Hypocotyl Region Research Articles

Auxin-regulated genes encoding cell wall-modifying proteins are expressed during early tomato fruit growth.

An expansin gene, LeExp2, was isolated from auxin-treated, etiolated tomato (Lycopersicon esculentum cv T5) hypocotyls. LeExp2 mRNA expression was restricted to the growing regions of the tomato hypocotyl and was up-regulated during incubation of hypocotyl segments with auxin. The pattern of expression of LeExp2 was also studied during tomato fruit growth, a developmental process involving rapid cell enlargement. The expression of genes encoding a xyloglucan endotransglycosylase (LeEXT1) and an endo-1, 4-beta-glucanase (Cel7), which, like LeExp2, are auxin-regulated in etiolated hypocotyls (C. Catalá, J.K.C. Rose, A.B. Bennett [1997] Plant J 12: 417-426), was also studied to examine the potential for synergistic action with expansins. LeExp2 and LeEXT1 genes were coordinately regulated, with their mRNA accumulation peaking during the stages of highest growth, while Cel7 mRNA abundance increased and remained constant during later stages of fruit growth. The expression of LeExp2, LeEXT1, and Cel7 was undetectable or negligible at the onset of and during fruit ripening, which is consistent with a specific role of these genes in regulating cell wall loosening during fruit growth, not in ripening-associated cell wall disassembly.

Differential accumulation of Aux/IAA mRNA during seedling development and gravity response in cucumber (Cucumis sativus L.).

The plant hormone auxin transcriptionally activates Aux/IAA genes. We have isolated three Aux/IAA cDNA from cucumber, two cDNAs (CS-IAA1 and CS-IAA2) containing the complete open reading frame (ORF), and one partial cDNA (CS-IAA3). Northern blotting analysis showed that Aux/IAA mRNAs were induced during the emergence of radicles from seed coats. After radicle emergence, their mRNAs accumulated in the basal part of the hypocotyl much more than in the apical part, and later in elongating region of hypocotyls. CS-IAAI and CS-IAA3 mRNA significantly accumulated in response to auxin, although the increment of the former mRNA accumulation by auxin application was much greater than that of the latter. CS-IAA2 did not show an apparent change by auxin treatment in our experiment. In horizontally germinating seedlings, the transition zone between hypocotyl and root curves was due to downward gravitropic growth. On the other hand, vertically germinating seedlings of cucumber do not curve in the early stage of seedling development. The CS-IAA1 mRNA accumulation in horizontally germinating seedlings was more than that in vertically germinating ones during radicle emergence. Furthermore, asymmetric distribution of CS-IAA1 mRNA was detected in the transition zone in in situ hybridization analysis. These results suggest that the CS-IAA1 gene product may be involved in the gravity response during early development of seedlings.

Auxin and brassinosteroid differentially regulate the expression of three members of the 1-aminocyclopropane-1-carboxylate synthase gene family in mung bean (Vigna radiata L.).

Indole-3-acetic acid (IAA) markedly increased ethylene production by inducing the expression of three 1aminocyclopropane-1-carboxylate (ACC) synthase cDNAs (pVR-ACS1, pVR-ACS6 and pVR-ACS7) in mung bean hypocotyls. Results from nuclear run-on transcription assay and RNA gel blot studies revealed that all three genes were transcriptionally active displaying unique patterns of induction by IAA and various hormones in etiolated hypocotyls. Particularly, 24-epibrassinolide (BR), an active brassinosteroid, specifically enhanced the expression of VR-ACS7 by a distinct temporal induction mechanism compared to that of IAA. In addition, BR synergistically increased the IAA-induced VR-ACS6 and VR-ACS7 transcript levels, while it effectively abolished both the IAA- and kinetin-induced accumulation of VR-ACS1 mRNA. In light-grown plants, VR-ACS1 was induced by IAA in roots, and VR-ACS6 in epicotyls. IAA- and BR-treatments were not able to increase the VR-ACS7 transcript in the light-grown tissues. These results indicate that the expression of ACC synthase multigene family is regulated by complex hormonal and developmental networks in a gene- and tissue-specific manner in mung bean plants. The VR-ACS7 gene was isolated, and chimeric fusion between the 2.4 kb 5'-upstream region and the beta-glucuronidase (GUS) reporter gene was constructed and introduced into Nicotiana tabacum. Analysis of transgenic tobacco plants revealed the VR-ACS7 promoter-driven GUS activity at a highly localized region of the hypocotyl-root junction of control seedlings, while a marked induction of GUS activity was detected only in the hypocotyl region of the IAA-treated transgenic seedlings where rapid cell elongation occurs. Although there was a modest synergistic effect of BR on the IAA-induced GUS activity, BR alone failed to increase the GUS activity, suggesting that induction of VR-ACS7 occurs via separate signaling pathways in response to IAA and BR. A scheme of the multiple regulatory pathways for the expression of ACC synthase multigene family by auxin and BR is presented.

Overlapping Yet Distinct Promoter Activities of Two Arabidopsis Genes Coding for Nearly Identical Isoforms of the Acyl Carrier Protein

Acyl carrier protein is encoded by at least five genes in Arabidopsis. Isoforms encoded by genes Acl1.2 and Acl1.3 differ by a single amino acid. We have examined the expression patterns directed by the Acl1.2 and Acl1.3 gene promoters in transgenic Arabidopsis using the reporter gene coding for ß-glucuronidase (GUS). Fluorometric and histochemical analyses showed that the two promoters have overlapping yet distinct patterns of activity in both vegetative and reproductive tissues. In germinating seeds, the Acl1.2 promoter directed expression in all tissues at 2 days after imbibition (DAI) and then gradually became restricted to the elongation zone of the root apex, the root-hypocotyl transition zone, shoot apex, and cotyledon vasculature by 6 DAI. Acl1.3 promoter down-regulation was evident only in the basal hypocotyl region during this same time period. In young plants, Acl1.2 promoter activity was restricted to apical regions and within lateral root primordia. In anthesis-stage flowers, Acl1.2 was expressed predominantly in the stigmatic papillae and mature pollen. Distinct expression profiles were also observed during embryogeny, where Acl1.2 activity declined prior to stages associated with cotyledon cell expansion and storage reserve deposition, whereas Acl1.3 promoter activity increased during this time. Overall, the Acl1.3 promoter exhibited a more constitutive pattern of expression when contrasted with Acl1.2, which tended to be expressed in meristematic or expanding tissues, and specific reproductive structures.

Higher activity of an aldehyde oxidase in the auxin-overproducing superroot1 mutant of Arabidopsis thaliana.

Aldehyde oxidase (AO; EC 1.2.3.1) activity was measured in seedlings of wild type or an auxin-overproducing mutant, superroot1 (sur1), of Arabidopsis thaliana. Activity staining for AO after native polyacrylamide gel electrophoresis separation of seedling extracts revealed that there were three major bands with AO activity (AO1-3) in wild-type and mutant seedlings. One of them (AO1) had a higher substrate preference for indole-3-aldehyde. This AO activity was significantly higher in sur1 mutant seedlings than in the wild type. The difference in activity was most apparent 7 d after germination, the same time required for the appearance of the remarkable sur1 phenotype, which includes epinastic cotyledons, elongated hypocotyls, and enhanced root development. Higher activity was observed in the root and hypocotyl region of the mutant seedlings. We also assayed the indole-3-acetaldehyde oxidase activity in extracts by high-performance liquid chromatography detection of indole-3-acetic acid (IAA). The activity was about 5 times higher in the extract of the sur1 seedlings, indicating that AO1 also has a substrate preference for abscisic aldehyde. Treatment of the wild-type seedlings with picloram or IAA caused no significant increase in AO1 activity. This result suggested that the higher activity of AO1 in sur1 mutant seedlings was not induced by IAA accumulation and, thus, strongly supports the possible role of AO1 in IAA biosynthesis in Arabidopsis seedlings.

Specific localization of the respiratory alternative oxidase in meristematic and xylematic tissues from developing soybean roots and hypocotyls.

We used tissue printing and specific immunostaining to examine the localization of the alternative oxidase (AOX) protein in correlation with measurements of AOX capacity. Selected root and hypocotyl regions were analyzed during the first 14 d of growth. It is shown that AOX protein is localized in the apical meristem and in developing xylem. The temporal pattern of expression is coincident with the evolution of AOX capacity. Data suggest that AOX expression is linked to xylem differentiation. Since heat is a major product of the alternative pathway, we speculate that thermogenesis is implicated in morphogenesis.

In vitro plant regeneration via organogenesis of cowpea [Vigna unguiculata (L.) Walp.

Shoot regeneration via organogenesis was achieved from axenic cowpea [Vigna unguiculata subsp. unguiculata L. (Walp.) Verde.] hypocotyls and cotyledons of advanced breeding lines and varieties. Cotyledons and embryos were excised from green immature pods. The apical parts of the embryos were removed and the hypocotyls were transferred to regeneration media. Cotyledons and hypocotyls were tested on media with gradients of several hormonal and putrescine combinations. Cowpea cotyledons and hypocotyls exhibited a pattern of shoot formation that occurred in three distinct phases. Multiple shoots developed within 45 days from the wounded region of the primary hypocotyl and cotyledons in different media containing a high cytokinin concentration. The induced plant explants were then grown for 20 days in low-intensity light (10 µmol m-2 s-1) on the same medium and numerous shoot buds emerged de novo from the upper part of the hypocotyl and the wounded part of the cotyledons. These buds had no apparent vascular connection with the parent tissues. The plant regeneration capability of this procedure was tested with several cowpea genotypes, five of which (83D-442, 86D-1010, 93K-624, Vita 3 and Ife Brown) responded positively with shoot development and were able to form roots and whole plants. Some somaclonal variation was observed.

Auxin regulation and spatial localization of an endo-1,4-beta-D-glucanase and a xyloglucan endotransglycosylase in expanding tomato hypocotyls.

Xyloglucan, the primary hemicellulosic cell wall polysaccharide in dicotyledons, undergoes substantial modification during auxin-stimulated cell expansion. To identify candidates for mediating xyloglucan turnover, the expression and auxin regulation of tomato Cel7 and LeEXT, genes encoding an endo-1,4-beta-glucanase (EGase) and a xyloglucan endotransglycosylase (XET), respectively, were examined. LeEXT mRNA was present primarily in elongating regions of the hypocotyl and was induced to higher levels by hormone treatments that elicited elongation of hypocotyl segments. Cel7 mRNA abundance was very low in both elongating and mature regions of the hypocotyl but was induced to accumulate to high levels in both hypocotyl regions by auxin application. Analysis of the time dependence of expression of Cel7 and LeEXT during auxin treatment suggested that induction of these genes is not required for rapid growth responses but may participate in the cell wall changes involved in sustained cell elongation. Localization of Cel7 and LeEXT mRNA by in situ hybridization revealed that both genes are expressed in outer cell layers of the hypocotyl. In untreated etiolated seedlings, LeEXT mRNA was detected in epidermal cells of the elongating region, a tissue considered to play a key role in auxin-induced elongation. After auxin treatment, Cel7 and LeEXT mRNA showed an overlapping spatial distribution in the epidermis and outer cortical cell layers. We conclude that LeEXT and Cel7 exhibit both unique and overlapping patterns of expression and have the potential to act cooperatively in mediating cell wall disassembly associated with expansive growth.

Developmental and pathogen-induced accumulation of transcripts of polygalacturonase-inhibiting protein in Phaseolus vulgaris L.

Expression of the polygalacturonase-inhibiting-protein gene ( pgip) during development of French bean (Phaseolus vulgaris L. cv. Red Kidney) was analysed in various organs, including leaves, stems and roots of etiolated and light-grown seedlings, light-grown adult plants, as well as flowers, pods, and seeds. In light-grown seedlings and plants, pgip transcripts were detected in all tissues examined, with higher levels found in the transition zone between the elongating and mature region of young hypocotyls, and in the basal region of the stem of adult plants. In etiolated hypocotyls, levels of pgip transcripts in the elongating region were five- to six fold higher than in light-grown hypocotyls. High levels of transcripts were also observed in pods. Accumulation of pgip mRNA was also followed in both compatible and incompatible race-cultivar interactions between Colletotrichum lindemuthianum races α and γ, respectively, and P. vulgaris by Northern blot analysis and in situ hybridisation. In situ experiments showed, in incompatible interactions, a rapid, intense and transient accumulation of pgip mRNA in epidermal cells proximal to the site of infection, and, less intense, within the cortical parenchyma underneath. In compatible interactions, no substantial accumulation of pgip mRNA was observed in hypocotyls; a very weak accumulation was observed in leaves during lesion formation.

Sweet Pepper (Capsicum annuum) Transformation using Agrobacterium rhizogenes

In vitro genetic transformation of chile pepper, Capsicum annuum var. New Mexico 6-4, was achieved. Seeds of `New Mexico 6-4' were grown aseptically on Murashige and Skoog medium. Seedlings 22 days old were wounded on the hypocotyl region using a sterile hypodermic needle. A. rhizogenes strain K 599 harboring the plasmid p35S GUS Intron was inoculated on the wound site. Three days later the seedlings were transferred onto MS media with antibiotics (Cefotaxime, Carbenicillin, Amoxicillin, Clavulanic acid, and Kanamycin). New roots were seen to initiate from the wound site 15 to 20 days after inoculation. The roots were morphologically identified as “hairy roots.” Glucuronidase (Gus) assay performed 40 days after inoculation on randomly chosen roots that had grown into the selection medium, showed that 6/25 (24%) of the inoculated seedlings had roots that showed intense blue coloration. Presence of an intron makes it impossible for the bacteria to express the reporter gene. The seedlings that had transformed roots had a different morphology with wrinkled leaves and short internodes. The pattern of expression of the introduced gene varied greatly. Some positive tissues had the root tips alone being blue; a few had the vascular tissues and the root tips blue; and others had the vascular tissues, the surrounding parenchyma cells, root tips, and the root hairs turn very dark blue. The transformed roots did not need to grow into the selection media to be Gus positive. Isolated roots cultured on MS media supplemented with 0.2 mg/L IAA were maintained for 120 days and continued to express the reporter gene. Currently, methods to regenerate transformed shoots from roots are being tested. The “hairy root” transformation system in pepper could have application in the testing of root-expressible constructs for transgene expression assays.

An improved and reliable chili pepper (Capsicum annuum L.) plant regeneration method

In this work we report a new method forin vitro chili pepper (Capsicum annuum L.) plant regeneration based on shoot formation from wounded hypocotyls. Chili pepper seeds were surface sterilized and germinated on agar (0.8%) at 25 ± 2°C in the dark. Five factors that may influence shoot regeneration were studied: age of seedlings, hypocotyl wounding site, time elapsed between wounding the hypocotyls and decapitation of seedlings, culture media and cultivars. In order to study the influence of the first three factors on shoot regeneration, the apical, middle or basal hypocotyl regions of seedlings of cv. Mulato Bajio at different stages of development (9, 15, 16, 21 and 28 d old) were wounded with a syringe needle, and the seedlings were cultured on MS semisolid medium without growth regulators at 25 ± 2°C under a 16/8 h light/dark photoperiod (daylight fluorescent lamps; 35 μmol m-(2) s-(-1)) until decapitation. The seedlings were decapitated (3 mm below the cotyledons) at different times after wounding (0, 2, 4, 10, 12 and 14 d), and each explant was evaluated for bud and shoot formation (≥ 5 mm in length) at the wounded site after 30 d of incubation. In general, seedlings at the stage of curved hypocotyl (9 d old) wounded in the apical region of hypocotyl were the best explants for shoot regeneration when inoculated on culture medium without growth regulators. Decapitation after wounding also influenced the shoot regeneration efficiency, with 10-14 d being the best period. Up to 90% shoot regeneration in cv. Mulato Bajio was obtained under these conditions. Statistically significant differences were observed for shoot formation among 21 cultivars tested. Regeneration of whole plants was achieved by rooting the shoots with indole-3-butyric acid pulses of 60 mg L(-1) for 3 h and then subculturing on MS medium without growth regulators.

In Vitro Regeneration and Isozyme Patterns in Zizyphus mauritiana

Shoot regeneration has been achieved in Zizyphus mauritiana from juvenile explants (internodal and nodal segments) on MS medium containing 2% sucrose and 50 mg l−1 each of asparagine, arginine and glutamine, 5 mg l−1 cystelne hydrochloride and various combinations of IAA/zeatin, BAP, KN and Ad. The explants were most responsive on the medium containing zeatin followed by BAP. Callusing could be induced from all parts of the seedling viz. cotyledonary leaf, hypocotyl, epicotyl, leaf and nodal region on various media tried. Expression of peroxidase and esterase in the in vivo and in vitro grown tissues was also compared through starch gel electrophoresis.

Structural Heterogeneity of Phospholipase D in 10 Dicots

The occurrence of multiple forms of phospholipase D (EC 3.1.4.4) was investigated in different tissues of castor bean (Ricinus communis) and in other plant species. Phospholipase D variants were resolved by nondenaturing and isoelectric focusing polyacrylamide gel electrophoresis and detected by immunoblotting using anti-phospholipase D antibodies and by enzyme activity assay. Three phospholipase D variants were produced differentially in the roots, endosperm, cotyledons, and hypocotyl of 5-day postgermination seedlings of castor bean. Furthermore, different patterns of phospholipase D variants were found in the different regions of hypocotyl (elongated and hook). Multiple phospholipase D forms were found in florets of cauliflower and broccoli, leaves of cabbage, celery, tomato, and potato, and alfalfa sprouts, suggesting that structural heterogeneity of phospholipase D occurs widely in plants.

Effects of abscisic acid on somatic embryo maturation of hybrid larch

particular catechins and proanthocyanidins, were present in all embryos from the very onset of development. They were localized preferentially in the proximal suspensor cells and the basal region of the embryo. However, accumulation of polyphenols was generally much more pronounced in embryos matured without ABA, indicating a lack of biochemical regulatory competence in those embryos. Somatic embryos of hybrid larch (Larix x lepto-europaea) which had been matured for 4 weeks on maturation medium, developed normally on medium supplemented with 60 μM ABA, but abnormally on medium with no ABA. A comparative structural and histochemical investigation was carried out on these two types of mature embryos. At the light microscope level, differences between both treatments were visible only after 2-3 weeks of maturation. At around this time, abnormal development becomes evident macroscopically: ABA-minus embryos remain rather stubby as opposed to the more cylindrically shaped ABA-plus embryos. Whereas somatic embryos matured with ABA consist of densely cytoplasmic cells showing a high rate of cell division, ABA-minus embryos are largely made up of expanded and highly vacuolate cells, indicating that growth in the latter is mainly due to cell expansion and not division. After 4 weeks of maturation, ABA-minus embryos begin to elongate in the hypocotyl region, and precocious germination was observed frequently. Again, these morphogenetic events were largely due to abnormal timing of cell expansion. Histochemically, storage proteins were found only in somatic embryos matured for 4 weeks with ABA. This observation is in line with results obtained by total protein analysis, yielding significantly lower total protein contents in ABA-minus embryos both on a fresh weight and a per embryo basis after 4-5 weeks of maturation. Deposition of starch grains mainly in the cortex tissue of the hypocotyl region was observed within 2 weeks of maturation in varying amounts regardless of ABA supply. Polyphenols, in

Effects of hypergravity on growth and cell wall properties of cress hypocotyls.

Elongation growth of etiolated hypocotyls of cress (Lepidium sativum L.) was suppressed when they were exposed to basipetal hypergravity at 35 x g and above. Acceleration at 135 x g caused a decrease in the mechanical extensibility and an increase in the minimum stress-relaxation time of the cell wall. Such changes in the mechanical properties of the cell wall were prominent in the lower regions of hypocotyls. The amounts of cell wall polysaccharides per unit length of hypocotyls increased under the hypergravity condition and, in particular, the increase in the amount of cellulose in the lower regions was conspicuous. Hypergravity did not influence the neutral sugar composition of either the pectin or the hemicellulose fraction. The amount of lignin was also increased by hypergravity treatment, although the level was low. The data suggest that hypergravity modifies the metabolism of cell wall components and thus makes the cell wall thick and rigid, thereby inhibiting elongation growth of cress hypocotyls. These changes may contribute to the plants' ability to sustain their structures against hypergravity.

Identification of a Novel Divergent Calmodulin Isoform from Soybean Which Has Differential Ability to Activate Calmodulin-dependent Enzymes

Calmodulin plays pivotal roles in the transduction of various Ca(2+)-mediated signals and is one of the most highly conserved proteins in eukaryotic cells. In plants, multiple calmodulin isoforms with minor amino acid sequence differences were identified but their functional significances are unknown. To investigate the biological function of calmodulins in the regulation of calmodulin-dependent enzymes, we cloned cDNAs encoding calmodulins in soybean. Among the five cDNAs isolated from soybean, designated as SCaM-1 to -5, SCaM-4 and -5 encoded very divergent calmodulin isoforms which have 32 amino acid substitutions from the highly conserved calmodulin, SCaM-1 encoded by SCaM-1 and SCaM-3. SCaM-4 protein produced in Escherichia coli showed typical characteristics of calmodulin such as Ca(2+)-dependent electrophoretic mobility shift and the ability to activate phosphodiesterase. However, the extent of mobility shift and antigenicity of SCaM-4 were different from those of SCaM-1. Moreover, SCaM-4 did not activate NAD kinase at all in contrast to SCaM-1. Also there were differences in the expression pattern of SCaM-1 and SCaM-4. Expression levels of SCaM-4 were approximately 5-fold lower than those of SCaM-1 in apical and elongating regions of hypocotyls. In addition, SCaM-4 transcripts were barely detectable in root whereas SCaM-1 transcripts were as abundant as in apical and elongating regions of hypocotyls. In conclusion, the different biochemical properties together with differential expression of SCaM-4 suggest that this novel calmodulin may have different functions in plant cells.

Adventitious shoot production from immature embryos of white clover

Cotyledonary-stage embryos of Haifa white clover, collected 13 days after cross-pollination, were induced to form adventitious shoots primarily from the hypocotyl region. The culture medium used for the production of adventitious shoots contained 5 μM thidiazuron and 0.5 μM α-naphthaleneacetic acid. Numerous shoot meristems were produced within the first week, discrete shoots developed by week three, small plantlets by week eight, and whole plants in soil by week ten. 95–100% of all embryos, regardless of genotype, produced adventitious shoots within four weeks with an average production of 17.5 shoots per embryo. The majority of shoots (on average 77%) were easily converted to whole plants in soil. The white clover regeneration system described is prolific, rapid and effective on a large number of genotypes.

Absence of phyB inhibits hypocotyl elongation in dark‐grown Ih cucumber seedlings: an active role for PrB

ABSTRACTCucumber (Cucumis sativus L.) seedlings carrying the long hypocotyl (Ih) mutation, which confers a lack of B‐type phytochrome (phyB), were significantly shorter than their near‐isogenic wild‐type counterparts when grown in complete darkness. Relative growth rates determined for 5 mm hypocotyl regions were lower in Ih seedlings in all growing regions, and the zone of elongation was less extensive in Ih hypocotyls. Digital imaging microscopy revealed that the pattern of epidermal cell lengths along the stem axis differed between the Ih mutant and the iso‐genic wild‐type. These findings (and the fact that experiments were conducted under conditions where phytochrome photoconversion to the far‐red‐absorbing form does not occur) suggest that the red‐absorbing form of phyB (PrB) is an active positive regulator of development in etiolated plants.

Effect of temperature on virulence of Rhizoctonia solani on soybean leaves and seedlings

Isolates representing 11 anastomosis groups (AGs) of Rhizoctonia solani from various geographic locations and host plants were tested for virulence on soybean leaves at 15, 20, 25, 30, and 35°C, and on soybean seedlings at 20, 25, and 30°C. Numbers of infection cushions formed on soybean leaves were determined using light microscopy. Isolates of AG‐1 IA, AG‐1 IB and AG‐5 were more virulent on soybean leaves at 20, 25, and 30°C than isolates of AG‐1 IC and AG‐4. Maximum numbers of infection cushions were formed on soybean leaves by AG‐1 (IA, IB, and IC), AG‐4, and AG‐5 at 25 and 30°C. The other AGs tested did not form infection cushions on soybean leaves although some caused minimal disease severity. Isolates of AG‐1 IA formed significantly more infection cushions and caused greater disease severity than AG‐1 IB and other isolates at 35°C. Maximum seedling infection, based on per cent area of hypocotyl region covered by lesions occurred at 25 C for AG‐1 (IA, IB, and IC) and AG‐4. Isolates of AG‐5 caused greater seedling infection at 20°C than at 25 and 30°C. The other AGs tested caused only minimal damage to the seedlings. Isolates of AG‐4 and AG‐5 are not known to cause Rhizoctonia foliar blights of soybean in Louisiana, but their potential to be destructive foliar pathogens is confirmed.

Embryogenic protoplast cultures of orange jessamine (Murraya paniculata) and their regeneration into plants flowering in vitro

Embryogenic callus was induced from the hypocotyl region of seedlings germinated from immature embryos of orange jessamine (Murraya paniculata (L.) Jack) on Murashige & Tucker (1969) medium containing 50 g l-1 sucrose, 5.0 mg l-1 benzyladenine, 2.5 mg l-1 2,4-dichlorophenoxyacetic acid and 600 mg l-1 malt extract. Isolated protoplasts divided to produce callus on Murashige & Tucker (1969) medium containing 50 g l-1 sucrose, 0.01 mg l-1 gibberellin A4+7 and 600 mg l-1 malt extract. Callus developed to plantlets via somatic embryogenesis on Murashige & Tucker (1969) medium with 50 g l-1 lactose but no plant growth regulators. These plantlets flowered in vitro on half strength Murashige & Tucker (1969) medium containing 50 g l-1 sucrose after 2 months culture.