Nuclear DNA content of somatic and zygotic embryos ofVitis vinifera cv. Grenache noir at the torpedo stage

Summary A flow cytometric analysis and an in situ DNA microspectrophotometric study were made concomitantly to establish why somatic grapevine (Vitis viniferacv. Grenache noir) embryos showed a low level of conversion into plantlets. In somatic embryos at the torpedo stage and in zygotic embryos at the same stage of development, ploidy level, DNA content per 2 C nucleus, and the cell-cycle state of the shoot apical meristem were examined. The frequency distribution histograms of nuclear DNA values were similar in the two types of embryos. At the torpedo stage both types of embryos had a majority of nuclei with 2 C DNA content equal to 1.6pg. In the shoot apices of somatic and zygotic embryos, DNA microspectrophotometry showed preferential blockage of the cell cycle at the G_0–1 stage; however, 20% of somatic embryo shoot apices were blocked at the G_0–2 stage. Analogies between somatic embryos and their zygotic homologues were shown. The genetical and environmental causes of the low level of conversion of grapevine somatic embryos into plantlets are discussed. Our work suggests that the in vitro culture conditions which were used could be incompatible with normal morphogenesis from the torpedo stage.     

Effect of dissolved oxygen concentration on differentiation of somatic embryos of Coffea arabica cv. Catimor 9722

The effect of two different dissolved oxygen (DO) concentrations (50 and 80%) on differentiation of somatic embryos (SE) from cell suspensions of coffee (Coffea arabica cv. Catimor 9722) was analyzed. Two bioreactors CMF-100 (CHEMAP AG) designed for the culture of cells, with 2-l glass vessels and a maximum work volume of 1.8 l were used. Each one was equipped with a gas blending unit (air, O₂, N₂, CO₂) for the control of DO concentration. The inoculation density of embryogenic cells was 1.0 gram of fresh weight per liter (g FW l^–1). The number of somatic embryos was greater (71 072 SE l^–1) with 80% DO, but the major proportion were globular and heart shaped SE (66 399 SE l^–1) and only 6.6% with regard to total was torpedo shaped SE. However, the 50% DO produced the higher number in the torpedo shaped SE (7389 SE l^–1) what represented 20.0% with regard to total. Thus, higher concentrations of DO induced globular and heart shaped SE differentiation, but for production of torpedo shaped SE lower concentrations DO are needed. The somatic embryos obtained in the bioreactor with 50% DO showed similar behavior to the somatic embryos obtained in the rotary shaker. After 8 weeks of culture, 49.2% germination was obtained, which allowed a total of 1725 plantlet to be transferred to conditions ex vitro. After 6 months of culture, 89.2% of conversion was achieved and 1539 plants obtained were transferred to field conditions.     

Photoautotrophic culture of Coffea arabusta somatic embryos: photosynthetic ability and growth of different stage embryos

Coffea arabusta somatic embryos were cultured and development of stomata, rate of CO2 fixation or production, chlorophyll content and chlorophyll fluorescence were studied in embryos at different stages of development. Cotyledonary and germinated embryos have photosynthetic capacity, although pretreatment at a high photosynthetic photon flux (PPF) (100 micromol m(-2) s(-1)) for 14 d increased photosynthetic ability. Except in a very small number of cases, stomata did not develop fully in precotyledonary stage embryos and were absent in torpedo stage embryos. Low chlorophyll content (90-130 microg g(-1) fresh mass) was noted in torpedo and precotyledonary stage embryos compared with cotyledonary and germinated embryos (300-500 microg g(-1) fresh mass). Due to the absence of stomata and low chlorophyll content in the torpedo and precotyledonary stage embryos, the photosynthetic rate was low and, in some cases, CO2 production was observed. These data suggest that the cotyledonary stage is the earliest stage that can be cultured photoautotrophically to ensure plantlet development. When grown photoautotrophically (in a sugar-free medium with CO2 enrichment in the culture headspace and high photosynthetic photon flux), torpedo and precotyledonary stage embryos lost 20-25% of their initial dry mass after 60 d of culture. However, in cotyledonary and germinated embryos, the dry mass of each embryo increased by 10 and 50%, respectively. By using a porous supporting material, growth (especially root growth) was increased in cotyledonary stage embryos. In addition, photoautotrophic conditions, high PPF (100-150 micromol m(-2) s(-1)) and increased CO2 concentration (1100 micromol mol(-1)) were found to be necessary for the development of plantlets from cotyledonary stage embryos.     

Classification of plant somatic embryos by computer vision

This article deals with the automation of the process of somatic embryogenesis for the propagation of plants. An important problem is the monitoring of the embryo production process in order to decide the time to start harvesting embryos for further processing. The classification algorithm development for somatic embryos of birch (Betula pendula Roth) showed that automated recognition of embryos at different developmental stages is possible. No globular stage embryos were classified to be heart or torpedo stage and no heart or torpedo stage embryos were classified to be at globular stage. Heart and torpedo stage embryos were classified into three developmental classes by a new index that describes the relation of embryo breadth to the length of the root. The probability of classifying a nonembryo as an embryo was less than 1%, and 14% of the object classified as embryos by a human expert were discarded by the algorithm. A computer vision system suitable for automated monitoring of samples from the bioreactor was constructed. (c) 1993 John Wiley & Sons, Inc.     

Alfalfa embryo production in airlift vessels via direct somatic embryogenesis

A procedure for the development of alfalfa (Medicago falcata L.) somatic embryos to the torpedo stage in air-lift vessels is described. Embryos were initiated from chopped leaf explants and were formed by direct somatic embryogensis. The system produced a high number of torpedo stage embryos. The effect of various inoculation densities on embryo development was studied. A procedure for the development and maturation of embryos in aerated liquid media was established. The rate of conversion of the torpedo stage embryos formed in the vessels was 83%.Abbreviations ABA abscisic acid - B5 Gamborgs B5 medium (Gamborg et al. 1968) - COT cotyledon embryo state - 2,4-d 2,4-dichlorophenoxyacetic acid - FW fresh weight - ID internal diameter - MS Murashige and Skoog medium (Murashige & Skoog 1962) - PEG polyethylene glycol - POLY polyembryos - VVM volume of gas/volume of bioreactor     

Cycle characteristics in a temporary immersion bioreactor affect regeneration,morphology, water and mineral status of coffee (<Emphasis Type="Italic">Coffea arabica</Emphasis>) somatic embryos

Mass regeneration of Coffea arabica L. somatic embryos using a temporary immersion bioreactor was improved by optimizing the immersion cycles, i.e. both the duration and the frequency of immersions. It was demonstrated that increasing the frequency of short immersions (1 min immersions every 24, 12 and 4 h) stimulated embryo production (480, 2,094 and 3,081 embryos/1-l bioreactor, respectively) and improved quality (60, 79 and 85 of torpedo shaped embryos, respectively). On the other hand, an increase in the immersion duration (1, 5 and 15 min) inhibited embryo regeneration (from 2,094 to 428 embryos per 1-l bioreactor) and negatively affected their morphological quality (from 79 to 49 torpedo-shaped embryos) and the conversion of embryos into plants (from 70 to 33). A 15 min immersion duration applied every 4 h produced hyperhydric symptoms in 90 of the embryos. Hyperhydric embryos were characterized by higher fresh weight and water content, more negative values for water potential and higher K⁺ content when compared to normal torpedo-shaped embryos. Micrographs showed structural problems in the globular stage, such as the existence of an irregular epidermis and an absence of reserves. Whatever the immersion cycle used, the somatic embryos exhibited water and mineral characteristics very different from those of their zygotic counterparts. The use of 1 min immersions every 4 h led to the production of the largest quantities of torpedo-shaped embryos without hyperhydricity that succeeded in regenerating plants (75 conversion).     

In vitro propagation and conservation of Indian sarsaparilla, Hemidesmus indicus L. R. Br. through somatic embryogenesis and synthetic seed production

A protocol has been developed for achieving somatic embryogenesis from callus derived from nodal cuttings and production of synthetic seeds in Hemidesmus indicus L. R. Br. a highly traded ethnomedicinal plant. Proembryogenic, friable, light yellowish callus was induced from the basal cut end of the nodal cuttings on Murashige and Skoog (MS) medium supplemented with 3 μM indole-3-butyric acid (IBA). The highest rate of somatic embryogenesis (92 %) was observed when the callus was subcultured on half strength MS medium supplemented with 2 μM IBA. On induction medium somatic embryos were developed up to the torpedo stage. Further elongation and germination of somatic embryos were obtained in MS medium supplemented with 4 μM 6-benzylaminopurine (BA) in combination with 1.5 μM gibberellic acid (GA₃). Somatic embryos were collected and suspended in a matrix of MS medium containing sodium alginate (3 % W/V) dropped into 75 mM calcium chloride (CaCl₂·2H₂O) solution for the production of synthetic seeds and later transferred to MS medium for germination. The synthetic seeds were successfully germinated on medium even after 120 days of storage at 4 °C. The plantlets were eventually transferred to soil with 92 % success.     

Recurrent somatic embryogenesis and plant regeneration from seedlings of <Emphasis Type="Italic">Hepatica nobilis</Emphasis> Schreb.

A method for secondary somatic embryogenesis was developed on embryos derived from embryogenic callus formed on Hepatica nobilis seedlings. Somatic embryogenesis (SE) was induced on seedlings (on the hypocotyl and epicotyl parts) grown on the Murashige and Skoog (1962) medium (MS) supplemented with 1 µM naphthaleneacetic acid (NAA), and/or 0.1 µM 6-benzyladenine (BA) and on medium without plant growth regulators (PGR). The best response of embryogenic callus formation was observed on the medium containing 1 µM NAA alone or with 0.1 µM BA. Individual somatic embryos, formed on embryogenic callus on the medium without PGR (MS0), at heart, torpedo and cotyledonary stage, were transferred to the media where secondary somatic embryo formation and development into plantlets occurred. Although the most efficient repetitive cycles of secondary SE were recorded for all stages of somatic embryos (heart, torpedo, cotyledonary) on the MS0 medium (77.8–87.4 %), secondary somatic embryos were also obtained on all media supplemented with cytokinins. The best rate of somatic embryos germination was achieved on MS media with 0.2 µM NAA and 2 µM BA, and 0.1 µM NAA and 1 µM BA (48.8–52.0 %) when more mature embryos (cotyledonary stage) were used. Plantlets grown from somatic embryos were successfully acclimatized to greenhouse conditions.     

The histodifferentiation events involved during the acquisition and development of somatic embryogenesis in oil palm (Elaeis guineensis Jacq.)

Understanding the fate and dynamics of cells during callus formation is essential to understanding totipotency and the somatic embryogenesis (SE) mechanisms. In the present study, the histodifferentiation events involved during the acquisition and development of somatic embryogenesis in oil palm (Elaeis guineensis Jacq.) was investigated. Zygotic embryos were inoculated on SE induction medium, and at 14 days the first divisions of the procambial and perivascular cells were observed. This region progressed to the formation of meristematic masses at 21 days, indicating their procambial and perivascular origin. Primary calli emerged at 45 days of culture, followed by progression to embryogenic calli at 90 days. The formation of proembryos (PE) from the meristematic cells occurred at 135 days of cultivation. The PE were isolated from the tissue of origin by the slight thickening of the cell wall, indicating their unicellular origin. When transferred to the maturation phase, differentiation of the somatic embryos at different developmental stages (globular and torpedo) was observed. The differentiated somatic embryos presented protoderm, procambial strands and plumules. Afterwards, they were transferred to culture medium without growth regulators in which conversion of the somatic embryos from torpedo stage into plants was observed. These results enable a greater understanding of the SE process and plantlet formation in E. guineensis.     

Plant regeneration through somatic embryogenesis from zygotic embryo-derived callus of <Emphasis Type="Italic">Areca catechu</Emphasis> L. (Arecaceae)

Summary An in vitro culture procedure was established for somatic embryogenesis and plant regeneration from callus cultures of the important palm ‘betel nut’ (Areca catechu L.). Segments of zygotic embryos of Areca catechu L. were cultured on Murashige and Skoog basal medium supplemented with dicamba (9.05, 18.1, 27.15, and 36.2 μM). After 7–8 wk in darkness, wounded regions of explants formed callus with yellow, soft, glutinous structures. Proliferation and maintenance of callus was on the same dicamba-containing medium. With regular subculture every 8 wk, the callus showed pale yellow, compact and nodular structures. During subculture, somatic embryos were formed spontaneously from nodular callus tissues within 2–4 mo. The embryos developed into plantlets after 10 wk of culture on basal medium free of plant growth regulators. After subculturing every month for 3 mo., the plantlets were transferred to containers for acclimatization in the greenhouse. The survival rate was 24%.     

花楸体细胞胚发生过程中抗氧化酶活性的变化

花楸体细胞胚发生过程中,胚性愈伤组织可溶性蛋白含量高于其他类型的愈伤组织,非胚性愈伤组织中超氧化物歧化酶(SOD)、过氧化物酶(POD)和过氧化氢酶(CAT)活性均高于其他类型的愈伤组织;SOD、POD活性均在胚性细胞向球形胚转化时下降,球形胚向心形胚发育时下降,心形胚向鱼雷形胚和鱼雷形胚向子叶形胚发育时再升高;CAT活性变化规律与SOD和POD活性变化不同,从胚性细胞到鱼雷形胚的3个发育时间内表现为下降-升高-下降的趋势,鱼雷形胚向子叶胚发育时略有回升。据此认为,SOD酶活性降低似可作为花楸胚性细胞分化以及胚胎早期发育的一个判断指标。     

Efficient somatic embryogenesis and plant regeneration from long-term cell suspension cultures of Medicago Truncatula cv. Jemalong

Summary Plants were suecessfully régenerated via somatic embryos from 3-yr-old cell suspension cultures of Medicago truncatula Gaertin. cv. Jemalong line M9-10a. The cultures were originally initiated from callus induced in well-expanded leaflets of 30 d in vitro-grown plants, Suspension cultures were established in stirred-liquid Murashige and Skoog (MS) basal salts and vitamins supplemented with 2.3 μM 2.4-dichlorophenoxyacetic acid (2,4-D) and 2.3 μM kinetin (Kin) and subeultured weekly. Somatic embryogenesis induction step was conducted in liquid MS medium containing 0.45 μM 2,4-D and 0.91 μM zeatin (Zea), during 1,2, and 3wk after subculture. Induced and non-induced cultures were transferred to solid embryo proliferation medium [EPM-MS basal salts and vitamins solidified with 0.2% (w/v) gelrite]. Somatic embryos developed until the late torpedo/dicotyledonary stages. We found that the best condition for the development of somatic embryos was achieved when suspension cultures were not subjected to the induction step. Induction of 1 and 2 wk led to a decrease in the recovery of somatic embryos and the 3-wk treatment resulted in no differentiation of somatic embryos. Plant regeneration was obtained in all conditions (except for 3wk induction) when embryos were transferred to an embryo conversion medium [ECM, similar to EPM but solidified with 0.7% (w/v) agar]. Embryo conversion rates were 54.5±1.6, 52.5±18.5, and 41.6±8.4% for 0, 1, and 2 wk induction treatments, respectively. These plants were successfully transferred to the greenhouse where they matured and produced seeds.     

Somatic embryogenesis in Vigna radiata (L.) Wilczek.

Somatic embryogenesis was achieved from immature cotyledon derived callus of mungbean, V.radiata (L.) Wilczek in MS liquid medium. Embryogenic callus was induced on MS medium with NAA (5 mg/L). Differentiation of somatic embryos was observed when embryogenic callus was transferred to MS liquid medium containing 2,4-D (1.5 mg/L) and L-proline (50 mg/L). The torpedo shaped embryos were transferred to MS liquid medium with BAP and ABA (1 mg/L each) for maturation and germination. Fifty per cent of torpedo shaped embryos were converted into tiny plants (8-9 plants out of 17) after one week of culture. The germinated embryos were isolated and transferred to MS half strength basal (solid) medium for further development.     

Role of phytohormones and nitrogen in somatic embryogenesis induction in cell culture derived from leaflets of <Emphasis Type="Italic">Azadirachta indica</Emphasis>

A protocol for somatic embryogenesis in Azadirachta indica A Juss. has been standardized using in vivo leaflets. Experiments were carried out to examine the effect of various auxins, cytokinins, sucrose, inorganic and organic salts on subsequent somatic embryo induction and maturation. Embryogenic calli were induced on Murashige and Skoog (MS) medium supplemented with 1.5 mg dm⁻³ kinetin and 1.5 mg dm⁻³ indole-3-acetic acid and subsequently all the stages of somatic embryo development (globular, cordate, torpedo and cotyledonary) were observed. Maturation of these embryos was accomplished with the same growth regulators after three subcultures. The histological study of somatic embryos showed resembles to zygotic embryos. The matured somatic embryos were transferred onto half strength MS-medium devoid of growth regulators for their germination (82 %). Plantlets were acclimatized in the field with a survival rate of 80–83.5 %.     

Expression of photosynthesis-related genes and their regulation by light during somatic embryogenesis in<Emphasis Type="Italic"> Daucus carota</Emphasis>

To clarify the spatial and temporal pattern of gene expression for photosynthesis-associated proteins during somatic embryogenesis in Daucus carota L., the localization of mRNAs for three genes, rbcL, Lhcb and por, was examined in dark-grown and light-irradiated somatic embryos by in situ hybridization. The three mRNAs were expressed in common in the mesophyll precursor cells of light-irradiated embryos at the late torpedo and plantlet stages, but characteristic expression patterns of each photosynthesis-related gene were also observed. Expression of rbcL mRNA first occurred throughout the embryo but gradually became localized in the mesophyll precursor cells and cortex during early embryogenesis. Localization of Lhcb mRNA in the mesophyll precursor cells and shoot apical meristem became clear in the early torpedo stage. Expression of Lhcb mRNA was not affected by light during early embryogenesis, but could be induced by light in the torpedo stage, suggesting that light-inducible expression of Lhcb mRNA arises within the torpedo stage. At the late torpedo stage, clear localization of por mRNA started in mesophyll precursor cells of the cotyledon in light-irradiated embryos. Greening potency of the embryo also appeared first at this stage. Therefore, greening and initial differentiation of photosynthetic tissues during somatic embryogenesis seem to be associated with coordinated expression of mRNA for rbcL, Lhcb and por in late torpedo-shaped embryos.Abbreviations DIG Digoxigenin - Lhcb3 Gene encoding a type-III light-harvesting chlorophyll a/b-binding protein of photosystem II - LHCII Light-harvesting chlorophyll a/b-binding protein of photosystem II - POR Protochlorophyllide oxidoreductase - rbcL Gene encoding the large subunit of Rubisco - Rubisco Ribulose-1,5-bisphosphate carboxylase/oxygenase     

Dynamics of morphological and anatomical changes in leaf tissues of an interspecific hybrid of oil palm during acquisition and development of somatic embryogenesis

Oil palm is an economically important plant species due to its high oil production per unit area. Large-scale clonal propagation of the species’s elite specimens is only possible through somatic embryogenesis, although methodology is partially still unknown and insufficiently understood. Current study characterizes in morphological and anatomical terms the acquisition and development stages of somatic embryogenesis of the oil palm’s immature leaves. The respective embryogenic stages were analyzed and characterized: immature leaves (initial explants); leaves with calli formation; leaves which failed to respond to calli formation; leaves with formation of root structures; primary calli; primary calli with differentiation of embryogenic calli; embryogenic calli; pro-embryogenic calli; calli with differentiated somatic embryos; somatic embryos at globular and torpedo stage; and mature fruit zygotic embryos. Cell masses emerged after approximately 60 days of cultivation through the proliferation of cells associated to initial explants´ vascular bundles. Consequently, the formation of two different types of calli was identified, namely, primary and embryogenic, respectively consisting partially and completely of meristematic cell clusters. After 420 days of cultivation, the propagules formed somatic embryos with no connection to source tissues, initially composed (globular stage) of a very characteristic ground meristem and protoderm. After 480 days of cultivation, as the cultures matured (torpedo stage), procambial strands, a structural characteristic also observed in mature zygotic embryos, were reported. The results provide an in-depth understanding of somatic embryogenesis of immature leaves of oil palm. Further, current analysis develops morphological markers at different stages of development obtained during the process.     

Somatic embryogenesis,encapsulation, and plant regeneration of <Emphasis Type="Italic">Rotula aquatica</Emphasis> lour., a rare rhoeophytic woody medicinal plant

Summary Indirect somatic embryogenesis, encapsulation, and plant regeneration was achieved with the rare rhoeophytic woody medicinal plant Rotula aquatica Lour. (Boraginaceae). Friable callus developed from leaf and internode explants on Murashige and Skoog (MS) medium with 0.45 μM 2,4-dichlorophenoxyacetic, acid (2,4-D) was most effective for the induction of somatic embryos. Subculture of the callus onto half-strength MS medium with the same concentration of 2,4-D resulted in highly embryogenic callus. Suspension culture was superior to solid medium culture for somatic embryogenesis. Embryogenic callus.during subsequent transfer to suspension cultures of half-strength MS medium having 0.23 μM 2,4-D induced the highest number of somatic embryos (a mean of 25.6 embryos per 100 mg callus) and the embryos were grown up to the torpedo stage. Transfer of embryos to half-strength MS basal solid medium allowed development, of 50% of the embryos to the cotyledonary stage. Of the cotyledonary embryos, 90% underwent conversion to plantlets on the same medium. Encapsulated cotyledonary embryos exhibited 100% conversion to plantlets. Ninety-five percent of the plantlets established in field conditions survived, and were morphologically identical to the mother plant.     

Plant regeneration protocol of medicinally important <Emphasis Type="Italic">Andrographis paniculata</Emphasis> (Burm. F.) Wallich <Emphasis Type="Italic">ex</Emphasis> Nees via somatic embryogenesis

Summary In vitro propagation of Andrographis paniculata (Burm. f.) Wallich ex Nees through somatic embryogenesis, and influence of 2,4-dichlorophenoxyacetic acid (2,4-1) on induction, maturation, and conversion of somatic embryos were investigated. The concentration of 2,4-D in callus induction medium determined the induction, efficacy of somatic embryogenesis, embryo maturation, and conversion. Friable callus initiated from leaf and internode explants grown on Murashige and Skoog (MS) medium supplemented with 2.26, 4.52, 6.78, and 9.05μM 2,4-D started to form embryos at 135, 105, 150, and 185d, respectively, after explant establishment. Callus initiated at 13.56μM 2,4-D did not induce embryos even after 240 d, whereas those initiated on MS medium with 4.52μM 2,4-D was most favorable for the formation and maturation of somatic embryos. Callus subcultured on the medium with reduced concentration of 2,4-D (2.26μM) became embryogenic. This embryogenic callus gave rise to the highest number of embryos (mean of 312 embryos) after being transferred to half-strength MS basal liquid medium. The embryos were grown only up to the torpedo stage. A higher frequency of embryos developed from callus initiated on 2.26 or 4.52 μM 2,4-D underwent maturation compared to that initiated on higher concentrations of 2.4-D. The addition of 11.7μM silver nitrate to half-strength MS liquid medium resulted in 71% of embryos undergoing maturation, while 83% of embryos developed into plantlets after being transferred to agar inedium with 0.44 μMN⁶-benzyladenine and 1.44 μM gibberellic acid. Most plantlets (88%) survived under field conditions and were morphologically identical to the parent plant.     

Anatomical study of zygotic and somatic embryos of Tilia cordata

A comparative anatomical study was carried out on zygotic and somatic embryos of Tilia cordata Mill. to evaluate the effect of growth conditions on their development. Zygotic embryos (heart-shaped, torpedo, cotyledonary), collected during two autumn periods, were examined to investigate the effect of growing season on embryo development. In comparison, the influence of growth conditions on the development of somatic embryos in vitro was also studied. Treatment with abscisic acid (ABA) and polyethylene glycol-4000 induced the development of somatic cotyledonary embryos similar to zygotic embryos with respect to morphology and anatomy, as illustrated by the differentiation of the apical meristems and of the procambium. The pattern of accumulation of starch and protein was also similar in these embryos. Somatic cotyledonary embryos that developed spontaneously without ABA showed defective accumulation of storage material and a general failure to form the shoot apical meristem, leading to very low germination rates. Vacuolar phenolic deposits were observed along the procambium of both zygotic and somatic embryos regardless of the maturation stage. Tracheid formation was observed only in somatic embryos formed without ABA in the medium and in precociously germinated somatic embryos. Phenolic vacuolar inclusions were frequently observed in epidermal cells of these embryos. This revised version was published online in June 2006 with corrections to the Cover Date.     

ISSR marker‐based detection of genomic stability in Cassia occidentalis L. plantlets derived from somatic embryogenesis

An efficient in vitro technology has been designed for mass multiplication of Cassia occidentalis (coffee senna) through somatic embryogenesis. Genetic stability of both regenerants and mother plant was evaluated. Embryogenic calli were produced on Murashige and Skoog (MS) medium supplemented with 20.0 μM 2,4‐dichlorophenoxy acetic acid (2,4‐D). Induction of somatic embryos occurred after transfer of calli to medium with reduced concentration of 2,4‐D (10.0 μM) fortified with 1.0 μM abscisic acid (ABA). Subculturing of these embryos onto the maturation medium (1.5 μM 6‐benzyladenine + 1.0 μM ABA + 0.3 μM α‐naphthalene acetic acid) favored progression of the embryos through torpedo, heart‐shaped, and cotyledonary stages; one‐half MS medium was considered the best for conversion of cotyledonary stage embryos to young plantlets. The plantlets were acclimatized to autoclaved soil rite, after which they were transferred to the green house. Among the survived plantlets, 10 plants for each primer were randomly selected for inter‐simple sequence repeat (ISSR) analysis. Of the 10 primers tested, 5 produced reproducible and monomorphic bands, 2 led to minor variation with the appearance of unique bands, and the remaining 3 did not show any banding pattern. The majority of the regenerants had similar characteristics to the donor plant, suggesting genetic homogeneity of in vitro raised plants.