In vitro culture promotes partial autonomous endosperm development in unfertilized ovules of wild-type Arabidopsis thaliana var. Columbia

Partial endosperm development without paternal genome involvement was induced in unpollinated ovaries of wild-type Arabidopsis thaliana cultured in vitro. Unpollinated pistils were cultured on hormone-free Murashige and Skoog (MS) medium with addition of 6% sucrose and supplemented with: benzylaminopurine (BAP; 2 mg l^–1) combined with naphthylacetic acid (NAA; 0.1 mg l^–1), 2,4-dichlorophenoxyacetic acid (2,4-D; explants exposed to 1-h auxin shock 20 or 40 mg l^–1, and transferred to hormone-free MS medium). Initiation of autonomous endosperm (AE) development was induced on all media used in 54 ovules from 39 cultured ovaries (26%), with an average frequency of 1.4 ovules/ovary. The highest frequency of partial endosperm formation occurred on media combining the two growth regulators BAP and NAA (59% of ovaries had ovules with AE), although endosperm development was also induced on hormone-free medium (in 20.5% of ovaries). The number of AE nuclei ranged from 2 to ~50, depending on the day of culture and medium; neither cellularization nor differentiation on specific regions typical for endosperm of wild-type Arabidopsis, were noted. Fertilization independent endosperm most probably originated from the secondary nucleus, but involvement of the polar nuclei could not be excluded, as indicated by nuclear size and structure. In vitro conditions did not influence egg cell proliferation. Gynogenic embryos were observed neither in the ovules with autonomous endosperm nuclei nor in ovules without endosperm induction.     

Induction of autonomous endosperm in Lupinus luteus,Helleborus niger and Melandrium album by in vitro culture of unpollinated ovaries

Autonomous division of the endosperm was induced by in vitro culture of unpollinated ovaries or placenta-attached ovules in Helleborus niger, Lupinus luteus and Melandrium album. The induction frequencies for the three species were 50%, 10–20% and 0.1%, respectively. The endosperms contained up to 20 free nuclei; only a few ovules with 80–420 endosperm nuclei were found. Induction of autonomous division of the endosperm, which is unusual in amphimictic plants, was observed in three new species. No embryos appeared in the ovules. This suggests a developmental independence of the endosperm from the embryo in the culture of unpollinated ovaries or ovules.     

Pseudogamous embryogenesis and the degree of parthenogenesis inAllium tuberosum

Summary Early stages of egg embryony and antipodal embryony in ovules from both pollinated and unpollinated flowers ofAllium tuberosum, a diplosporous apomict, were observed. Whether flowers were pollinated or not, autonomous egg and antipodal embryonies occurred at nearly equal frequencies and progressed almost synchronously for several days. An electrophoretic analysis of endosperm esterase demonstrated the fertilization of polar nuclei. It was thus confirmed thatA. tuberosum is a pseudogamous apomict. The degree of parthenogenesis, calculated as the percentage of ovules in which egg cells showed autonomous embryonic development, ranged between 62% and 94% among six cultivars and was nearly equal to the degree of diplospory previously reported. The present results show that most unreduced egg cells and egg-like antipodal cells ofA. tuberosum have the ability to start embryogenesis independently of pollination.     

百合未授粉子房离体培养胚胎形成及植株再生

未受精子房离体培养是诱导雌核产生单倍体的技术之一。以1个野生种和3个杂种系共7个百合(Lilium)基因型为实验材料, 探讨了基础培养基、花蕾取样时期和外源激素等因素对百合未授粉子房离体培养胚胎形成的影响。结果表明, CBM、MS和BDS三种基础培养基均可诱导百合未授粉子房胚胎形成, 但以BDS培养基诱导效果最佳; 开花前1天的花蕾较适于百合未授粉子房离体培养; 2 mg·L^-1 2,4-D + 2 mg·L^-1 6-BA和2 mg·L^-1 2,4-D + 2 mg·L^-1 KT两种外源激素配方均适用于百合未授粉子房离体培养诱导胚胎形成。在培养过程中, 大多数胚性胚珠中只含有1个胚胎, 位于珠孔端、合子端或极核处, 少数胚性胚珠中含有双胚胎。通过百合未授粉子房离体培养, 从5个基因型材料中共获得146株再生植株。采用根尖染色体计数法对其中的62株进行了倍性测定, 其中43株与母体植株染色体倍性不同。     

An ultrastructural study of early endosperm development and synergid changes in unfertilized cotton ovules

Excised, unfertilized cotton (Gossypium hirsutum L.) ovules were cultured for 1–5 days postanthesis and embryo-sac development was studied with the electron microscope. In some ovules the two polar nuclei fuse and the diploid endosperm nucleus goes through a limited number of free nuclear divisions after 2–3 days in culture. Each nucleus has two nucleoli, in contrast to nuclei of fertilized triploid endosperm which have three nucleoli. Precocious cell walls form between the endosperm nuclei on the 3rd day in culture. The morphology of the plastids, mitochondria, rough endoplasmic reticulum (RER), dictyosomes and microbodies, and the amount of starch and lipid in the diploid cellular endosperm are similar to those of the central cell. A few large helical polysomes appear close to plastids and mitochondria. After 2 days in culture, one of the two synergids in the unfertilized cultured ovules shows degenerative changes which in fertilized ovules are associated with the presence of the pollen tube, i.e., increase in electron density, collapse of vacuoles, irregular darkening and thickening of mitochondrial and plastid membranes, disappearance of the plasmalemma and the membranes of the plasmalemma and the membranes of the RER. The second synergid remains unchanged in appearance. The egg cell does not shrink or divide or show structural changes characteristic of the cotton zygote. Embryo-sac development is arrested on the 4th and 5th days in culture. The nucellus continues growth and at 14 days crushes the degenerate embryo sac.     

Plant regeneration from ryegrass ovules cultivated on endosperm-derived feeder cells

An efficient method for the regeneration of zygote-derived plants via ovule culture is desirable for overcoming postzygotic cross incompatibility as well as for the development of certain methods for genetic manipulation. High-frequency plantlet regeneration from ovules of Italian ryegrass (Lolium multiflorum Lam.) and a hybrid Italian/perennial ryegrass excised 1 to 4 days post pollination was obtained by culture on endosperm-derived feeder cells. Ovules excised 3 or 4 days after anthesis and grown on feeder cells generally regenerated about twice as frequently as ovules grown directly on nutrient medium. In one of the genotypes tested, ovules excised 1, 2 and 3 days post pollination developed into plantlets at percentages of 38.1, 52.0 and 52.8, respectively, using the feeder-cell system.Abbreviations EM endosperm multiplications - OC ovule culture - R regeneration - 2,4-d 2,4-dichlorophenoxyacetic acid     

Embryological study on gynogenesis in onion (Allium cepa L.)

Haploid induction in onion can, to date, be induced only via gynogenesis by culturing unfertilized flowers, ovaries or ovules. The process of haploid embryo induction has been macroscopically well studied, but only limited data exist from microscopic examination of ovule development status at the inoculation stage and of the origin of gynogenic embryos. Microscopic studies were carried out using individual donor plants with relatively high embryo induction frequencies (45.9 embryos formed per 100 flowers, on average, for 2 years). Ovaries from flower bud culture were fixed at 1 week intervals up to the 7th week of culture. These were compared with pollinated ovaries at 1 or 2 weeks after pollination. In total, 1428 unfertilized embryo sacs were examined. The results indicate that, at the time of inoculation, ovules within ovaries 2.0–3.0 mm in diameter contained two- or four-nucleate embryo sacs in the smallest ovaries to mature embryo sacs in the largest ovaries. It seems likely that the embryos are actually induced from ovaries cultured at the immature stage. After 1 or 2 weeks in culture, the egg apparatus primarily consisted of distinctly enlarged synergids and the egg cell, which was often detached from the micropylar pole. But free nuclear endosperm was also formed. From the 2nd to 7th week in culture, formation of haploid embryos (from globular to the almost mature cylindrical stage) was detected in 5.7% of the ovules. Their origin, for several reasons, was most likely the egg cell. In addition, ovules containing endosperm only (3.6%) and ovules containing the egg apparatus (0.5%) or both endosperm and embryo (0.4%) were detected. This observation is probably unique and has not yet been reported in other species studied. Received: February 2001 / Revision accepted: 20 April 2001     

The development of onion (<Emphasis Type="Italic">Allium cepa</Emphasis> L.) Embryo sacs <Emphasis Type="Italic">in vitro</Emphasis> and gynogenesis induction in relation to flower size

Summary The development of embryo sacs (ES) in vitro and induction of gynogenesis were studied in onion flower bud culture. Explants were divided into three groups according to their size at inoculation: (a) small flower buds (2.3–3.0 mm in diameter); (b) medium flower buds (3.1–3.7 mm); and (c) large flower buds (3.8–4.4 mm). For histological study, excised ovaries were fixed at inoculation and then at 3-d intervals until day 12, and after 2 and 3 wk of culture. Some explants were cultured until embryo emergence, i.e., 3–5 mo. In total, 2592 ovules were examined histologically. At inoculation, 83% of ovules in small flower buds contained a megaspore mother cell; in 17% of ovules, two-nucleate ES occurred. In medium flower buds two-nucleate, four-nucleate, and mature ES were present at frequencies of 15%, 46%, and 40%, respectively. In large flower buds, only mature ES occurred. In vitro conditions did not disturb meiosis and megagametophyte development in non-degenerated ovules. Regardless of the developmental stage at inoculation, only mature ES occurred on day 12. Gynogenic embryos were found after 2 wk of culture, indicating that embryos developed in mature ES exclusively. Embryos were detected in 5.4% of histological studied ovules; however, the number of embryos after 3–5 mo. was higher (12.4%). The parthenogenetic origin of the embryos is discussed. In addition, ES containing endosperm only (6.5%) and both endosperm and embryo (0.4%) were observed.     

Regeneration of tepals,stamens and ovules in explants from perianth of Hyacinthus orientalis L. importance of explant age and exogenous hormones

Regeneration of tepals, stamens and ovules from perianth explants of Hyacinthus orientalis L. in different developmental stages could be controlled by means of exogenous hormones. Perianth explants in a relatively early stage of development were competent for differentiation of tepals on Murashige and Skoog (MS) medium supplemented with 2 mg·1^-1 N⁶-benzylaminopurine (BAP) or zeatin and 0.1 mg·1^-1 2,4-dichlorophenoxyacetic acid (2,4-D). Perianth explants in a later stage of development regenerated stamens and ovules, and marked difference was observed in the activity of BAP and zeatin in this regard. Zeatin stimulated more strongly stamen formation, while BAP enhanced ovule formation. Thus, stamens were formed when the explants were cultured for four months on medium with 2 mg·1^-1 BAP and 0.1 mg·1^-1 2,4-D and then transferred to medium with 0.2 mg·1^-1 zeatin and 0.005 mg·1^-1 1-naphthaleneacetic acid. On the other hand, differentiation of ovules occured in explants cultured for two weeks on the former medium and then transferred to medium with 0.1 mg·1^-1 BAP and 0.01 mg·1^-1 2,4-D. Although ovule formation could also be obtained with 2 mg·1^-1 BAP alone, it was substantially enhanced by the presence of 0.1 mg·1^-1 2,4-D in the medium in the early stages of culture. The results demonstrate the importance of both the developmental stage of the source organ from which explants are excised and of the hormone composition of the medium for the regeneration of different floral organs by perianth explants of Hyacinthus.Abbreviations BAP N⁶-benzylaminopurine - 2,4-D dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid     

Plant regeneration through somatic embryogenesis in Quercus suber

Cork oak ( Quercus suber L.) zygotic embryos, endosperm and ovules were treated with different concentrations of 2,4-D for induction of somatic embryos. Plant material was collected during the embryo development season, from June to September. Immature embryos proved to be the most reactive initial explant. Callus and somatic embryos developed a few weeks after the beginning of the 2,4-D treatment. For embryo development experiments, different growth regulators and cold and desiccation treatments were tested. Cold storage of somatic embryos matured in vitro at 5°C was the best treatment for breaking dormancy.     

Development of fertilized ovules and their role in the growth of the pea pod

The histological development of fertilized ovules during fruit-set and development in pea ( Pisum sativum L. cv. Alaska) has been investigated. Killing the ovules on day 0 (anthesis) or day 1 prevented fruit-set and resulted in ovary degeneration. When the ovules were destroyed at later stages the ovaries developed, though the rate of growth of the pod was reduced significantly. Pollination in pea occurs normally the day before anthesis, and fertilization of the egg cell 32 to 48 h later. The first divisions of the zygote and endosperm nuclei started simultaneously (ca 48 h after pollination) but the endosperm developed more rapidly than the embryo; the embryo sac cavity was lined with free endosperm nuclei at the time of beginning suspensor elongation. Extracts of endosperm and ovule coats from ovules at day 7 after anthesis showed fruit-set activity in pea, the latter material having about 3 times more activity than the former per ovule basis. These results indicate that fertilization of the ovule is necessary for fruit-set in pea, and that compounds which induce fruit-set are probably synthesized in the ovules following fertilization.     

Endosperm response to pollen irradiation in kiwifruit

 Cytological details of endosperm development after pollination with irradiated pollen were studied in Actinidia deliciosa (kiwifruit) cultivar Hayward. Pollinations were carried out involving five different sources of pollen (Matua, Tomuri, Burt, Berryman, and fruiting male) irradiated with gamma rays at doses of 700 and 900 Gy. Non-irradiated crosses were used as controls. Irradiated pollen induced development of approximately 25–30% of the ovules. Two types of ovules were observed: (1) with both embryo and endosperm and (2) with endosperm only. No mitotic abnormalities were found in control or irradiated endosperms. Mitotic divisions were regular and nuclei spherical and evenly spaced. However, the cells of irradiated endosperm usually contained low amounts of storage products. Ploidy level of the endosperms was evaluated by nuclear size (volume) with the use of image analyzis. Mean nuclear size in control and irradiated endosperms was 1598.3 and 750.9 μm³, respectively. It is concluded that endosperm produced after pollination with irradiated pollen is autonomous and represents the 2n level. Received: 14 October 1998 / Revision accepted: 10 March 1999     

Developmental events in ovules of the ornamental plant Rudbeckia bicolor Nutt

Microscopic observations of R. bicolor ovules showed that tetrasporic embryo sacs of Fritillaria type are formed. In the mature female gametophytes modifications in antipodal cell formation and egg apparatus organization were observed, e.g. morphological resemblance was evident of antipode or synergid to the egg cell. In the central cell cytoplasm of the mature gametophytes the presence of small bodies was a characteristic feature. Development of both embryo and endosperm was observed in ∼73% of ovules at the embryo stage, while retarded or arrested development of the endosperm was found in ∼26% of them. Occasionally, two embryos occurred in the embryo sac. This is the first record of polyembryony in this species. Although hemigamy has been previously described in Rudbeckia bicolor Nutt., in the present investigations mosaic structure of embryos was not detected. Measurements of the C-DNA amount (flow cytometry) revealed embryo nuclei with 2C DNA content only, and endosperm nuclei with 5C DNA content in the mature seeds. No peak corresponding to 1C nuclei was detected in the histograms obtained from the nuclear preparation of seeds or seedling parts. These results suggest that hemigamy is not an obligatory phenomenon in R. bicolor. The mean 2C DNA value was determined as 14.51 pg (the first estimation for this species).     

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.     

Somatic embryogenesis from immature peach palm inflorescence explants: towards development of an efficient protocol

Various factors affect the induction of somatic embryogenesis in peach palm (Bactris gasipaes Kunth). Among these, both the type and level of auxins had the greatest influence on in vitro responses, although the genotype and the developmental stage of the explants also influenced results. Younger inflorescences were more competent to respond to SE induction than more mature inflorescences and the use of a pre-treatment with 2,4-D (200 μM) in liquid MS culture medium also increased the embryogenic capacity, and diminished the development of flower buds. Higher oxidation rates were observed in explants maintained on 2,4-D-supplemented culture medium, while on 300 μM or 600 μM Picloram and Dicamba lower oxidation rates were observed. The progression from floral meristem to flower bud occurred at high frequency when low concentrations of auxins were used, independent of the type. Higher concentrations of Picloram or Dicamba reduced or even inhibited flower bud development. Picloram also enhanced the embryogenic induction rate more than 2,4-D and Dicamba, and among the concentrations evaluated 300 μM Picloram enhanced induction for both genotypes, with significant differences between genotypes. The best combination of variables used the least mature inflorescence (Infl1) from genotype I with the 2,4-D pre-treatment and 300 μM Picloram to generate 5 embryogenic calli from 18 explants; 26 embryos were obtained on average from each embryogenic callus. From these, eighteen embryos converted to plantlets and six of these survived transfer to the greenhouse.     

COORDINATED TIMETABLES FOR MEGAGAMETOPHYTE DEVELOPMENT AND POLLEN TUBE GROWTH IN RHODODENDRON NUTTALLII FROM ANTHESIS TO EARLY POSTFERTILIZATION

Rhododendron nuttallii T. W. Booth (Ericaceae) was used to derive concurrent timetables for megagametophyte, pollen tube and early postfertilization development from anthesis through 3 wk after pollination, based on timed collections of self-pollinated pistils. Stages of development were determined for over 33,500 cleared ovules, including, for selected collection dates, stages on different portions of the placenta. Pollen tube information was obtained by fluorescence microscopy of pistil squashes stained with aniline blue. Because of the very large number of ovules observed, it was possible to recognize a much more closely graded series of stages in megagametophyte development than is usually the case. While a range of stages occurred on all days, development progressed steadily from a majority of functional spores and 2-nucleate gametophytes on the day of anthesis to mostly a late zygote-primary endosperm stage at 18 days, and some 2-celled endosperm stages at 21 days, after pollination. At all times the most advanced stages, including first pollen tube entries, occurred on the outer surface of the lower half of the placenta, and the youngest on the inner surface of the uppermost portion. Fertilizable ovules were not found in any frequency until 8 days after pollination (then in only about 34% of the ovules); a few fertilized ones were seen after 10 days but constituted less than 5% until 12 days after pollination, thereafter increasing to about 60%. Fertilization occurred in any one of three morphologically recognizable stages distinguished by position and state of fusion of polar nuclei. Pollen germinated on the stigma 1–2 hr after pollination, and pollen tubes grew at a rate of about 1–1.25 cm/day, reaching the top of the ovary in 8–9 days with the first ovule entries seen after 10 days. There was a close correlation between megagametophyte development and pollen tube growth, with large numbers of functionally mature ovules not being found until pollen tubes had reached the ovary. While nuclei within ovules could not be distinguished in the squashes, three gametophyte stages that could be recognized—unelongated, elongated either without or with a pollen tube—were tallied for almost 29,000 ovules. The progression in these general stages corresponded well with that documented in more detail from cleared ovules. Unpollinated pistils showed a similar progression of gametophyte stages until the time fertilization would start to occur, after which there was continued accumulation of functionally mature ovules. A variety of abnormally developed and/or collapsing(ed) ovules or gametophytes were seen; collectively, they averaged over 8.6% of all ovules.     

速生湿地松良种胚性愈伤组织诱导与增殖

为使速生湿地松良种快速大规模繁殖,对其胚性愈伤组织进行诱导和增殖优化研究.该文以1代湿地松种子园中10个速生湿地松优良无性系(基因型)的未成熟合子胚为外植体,系统研究基因型、合子胚发育阶段、基本培养基、植物生长调节剂种类和浓度等不同因子对胚性愈伤组织诱导效率的影响,探讨胚性愈伤组织的增殖条件.结果表明:基因型、合子胚发...     

龙须草无融合生殖的胚胎学证据

采用石蜡切片技术对龙须草(Eulaliopsis binata(Rotz)C.E.Hubb)进行了系统的胚胎学研究,证明龙须草为禾本科植物中一种新的无融合生殖材料.龙须草无融合生殖方式为无孢子生殖,在胚珠发育早期,多个珠心细胞特化为无孢子生殖原始细胞,由原始细胞发育为单核胚囊,经两次有丝分裂形成4核胚囊,进一步分化形成两种类型的成熟胚囊:(1)具1个卵细胞,1个助细胞和2个极核,占观察总数的67.6%;(2)具1个卵细胞,2个助细胞和1个极核,占观察总数的32.4%.胚囊发育属大黍型.多个无孢子生殖原始细胞可以同时发育,最后形成2个或多个胚囊,其比例为17.7%.胚珠内没有有性胚囊的发育.胚的发生有两种类型:(1)早发生胚(74%),开花前1～2 d,极核未分裂前卵细胞分裂形成胚;(2)迟发生胚(26%),开花后2～3 d,极核分裂形成多个胚乳游离核后,卵细胞启动分裂形成胚.存在多胚现象,多胚来自不同胚囊内卵细胞的孤雌生殖,多胚发生率为13%.胚乳由极核不经受精自发分裂产生.     

Co-culture with Daucus carota somatic embryos reveals high 2,4-D uptake and release rates of Arabidopsis thaliana cultured cells

By means of co-culture in growth regulator-free medium we analysed whether factors secreted into the medium of Daucus carota (carrot) somatic embryo cultures would be able to overcome the developmental arrest of globular Arabidopsis thaliana somatic embryos. Instead of Arabidopsis embryogenesis being promoted the development of carrot somatic embryos was inhibited at the globular stage in the presence of Arabidopsis suspension culture aggregates with attached globular embryos. Several experiments showed that this was due to the release of previously accumulated 2,4-D by the Arabidopsis cultures. (1) In addition to arresting carrot embryogenesis, co-culture with Arabidopsis cell suspensions also induced callus formation on Arabidopsis root segments. (2) Both effects only occurred with Arabidopsis suspensions grown in the presence of 2,4-D and not with those grown in the presence of NAA, demonstrating that Arabidopsis is not segregating a “general” inhibiting factor. (3) Both effects could be prevented by either binding 2,4-D to active charcoal or by washing it away by changing the medium daily. (4) Uptake of 2,4-D into Arabidopsis cells during culture in 2,4-D containing medium and subsequent release of 2,4-D after transfer to growth regulator-free medium was measured. (5) These low levels of released 2,4-D (0.2– 0.5 μm) could mimic the observed effects. Taken together these data suggest that the high intracellular 2,4-D content of Arabidopsis cultures may interfere with Arabidopsis somatic embryo development beyond the globular stage. Received: 13 November 1997 / Revision received: 2 February 1998 / Accepted: 16 November 1998     

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.