derived from nucellar tissue of cashewnut (Anacardium occidentale L.). Callus was obtained from nucellar tissue after 3 wk of culture on semisolid Murashige and Skoog (MS) basal medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D, 5 μM)+gibberellic acid (GA₃, 15 μM)+N⁶-benzyladenine (BA, 5 μM). This callus gave rise to an embryogenic mass after 9 wk on maintenance medium containing 2,4-D (10 μM)+GA₃ (15 μM)+4% sucrose +0.5% activated charcoal +10% coconut water (CW) +0.05% casein hydrolysate (CH). The embryogenic mass, after transfer to medium supplemented with 2,4-D (5 μM)+GA₃ (30 μM)+4% sucrose +0.5% activated charcoal +10% CW +0.05% CH, gave rise to somatic embryos. The developmental stages of somatic embryos were observed using light and stereo microscopes. Histological study of somatic embryo development was also carried out. The present study would be useful for clonal propagation, and variety improvement in cashewnut, which is essential due to its increasing demand and export potential.     

Efficient plant regeneration in <Emphasis Type="Italic">Holarrhena antidysenterica</Emphasis> Wall., from shoot segment-derived callus

Summary A procedure has been outlined for plant regeneration of an important medicinal shrub, Holarrhena antidysenterica, through shoot segment-derived callus. Explants used for callus induction were shoot segments derived from 14-d-old axenic plants on Murashige and Skoog (MS) medium supplemented with 15 μM N⁶-benzyladenine (BA). A white friable type of callus was obtained in 4.52 μM 2,4-dichlorophenoxyacetic acid and 2.32 μM kinetin which did not have the potentiality to regenerate. High-frequency shoot differentiation was achieved on transferring the friable callus to MS medium supplemented with 17.8 μM BA and 8.0 μM naphthaleneacetic acid. The highest percentage of calluses forming shoots (65.06±2.26) was achieved in this medium. The organogenetic potential of the regenerating callus was influenced by the age of the culture. Rooting was achieved on the shoots using MS medium with 25 μM indolebutyric acid. The plantlets were acclimatized and established in soil. The regenerated plants were morphologically uniform and exhibited similar growth characteristics and vegetative morphology to the donor plants.     

Antimycobacterial activity of two natural alkaloids,vasicine acetate and 2-acetyl benzylamine,isolated from Indian shrub <Emphasis Type="Italic">Adhatoda vasica</Emphasis> Ness. leaves

In folk medicine, Adhatoda vasica Ness. (Acanthaceae) is used to treat asthma and cough. The leaves of A.vasica were powdered and extracted with hexane, ethyl acetate and methanol. The hexane extract showed 97% reduction in colony-forming units (CFU) at 100 μg/ml. The hexane extract was subjected to column chromatography. Two natural compounds, vasicine acetate and 2-acetyl benzylamine, were isolated from it. They were bioassayed against Mycobacterium tuberculosis. The two compounds showed strong antimycobacterial activity. Vasicine acetate and 2-acetyl benzylamine isolated from hexane extract of A.vasica leaves, significantly inhibited M. tuberculosis and one multi-drug-resistant (MDR) strain and one sensitive strain at 200 and 50 μg/ml, respectively. Our study demonstrated that both the compounds, vasicine acetate and 2-acetyl benzylamine, could be evaluated further for developing a drug to control M. tuberculosis.     

Somatic embryogenesis in <Emphasis Type="Italic">Schisandra chinensis</Emphasis> (Turcz.) Baill.

Summary Regeneration of plants via somatic embryogenesis was achieved from zygotic embryo explants isolated from mature seeds of Schisandra chinensis. Merkle and Sommer's medium, fortified with 2,4-dichlorophenoxyacetic acid (2,4-D; 9.04 μM) and zeatin (0.09 μM), was effective for induction of embryogenic callus. The development of a proembryogenic mass and somatic embryos occurred on Murashige and Skoog medium (MS) free of plant growth regulators. The embryogenic callus induced on Merkle and Sommer's medium supplemented with 2,4-D (9.04 μM) and zeatin (0.09 μM) showed development of the maximum number of somatic embryos when transferred to MS medium free of plant growth regulators. The maximum maturation and germination of cotyledonary somatic embryos (46.3%) occurred on MS medium supplemented with 2,4-D (0.45 μM) and N⁶-benzyladenine (1.11 μM). The somatic embryo-derived plants were successfully hardned, with a survival rate of approximately 67%, and established in the field.     

Plant regeneration from protocorm-derived callus of <Emphasis Type="Italic">Cypripedium formosanum</Emphasis>

Summary Totipotent callus of Cypripedium formosanum, an endangered slipper orchid species, was induced from seed-derived protocorm segments on a quarter-strength Murashige and Skoog medium containing 4.52 μM 2,4-dichlorophenoxyacetic acid and 4.54 μM 1-phenyl-3-(1,2,3-thiadiazol-5-yl)-urea (thidiazuron). This callus proliferated well and was maintained by subculturing on the same medium. On average, 13 protocorm-like bodies could be obtained from a piece of 4 mm callus after being transferred to the medium with 4.44 μM N⁶-benzyladenine after 8 wk of culture. The regenerated protocorm-like bodies formed shoots and roots on medium containing 1 g l⁻¹ activated charcoal and 20 g l⁻¹ potato homogenate. After 24 wk of culture on this medium, well-developed plantlets ready for potting were established.     

Thidiazuron-induced high-frequency shoot organogenesis from leaf-derived callus of ia medicinal climber, <Emphasis Type="Italic">Tylophora Indica</Emphasis> (Burm. F.) merrill

Summary Tylophora indica (Burm. f.) Merrill is a threatened medicinal climber distributed in the forests of northern and peninsular India. An efficient and reproducible protocol for high-frequency callus regeneration from immature leaf explants of T. indica was developed. Organogenic callus formation from immature leaf pieces was obtained by using Murashige and Skoog (MS) medium supplemented with 7 μM 2,4-dichlorophenoxyacetic acid and 1.5 μM 6-benzyladenine. On this medium 92% explants produced callus. The optimal hormone combination for plantlet regeneration was 8 μM thidiazuron, at which shoot regeneration was obtained from 100% of the cultures, with an average of 66.7 shoots per culture. Histological studies of the regenerative callus revealed that shoot buds were originated from the outermost regions. For root formation, half-strength MS medium supplemented with 3 μM indole-3-butyric acid was used. Plants were transferred to soil, where 92% survived after 3 mo. of acclimatization.     

Shoot-bud regeneration in subcultured callus of engelmann spruce

Summary Callus cultures ofPicea engelmannii (Parry, Engelmann spruce) were initiated and established from mature embryos cultured on von Arnold and Eriksson’s medium (AE) supplemented with N⁶-benzyladenine (10μM) and naphthalene acetic acid (10 μM). Cultures were maintained by subculture at 3-to-4-wk intervals. After three subcultures, callus was transferred to AE medium with only N⁶-benzyladenine (25 μM). Adventitious buds appeared on the surface of the callus after 2-to 4-wk and grew to adventitious shoots on AE medium without growth hormones or on AE medium with kinetin (0.1 μM). Shoot-forming capacity was maintained through 7 further subcultures. This study was supported by the Natural Sciences and Engineering Research Council of Canada grant G1438 to T. A. Thorpe and D. I. Dunstan.     

Somatic embryogenesis,organogenesis, and regeneration from leaf callus culture of <Emphasis Type="Italic">Decalepis hamiltonii</Emphasis> Wight &; Arn., an endangered shrub

Summary A novel protocol has been developed for inducing somatic embryogenesis from leaf cultures of Decalepis hamiltonii. Callus was obtained from leaf sections in Murashige and Skoog (MS) medium supplemented with α-naphthaleneacetic acid (NAA)+N⁶-benzyladenine (BA) or 2,4-dichlorophenoxyacetic acid (2,4-D)+BA. Nodular embryogenic callus developed from the cut end of explants on media containing 2,4-D and BA, whereas compact callus developed on media containing NAA and BA. Upon subsequent transfer of explants with primary callus onto MS media containing zeatin and/or gibberellic acid (GA₃) and BA, treatment with zeatin (13.68μM) and BA (10.65 μM) resulted in the induction of the highest number of somatic embryos directly from nodular tissue. The maturation of embryos took place along with the induction on the same medium. Embryogenic calluses with somatic embryos were subcultured onto MS basal medium supplemented with 4.56μM zeatin+10.65 μM BA. After 4wk, more extensive differentiation of somatic embryos was observed. The mature embryos developed into complete plantlets on growth regulator-free MS medium. A distinct feature of this study is the induction of somatic embryogenesis from leaf explants of Decalepis hamiltonii, which has not been reported previously. By using this protocol, complete plantlets could be regenerated through indirect somatic embryogenesis or organogenesis from leaf explants in 12–16 wk.     

Organogenesis,embryogenesis, and synthetic seed production in <Emphasis Type="Italic">Arnebia euchroma</Emphasis>—A critically endangered medicinal plant of the Himalaya

Summary This is the first report of simultaneous organogenesis and somatic embryogenesis in Arnebia euchroma, a highly valued, critically endangered medicinal plant of the Himalaya. Root-derived callus showed only rhizogenesis, whereas leaf-derived callus showed simutaneous organogenesis and somatic embryogenesis. Organogenesis was optimal (12.2 shoots per culture) in 1 μM indole-3-butyric acid combined with 2.5 μM 6-benzyladenine and induction of somatic embryogenesis (16.3 embryos per culture) occurred in 2.5 μM indole-3-butyric acid combined with 2.5 μM 6-benzyladenine. Shoots rooted (100%) best in half-strength Murashige and Skoog (MS) medium supplemented with 2.0 μM indole-3-butyric acid. Early cotyledonary-stage embryos encapsulated with 3% sodium alginate and calcium nitrate (100 mM for 25 min) showed 60.6% germination in MS medium. Rooted shoots transferred to a mixture of sterile soil, sand, and peat (1∶1∶1 by volume) showed 72% survival ex vitro. Application of these protocols would be helpful in reducing pressure in natural populations, in genetic transformation studies, and in long-term storage of elite genotypes through synthetic seed production.     

Tissue culture and <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of hemp (<Emphasis Type="Italic">Cannabis sativa</Emphasis> L.)

Summary Hemp (Cannabis sativa L.) is cultivated in many parts of the world for ils fiber, oil, and seed. The development of new hemp cultivars with improved traits could be facilitated through the application of biotechnological strategies. The purpose of this study was to investigate the propagation of hemp in tissue culture and to establish a protocol for Agrobacterium-mediated transformation for foreign gene introduction. Stem and leaf segments from seedlings of four hemp varieties were placed on Murashige and Skoog medium with Gamborg B5 vitamins (MB) supplemented with 5 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 1 μM kinetin, 3% sucrose, and 8 gl⁻¹ agar. Large masses of callus were produced within 4 wk for all cultivars. Suspension cultures were established in MB medium containing 2.5 μM 2,4-D. To promote embryogenesis or organogenesis, explants, callus, and suspension cultures derived from a range of explant sources and seedling ages were exposed to variations in the culture medium and changes to the culture environment. None of the treatments tested were successful in promoting plantlet regeneration. Suspension cells were transformed with Agrobacterium tumefaciens strain EHA101 carrying the binary vector pNOV3635 with a gene encoding phosphomannose isomerase (PMI). Transformed callus was selected on medium containing 1–2% mannose. A chlorophenol red assay was used to confirm that the PMI gene was expressed. Polymerase chain reaction and Southern hybridization detected the presence of the PMI gene. Copy number in different lines ranged from one to four.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of the rare medicinal plant <Emphasis Type="Italic">Ceropegia candelabrum</Emphasis> L. through somatic embryogenesis

Summary Efficient in vitro propagation of Ceropegia candelabrum L. (Asclepidaceae) through somatic embryogenesis was established. Somatic embryogenesis depended on the type of plant growth regulators in the callus-inducing medium. Friable callus, developed from leaf and internode explants grown on Murashige and Skoog (MS) medium supplemented with 4.52μM2,4-dichlorophenoxyacetic acid (2,4-D), underwent somatic embryogenesis. Compared to solid media, suspension culture was superior and gave rise to a higher number of somatic embryos. Transfer of the friable callus developed on MS medium containing 4.52μM 2,4-D to suspension cultures of half- or quarter-strength MS medium with lower levels of 2,4-D (0.23 or 0.45 μM) induced the highest number of somatic embryos, which developed up to the torpedo stage. Somatic embryogenesis was asynchronous with the dominance of globular embryos. About 100 mg of callus induced more than 500 embryos. Upon transfer to quarter-strength MS agar medium without growth regulators, 50% of the somatic embryos underwent maturation and developed into plantlets. Plantlets acclimatized under field conditions with 90% survival.     

Influence of endophytic fungal elicitation on production of inophyllum in suspension cultures of <Emphasis Type="Italic">Calophyllum inophyllum</Emphasis> L.

The influence of dried cell powder and culture filtrates of endophytic fungi on production of inophyllum in cell suspension cultures of leaf- and stem-derived callus of Calophyllum inophyllum was investigated. Two fungi, Nigrospora sphaerica and Phoma spp., endophytic to C. inophyllum, were isolated from leaf tissues, and were identified by both 18S rRNA gene amplification and sequencing. Elicitation of suspension cultures of both callus types of C. inophyllum with dried cell powder and culture filtrates of both fungi consistently elicited production of inophyllum A, B, C, and P. In comparison to stem-derived callus, suspension cultures of leaf-derived callus enhanced production of most inophyllum. Of the four inophyllum studied, the highest production of inophyllum A, C, and P was achieved in elicited suspension cultures of leaf-derived callus. Suspension cultures of stem-derived callus enhanced production only of inophyllum B. When suspension cultures of leaf-derived callus were elicited with 40 mg dried cell powder of Phoma spp., a level of 751-fold (6.84 mg/100 g elicited biomass) of inophyllum A was produced, compared to control. Whereas, a level of 414-fold (6.22 mg/100 g elicited biomass) of inophyllum B was produced when suspension cultures of stem-derived callus were elicited with 20 mg dried cell powder of N. sphaerica. When compared to control, a 10% culture filtrate of N. sphaerica in suspension cultures of leaf-derived callus elicited inophyllum C and P production by 928-fold (7.43 mg/100 g elicited biomass) and 750-fold (1.5 mg/100 g elicited biomass), respectively.     

Shoot organogenesis and plantlet regeneration from hypocotyl-derived cell suspensions of a tree legume, Dalbergia sissoo Roxb

Summary A complete and efficient protocol is presented for plant regeneration from cell-suspension cultures of Dalbergia sissoo Roxb., an economically important leguminous tree. Factors influencing callus initiation, establishment of cell-suspension culture, callus formation from embredded microcolonies, and shoot organogenesis from suspension-derived callus were identified. Of the two different auxins tested, callus induction was better on a medium containing naphthalene acetic acid (NAA). The percentage of callus induction increased considerably when NAA at 2.0 mg l⁻¹ (10.8 μM) was added in conjunction with 0.5 mg l⁻¹ (2.2 μM) N⁶-benzyladenine (BA). Of the three different explants evaluated for callus induction, hypocotyl segments were most responsive. Friable hypocotyl-derived callus from the second subculture passage was used to initiate the cell-suspension culture. Optimum growth of the cell suspension was observed in MS medium supplemented with the same growth regulators as described above for callus induction, with an initial inoculum cell density of 1%. The plating efficiency of the microcolonies was greatly influenced by harvesting time and the gelling agent used for plating. Efficiency was highest (93%) with cells harvested at their exponential growth phase and plated in 1.2 g l⁻¹ Phytagel. Shoot organogenesis from callus cultures was higher on a medium supplemented with a combination of BA and NAA than on BA alone. Seventy-one per cent of cultures exhibited shoot-bud differentiation on a medium containing 3.0 mg l⁻¹ (13.3 μM) BA and 0.5 mg l⁻¹ (2.7 μM) NAA. Regenerated shoots were rooted on half-strength MS medium containing 1 mg l⁻¹ each of indole-3-acetic acid (5.7 μM), indole-3-butyric acid (4.9 μM) and indole-3-propionic acid (5.3 μM). Plantlets were acclimated and established in soil.     

Effects of salinity on growth and compatible solutes of callus induced from <Emphasis Type="Italic">Populus euphratica</Emphasis>

Summary The present study aimed to evaluate the response to salinity of Populus euphratica, which is more salt-resistant than other poplar cultivars, at the cellular level. To this purpose, callus was induced from shoot segments of P. euphratica on Murashige and Skoog (MS) medium supplemented with 0.5 mg l⁻¹ (2.2 μM) 6-benzyladenine (BA) and 0.5 mg l⁻¹ (2.7 μM 1-naphthaleneacetic acid (NAA). Callus was transferred to MS medium supplemented with 0.25 mg l⁻¹ (1.1 μM) BA and 0.5 mg l⁻¹ NAA. The relative growth rate of callus reached a maximum in the presence of 50 mmol l⁻¹ NaCl and growth was inhibited with increasing NaCl concentrations. Examination of the changes of osmotic substances under salt stress showed that accumulation of proline, glycine betaine, and total soluble sugars increased with increasing salt concentrations. The results indicate that the response of the callus of P. euphratica to salt stress is similar to that of the whole plant.     

Plant regeneration through somatic embryogenesis in medicinally important <Emphasis Type="Italic">Centella asiatica</Emphasis> L.

Summary High-frequency somatic embryogenesis and plant regeneration was achieved on callus derived from leaf (petiole and lamina) and internode explants of Centella asiatica L. Growth regulators significantly influenced the frequency of somatic embryogenesis and plant regeneration. Calluses developed on Murashige and Skoog (MS) medium fortified with 4.52 μM 2,4-dichlorophenoxyacetic acid (2,4-D) or 5.37 μM α-naphthaleneacetic acid (NAA), both with 2.32 μM kinetin (Kn), were superior for somatic embryogenesis. Callus developed on NAA and Kn-supplemented medium favored induction and maturation of embryos earlier compared to that on 2,4-D and Kn. Embryogenic callus transferred from NAA and Kn-supplemented medium to suspension cultures of half-strength MS medium with NAA (2.69 μM) and Kn (1.16 μM) developed a mean of 204.3 somatic embryos per 100 mg of callus. Embryogenic callus transferred from 2,4-D and Kn subsequently to suspension cultures of half-strength MS medium with 2,4-D (0.45 μM) and Kn (1.16 μM) developed a mean of 303.1 embryos per 100 mg of callus. Eighty-eight percent of the embryos underwent maturation and conversion to plantlets upon transfer to half-strength MS semisolid medium having 0.054 μM NAA with either 0.044 μM BA or 0.046 μM Kn. Embryo-derived plantlets established in field conditions displayed morphological characters identical to those of the parent plant.     

Efficient plant regeneration from embryogenic suspension cultures of sweetpotato

Summary Using 15 Chinese and Japanese cultivars of sweetpotato, Ipomoea batatas (L.) Lam., we succeeded in developing an efficient plant regeneration system from embryogenic suspension cultures. The embryogenic callus derived from shoot apices of the 15 cultivars was used to initiate embryogenic suspension cultures in Murashige and Skoog (MS) medium containing 9.05 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Rapidly proliferating and well-dispersed embryogenic suspension cultures were established. Cell aggregates 0.7–1.1 mm in size from embryogenic suspension cultures were transferred to solid MS medium supplemented with 9.05 μM of 2,4-D and formed embryogenic callus with somatic embryos. The embryogenic callus with somatic embryos was further transferred to MS medium supplemented with 3.78 μM of abscisic acid, resulting in the germination of somatic embryos. Within 20 wk after the initiation, the frequencies of cell aggregates forming plantlets reached approximately 100% for the 15 tested cultivars. These plantlets, when transferred to soil, showed 100% survival. No morphological variations were observed.     

Valepotriates accumulation in callus, suspended cells and untransformed root cultures of Valeriana glechomifolia

Summary Valeriana glechomifolia is an endemic species of southern Brazil, capable of accumulating, in all of its organs, the terpene derivatives known as valepotriates, the presumed sedative components of the roots of pharmaceutically used species of Valeriana. In vitro cultures of the plant were established and the accumulation of acevaltrate, didrovaltrate, and valtrate in callus, cell suspension, and untransformed root cultures was studied. Leaves of in natura plants and roots of micropropagated plantlets were used as the explants for callus induction and root culture establishment, respectively, on Gamborg B5 basal medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) alone or with kinetin (KIN). Culture growth and secondary metabolite yields were enhanced with 2,4-D (4.52μM) and KIN (0.93μM). Maximum valepotriate contents, quantified by HPLC, of acevaltrate (ACE) 2.6mg g⁻¹ DW, valtrate (VAL) 10.2mgg⁻¹ DW, and didrovaltrate (DID) 2.9mg g⁻¹ DW were observed in root cultures after 7–8wk of culture.     

<Emphasis Type="Italic">In vitro</Emphasis> selection of NaCl-tolerant callus lines and regeneration of plantlets in a bamboo (<Emphasis Type="Italic">Dendrocalamus strictus</Emphasis> Nees)

Summary Sodium chloride-tolerant plantlets of Dendrocalamus strictus were regenerated successfully from NaCl-tolerant embryogenic callus via somatic embryogenesis. The selection of embryogenic callus tolerant to 100 mM NaCl was made by exposing the callus to increasing (0–200 mM) concentrations of NaCl in Murashige and Skoog medium having 3% (w/v) sucrose, 0.8% (w/v) agar, 3.0 mg l⁻¹ (13.6 μM) 2,4-dichlorophenoxyacetic acid (2,4-D), and 0.5mg l⁻¹ (2.3μM) kinetin (callus initiation medium). The tolerance of the selected embryogenic callus to 100 mM NaCl was stable through three successive transfers on NaCl-free callus initiation medium. The tolerant embryogenic callus had high levels of Na⁺, sugar, free amino acids, and proline but a slight decline was recorded in K⁺ level. The stable 100 mM NaCl-tolerant embryogenic callus differentiated somatic embryos on maintenance medium [MS medium +3% sucrose +0.8% agar +2.0 mg l⁻¹ (9.0 μM) 2,4-D+0.5 mg l⁻¹ (2.3 μM) kinetin] supplemented with different (0–200 mM) concentrations of NaCl. About 39% of mature somatic embryos tolerant to 100 mM NaCl germinated and converted into plantlets in germination medium [half-strength MS+2% sucrose+0.02 mg l⁻¹ (0.1 μM) α-naphthaleneacetic acid +0.1 mg l⁻¹ (0.49 μM) indole-3-butyric acid] containing 100 mM NaCl. Of these plantlets about 31% established well on transplantation into a garden soil and sand (1:1) mixture containing 0.2% (w/w) NaCl.