Direct and indirect somatic embryogenesis on cotyledon explants of <Emphasis Type="Italic">Quassia amara</Emphasis> L., an antileukaemic drug plant

Summary In vitro propagation of Quassia amara L. (Simaroubaceae) was attempted using mature and juvenile explants. Attempts to establish in vitro culture using leaf and internode explants from a plant more than 15yr old were unsuccessful due to severe phenolic exudation. Plant regeneration through direct and indirect somatic embryogenesis was established from cotyledon explants. Murashige and Skoog (MS) medium with 8.9 μM N⁶-benzyladenine (BA) and 11.7 μM silver nitrate induced the highest number (mean of 32.4 embryos per cotyledon) of somatic embryos. Direct somatic embryogenesis as well as callus formation was observed on medium with BA (8.9–13.3 μM). Semi-mature pale green cotyledons were superior for the induction of somatic embryos. Embryos developed from the adaxial side as well as from the point of excision of the embryonic axis. More embryos were developed on the proximal end compared to mid and distal regions of the cotyledons. Subculture of callus (developed along with the somatic embryos on medium with BA alone) onto medium containing 8.9 μM BA and 11.7 μM silver nitrate produced a mean of 17.1 somatic embryos. Primary somatic embryos cultured on MS medium with 8.9 μM BA and 11.7μM silver nitrate produced a mean of 9.4 secondary somatic embryos. Most of the embryos developed up to early cotyledonary stage. Reduced concentration of BA (2.2 or 4.4 μM) improved maturation and conversion of embryos to plantlets. Ninety percent of the embryos converted to plantlets. The optimized protocol facilitated recovery of 30 plantlets per cotyledon explant within 80d. Plantlets transferred to small cups were subsequently transferred to field conditions with a survival rate of 90%.     

Study of aberrant morphologies and lack of conversion of somatic embryos of chickpea (Cicer arietinum L.)

Summary Somatic embryos which originated from mature embryo axes of the chickpea (Cicer arietinum L.) showed varied morphologies. Embryos were classified based on shape of the embryo and number of cotyledons. “Normal” (zygotic-like) embryos were bipolar structures with two cotyledons and a well-developed shoot and root apical meristem, whereas “aberrant” embryos were horn-shaped, had single and multiple cotyledons, and were fasciated. Histological examination revealed the absence of a shoot apical meristem in horn-shaped embryos. Fasciated embryos showed diaxial fusion of two embryos. Secondary embryogenesis was also observed, in which the embryos emerged from the hypocotyl and cotyledonary region of the primary somatic embryo. This report documents the absence of an apical meristem as a vital factor in the lack of conversion of aberrant somatic embryos.     

Effects of type of explant and age,plant growth regulators and medium strength on somatic embryogenesis and plant regeneration in <Emphasis Type="Italic">Eucalyptus camaldulensis</Emphasis>

Plant regeneration was achieved through direct and indirect somatic embryogenesis in Eucalyptus camaldulensis. Callus was induced from mature zygotic embryos and from cotyledon explants collected from 10, 15, 25, and 30-day-old seedlings cultured on Murashige and Skoog (MS) basal medium supplemented with different concentrations of naphthaleneacetic acid (NAA). Maximum callus induction from mature zygotic embryos was obtained on MS basal medium containing 1 mg l⁻¹ NAA. The frequency of callus development varied based on the age of the cotyledon explants 10-day-old explants giving highest percentage on MS basal medium supplemented with 1 mg l⁻¹ NAA. Callus obtained from mature zygotic embryos gave highest frequency of somatic embryogenesis on MS basal medium containing 0.5 mg l⁻¹ benzyladenine (BA) and 0.1 mg l⁻¹ NAA. Separate age wise culture of the calli, obtained from cotyledons of different ages cultured separately, revealed high somatic embryogenic potential on callus from 10-day-old cotyledons. Direct somatic embryogenesis too was obtained from hypocotyl explants without an intervening callus phase on MS basal medium containing 0.5 mg l⁻¹ BA. The effects of abscisic acid (ABA), sucrose, and different strengths of MS medium on somatic embryo maturation and germination were also investigated. Number of mature somatic embryos increased with lower concentrations (0–1 mg l⁻¹) of ABA while no significant differences were observed at higher concentrations (2–5 mg l⁻¹) of ABA. Compared to basal medium containing lower concentrations of sucrose (1%), the MS medium supplemented with higher levels of sucrose (4%) showed significantly lower frequency of mature somatic embryos. Basal medium without any dilution gave the highest number of immature embryos. However, the number of mature embryos was high at higher medium dilutions.     

Influence of Irradiated Pollen on Embryo and Endosperm Development in Kiwifruit

Development of seeds following pollination with irradiated pollenwas studied inActinidia deliciosa(kiwifruit) ‘Hayward’.Pollinations were carried out using two different sources ofpollen (‘Tomuri’ and ‘Matua’) irradiatedwith gamma rays at doses of 700 and 900 Gy. Non-irradiated crosseswere used as controls. Pollen irradiation had little effectonin vitropollen germination. Irradiated pollen affected seedset and seed content, and induced the formation of parthenogeneticembryos. In comparison to the control, the embryo growth ratewas slower and the endosperm contained very low amounts of storageproducts. Seed set was significantly reduced following bothdoses of irradiation. Two types of seeds were observed: (1)seeds with endosperm only; and (2) seeds with both embryo andendosperm. The proportion of seeds containing endosperm onlywas almost ten-fold higher than those containing both embryoand endosperm. Embryo production by gamma-irradiated pollenwas genotype- and dose-dependent. The induction of parthenogenesiswas higher following gamma ray doses of 900 Gy than 700 Gy,which suggests the ‘Hertwig Effect’; the best efficiencywas obtained with ‘Tomuri’ pollen. Ploidy levelof parthenogenetic embryos was evaluated by nuclear size (area)with the use of image analysis. There was a large differencein embryo nuclei size between control and parthenogenetic embryos(mean size 90.8 and 49.1 µm², respectively). It is concludedthat parthenogenetic embryos represent trihaploids.Copyright1998 Annals of Botany Company. Actinidia deliciosa, kiwifruit, pollen irradiation, induced parthenogenesis.     

Plant regeneration via somatic embryogenesis from in vitro tissue culture in two Tunisian Cucumis melo cultivars Maazoun and Beji

Hypocotyl, cotyledon and zygotic embryo explants from two Tunisian Cucumis melo L. cultivars Beji and Maazoun, cultured on the MS medium added with 2,4-D (0.25–1 mg l⁻¹) and BA (0.10–0.50 mg l⁻¹), produce calluses with somatic embryos after 3 weeks of culture. For Beji c.v. the highest percentage (62.50%) of embryogenesis was observed for cotyledons. The average embryo number per callus was 10.40. Embryogenesis induction for zygotic embryos reached 33.50% with 29 embryos per callus. The embryogenesis ability of hypocotyls did not exceed 12.50% (2.50 embryos per callus). Somatic embryogenesis for Maazoun c.v. explants was less efficient. Embryos formation was observed only for cotyledons (29%) and zygotic embryos (25%). Cotyledonary staged embryos, when transferred to hormone free MS medium, germinated. The maximum germination rates were 51.50 and 44.50%, respectively for Maazoun and Beji c.v. The highest percentage (36.50%) of survival plants was noted for Beji c.v. Regenerants were diploids (2n = 2x = 24) and morphologically similar to their parents issued from seeds.     

Effect of Gibberellin on Mature Euonymus europaeus L. Seeds

The release from dormancy of Euonvmus europaeus L embryos bya brief treatment with GA₃ has been studied During 48 h incubationof dormant embryos in GA-free medium, phospholipid levels increasedat first, then declined sharply over the last 6 h When the embryoswere placed in GA₃ medium during this 6 h period levels of totalphospholipids as well as of phosphatidylethanolamine increasedwhilst phosphatidylinositol and phosphatidylcholine declinedslightly Fine structural changes stimulated by a brief GA₃ treatmentwere of different character depending on tissue region (1) ‘destructive’changes occurred in the superficial procortical parenchyma onthe hypocotyl/radicle boundary, involving autolysis and decompartmentationof organelles, (2) ‘positive’ changes occurred inregions close to root and shoot apical meristems, involvingdegradation of protein bodies and their conversion into vacuoles,and the proliferation of various organelles A number of differenceswere noted when the changes in GA₃-treated embryos were comparedwith those induced by low temperature, which also overcomesdormancy The results suggest that germination is accompaniedby different cytological events depending on whether it is inducedby cold or GA₃ The growth of embryos in which dormancy was overcomeby GA3 was due to the activation of the apical root meristemclose to the quiescent centre, whilst in embryos in which germinationwas induced by low temperature, the periphery of hypotocotyl/radicleboundary was the site of activation Euonymus europaeus L, dormant embryo, fine structure, phospholipids, GA₃ and cold treatments     

Transient Expression of Exogenous DNA in Intact, Viable Wheat Embryos Following Particle Bombardment

Expression of foreign DNA has been detected in intact, germinatingwheat embryos (Triticum aestivum L.) following bombardment withtungsten particles complexed with a reporter gene encoding thebacterial enzyme ß-glucuronidase (‘GUS’:E.C.3.2.1.31). Expression was detected in situ in individualcells and groups of cells, by supplying the germinating embryoswith the chromogenic substrate of the GUS enzyme, ‘X-gluc’.Expression was dependent on the presence of a constitutive plantpromoter, the Cauliflower Mosaic Virus ‘35S’ promoter,fused to the GUS structural coding sequence. The relative simplicityof this technique recommends its future use for the assay ofregulatory elements which control the spatial and temporal specificityof genes expressed during embryo development. Key words: Transient expression, particle bombardment, wheat embryo, Triticum aestivum L.     

Vibrating probe measurement of ionic currents around developing embryo of oil palm (Elaeis guineensis Jacq.)

The current patterns around zygotic embryos of the oil palmembryo (Elaeis guineensis Jacq.) were investigated. Ionic currentsaround the embryo were measured at different stages in its developmentusing a two-dimensional vibrating probe system. The averagecurrent density was found to be in the order of 1–µAcm^–2. In the early developmental stage, the current wasfound entering the cotyledon and leaving the radicle. Both themagnitude and direction of this current changed when embryosat different developmental stages were observed. The currentswere found to enter the region in the process of differentiationor elongation, and in some cases this could be the reverse ofthe current direction in the preceding stage. Within one ofthe stages of the embryos, current density was correlated withthe rate of elongation. A very large inward current (up to 20µAcm^–2) was also detected at the point where the shoot wasbreaking through the surface. This was probably caused by theso-called ‘injury current’. Key words: Ionic currents, vibrating probe, oil palm embryo, E. guineensis, injury current     

Direct somatic embryogenesis and plant regeneration from immature explants of chickpea

A protocol for plant regeneration via somatic embryogenesis was developed in two chickpea (Cicer arietinum L.) cultivars ICCV-10 and Annigeri. Somatic embryos were induced from immature cotyledons on Murashige and Skoog’s (MS) medium supplemented with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), α-naphthaleneacetic acid (NAA) and picloram alone or in combination with 0.5 — 2.0 mg dm⁻³ N⁶-benzylaminopurine (BA) or kinetin (KIN). NAA was better for somatic embryo induction compared to other auxins. The well formed, cotyledonary shaped embryos germinated into plantlets with 36.6 % frequency on MS medium supplemented with 2.0 mg dm⁻³ BA + 0.5 mg dm⁻³ abscisic acid (ABA). The frequency of embryogenesis and plantlet regeneration was higher in cv. ICCV-10 as compared to cv. Annigeri. Regenerated plants were transferred to soil (40 % survival) and grown to maturity. Histological studies of explants at various developmental stages of somatic embryogenesis reveled that somatic embryos developed directly from the cotyledon cells and they were single cell origin.     

High-efficiency plant production via direct somatic single embryogenesis from preplasmolysed cotyledons of Panax ginseng and possible dormancy of somatic embryos

Cotyledon explants of immature ginseng zygotic embryos cultured on Murashige and Skoog medium lacking growth regulators formed somatic embryos directly, most in a multiple state, fused together and to the parent cotyledon explants. When the cotyledon explants of ginseng were pretreated with 1.0 m sucrose for 24–72 h, all the somatic embryos developed individually from all surfaces of the cotyledons and the number of somatic embryos per explant was enhanced fourfold. Histological observation revealed that all the single somatic embryos from preplasmolysed cotyledons originated from epidermal single cells, whereas all the multiple embryos from cotyledons without pretreatment originated from epidermal and subepidermal cell masses. When the somatic embryos matured to the cotyledonary stage, further growth ceased and they remained white, probably indicating dormancy. Gibberellic acid (GA₃) (over 1.0 mg/l) or chilling treatment (–2°C for over 8 weeks) were prerequisites for the germination of somatic embryos. Ultrastructural observation revealed that the cotyledon cells of somatic embryos without chilling or GA₃ treatment contained numerous lipid reserves, dense cytoplasm, proplastids and non-activated mitochondria, whereas the cotyledon cells of somatic embryos after chilling or GA₃ treatment were highly vacuolated and contained well-developed chloroplasts and active-state mitochondria enclosing numerous cristae, indicating that in-vitro-developed somatic embryos of P. ginseng may be dormant after maturing in a manner similar to zygotic embryos. Received: 8 July 1998 / Revision received: 31 August 1998 / Accepted: 23 September 1998     

Developmental and structural aspects of somatic embryos formed on medium containing 2,3,5-triiodobenzoic acid

Cotyledon explants of ginseng (Panax ginseng C.A. Meyer) zygotic embryos produced somatic embryos at a high rate (68%) on medium without any growth regulators. Under this culture condition, apparent polar somatic embryogenesis occurred near the basal-excised portion of the cotyledons. When the cotyledon explants were cultured on medium containing 2,3,5-triiodobenzoic acid (TIBA), an auxin polar-transport inhibitor, the frequency of somatic embryo formation markedly decreased and was completely inhibited on medium containing 20 μM TIBA. On medium containing 5–10 μM, somatic embryos developed sporadically on the surface of the cotyledons and had a normal embryo axis but jar-shaped cotyledons. Embryos with jar-shaped cotyledons were also observed to occur at a high frequency when the early globular embryos formed on hormone-free medium were transferred to medium containing 20 μM TIBA. From these results, it was deduced that endogenous auxin in the cotyledon explants plays an important role in the induction of somatic embryos and that the cotyledon development in somatic embryos is also related to the polar transport of endogenous auxin. Received: 11 October 1996 / Revised version received: 8 January 1997 / Accepted: 26 January 1997     

Screening of soybean, Glycine max L.) Merrill, lines for somatic embryo induction and maturation capability from immature cotyledons

Summary Seventeen breeding lines of soybean, Glycine max (L.) Merrill, and cv. Jack, from relative maturity groups 0.3–7.5 were assessed for their ability to undergo somatic embryogenesis. The goal of this study was to determine which lines had high embryogenic capacity. We also sought to understand the relationship between relative maturity and embryogenesis. Embryos from immature cotyledons were initiated on solid MS medium with varying levels of 2,4-dichlorophenoxyacetic acid (2,4-D). Qualitative and quantitative measures of initiation, proliferation, differentiation, and maturation were recorded. The breeding lines differed significantly with respect to percent induction, number of embryos induced, and quality of induced embryos. After 1 mo, of proliferation, two early maturing lines, the control, Jack, and NK-5, had the best overall performance. High percent response of proliferating embryos was positively associated with lower maturity groups. Relatively high concentrations of 2,4-D (compared with that used in prolifcrating medium, e.g., 226 μM; 50 mg l⁻¹) in the initiating medium reduced numbers of embryo clusters per cotyledon initiated and percent initiation, and the concentration of 2,4-D affected the proliferation of somatic embryos in a breeding line-dependent manner. The breeding lines differed significantly in the time to produce mature somatic embryos. There was a positive correlation between immature embryo quality and number of differentiated somatic embryos produced. Retired.     

Requirement of cotyledons for gibberellic acid-induced hypocotyl elongation in lettuce seedlings. Isolation of the cotyledon factor active in enhancing the effect of gibberellic acid

The role of cotyledons in hypocotyl elongation caused by gibberellicacid was studied using young seedlings of lettuce, Lactuca saliva,var. ‘Grand Rapids’. Removal of cotyledons fromintact seedlings resulted in a depression of hypocotyl elongationcaused by gibberellic acid. Gibberellic acid-induced hypocotylelongation in decotylized seedlings, was however, substantiallyenhanced by incubating the seedlings together with excised cotyledons.The exudate from excised cotyledons also enhanced the effectof gibberellic acid on hypocotyl elongation in decotylized seedlings.This active principle (named the cotyledon factor) in the cotyledonexudate was stable against heating at 100?C for 15 min, permeatedthe dialysis membrane, and was extractable with ethyl acetate.Biological activity of the cotyledon factor was not replacedby indole-3-acetic acid, kinetin, cyclic AMP, vitamins, sucroseor inorganic nutrients. The biological significance of the cotyledonfactor is discussed in relation to the action of gibberellicacid. (Received February 14, 1973; )     

Factors affecting plantlet regeneration from <Emphasis Type="Italic">in vitro</Emphasis> cultured immature embryos and cotyledons of <Emphasis Type="Italic">Prunus mume</Emphasis> “Xue mei”

Using immature embryos and cotyledons as explants, a successful system to culture immature embryos and induce direct regeneration from cotyledons was established for Prunus mume “Xuemei”. For immature embryo culture, a high frequency of plantlet formation (89.5%) from the embryonic axis was obtained using half-strength Murashige and Skoog (1/2 MS) medium supplemented with 13.2 μM 6-benzyladenine (BA) and 2.7 μM 1-naphthaleneacetic (NAA). Shoots formed directly from cotyledons with the embryo axis intact when explants were cultured on 1/2 MS medium containing 2.2 μM BA with different combinations of NAA (2.7, 5.4 μM) and indole-3-butyric acid (IBA) (0, 2.5, 5.0 μM). Better results were achieved when the embryonic axis was removed from the cotyledons and cultured on 1/2 MS medium supplement with 13.2 μM BA, 2.7 μM NAA or 2.2 μM BA, 2.2 μM thidiazuron (TDZ), and 2.7 μM NAA, respectively. Regenerated shoots were successfully rooted on 1/2 MS or Woody Plant medium (WPM) supplemented with 2.5–5.0 μM IBA. The effect of the embryonic axis, BA, and TDZ on cotyledon regeneration was investigated in detail. Rooted plantlets were transferred to soil successfully.     

Expression of gus in somatic embryo cultures of black spruce after microprojectile bombardment

Immature embryos (stage I) and cotyledonary somatic embryos(stage III) of black spruce [Picea mariana (Mill) B.S.P.] werebombarded with tungsten particles coated with a gene constructcontaining the fusion of gus:: nptll. GUS (ß-glucuronidase)activity was monitored histochemically with X-gluc giving ablue colour where transient gene expression was detected inthe bombarded tissues. A high transient expression of gus wasobserved in stage I embryo cultures 2 d after bombardment (202GUS foci per 300 mg tissue). GUS activity had substantiallydiminished in this material 14 d after bombardment, when grownin liquid LP maintenance medium containing BA (4.4µM),2,4-D (9µM) and 1% sucrose. However, when stage I embryoswere cultured on LP maturation medium containing BA (40 µM),IBA (1 µM), 3.4% sucrose and 0.8% agar, GUS activity after2 d was 335 GUS foci per 300 mg tissue, and the activity wasdetected until 30 d after bombardment. With stage III somaticembryos cultured on LP maintenance medium, 92% showed GUS activity2d after bombardment (16 GUS foci per embryo), and 31 % showedactivity 30 d after bombardment (4 GUS foci per embryo). GUSactivity was still evident in 12% of the embryos (2 GUS fociper embryo) 45 d after bombardment. Key words: Black spruce, gus = E. coli geneuid A encoding ß-glucuronidase, nptll = gene encoding neomycin phos-photransferase, somatic embryos     

Shoot Organogenesis from Immature Zygotic Embryo Cultures of Ginkgo biloba

Immature zygotic embryos were cultured on Murashige and Skoog's medium (MS) supplemented with various combinations of 2,4-dichlorophenoxyacetic acid (2,4-D), naphthaleneacetic acid (NAA), benzyladenine (BA) and zeatin or with various concentrations of 2,4-D alone. The maximum number (8 per embryo) of adventitious buds formed from cotyledons of heart stage embryos cultured on MS medium with 1 mg dm⁻³ BA and 0.01 mg dm⁻³ NAA. The adventitious buds originated from procambial strands of immature embryo cotyledons and then developed into adventitious bud primordia within 20 d. Adventitious buds transferred to hormone free MS medium grew into shoots, but did not produce plantlets because the shoots failed to root. This revised version was published online in July 2006 with corrections to the Cover Date.     

Somatic embryogenesis in soybean via somatic embryo cycling

Summary The objectives of the present research were: a) to develop an efficient soybean embryogenic regeneration system characterized by a high frequency of explant response and a large number of somatic embryos per explant; b) to evaluate the factors affecting somatic embryogenesis via somatic embryo cycling; and c) to identify the origin of somatic embryos in the system. A highly improved and efficient system for soybean somatic embryogenesis was established using somatic embryo cotyledons and somatic embryo hypocotyl/radicle explants plated on α-naphthaleneacetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-D) supplemented MS basal media. The system included somatic embryo cycling between liquid and solid medium and it consistently gave rise to a much higher frequency of explant response and a larger number of embryos per responding explant than those obtained from zygotic cotyledon explant tissues. Genotype, differences were observed for response in some of the treatments with cv “Fayette” being more responsive than “J103”. Histological studies revealed that somatic embryos induced in the somatic embryo cycling system originated almost exclusively from epidermal cells on both 2,4-D and NAA inductive media. The cells of the epidermis proliferated to produce somatic embryos directly without an intervening callus phase. A single-cell origin of somatic embryos was observed in cultures on a 40 mg/liter 2,4-D treatment. A large number of responding cells in the epidermis was also observed in the 10 mg/liter NAA treatment. The single-cell origin of somatic embryos from epidermal layers of the explant tissues should facilitate development of an efficient transformation system for soybean.     

Cotton somatic embryo morphology affects its conversion to plant

Somatic embryos differentiated from hypocotyl explant in cotton (Gossypium hirsutum L.) exhibited very divergent morphologies. Six different types of somatic embryos based on cotyledon development were observed. The growth hormones (2,4-dichlorophenoxyacetic acid and kinetin) used in induction and maintenance media did not affect embryo rooting and germination. The 95 % conversion of normal embryos (with two cotyledons) was achieved, while an overall conversion was only 38 %. Horn shaped embryos failed to exhibit shoot growth. Poorly developed apical meristems were responsible for lower conversion percentages in some of embryo classes. However, regenerated plants phenotypically resembled to seed grown control plants regardless of somatic embryo morphology.     

Somatic embryogenesis from callus cultures of <Emphasis Type="Italic">Terminalia chebula</Emphasis> Retz.: an important medicinal tree

Somatic embryogenesis was obtained from cotyledon and mature zygotic embryo callus cultures of Terminalia chebula Retz. Callus cultures of cotyledon and mature zygotic embryo were initiated on induction medium containing Murashige and Skoog (MS) nutrients with 1.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) either 0.01 or 0.1 mg/l Kinetin and 30 g/l sucrose. Induction of somatic embryogenesis, proliferation and development was obtained through different culture passages. Embryogenic cotyledon callus with globular somatic embryos was obtained on MS basal medium supplemented with 50 g/l sucrose. Globular somatic embryos were observed from mature zygotic embryo callus on induction medium. Different stages of somatic embryo development from cotyledon and mature zygotic embryo calluses were observed on MS basal medium supplemented with 50 g/l sucrose after 4 weeks of culture. Histological studies have revealed the developmental stages of somatic embryos. A maximum of 40.3±1.45 cotyledonary somatic embryos/callus was obtained from mature zygotic embryo compared to 7.70±0.37 cotyledonary somatic embryos/callus initiated from cotyledons. Germination of somatic embryos and conversion to plants were achieved. Highest frequency of germination (46.66±0.88) of somatic embryos was obtained on MS basal medium containing benzyladenine (0.5 mg/l) with 30 g/l sucrose.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration through somatic embryogenesis and organogenesis using cotyledons of <Emphasis Type="Italic">Cassia angustifolia</Emphasis> Vahl

In vitro regeneration through somatic embryogenesis as well as organogenesis using cotyledon of a woody medicinal legume, Cassia angustifolia is reported. The cotyledons dissected from semi-mature seeds, if inoculated on Murashige and Skoog’s medium (MS) supplemented with auxin alone or in combination with cytokinin, produced direct and indirect somatic embryos. A maximum of 14.36 ± 2.26 somatic embryos per 20 mg of explants including callus were produced in 70% cultures on MS medium with 2.5 μM benzyladenine (BA) + 10 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Although the percentage of embryogenic cultures was higher (83.33%) at 10 μM 2,4-D + 1 μM BA, the average number of somatic embryos was much less (7.6 ± 0.85) at this level, whereas at 2.5 μM BA and 5 μM 2,4-D, there was a simultaneous formation of both somatic embryos and shoots. The somatic embryos, although started germinating on the same medium, developed into full plantlets only if transferred to MS basal with 2% sucrose. Cytokinins alone did not induce somatic embryogenesis, but formed multiple shoots. Five micromolar BA proved optimum for recurrently inducing shoots in the competent callus with a maximum average of 12.04 ± 2.10 shoots and shoot length of 2.26 ± 0.03 cm. Nearly 91.6% shoots (2–2.5 cm in size) organized an average of 5.12 ± 0.58 roots on half strength MS + 10 μM indole-3-butyric acid. All the plantlets have been transferred successfully to soil. Types of auxin and its interaction with cytokinin significantly influenced somatic embryogenesis.