Non-mycorrhizal fungal endophytes in two orchids of Kaiga forest(Western Ghats), India

We used standard isolation protocols to explore the endophytic fungal communities in three tissue types of two dominant orchids （Bulbophyllum neilgherrense and Vanda testacea） of the Kaiga forest of the Western Ghats. We surface sterilized and assessed 90 segments of each orchid for the occurrence and diversity of endophytic fungal taxa. The 118 fungal isolates were obtained from root, bulb and leaves of B. neilgherrense, consisting of 17 anamorphic taxa （range, 10-15 taxa） with 1.3 fungal taxa per segment （range, 1.2-1.4 taxa）. Four taxa （Aspergillus flavus, A. niger, Penicillium sp. and morpho sp. 1） belonged to the core group （11.1%--32.2%）. The relative abundance of A. flavus and morpho sp. 1 was more than 10%. A total of 130 fungal isolates from roots, stems and leaves of V. testacea yielded 20 anamorphic taxa （range, 11-15 taxa） with 1.4 fungal taxa per segment （range, 1.4-1.5 taxa）. Aspergillusflavus, A. niger, A. ochraceus, Gliocladium viride, Penicillium sp. and morpho sp. 1 belonged to the core group. Relative abundance exceeded 10% for A. flavus, A. niger, and morpho sp. 1. The Simpson and Shannon diversity indices were higher in leaf than root or bulb/stem of both orchids. Jaccard＇s similarity coefficient was higher between root and leaf in both orchids （56.3%-60%） than between other pairs. Our study revealed that the endophytic fungal assemblage and diversity orB. neilgherrense and E testacea of Kaiga forest of the Western Ghats were relatively similar between orchids and their tissues.

Phylogenetic endemism of the orchids of Megamexico reveals complementary areas for conservation

Orchid diversity provides a unique opportunity to further our understanding of biotic and abiotic factors linked to patterns of richness,endemism,and phylogenetic endemism in many regions.However,orchid diversity is consistently threatened by illegal trade and habitat transformation.Here,we identified areas critical for orchid conservation in the biogeographic province of Megamexico.For this purpose,we evaluated orchid endemism,phylogenetic diversity,and phylogenetic endemism within Megamexico and characterized orchid life forms.Our results indicate that the majority of the regions with the highest estimates of endemism and phylogenetic endemism are in southern Mexico and northern Central America,mostly located on the Pacific side of Megamexico.Among the most important orchid lineages,several belong to epiphytic lineages such as Pleurothallidinae,Laeliinae and Oncidiinae.We also found that species from diverse and distantly related lineages converge in montane forests where suitable substrates for epiphytes abound.Furthermore,the southernmost areas of phylogenetic diversity and endemism of Megamexico are in unprotected areas.Thus,we conclude that the most critical areas for orchid conservation in Megamexico are located in southern Mexico and northern Central America.We recommend that these areas should be given priority by the Mexican system of natural protected areas as complementary conservation areas.

Efficiency of microsatellite isolation from orchids via next generation sequencing

Microsatellites, or simple sequence repeats (SSRs), are highly polymorphic, co-dominant genetic markers commonly used for population genetics analyses although de novo development of species specific microsatellites is cost-and time-intensive. Orchidaceae is one of the most species-rich families of angiosperms with more than 30,000 species estimated. Despite its high species-diversity, microsatellites are available only for a few species and all were developed by only using Sanger sequencing methods. For the first time in orchids, we used 454 GS-FLX sequencing to isolate microsatellites in two species (Cypripedium kentuckiense and Pogonia ophioglossoides), and report preliminary results of the study. From 1/16th plate that was subjected to sequencing, 32,665 reads were generated, from which 15,473 fragments contained at least one SSR. We selected 20,697 SSRs representing di-, tri-, and tetra-nucleotides. While 3,674 microsatellites had flanking regions on both sides, useable primer pairs could be designed for 255 SSRs. The mean numbers of reads, SSRs, and SSR-containing reads useful for primer design estimated for other 15 orchid species using Sanger sequencing method were 166, 78 and 31, respectively. Results demonstrate that the efficiency of microsatellite isolation in orchids is substantially higher with 454 GS-FLX sequencing technique in comparison to the Sanger sequencing methods.

Similar mycorrhizal fungal communities associated with epiphytic and lithophytic orchids of Coelogyne corymbosa

Mycorrhizal fungi are essential for the growth and development of both epiphytic(growing on trees)and lithophytic(growing on rocks)orchids.Previous studies indicate that in lowland tropical areas,orchid mycorrhizal fungal compositions are correlated with the life form(i.e.,epiphytic,lithophytic,or terrestrial)of their host plants.We therefore tested if a similar correlation exists in an orchid distributed at higher elevations.Coelogyne corymbosa is an endangered ornamental orchid species that can be found as a lithophyte and epiphyte in subtropical to subalpine areas.Based on high-throughput sequencing of the fungal internal transcribed spacer 2(ITS2)-rDNA region of mycorrhizae of C.corymbosa,we detected 73 putative mycorrhizal fungal Operational Taxonomic Units(OTUs).The OTUs of two dominant lineages(Cantharellales and Sebacinales)detected from C.corymbosa are phylogenetically different from those of other species within the genus Coelogyne,indicating that different orchid species prefer specific mycorrhizal fungi.We also found that the Non-metric multidimensional scaling(NMDS)plots of orchid mycorrhizal fungi were not clustered with life form,the variations among orchid mycorrhizal fungal communities of different life forms were not significant,and most of the OTUs detected from epiphytic individuals were shared by the lithophytic plants,suggesting that orchid mycorrhizal associations of C.corymbosa were not affected by life form.These findings provide novel insights into mycorrhizal associations with endangered ornamental orchids.

Photosynthetic Light Utilization Efficiency, Water Relations and Leaf Growth of C3 and CAM Tropical Orchids under Natural Conditions

Native orchid species of Singapore in their natural conditions experience stress from high irradiance, high temperatures and periods of extended low rainfall, which impact orchid plant physiology and lead to reduced growth and productivity. In this study, it was found that there was a reduction in photochemical efficiency of photosystem II (PSII) in 6 native orchid species under high light (HL) and Bulbophyllum membranaceum under low light (LL). There was chronic photoinhibition in these 6 orchid species over a period of 3 months after transplanting onto the tree trunks without watering and fertilization, especially in Coelogynes mayeriana and Bulbophyllum membranaceum under both HL and LL. This chronic photoinhibition caused by sustained period of water deficit in their natural conditions was later reversed by natural re-watering conditions from higher rainfall. These results indicate that water deficit has a greater impact on photosynthetic light utilization efficiency than excess light. The present study also showed that after natural rewatering, relative water content (RWC) of leaves and pseudobulbs generally increased. During the natural re-watering, total leaf area also gradually increased and reached maximum expansion after 7 weeks under both HL and LL, with some exceptions due to leaf abscission or decline in total leaf area, possibly a strategy for water conservation.

The antioxidant activity of selected wild orchids of Nepal

Objective:To assess the antioxidant activity as well as the total polyphenolics and flavonoids content of thirteen extracts from nine wild orchids of Nepal.Methods:First,a preliminary phytochemical screening of thirteen extracts of nine orchids was carried out using established protocols.Then,the total polyphenolic and flavonoid contents of each extract were evaluated using the Folin-Ciocalteu's phenol reagent and aluminium chloride methods respectively.Finally,antioxidant activity was determined by 2,2-diphenyl-1-picryl hydrazyl free radical scavenging assay and expressed in terms of half inhibition concentrations(IC50 values).Results:All the tested parameters showed significant variation at P=0.05.The total flavonoids varied with the greatest amounts found in the leaves of Rhynchostylis retusa and the smallest amounts found in the roots of Gastrochilus acutifolius(G.acutifolius).The amount of total polyphenolics was highest in the stems of Vanda cristata and lowest in the leaves of G.acutifolius.Antioxidant activity was highest in the stems of Vanda cristata and lowest in the leaves of G.acutifolius.Conclusions:Some of the orchid extracts studied exhibited levels of antioxidant activity which were comparable to or even higher than those found for extracts of medicinal plants and orchids studied previously.It is suggested that drugs and natural food additives can be extracted from those wild orchids with high levels of antioxidant activity.

Genome Sequencing Reveals the Role of MADS-box Gene Families in the Floral Morphology Evolution of Orchids

Orchid origin and evolution are common topics in evolutionary biology. Orchidaceae have approximately 30 000 orchid species distributed in diverse habitats and account for approximately 10% of the flowering plant species worldwide. Orchids provide us with materials to explore coevolution and organic evolution. In this review, we highlighted the genome study progress of orchids. In addition, we revealed the role of MADS-box gene families in the floral morphology and evolution of orchids. Genomics studies confirmed that all five subfamilies of existing orchids evolved from a common ancestor. Loss of Mβ MADS-box genes resulted in the endosperm from the seed of all existing orchids being absent. Perianth reversion to the ancestral state occurred because Apostasia and Apostasioideae lost B-AP3 and E class paralogous genes. Loss of P-subclade members of MIKC*-Type in Phalaenopsis equestris, Dendrobium catenatum, and Epidendroideae caused the formation of pollinium.In addition, the combined loss of AGL12 and contraction of ANR1 gave orchids the ability to be successfully epiphytic on trees or rocks and to develop a unique root system. Both pollinium and epiphytic production on trees are beneficial for orchid adaptations, and Epidendroideae evolved more species(~ 20 000) than Apostasioideae(16 species). Genome studies shed new light on determining the evolutionary history of orchids and understanding the genetic mechanisms of orchid morphological evolution.

Orchid diversity and distribution pattern in karst forests in eastern Yunnan Province,China

Global biodiversity loss and mass extinction of species have raised serious global concerns,especially in fragile ecosystems.Karst forest,a well-known fragile ecosystem,is served as a natural laboratory for biogeographical,ecological,evolutionary,and taxonomic research.However,species richness and distribution patterns in karst forests largely remain unclear.Orchidaceae has been regarded as the“flagship”group of plant diversity.In this study,we use Wanfengshan Nature Reserve in southeast Yunnan,China,as a model to determine the conservation status of orchids in the karst forest by conducting systematic field surveys in the past three years from 2019 to 2022.In total,we identified 78 orchid species in 35 genera in Wanfengshan Nature Reserve.The dominant species were Bulbophyllum andersonii,Eria coronaria,Nervilia mackinnonii and Paphiopedilum micranthum.Multiple life forms were also observed,including epiphyte(14.29%),facultative epiphyte(18.18%),lithophyte(23.38%),and terrestrial(44.15%).Orchid species richness showed a bimodal pattern varied with altitude,the higher richness was recorded at 1,500-1,600 and 1,200-1,300 m,while the minimum was below 1,100 m.Each independent hill is rich in orchid species including‘endemic’species.A total of 25(33.8%)orchid species were considered threatened by the Redlist of Chinese Vascular Plants(Qin et al.,2017),including 3 CR species,5 EN species and 17 VU species.We call for more research addressing the mechanisms of ecological adaption,mycorrhizal interactions,and pollination of orchids in karst forests,and effective ways for recovery and conservation of orchids.

Mycorrhizal Fungi Spore Abundance in Old-Growth Forest Soil

Soil samples were collected from the base of Aplectrum hymale individuals to assess mycorrhizal spores abundance. The hypothesis that mycorrhizal spore abundance would increase with proximity to the plant was not supported;however, spores increased significantly with distance from the Aplectrum hymale plants up to one meter.

Plant species with extremely small populations(PSESP) in China:A seed and spore biology perspective

Approximately one fifth of the world's plants are at risk of extinction.Of these,a significant number exist as populations of few individuals,with limited distribution ranges and under enormous pressure due to habitat destruction.In China,these most-at-risk species are described as 'plant species with extremely small populations'(PSESP).Implementing conservation action for such listed species is urgent.Storing seeds is one of the main means of ex situ conservation for flowering plants.Spore storage could provide a simple and economical method for fern ex situ conservation.Seed and spore germination in nature is a critical step in species regeneration and thus in situ conservation.But what is known about the seed and spore biology(storage and germination) of at-risk species? We have used China's PSESP(the first group listing) as a case study to understand the gaps in knowledge on propagule biology of threatened plant species.We found that whilst germination information is available for 28 species(23%of PSESP),storage characteristics are only known for 8%of PSESP(10 species).Moreover,we estimate that 60%of the listed species may require cryopreservation for long-term storage.We conclude that comparative biology studies are urgently needed on the world's most threatened taxa so that conservation action can progress beyond species listing.

Understanding the Phylomorphological Implications of Pollinia from <i>Dendrobium</i>(Orchidaceae)

We aim to evaluate the evolutionary trends of pollen ultra-structures and microstructures in the light of molecular phylogeny, and to test whether pollen micromorphology is a predictor of interspecific phylogenetic relationship or a consequence of ecological influences. Using scanning electron microscopy pollinia micromorphologies of 18 species of the genus Dendrobium were comprehensively examined. A phylogenetic tree from ribosomal-ITS2 sequences was constructed reflecting molecular phylogeny. In result, two major clusters were identified representing the sections Aporum, Formoase Dendrobium and Holochrysa. The section Dendrobium could be further classified, for the first time, into two clades sharing the same root of origin. Variations in the shape of pollinia were distinct and consistent constituting six novel groups. Other qualitative/quantitative keys to pollinia showed congruence with molecular phylogeny and supported the correlated evolution of these traits. Concurrently pollen sculpturing also showed large variation in exine morphology (rugular/psilate to psilate-scabrate/rugulate-scabrate) providing explicit clues for phylogenetic trend of exine evolution. For the characteristic sculpturing on the pollinia surface, a simple version of pre-pattern model is also hypothesized for deeper insight into pattern formation in other biological systems. Results indicated that distinct groups of Dendrobium species have parallel and independent evolution accompanied with genetic changes occurred during species diversification. Thus pollen micromorphology is a good candidate for inferring the genetic relationship at inter-specific level having phylogenetic implications even using abundant palynological records.

Importance of Quercus spp.for diversity and biomass of vascular epiphytes in a managed pine-oak forest in Southern Mexico

Background:Forestry management modifies the diversity,structure,and functioning of intervened forests.Timber extraction reduces tree density and basal area,leading to changes in the communities of vascular epiphytes.The objective of this study was to evaluate the diversity and biomass of vascular epiphytes in Quercus trees remaining in two pine-oak forest stands that have been subjected to two stages of the Silvicultural Development Method(release cutting,and thinning)in comparison with an unharvested old-secondary forest in southern Mexico.For each treatment,richness of epiphytes present on 60 oak trees was recorded and their dry biomass estimated.We calculated the true diversity(Hill numbers)and beta diversity using the Jaccard coefficient of similarity,and generated rank abundance curves per taxonomic epiphyte group(bromeliads,orchids,ferns and others).For each treatment,the relationships between overall diversity and epiphyte biomass to the host trees basal area were analyzed using log linear models.Results:We recorded a total of 67 species of epiphytes species belonging to 10 families hosted by five species of oaks.The greatest species richness(^(0)D)was recorded in the old-secondary forest.Fewer common(^(1)D)and dominant(^(2)D)species were recorded in the release cutting than in the other treatments.Epiphyte diversity and biomass were both slightly related to host tree basal area.Composition of epiphytes was similar(60%)among treatments,although orchids,bromeliads,and other families were more diverse in the old-secondary forest.Most bromeliad species were shared across all treatments,although orchids presented the most exclusive species in the unharvested forest.The bromeliad Tillandsia seleriana provided the greatest contribution to biomass in all treatments,followed by the orchid Camaridium densum.Generalized linear models indicated that epiphyte diversity was significantly related to treatment,and epiphyte biomass to basal area of host trees.Conclusions:Although forest management affects diversity,composition,and abundance of vascular epiphytes,most of their diversity and biomass can be maintained despite timber harvesting.This requires sparing some mature oaks during logging,as they contribute to conservation,establishment,and development of epiphytic communities,and maintaining untreated areas as a source of propagules for these communities.

A comparative study of Epipactis atrorubens in two different forest communities of the Middle Urals, Russia

The objective of this study was to compare ecophysiological and morphological parameters of a regionally endangered orchid species,Epipactis atrorubens(Hoffm.ex Bernh.)Bess.,growing in two forest communities(on serpentine and granite outcrops)of the Middle Urals,Russia.Biodiversity,dominance,and phytocoenosis studies showed the colonization of a wide range of plant species on both sites.The physicochemical properties of the soil,chemical composition and morphological features of E.atrorubens,growing under technogenic conditions(asbestos deposits),on serpentine outcrops and in the natural environment of the granite massif were studied for the first time.The serpentine substrate differed from the granite one by its greater stoniness,circumneutral pH and lower contents of available nitrogen and phosphorus.Extremely high concentrations of magnesium were found in the serpentine soil,some 79 times higher than in the granite substrate.High concentrations of nickel(94 times),chromium(59 times),cobalt(17 times),and iron(4 times)were found in the serpentine substrate,higher than in the granite substrate.The differences between the sites for available metal contents and for root and shoot metal contents were significantly less.Concentrations of most of the metals in the roots were higher than in the shoots.Despite higher metal concentrations and lower nitrogen and phosphorus levels in serpentine soils,E.atrorubens had a larger population and greater viability compared to those growing on granite.Plants on serpentine outcrops were characterized by the formation of a larger number of fruits,greater root lengths and thicker leaf blades,compared to plants on granites.The well-developed orchid mycorrhizae contributed to the survival of this species under unfavorable serpentine conditions.Hence,serpentine outcrops formed due to the mining of asbestos could be a suitable substrate for the light-demanding E.atrorubens due to its capacity to adapt to dry,rocky,nutrient-depleted soils and limited competition from other plants.

Orchid conservation in China from 2000 to 2020:Achievements and perspectives

We review achievements in the conservation of orchid diversity in China over the last 21 years.We provide updated information on orchid biodiversity and suggestions for orchid conservation in China.We outline national policies of biodiversity conservation,especially of orchid conservation,which provide general guidelines for orchid conservation in China.There are now approximately 1708 known species of Orchidaceae in 181 genera in China,including five new genera and 365 new species described over the last 21 years.The assessment of risk of extinction of all 1502 known native orchid species in China in 2013 indicated that 653 species were identified as threatened,132 species were treated as data-deficient,and four species endemic to China were classified as extinct.Approximately 1100 species(ca.65%)are protected in national nature reserves,and another~66 species in provincial nature reserves.About 800 native orchid species have living collections in major botanical gardens.The pollination biology of 74 native orchid species and the genetic diversity and spatial genetic structure of 29 orchid species have been investigated at a local scale and/or across species distributions.The mycorrhizal fungal community composition has been investigated in many genera,such as Bletilla,Coelogyne,Cymbidium,Cypripedium,and Dendrobium.Approximately 292 species will be included in the list of national key protected wild plants this year.Two major tasks for near future include in situ conservation and monitoring population dynamics of endangered species.

Transcriptome Analysis Reveals Clues into leaf-like flower mutant in Chinese orchid Cymbidium ensifolium

The floral morphology of Cymbidium ensifolium,a well-known orchid in China,has increasingly attracted horticultural and commercial attention.However,the molecular mechanisms that regulate flower development defects in C.ensifolium mutants are poorly understood.In this work,we examined a domesticated variety of C.ensifolium named‘CuiYuMuDan',or leaf-like flower mutant,which lacks typical characteristics of orchid floral organs but continues to produce sepal-to leaf-like structures along the inflorescence.We used comparative transcriptome analysis to identify 6234 genes that are differentially expressed between mutant and wild-type flowers.The majority of these differentially expre ssed genes are involved in membrane-building,anabolism regulation,and plant hormone signal transduction,implying that in the leaf-like mutant these processes play roles in the development of flower defects.In addition,we identified 152 differentially expre ssed transcription factors,including the bHLH,MYB,MIKC,and WRKY gene families.Moreover,we found 20 differentially expressed genes that are commonly involved in flower development,including MADS-box genes,CLAVATA3(CLV3),WUSCHEL(WUS),and PERIANTHIA(PAN).Among them,floral homeotic genes were further investigated by phylogenetic analysis and expression validation,which displayed distinctive spatial expression patterns and significant changes between the wild type and the mutant.This is the first report on the C.ensifolium leaf-like flower mutant transcriptome.Our results shed light on the molecular regulation of orchid flower development,and may improve our understanding of floral patterning regulation and advance molecular breeding of Chinese orchids.

Conservation implications of population genetic structure in a threatened orchid Cypripedium tibeticum

Cypripedium tibeticum is a threatened orchid which efficient conservation requires knowledge of its extent and structure of genetic variation. Using two chloroplast DNA fragments(rps16 and trnL-F), we analyzed 157 individuals from 9 populations representing the species range in China. Seven haplotypes were identified. C. tibeticum had high total genetic diversity(H_T=0.80) with major contribution to this diversity made by among-population component(G_ST=0.64, Ф_ST= 0.86). However, despite high population differentiation there was no clear phylogeographic structure. The populations CY and DC made the greatest contribution to the total gene diversity as well as allelic richness. The possible mechanisms and implications of these findings for conservation of the species are discussed.

Evolution of Two Ubiquitin-like System of Autophagy in Orchid

As an important horticultural plant,the orchid is widely distributed in its natural habitat and faces various environmental stresses,among which nutrient recycling and stress resistance are of great concern.During these processes,autophagy is an essential pathway,which is a conserved self-eating process that degrades macromolecular components and recycles cell materials or nutrients during developmental processes or under stress conditions.Two ubiquitin-like systems(UBLs)play a major role in the initiation of autophagy and are associated with two key proteins:ATG8 and ATG12.In this study,we identified and refined the UBL-related genes in orchids and performed phylogenetic reconstruction together with other plant species.We found that the orchid had unique domains in UBL-related genes,indicating potential functional diversification in the ATG8 system in plants.Transcriptome and protein tertiary structure prediction indicated that conserved domains that are vital for the canonical function of ATG12 are incomplete in orchids,in which a novel mechanism of autophagy may have evolved.

Fungal Isolate “KMI” Is a New Type of Orchid Mycorrhizal Fungus

We analyzed an isolate of fungus that seems to be a new type orchid mycorrhizal fungus, “KMI (refers to Kyoto-Ma- tsubara-Ishii)”, obtained from the roots of Paphiopedilum thailandense Fowl. KMI has hyphal branching into right angle similar to Rhizoctonia-like fungi, but its spore formation resembled Nectoria, which is known as a teleomorph of Fusarium rather than Rhizoctonia. Its ribosomal DNA sequences of 18S and ITS have no similarity with any known fungal species. Proteins in molecular weight of 53 and 24 kDa, which are common to mycorrhizal fungi, were detected in KMI. When KMI was inoculated onto orchids, however it didn’t form pelotons or coils, but the hyphae in the root tissues were observed and the tissues were not decayed. No significant symptoms of Rhizoctonia or Fusarium disease, however, were developed on tomato and cucumber plants. The evidences suggest that KMI is a new type of orchid mycorrhizal fungus.

Embryo and Protoplast Isolation from <i>Barlia robertiana</i>Seeds (Orchidaceae)

In the present study, it is aimed to investigate embryo and protoplast isolation from orchid seeds, because of know-how deficiency. As material Barlia robertiana seeds were subjected enzymatic process. Globular embryos with suspensor were fully removed by enzymatic maceration for between 100-180 min of incubation. The highest obtained embryo yields were found in Rapidase EX Color and Rapidase Vino Super, with degree of 89.2-90.3 × 103·g-1 MF and 86.4-91.3 × 103·g-1 MF, respectively. According to multicomponent maceration tests, a treatment comprising 150 min of the enzymatic reaction with 0.18% (E/S) at 50℃ was the most optimal for obtaining high embryo yields. Regarding protoplast isolation tests, enzyme combinations C (1% Cellulase “Onozuca” R-10 + 0.2% Macerozyme R-10 and 0.1% Driselase) and F (1% Cellulase “Onozuka” + 1% Macerozyme + 0.5% Driselase) after 15 hours produced the best results for protoplast isolation and were significantly different compared to other enzyme combinations. Greater protoplast yields were obtained using a rotatory system with protoplasts incubated in the dark. Also the present findings were discussed from the point of view of in vitro plant manipulation and orchid cultivation in cultural media and then their importance emphasized in molecular biological/genetical studies.

Plant Regeneration Through Indirect Somatic Embryogenesis in Coelogyne Cristata Orchid

A newly efficient protocol has been established for high frequency somatic embryogenesis through callus culture of Coelogyne cristata. The best frequency of callusing was obtained from leaf segments (3-5 mm) cultured on the MS medium supplemented with 2 mg?L–1 2,4-D and 2 mg?L–1 BA combination. A negative effect of coconut water was ob-served on the callus induction medium. When callus lines no. 4, 6 and 8 induced from leaf segments were sub-cultured separately on ? MS and MS media containing AC (1-3 g?L–1), formation of somatic embryos was found. However, percentages of embryo formation and the number of embryos per explants were strongly affected by media and callus lines used. The effect of 1/2 MS media is definitely better than MS medium for somatic embryogenesis from the selected lines of leaf derived callus. Among the callus lines, line no. 4 is the best for somatic embryogenesis followed by line no. 6 and 8. The somatic embryos converted into healthy plants with well developed shoots on the same media. The plant-lets were transferred to 1/2 MS medium containing 1 g?L–1 AC for plant regeneration until 8 weeks of culture and successfully acclimatized in the greenhouse.