Globular Embryo Stage Research Articles

Fruit and Seed Development of <i>Sorbus pohuashanensis</i> Hedl

Seed development traits of Sorbus pohuashanensis are discussed with special regard to embryo formation. The purpose of this research was to provide additional data for understanding embryo developmental processes and identifying the appropriate developmental stage for seed collection in S. pohuashanensis. A correlation was found between the embryo developmental stages and the time after pollination. The developmental stages cover globular, heart, torpedo and cotyledonary embryo stages within 30 days post-pollination. There were significant differences in embryo length, width and length to width ratio at different development stages. A significant negative correlation between embryo dry weight (DW) and moisture content (MC) was also observed.

Fertilization and embryogeny in Agapanthus praecox ssp. orientalis (Leighton) Leighton

Fertilization and embryogeny in Agapanthus praecox ssp. orientalis are described for the first time, and embryogenic characters of Agapanthus are discussed. The main results are: (1) The pollen tube enters the embryo sac and discharges two sperm 44–48 h after pollination. (2) The sperm fuse with the egg cell and polar nuclei, forming zygote and primary endosperm nucleus, approximately 50 h after pollination. The zygote then enters a short period of dormancy. (3) Seven days after pollination, the zygote starts division. The first division of the zygote is transversal. (4) The embryo undergoes globular stage, rod-shaped stage, and finally forms a monocotyledonous embryo. (5) The suspensor cells are ephemeral and degenerate at the globular embryo stage. (6) Endosperm cells contain massive starch grains as nutrition for embryo development. (7) Embryogeny conforms to the Onagrad type, and endosperm formation is of the nuclear type; the whole process of embryogeny and endosperm development needs approximately 60 days in A. praecox ssp. orientalis. (8) Dicotyledonous together with monocotyledonous forms of embryo morphogenesis in Agapanthus supports the concept of homology of monocots and dicot cotyledons.

Identification of SSR markers using soybean (Glycine max) ESTs from globular stage embryos

Microsatellites, or simple sequence repeats (SSRs), in expressed sequence tags (ESTs) provide an opportunity for low cost SSR development. We looked for EST-SSRs in 403,511 ESTs (generated by 454 sequencing and representing 70,654 contigs and 52,082 singletons) from soybean globular stage embryos. Among 122,736 unique ESTs, 3,729 contained one or more SSRs. In total, 3,989 SSRs were identified including 304 mono, 1,374 di, 2,208 tri, 70 tetra, 13 penta and 20 hexanucleotide SSRs. Thirty three EST-SSRs were selected for primer design and polymorphism analysis using twenty soybean cultivars and one wild-type soybean. Successful amplification was obtained using 21 of these primer pairs, 11 of which detected polymorphisms in these soybean cultivars. These results demonstrated that 454 high throughput sequencing is a powerful tool for molecular marker development. From the 3,989 identified SSRs we expect to obtain a large number of makers with polymorphism among different soybean cultivars, which would be useful for analysis of genetic diversity and maker assisted selection in the soybean breeding programs.

Studies on reproductive characteristics of an interspecific chimera between Brassica juncea and Brassica oleracea

Periclinal chimera plants of red cabbage and tuber mustard were studied for investigating reproductive processes. In the shoot apical meristem of chimera TCC (L1-L2-L3, L1 being the outermost layer of shoot apical meristem, L2 the middle layer, L3 the innermost layer; T = tuber mustard, C = red cabbage), the outermost layer was derived from tuber mustard, while the middle and the innermost layers were from red cabbage. In the present study, displacement of the L1 cell layer was found to result in infertile plants. The viability of TCC’s pollen from the L2 layer became significantly lower than that of red cabbage, and the self-pollinated embryo aborted at an early stage. Anatomical observation of ovules showed that the cellularized endosperm developed partially and degraded prematurely at the globular-embryo stage. Ovule culture was carried out to rescue the selfed embryos of TCC, and the results showed that ovules at 16 days after pollination could be rescued on Murashige and Skoog (MS) medium supplied with 3% sucrose, 0.2% active carbon, 400 mg/l glutamine, and casein hydrolysate. Morphological, chromosomal, and molecular identifications consistently showed that the selfed progeny of TCC was red cabbage. These investigations indicated that the heterologous L1 cell layer greatly influenced the reproductive characteristics of the plants, although it did not participate directly in gametogenesis and embryo ontogenesis.

Somatic embryogenesis and plant regeneration in Pterocarpus marsupium Roxb.

Somatic embryogenesis (SE) has been achieved from hypocotyl-derived callus culture in Pterocarpus marsupium. Ninety percent of hypocotyl explants (excised from 12-day-old in vitro germinated axenic seedlings) produced callus on Murashige and Skoog medium supplemented with 5 μM 2,4-dichlorophenoxyacetic acid and 1 μM a 6-benzyladenine (BA). Induction of SE occurred after transfer of callus clumps (200 ± 20 mg fresh mass) to MS medium supplemented with BA at 2.0 μM, where a maximum of 23.0 ± 0.88 globular stage embryos per callus clump were observed after 4 weeks of culture. Subculturing of these embryos on MS medium supplemented with 0.5 μM BA, 0.1 μM α-naphthalene acetic acid and 10 μM abscisic acid significantly enhanced the maturation of somatic embryos to early cotyledonary stage, where 21.4 ± 0.32 embryos per callus clump were recorded after 4 weeks of culture. Of 30-well developed somatic embryos, 16.6 ± 0.33 germinated and subsequently converted into plantlets on half-strength MS medium supplemented with 1.0 μM BA. The morphologically normal plantlets with well-developed roots were first transferred to 1/4-liquid MS medium for 48 h and then to pots containing autoclaved soilrite and acclimatized in a culture room. Thereafter, they were transferred to a greenhouse, where 60% of them survived.

Somatic embryogenesis and regeneration from shoot primordia of Crocus heuffelianus

Crocus heuffelianus belongs to the C. vernus (Iridaceae) species aggregate. In the Carpathian Basin and particularly in Hungary it is considered an endangered species. Therefore our aim was to establish a tissue culture system with potential of germplasm preservation of this taxon. For in vitro culture experiments, shoot primordia from corms were the most suitable. We induced an embryogenic callus line from those explants on basal Murashige-Skoog (MS) medium supplemented with Gamborg’s vitamins, 2% (w/v) sucrose, 10 mg l−1 (53.7 μM) α-naphthaleneacetic acid (NAA) and 1 mg l−1 (4.44 μM) 6-benzyladenine (BA). Globular stage embryos developed on this medium and several culture conditions were used in an attempt to obtain mature embryos and plant regeneration. Firstly a decrease of auxin/cytokinin concentration and ratio, then secondly a decrease in the strength of culture medium and the concentration of carbon source was used, which was effective in embryogenesis and the production of plants. Regeneration medium used in the second step was fourfold diluted MS medium and Gamborg’s vitamins supplemented with 1% (w/v) sucrose, 0.05 mg l−1 (0.26 μM) NAA and 0.5 mg l−1 (2.22 μM) BA, with a 14/10 h photoperiod. Under these conditions we could detect all the stages of somatic embryo development characteristic for Iridaceae. This is the first report demonstrating the production of stable tissue culture of C. heuffelianus with potential use in germplasm preservation via plant regeneration. This study could also contribute to a better understanding of somatic embryogenesis in the Crocus genus.

Confocal Microscopy Study of Arabidopsis Embryogenesis Using GFP:mTn

Embryogenesis in transgenic Arabidopsis plants with GFP:mTn, a chimeric fusion of soluble shifted green fluorescent protein and a mouse actin binding domain, was studied. Confocal laser scanning microscopy was used to determine patterns of formation and cellular responses during asymmetric cell division. Before such cells divide, the nucleus moves to the position where new cell walls are to be formed. The apical–basal axis of the embryo develops mainly at the zygote to octant stage, and these events are associated with asymmetric divisions of the zygote and hypophyseal cells. Formation of the radial axis is established from the dermatogen to the globular-stage embryo via tangential cell division within the upper tiers. Bilateral symmetry of the embryo primarily happens at the triangular stage through zig-zag cell divisions of initials of the cotyledonary primordia. All stages of embryogenesis are described in detail here.

Integration of tomato reproductive developmental landmarks and expression profiles, and the effect of SUN on fruit shape

BackgroundUniversally accepted landmark stages are necessary to highlight key events in plant reproductive development and to facilitate comparisons among species. Domestication and selection of tomato resulted in many varieties that differ in fruit shape and size. This diversity is useful to unravel underlying molecular and developmental mechanisms that control organ morphology and patterning. The tomato fruit shape gene SUN controls fruit elongation. The most dramatic effect of SUN on fruit shape occurs after pollination and fertilization although a detailed investigation into the timing of the fruit shape change as well as gene expression profiles during critical developmental stages has not been conducted.ResultsWe provide a description of floral and fruit development in a red-fruited closely related wild relative of tomato, Solanum pimpinellifolium accession LA1589. We use established and propose new floral and fruit landmarks to present a framework for tomato developmental studies. In addition, gene expression profiles of three key stages in floral and fruit development are presented, namely floral buds 10 days before anthesis (floral landmark 7), anthesis-stage flowers (floral landmark 10 and fruit landmark 1), and 5 days post anthesis fruit (fruit landmark 3). To demonstrate the utility of the landmarks, we characterize the tomato shape gene SUN in fruit development. SUN controls fruit shape predominantly after fertilization and its effect reaches a maximum at 8 days post-anthesis coinciding with fruit landmark 4 representing the globular embryo stage of seed development. The expression profiles of the NILs that differ at sun show that only 34 genes were differentially expressed and most of them at a less than 2-fold difference.ConclusionThe landmarks for flower and fruit development in tomato were outlined and integrated with the effect of SUN on fruit shape. Although we did not identify many genes differentially expressed in the NILs that differ at the sun locus, higher or lower transcript levels for many genes involved in phytohormone biosynthesis or signaling as well as organ identity and patterning of tomato fruit were found between developmental time points.

A gain-of-function mutation inIAA18altersArabidopsisembryonic apical patterning

Lateral organ emergence in plant embryos and meristems depends on spatially coordinated auxin transport and auxin response. Here, we report the gain-of-function iaa18-1 mutation in Arabidopsis, which stabilizes the Aux/IAA protein IAA18 and causes aberrant cotyledon placement in embryos. IAA18 was expressed in the apical domain of globular stage embryos, and in the shoot apical meristem and adaxial domain of cotyledons of heart stage embryos. Mutant globular embryos had asymmetric PIN1:GFP expression in the apical domain, indicating that IAA18-1 disrupts auxin transport. Genetic interactions among iaa18-1, loss-of-function mutations in ARF (Auxin response factor) genes and ARF-overexpressing constructs suggest that IAA18-1 inhibits activity of MP/ARF5 and other ARF proteins in the apical domain. The iaa18-1 mutation also increased the frequency of rootless seedlings in mutant backgrounds in which auxin regulation of basal pole development was affected. These results indicate that apical patterning requires Aux/IAA protein turnover, and that apical domain auxin response also influences root formation.

Proteomic analysis of somatic embryogenesis in Valencia sweet orange (Citrus sinensis Osbeck)

Two dimensional gel electrophoresis combined with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) was employed to study the somatic embryogenesis (SE) in Valencia sweet orange (Citrus sinensis Osbeck). Twenty-four differentially expressed proteins were identified at five time points of citrus SE (0, 1, 2, 3, 4 weeks after embryo initiation) covering globular, heart/torpedo and cotyledon-shaped embryo stages. The general expression patterns for these proteins were consistent with those appeared at 4 weeks of citrus SE. The most striking feature of our study was that five proteins were predicted to be involved in glutathione (GSH) metabolism and anti-oxidative stress, and they exhibited different expression patterns during SE. Based on that oxidative stress has been validated to enhance SE, the preferential representation for anti-oxidative proteins suggests that they could have a developmental role in citrus SE. Some proteins involved in cell division, photosynthesis and detoxification were also identified, and their possible roles in citrus SE were discussed.

BIM1, a bHLH protein involved in brassinosteroid signalling, controls Arabidopsis embryonic patterning via interaction with DORNRÖSCHEN and DORNRÖSCHEN-LIKE

The BIM1 protein which has been implicated in brassinosteroid (BR) signal transduction was identified from a two hybrid screen using the N-terminus, including the AP2 domain, of the transcription factors DORNROESCHEN (DRN) and DORNROESCHEN-LIKE (DRNL) which control embryonic patterning. The protein-protein interaction between BIM1 and DRN or DRNL was confirmed by co-immunoprecipitation and for DRN also in vivo by bimolecular fluorescence complementation. BIM1 can also physically interact with PHAVOLUTA (PHV), another interaction partner of DRN and DRNL. Loss of BIM1 function results in embryo patterning defects at low penetrance, including cell division defects in the hypophyseal region and apical domain defects such as cotyledon fusion and polycotyledony, in addition to polyembryony. BIM1 expression overlaps with that of DRN and DRNL from early globular embryo stages onwards. Higher order mutants between bim1, drn, drnl and phv suggest that although BIM1 may act partially redundantly with DRN in early embryo development, all genes function within the same pathway determining cotyledon development, supporting the hypothesis that they participate in a multimeric transcription factor complex. A role of BIM1 in embryonic development not only implicates a function for brassinosteroids in this process, but the interaction of BIM1 with DRN, involved with auxin signalling, represents a possible point of hormonal crosstalk in embryonic patterning and the first example of an interaction of components of the auxin and BR signalling pathways.

Identification of genes specifically expressed in androgenesis-derived embryo in rapeseed (Brassica napus L.)

Microspore culture is an efficient system to produce haploid and double-haploid plants, and is a useful model system in developmental research. Although many studies have been carried out to understand the mechanism of microspore embryogenesis, the details of molecular events occurring during its developmental process are unclear. During embryogenesis, a marked morphological change occurs just after globular stage embryos. To gain insight into the developmental process of androgenesis in rapeseed (Brassica napus), we isolated the genes expressed differentially during globular stage by suppression subtractive hybridization (SSH). Of 254 ESTs isolated and sequenced, 82 were singletons and the remaining 172 were assembled into 28 groups based on homology to the identical gene, resulting in the identification of 110 non-redundant EST groups, which are potentially up-regulated genes in androgenesis. Survey of the potential function of the genes showed that 96.4% of these genes were homologous to known genes with 42.7% of unclassified proteins and 13.6% of metabolism. To confirm the expression profiles of isolated genes during androgenesis and zygotic embryogenesis, six selected genes were examined by quantitative RT-PCR analysis. All genes were highly expressed in globular and other stages of androgenesis. The expressions of these genes were confirmed in zygotic embryogenesis. These results serve as a starting point for understanding the mechanism of androgenesis and zygotic embryogenesis.

Localization of arabinogalactan-proteins in different stages of embryos and their role in cotyledon formation of Nicotiana tabacum L.

Arabinogalactan proteins (AGPs) have been implicated in plant development including sexual plant reproduction. In this paper, the expression of AGPs and the effects of β-glucosyl Yariv reagent (βGlcY, which binds arabinogalactan proteins) in embryo development and cotyledon formation were investigated. Immunofluorescence assay displayed that the expression of AGPs labeled with antibody JIM13 was developmentally regulated. In early stages, AGPs were evenly distributed in the whole embryo, except for a short polar expression in the basal suspensor cell. In the globular stage of embryo, AGPs were condensed in the embryo proper (EP), apex of the EP, and at the juncture of the EP and suspensor. In heart-shaped embryo, APGs were only present at the juncture of the EP and suspensor. Immunogold labeling assay showed that the strong expression of AGPs at the juncture of the EP and suspensor was localized in the cell wall. Provision of βGlcY to the in vitro ovule culture medium caused delayed growth of embryos, cotyledon defect and abnormal venation pattern. Consequently, βGlcY induced the death of defective seedlings with the characteristics of deformed or irregular single cotyledon. Our results suggested that AGPs play functional roles in embryo development, cotyledon formation and seedling morphology establishment in Nicotiana tabacum L.

Establishment of an in vitro fertilization system in rice (Oryza sativa L.)

In vitro fertilization (IVF) systems using isolated male and female gametes have been utilized to dissect fertilization-induced events in angiosperms, such as egg activation, zygote development and early embryogenesis, as the female gametophytes of plants are deeply embedded within ovaries. In this study, a rice IVF system was established to take advantage of the abundant resources stemming from rice research for investigations into the mechanisms of fertilization and early embryogenesis. Fusion of gametes was performed using a modified electrofusion method, and the fusion product, a zygote, formed cell wall and an additional nucleolus. The zygote divided into a two-celled embryo 15-24 h after fusion, and developed into a globular-like embryo consisting of an average of 15-16 cells by 48 h after fusion. Comparison of the developmental processes of zygotes produced by IVF with those of zygotes generated in planta suggested that zygotes produced by IVF develop and grow into early globular stage embryos in a highly similar manner to those in planta. Although the IVF-produced globular embryos did not develop into late globular-stage or differentiated embryos, but into irregularly shaped cell masses, fertile plants were regenerated from the cell masses and the seeds harvested from these plants germinated normally. The rice IVF system reported here will be a powerful tool for studying the molecular mechanisms involved in the early embryogenesis of angiosperms and for making new cultivars.

Development of megagametophyte, embryo, and seed in Senna corymbosa (Lam.) H.S. Irwin & Barneby (Leguminosae – Caesalpinioideae)

The main aspects of seed ontogeny in Senna corymbosa were studied by standard anatomical microtechniques for light microscope observations. The results revealed an ana-campylotropous, bitegmic, and crassinucelate mature ovule. A single archesporocyte developed by an archesporial cell enlargement from the subhypodermal multicellular archesporium. Meiosis originated linear or T-shaped megasporic tetrads. The functional megaspore was the chalazal one. Megagametophytic development conformed to the Polygonum type. Fertilization was porogamic. Endosperm development was free nuclear and conformed to a chalazal haustorium. Cellular endosperm was initiated from the micropylar end during the globular embryo stage. Embryogeny derived from a linear proembryonal tetrad. The mature embryo showed an oblique axis. The testa derived from the outer ovular integument. Nucellar and endosperm remnants, and the micropylar region of the inner ovular integument, persisted at embryo maturity. The absence of a pleurogram would be adaptative to wetland habitats. The taxonomic use of the mature embryo axis in the Cassieae and the phylogenetic employment of megasporic arrangements in Leguminosae needs some reinterpretation. © 2007 The Linnean Society of London, Botanical Journal of the Linnean Society, 2007, 153, 169–179.

Transcriptional Profiling of the Arabidopsis Embryo

We have used laser-capture microdissection to isolate RNA from discrete tissues of globular, heart, and torpedo stage embryos of Arabidopsis (Arabidopsis thaliana). This was amplified and analyzed by DNA microarray using the Affymetrix ATH1 GeneChip, representing approximately 22,800 Arabidopsis genes. Cluster analysis showed that spatial differences in gene expression were less significant than temporal differences. Time course analysis reveals the dynamics and complexity of gene expression in both apical and basal domains of the developing embryo, with several classes of synexpressed genes identifiable. The transition from globular to heart stage is associated in particular with an up-regulation of genes involved in cell cycle control, transcriptional regulation, and energetics and metabolism. The transition from heart to torpedo stage is associated with a repression of cell cycle genes and an up-regulation of genes encoding storage proteins, and pathways of cell growth, energy, and metabolism. The torpedo stage embryo shows strong functional differentiation in the root and cotyledon, as inferred from the classes of genes expressed in these tissues. The time course of expression of the essential EMBRYO-DEFECTIVE genes shows that most are expressed at unchanging levels across all stages of embryogenesis. We show how identified genes can be used to generate cell type-specific markers and promoter activities for future application in cell biology.

GhSEM-1 Marker Potentially Associated with Regeneration Ability in Cotton

A marker protein for embryogénie potential could be useful in determining if target tissue for Agrobacterium tumefaciens or microprojectile bombardment has the ability to regenerate plants. Certain varieties of cotton, especially Coker 312, are known to form somatic embryos readily, while others are more recalcitrant. Callus induced from Coker 312–17wa s found to be more embryogénie than that from TM-1. To investigate the molecular basis for this difference in somatic embryo potential, total RNA was isolated from globular stage callus embryos, calli with heart-stage embryos, and non-embryogenic calli. Northern analyses were performed using a developmentally regulated cotton fiber gene as a probe. This probe hybridized to a single band, GhSEM-1 (Gossypium hirsutum somatic embryogénie marker 1), only in globularstage embryos. Genomic DNA was also isolated from young leaf tissue of both embryogénie (Coker 312–17) and non-embryogenic (TM-1) lines and a Southern analysis using the same probe was performed. No differences were found between the genomic DNA of both embryogenie and non-embryogenic lines, indicating that this gene was structurally present in both lines but only expressed in Coker 312–17 at the globular stage. Antibodies against GhSEM-1 were developed and used in immunoblots after SDS-PAGE separation of proteins from embryogenie and non-embryogenic calli. The antibody recognized a -15 kDa protein in the globular and heart-stage somatic embryos from Coker 312–17 and was designated as GEP15 (15 kDa Gossypium embryogénie protein). This protein could be used as a marker for determining if tissue has embryogénie potential. Our results represent the first reported potential association of a marker gene (GhSEM-1) and protein (GEP15) with somatic embryogenesis in cotton.

Physiological influences in the development and function of the shoot apical meristem of microspore-derived embryos ofBrassica napus‘Topas’

The effect of auxins and abscisic acid (ABA) on shoot apical meristem development and function was analyzed in microspore-derived embryos of Brassica napus L. ‘Topas’. Embryos were treated with an auxin transport inhibitor, tri-iodobenzoic acid (TIBA) or exogenous indole-3-acetic acid (IAA) at various stages during their development. If embryos were treated at the preglobular or globular stages of embryogenesis, they developed one fused cotyledon indicating a continuation of radial symmetry. However, if treated later in development, embryos were comparable to the control and formed two separate cotyledons. To demonstrate that this was not a pharmacological effect, an auxin antagonist, 2-(p-chlorophenoxy)-2-methylpropionic acid, included with the TIBA treatment produced normal embryos with two separate cotyledons. Histological observations revealed that the shoot apical meristem of the embryos treated with TIBA or IAA at the preglobular and globular stages was altered. Preglobular and globular stage embryos that were treated with TIBA exhibited a dramatic decline in conversion frequency compared with their controls. Embryos that were treated later in development had conversion frequencies comparable to their controls, and their shoot apical meristems also were similar to controls. Application of ABA at the preglobular and globular stages maintained meristem integrity and improved embryo conversion. However, ABA could not reverse the TIBA effect.

Cell-to-Cell Movement of Green Fluorescent Protein Reveals Post-Phloem Transport in the Outer Integument and Identifies Symplastic Domains in Arabidopsis Seeds and Embryos

Developing Arabidopsis (Arabidopsis thaliana) seeds and embryos represent a complex set of cell layers and tissues that mediate the transport and partitioning of carbohydrates, amino acids, hormones, and signaling molecules from the terminal end of the funicular phloem to and between these seed tissues and eventually to the growing embryo. This article provides a detailed analysis of the symplastic domains and the cell-to-cell connectivity from the end of the funiculus to the embryo, and within the embryo during its maturation. The cell-to-cell movement of the green fluorescent protein or of mobile and nonmobile green fluorescent protein fusions was monitored in seeds and embryos of plants expressing the corresponding cDNAs under the control of various promoters (SUC2, SUC3, TT12, and GL2) shown to be active in defined seed or embryo cell layers (SUC3, TT12, and GL2) or only outside the developing Arabidopsis seed (AtSUC2). Cell-to-cell movement was also analyzed with the low-molecular-weight fluorescent dye 8-hydroxypyrene-1,3,6-trisulfonate. The analyses presented identify a phloem-unloading domain at the end of the funicular phloem, characterize the entire outer integument as a symplastic extension of the phloem, and describe the inner integument and the globular stage embryo plus the suspensor as symplastic domains. The results also show that, at the time of hypophysis specification, the symplastic connectivity between suspensor and embryo is reduced or interrupted and that the embryo develops from a single symplast (globular and heart stage) to a mature embryo with new symplastic domains.

In vitro somatic embryogenesis in callus cultures of Azadirachta indica A. Juss.—a multipurpose tree

Plant regeneration via somatic embryogenesis was achieved in embryogenic callus cultures derived from immature zygotic embryos 40 days after anthesis of Azadirachta indica A. Juss. on semisolid basal Murashige and Skoog (MS) salts and vitamins supplemented with 1.11μM 6-benzylaminopurine (BA) and 4.52–6.78μM 2,4-dichlorophenoxyacetic acid (2,4-D). The globular-stage embryos were induced when the callus was transferred to medium with 1.11μM BA and 0.45μM 2,4-D. The highest average number of somatic embryos per 200mg of callus was 152.8 after 8 weeks of culture on the medium. Maturation and germination of the somatic embryos were achieved on half-strength MS salts and vitamins supplemented 0.38–0.94μM abscisic acid (ABA) and 2% (w/v) sucrose. The maximum percentage (64.2%) of germination was obtained with 0.94μM ABA within 2 weeks of culture. Somatic embryo-derived plantlets were acclimatized in a greenhouse and subsequently showed normal growth. This efficient protocol will be helpful for propagating elite clones on a mass scale and will also be useful for genetic transformation study.