Early,non-destructive selection of microspore-derived embryo genotypes in oilseed rape (<Emphasis Type="Italic">Brassica napus</Emphasis> L.) by molecular markers and oil quality analysis

In oilseed rape (Brassica napus L.) breeding, microspore culture is frequently applied for the immediate regeneration of homozygous doubled haploid (DH) plants. From the regenerated microspore-derived embryos (MDEs), usually only a smaller subset of around 200 are used for plantlet regeneration and cultivation in the greenhouse until seed harvest, without there being any knowledge about their quality traits and agronomic performance. The random selection of MDEs implies that valuable rare recombinant genotypes may be discarded at an early stage of in vitro culture. We report here on the development of a simple protocol for simultaneously extracting lipids (for oil quality analysis) and the isolation of DNA (for marker-assisted selection) from single cotyledons dissected from MDEs under aseptic conditions, thus keeping the rest of the embryo in vitro for plantlet regeneration. Neither the fatty acid extraction nor the transmethylation with sodium methylate at high pH interfered with subsequent DNA isolation. The feasibility of the protocol was tested using MDEs from a cross segregating for two linked transgenes, fae1 and plsC, affecting the fatty acid composition. Multiplex PCR was performed with specific PCR primers for the plsC gene and with locus-specific primers for a resident single copy fad2 gene. The amplification of the fad2 gene provided a control for the presence of DNA in sufficient quantity and quality, whereas the amplification of the plsC gene showed a 1:1 segregation expected for a single copy transgene in a segregating DH population. The early identification of the 50% MDE genotypes carrying the desired transgenes, along with a high expression of the trait, allows their early selection for plantlet regeneration.     

Efficient callus induction and plant regeneration from anther of Chinese narcissus (Narcissus tazetta L. var. chinensis Roem)

Callus culture has, to date, been reported only in a few species of Narcissus. We used anthers of Chinese narcissus (Narcissus tazetta L. var. chinensis Roem) as explants for callus induction and plant regeneration. A high percentage of anthers at the early- to mid-uninucleate microspore stage were responsive on the basal MS medium supplemented with 0.5–1 mg l^–1 2,4-dichlorophenoxyacetic acid and 0.5–2 mg l^–1 6-benzyladenine under dark conditions. Calli were initiated from anther connective tissue or anther wall tissue, and no division of microspores occurred during callus formation, as determined by histological observation. Using 20 random amplified polymorphic DNA primers, we verified the genetic integrity of the anther-derived plants of Chinese narcissus with respect to the donor plants. These results suggest that anther culture in vitro can provide an efficient new micropropagation technique for Chinese narcissus as well as a new strategy for in vitro mass propagation of other daffodils.     

Prospects of androgenetic induction in <Emphasis Type="Italic">Lupinus</Emphasis> spp.

Anthers cultures of six Polish cultivars of pasture lupin (Lupinus L.) were examined for their androgenic response. Anthers with microspores at the uninucleate stage were isolated from flower buds and cultured in liquid media. Better viability of androgenetic structures was obtained when donor plants had grown under field as opposed to greenhouse conditions. A density of five anthers per 0.5 ml medium was more conducive to androgenetic induction than 25 anthers per 0.5 ml medium. Addition of 5% maltose to the induction medium and culture at 25°C without pre-treatment of flowers, buds or anthers promoted microspore release and division. The greatest frequency of androgenic callus, ~70% was developed from cvs. Katon, Wat (white lupin), in contrast to cvs. Legat, Juno (yellow lupin), Polonez and Sonet (narrow-leafed lupin) with callus induction ~30–40%. Despite various combinations of media tested, plant regeneration was not obtained from anther derived callus.     

Optimisation of procedures for microprojectile bombardment of microspore-derived embryos in wheat

Using the PDS-1000/He Biolistic® Particle Delivery System, the microprojectile travel distance, rupture disk pressure and DNA/gold particle concentrations were assessed in order to optimise short and longer-term β-glucuronidase reporter gene expression in microspore-derived embryos of wheat. The effects were also evaluated of using sterile filter paper to support explants and treatment with a high osmoticum medium (0.2 M mannitol/0.2 M sorbitol or 0.4 M maltose). In the optimised procedure, wheat microspore-derived embryos (MDEs), were placed on filter paper and incubated on medium containing 0.4 M maltose, for 4 h pre- and 45 h post-bombardment. Five μl pAHC25 (0.75 mg ml^-1 in TE buffer) was precipitated onto 25 μl gold particles (60 mg ml^-1 in sterile water), using 20 μl spermidine (0.1 M) and 50 μl CaCl₂ (2.5 M). The particles were centrifuged and resuspended in 75 μl absolute ethanol prior to the preparation of 6 macrocarriers. A microprojectile travel distance of 70 mm, a rupture pressure of 1300 p.s.i., and a vacuum of 29′′ Hg were employed. Maltose at 0.4 M in the support medium was the most important factor influencing GUS activity in bombarded tissues. GUS activity, 1 day post-bombardment, reached 52 ± 17 GUS-positive foci/MDE (mean ± s.e.m, n=3), with 17 ± 4 foci/MDE at 15 days, giving a 3.0-fold increase (p<0.05) compared to expression in MDEs bombarded on medium without a high osmoticum treatment.     

In vitro induction of haploid,diploid and triploid plantlets by anther culture of Iochroma warscewiczii Regel

Pollen of Iochroma warscewiczii Regel (Solanaceae) produced embryogenic calli or embryos inside anthers cultured on Nitsch & Nitsch medium. Two distinct pathways could be recognized in this process, one involving mainly the vegetative cell, and the second starting with two equal cells in the pollen grains.In all media tested, androgenesis initiation was highest when anthers contained pollen at the first mitosis, or close to it, at inoculation. High sucrose (7%) and calcium (11.3 mM) concentrations were found to be highly desirable for the induction of androgenesis in this species. Addition of benzylaminopurine (0.5 mg l^–1) to the culture medium seems to slightly improve callus or embryo production. When all three factors were present at optimal concentrations as much as 13.9% of inoculated anthers were found to be embryogenic.Plantlet development from pollen embryos required lower sucrose (3%) and a combination of 0.1 mg l^–1 benzylaminopurine and 0.5 mg l^–1 gibberellic acid in the culture medium. Cytological analysis of 55 regenerated plantlets showed that about 49% were haploids, but diploid (ca. 49%) and triploid (ca. 2%) plants were also obtained.     

Colchicine Induced Embryogenesis in Maize

The present study involves in vitro androgenesis of Zea mays L. using anther culture. We tested combinations of single factors and their influence on microspore induction. Embryogenic induction of microspores within anthers in in vitro conditions was the best when combination of cold treatment, TIBA (0.1 mg l^–1) in media and colchicine (0.02% during first 3 days of culture) was applied, but colchicine alone can be factor, which can stimulate or initiate embryogenesis in anther culture of maize.     

Micrografting and <Emphasis Type="Italic">ex vitro</Emphasis> grafting for somatic embryo rescue and plant recovery in avocado (<Emphasis Type="Italic">Persea americana</Emphasis>)

To improve plant regeneration from oat (Avena sativa L.) anther culture, the effects of induction medium supplements and culture conditions were studied. Significantly better plant regeneration rates were obtained with cultivars Lisbeth (naked type) and Aslak when a medium containing W₁₄ salts and vitamins, supplemented with 2,4-d, BAP, Ethephon, l-cysteine and myo-inositol, was used for induction in the dark compared with a medium containing only 2,4-d and kinetin. Genotypes reacted differently on the light during the induction phase. Use of dim light significantly decreased the green plant regeneration rates in cv. Lisbeth, while in cv. Aslak the difference was not so clear. Up to 30 green plants per 100 anthers were recovered from Aslak × Lisbeth progeny and in total, over 500 oat regenerants were produced. With these numbers, acceptable rates of DH-production for cultivar breeding and genetic study purposes are approached. The agronomic performance of some DH lines was compared with that of the plants derived from commercial seeds of the same cultivars in the field experiment. A few differences were found, but generally DH lines yielded the same or more as the commercial cultivars.     

Organogenesis and embryogenesis inHelianthus tuberosus and in the interspecific hybridHelianthus annuus ×Helianthus tuberosus

A method of plant regeneration from cotyledons ofHelianthus tuberosus, Helianthus annuus ×Helianthus tuberosus and for the backcross of the interspecific hybrids onH. annuus was developed. Induction of somatic embryogenesis and plantlet regeneration from anther culture of the interspecific hybridsH. annuus ×H. tuberosus is reported.Cotyledons were cultured on Murashige and Skoog basal medium (MS) supplemented with indole-3-acetic acid (IAA) and 6-furfurylaminopurine (kinetin) or N⁶-benzylaminopurine (BAP). Shoot regeneration occurred on most of the media tested, but the best results were obtained on media with a high concentration of cytokinins (BAP or kinetin: 4 mg l^–1) and lower concentration of auxin (IAA: 0.5–1 mg l^–1).Embryogenic callus and adventitious buds were initiated from only two anthers of the hybridH. annuus ×H. tuberosus cultured on the MS medium containing BAP (0.2 mg l^–1) and 1-naphtalenacetic acid (NAA: 0.1 mg l^–1). Prolonged culture of these embryogenic calli and buds on the original medium with successive subculture on MS basal medium without growth regulators resulted in embryo formation and shoot differentiation. The plantlets, after rooting, were established in soil.     

The influence of bud length,age of the tree and culture media on androgenesis induction inAesculus carnea Hayne anther culture

Statistical analyses of the data revealed very significant differences in androgenesis induction ofA. carnea Hayne anther culture depending on the bud length, nutrient medium composition and age of the parental tree. Significant mutual influence of all these factors was also observed. The highest number of androgenic anthers was obtained when 4 mm long buds were used. Older trees (60 and 100 yrs) gave a higher number of androgenic anthers than the younger ones (20 and 40 yrs). MS medium supplemented with 2,4-d and Kin (1 mg l^–1, each) was the most favourable for androgenesis induction. Pollen embryos (haploids and aneuploids) were formed by the division of uninuclear microspores.The highest percentage of germinated embryos and further synchronous development of the shoot and root was achieved in MS medium supplemented with IAA, GA₃ (1 mg l^–1) and activated charcoal (1%). When other germination media were used, malformations of androgenic embryos were observed.Abbreviations AC activated charcoal - H casein hydrolysate - 2,4-d 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - BAP 6-benzylaminopurine - GA₃ gibberelic acid - Kin 6-furfurylaminopurine - MS Murashige and Skoog - T thidiazurone - N phenyl-N'-1,2,3-thiadiazol-5-ylurea - Z zeatin-6-(4-hydroxy-3-methyl-trans-2-butenylamino)purine     

Somatic embryogenesis and plant regeneration in Medicago arborea L.

Summary An efficient plant regeneration system employing cotyledons, hypocotyls, petioles and leaves as explants and characterized by continuous and prolific production of somatic embryos, has been developed with Medicago arborea ssp. arborea. The optimal somatic embryogenic response was obtained using a two-step protocol, where explants were incubated under a 16 h photoperiod for 2 mo. on Murashige and Skoog (MS) medium containing 2,4-dichlorophenoxyacetic acid (2,4-D; 9 μM) and kinetin (9 μM), and followed by transfer to kinetin-free MS medium with 2,4-D (2.25 μM). Removal of the cytokinin and a reduction in the concentration of auxin (2.25 μM) in the second step of culture were critical for enhanced production of somatic embryos. The best explants proved to be cotyledons and petioles (i.e. a mean of 18.0±0.70 somatic embryos at 3 mo. for petiole culture). Somatic embryos were converted into normal plantlets (8.0±0.89%) when cultured on basal MS medium with 5 μM indolebutyric acid. No somatic embryos were obtained when thidiazuron was used in the culture media. Using petioles as explants and N⁶-benzyladenine (BA), embryogenesis was induced in the second step of culture when BA was removed from the medium and the concentration of 2,4-D was decreased to 2.25 μM.     

Callus induction and plantlet regeneration in Bixa oreliana L., an annatto-yielding tree

Summary A protocol has been developed for plantlet regeneration from seed callus of Bixa orellana L. Seeds demonstrated a high percentage of callus induction (63±7.3%) and a high yield (356±14.7 mg per seed) of white friable callus on Murashige and Skoog (MS) medium containing 5.0 μM l-naphthaleneacetic acid (NAA) and 2.5μM N ⁶-benzyladenine (BA) within 6 wk of culture in the dark. Callus induction frequency was greater under 24h dark as compared to 16h light/8h dark photoperiod or 24h light photoperiod. Increased myo-inositol (MI: 200mgl⁻¹) and addition of ascorbic acid (AA: 200 mgl⁻¹) to the culture medium positively improved callus induction frequency and growth. Shoot differentiation from white friable seed callus was best using 10.0 μM BA and 5.0 μM NAA, where the highest percentage of calluses forming shools (74.9±4.8%), the highest number of shoots per callus (six or seven) and the highest shoot-forming index (5.0) were obtained within 6 wk. Shoots elongated to 4 cm within 4 wk of transfer onto MS medium devoid of growth regulators. Shoots were rooted using half-strength MS medium containing 5.0 μM indole-3-butyric acid (IBA). About 85% of these plants were established in pots containing pure garden soil and organic manure after 3 wk of hardening. Regenerated plants were morphologically uniform with normal leaf, shape and growth patterns. These plants are currently being screened for the presence of agronomically useful genetic variants.     

Micropropagation of <Emphasis Type="Italic">Vitex agnus-castus</Emphasis>, (Verbenaceae)—a valuable medicinal plant

This study describes in vitro shoot induction and plant regeneration from a mature apical meristem and nodal explants of the endangered medicinal shrub Vitex agnus-castus. Multiple shoots were induced directly from the axis of nodal and apical meristem explants on Murashige and Skoog (MS) medium containing 3% sucrose and different concentrations (1.0, 1.5, 2.0, and 2.5 mg/l) of 6-benzyl aminopurine (BAP) in combination with Kinetin (Kin) and α-naphthalene acetic acid (NAA), both at 0.1 mg/l. BAP and Kinetin were used as supplements to MS basal medium, either individually or in combination with auxins. The optimal concentration of BAP for inducing bud break was found to be 2.0 mg/l when Kinetin was at 0.1 mg/l. Regeneration frequency was highest for both apical meristem and nodal explants (94.5% and 90.3%, respectively) when explants were cultured on MS medium supplemented with BAP (2.0 mg/l) and Kin (0.1 mg/l). A maximum of 7.7 ± 0.4 and 6.7 ± 0.2 shoots were obtained per explant for apical meristem and nodal explants, respectively. Regenerated shoots, transferred to MS medium supplemented with either 1.0 or 1.5 mg/l BAP combined with 0.1 mg/l GA₃, showed maximum elongation of 6.7 ± 0.4 and 6.0 ± 1.3 cm in apical meristem and nodal explants, respectively. In vitro regenerated shoots transferred to half-strength MS medium supplemented with 0.1 mg/l IBA induced 90.4% of the shoots to form roots after 30–35 d of culture. Up to 80% of the regenerated shoots were successfully established in soil in the greenhouse.     

Micropropagation of Lepidium virginicum (Brassicaceae), a plant with antiprotozoal activity

Summary Micropropagation is a technique to ensure a constant and uniform source of medicinal plants. In this report, we describe the micropropagation of Lepidium virginicum L. (Brassicaceae), a wild plant used as an antiamoebic in traditional Mexican medicine. In vitro-germinated seeds were cultured in Murashige and Skoog (MS) medium to obtain pathogen-free cotyledons, hypocotyls, and apical bud (AB) explants. For induction of morphogenesis, the effect of cytokinins, benzyladenine (BA) and kinetin (KN), combined with auxin, indole-3-acetic acid (IAA) was evaluated. The best rate of shoot proliferation was induced 15 d after culture on MS mineral medium supplemented with IAA∶KN (0.57∶13.94 μM) from AB explants. Maximum shoot elongation was achieved without plant growth regulators. The effect of indole-3-butyric acid (IBA) (14.76 μM) was evaluated for in vitro root induction; 60 d after culture all the shoots had developed roots. All rooted plants were successfully transferred to pots and 100% acclimatized in ex vitro conditions. The methanol extracts from the micropropagated active explants of L. virginicum showed and IC₅₀ antiprotozoal value between 141.90 and 268.53 μg ml⁻¹.     

A study of factors affecting embryo yields from anther culture of cabbage

In cabbage (Brassica oleracea var. capitata), a thermal shock treatment of 24 h at 35 °C at the start of the culture period resulted in higher embryos per 100 anthers (30.0) compared to a treatment of 48 h. Similarly , a chilling treatment of 24 h at 4 °C resulted in a higher embryo yield (6.0) per 100 anthers compared to a treatment of 48 h. However, the embryo yields were significantly higher (p> 0.01) in thermal shock than chilling treatments in all experiments. Treatments of 6 days at either 35 °C or 4 °C gave no embryos. The most responsive cultivar was the F₁ hybrid , Hercules, in all experiments. Although anther culture was successful in the other genotypes, the open pollinated ones, the highest number of embryo yields per 100 anthers was obtained in the hybrid. High temperature treatment before culture had a beneficial effect on the embryo yields. The responsiveness of anthers to addition of increasing concentration of silver nitrate (AgN0₃) (the ethylene inhibitor) to the culture medium, showed a progressive increase in the embryo yields in all the genotypes. Since embryos were also formed in the absence of silver nitrate, probably, due to a greater genotype × medium interaction, it is noted that the presence of silver nitrate in the medium may not be essential for cabbage anther culture as reported earlier. The findings of this study may be recommended for large production of cabbage embryos in culture.     

Improved androgenesis of interspecific potato and efficiency of SSR markers to identify homozygous regenerants

Three interspecific diploid potato hybrids between selections of Solanum phureja Juz. & Buk. and S. chacoense Bitt. were used in anther culture experiments to construct a monoploid family. Different aspects of the anther culture process were affected by the treatments, such as: growing conditions of donor plants, ways of preparing the anther culture medium, and conditions of anthers in culture. Genotype and date of culture initiation were among the most significant sources of variation. Significant improvements in anther culture response were achieved by growing plants at 30°C and by a heat shock of 35°C for 12 h given to anthers in culture, which gave an increase of up to 40% in embryo yield. However, the heat shock reduced the plant regeneration rate. The majority of regenerated plants was diploid, suggesting that there were several recessive lethal alleles in heterozygous status in the anther-donor. Among the regenerants, the homozygotes could be successfully identified by simple sequence repeat analysis, using eight polymorphic primer pairs. This revised version was published online in June 2006 with corrections to the Cover Date.     

A simple wheat haploid and doubled haploid production system using anther culture

Summary Wheat (Triticum aestivum L.) haploids and doubled haploids have been used in breeding programs and genetic studies. Wheat haploids and doubled haploids via anther culture are usually produced by a multiple step culture procedure. We improved a wheat haploid and doubled haploid production system via anther culture in which plants are produced from microspore-derived embryos using one medium and one culture environment. In the improved protocol, tillers of donor plants were pretreated at 4°C for 1–2 wk before anthers were plated on a modified 85D12 basal medium with phenylacetic acid (PAA) and zeatin and cultured at 30°C with a 12-h daylength (43 μEs⁻¹m⁻²) in an incubator. Microspore-derived embryos developed in 2–3 wk and the plants were produced 3–4 wk after anther plating. In the improved system, as much as 53% of the anthers of Pavon 76 were responsive with multiple embryos. For plant regeneration, as many as 22 green and 25 albino plants were produced from 100 anthers. Sixty-five green plants were grown to maturity and 32 (49%) plants were fertile and produced seeds (indicating spontaneous chromosome doubling) while 33 plants did not produce seed. Of five Nebraska breeding lines tested using the protocol, NE96675 was very responsive and the other lines less so, indicating that the protocol is genotype-dependent.     

Production of doubled haploid rice plants (Oryza sativa L.) by anther culture

The results of anther culture of F₂ pollen issued from 23 single crosses are presented. A relation between the morphology of the panicle and the microspore stage was established. After cold-pretreatment (8 days at 4°C), the anthers were cultured on the callus-induction medium N6 supplemented with 1 mg l^–1 naphthaleneacetic acid. The calli were transferred to MS plant regeneration medium supplemented with 3 mg l^–1 kinetin + 0.5 mg l^–1 naphthaleneacetic acid. The induction frequency varied from 0.22% to 29% and the regeneration frequency from 0% to 144.4%, dependent upon the crosses used. On average, 27% of the plants obtained were albinos and 59% of the green plants underwent spontaneous chromosome doubling. Thirtynine doubled haploid lines were evaluated and multiplied in the field. Lines with an excellent behaviour in upland culture conditions were selected from two crosses.     

Liquid culture for efficient micropropagation of Wasabia Japonica (MIQ.) matsumura

Summary An efficient protocol for in vitro propagation of the valuable medicinal plant, Wasabia japonica (Miq.) Matsumura is described through shoot tip proliferation and direct regeneration. Multiple shoots were induced from shoort tips cultured on Murashige and Skoog (MS) semi-solid medium containing various concentrations (0.5–50 μM) of N⁶-benzyladenine (BA), thidiazuron, kinetin, and zeatin. A comparison was made on shoot multiplication between semi-solid and liquid culture media. Well-developed shoots were obtained using full-strength MS semi-solid medium containing 5.0 μM BA. However, the greatest shoot proliferation was achieved on either full- or half-strength MS liquid media supplemented with 5.0 μM BA for 4 wk (15.3±0.9 and 15.0±0.7 shoots per explant, respectively), and on half-strength MS liquid medium for 6 wk (25.8±1.3 shoots per explant) in culture. In contrast, the maximum number of shoots per explant on full-strength MS semi-solid medium was achieved with either 5.0 μM BA (10.4±0.6 shoots per explant) or 10.0 μM kinetin (10.9±0.8 shoots per explant). Fresh weight of explants and length of shoots derived from full-strength MS liquid medium (1055±77 mg and 34.2±1.0 mm, respectively) were significantly higher than those derived from full-strength MS semisolid medium (437.6±17.3 mg and 15.4±0.7 mm, respectively). Quarter-strength MS liquid medium had no significant difference in shoot proliferation when compared to quarter-strength MS semi-solid medium. Elongated shoots were separated and rooted on half-strength MS semi-solid media fortified with 1-naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA), or indole-3-acetic acid (IAA) ranging from 0.1 to 10.0 μM. Root formation was greatest with IBA when compared with IAA and NAA. One hundred percent of shoots were rooted on half-strength MS medium with 5.0 μM IBA, while vigorous roots were obtained with 10.0 μM IBA. Micropropagated plantlets were successfully established in soil with 95% survival rate after heardening.     

Hormones in zygotic and microspore embryos of Brassica napus

A number of studies have used microspore-derived embryos (MDEs) as amodel for examining a range of processes, including hormonal regulation ofembryo development. We examined the hormonal physiology of MDEs with theprimaryobjective of testing the validity of using the MDE system as a model forhormonally-regulated development in zygotic embryos, through late stages. To dothis we identified and quantified endogenous levels of abscisic acid (ABA),indole-3-acetic acid (IAA) and a number of gibberellins (GAs), includingGA₁₉, GA₂₀, GA₁ and GA₈ in bothMDEsand zygotic embryos. The presence of GA₁₉, together with itsC₁₉ metabolites indicates that the early-13 hydroxylation pathway isoperative in both embryo systems. Gibberellins A₄ and GA₉were also identified, thereby confirming the presence of the earlynon-hydroxylation pathway in B. napus MDEs and zygoticembryos. In general, the pattern of change of hormone (ABA, IAA, GA₁and GA₂₀) content per embryo through embryogenesis was similar forMDEs and zygotic embryos. Indole-3-acetic acid and GA₁ increased toamaximum at day 30 after culture (DAC) before decreasing. Abscisicacid levels increased to a maximum at day 35, and declined in zygoticembryos but not in MDEs. GA₂₀ increased to the final harvest atmaturity, or day 40. The absolute content (g/embryo) of each hormone, howeverwas appreciably lower (5- to 15- fold) in the MDEs. This was not the result ofdilution into surrounding medium for ABA or IAA; GA₁, however, didaccumulate in the medium. Although there were absolute quantitative differencesin the levels of IAA and ABA found in the two embryo systems, the similaritiesin the pattern of hormone changes suggests that the MDE system can serve as auseful model for examining the physiological roles of hormones duringembryogenesis.     

Microspore embryogenesis and the development of a double haploidy protocol for cow cockle (Saponaria vaccaria)

Factors affecting microspore embryogenesis of cow cockle (Saponaria vaccaria) were evaluated including donor plant growing conditions, genotype, bud size, density, medium composition, and culture conditions. Of the two donor plant (day/night) temperature regimes evaluated (10/5°C and 20/15°C), plants grown at 20/15°C were the most embryogenic. An embryogenic frequency of greater than 350 embryos/100 buds was observed in the most embryogenic genotype, cv. ‘White Beauty’. Buds from 3–9 mm in length were evaluated for their embryogenic potential; buds that were 4–7.9 mm produced the most embryos/100 buds. Of all the media compositions evaluated, NLN medium with 15% sucrose resulted in the most embryos. Cow cockle microspores required an initial period of 32°C for 3 days for production of microspore-derived embryos (MDEs).