Cryopreservation of non-encapsulated embryogenic tissue of sweet potato (Ipomoea batatas)

Embryogenic tissue of the sweet potato (Ipomoea batatas (L) LAM) genotype TIB 10 was established from in vitro axillary shoot tips on Murashige and Skoog (1962) medium supplemented with 5 M 2,4-dichlorophenoxyacetic acid. Embryogenic aggregates of fresh mass 9.0–12 mg were subjected to a rapid freezing protocol in liquid nitrogen following sucrose preculture and varying degrees of dehydration. Up to 50% of embryogenic explants survived rapid freezing after preculture on 0.4 or 0.7M sucrose only. Dehydration with silica gel to moisture contents in the range 18–41% improved the survival after cryopreservation of embryogenic tissue. Tissue dehydrated for intermediate periods exhibited poor survival. Following freezing, embryogenic tissue appeared to develop normally, retaining its competence to produce mature embryos and plantlets.Abbreviations BA 6-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog (1962) medium     

Cryopreservation of embryogenic tissue of sweet potato (Ipomoea batatas): use of sucrose and dehydration for cryoprotection

Embryogenic tissue of two sweet potato (Ipomoea batatas (L) LAM) genotypes, TIB 10 and Nemanete (Nem), was established from in vitro axillary meristems on Murashige and Skoog (1962) media supplemented with 2,4-dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxyacetic acid respectively. Embryogenic aggregates of approximately 1.5–2.0 mm in diameter were subjected to a rapid or a two-step freezing protocol in liquid nitrogen following alginate encapsulation, sucrose preculture and varying degrees of dehydration. Up to 28% of encapsulated embryogenic aggregates of TIB 10 survived rapid freezing without dehydration. This was not enhanced by dehydration prior to freezing. However, survival after dehydration was enhanced up to 74% by incorporating an initial slow cooling step prior to plunging the tissue into liquid nitrogen. Following freezing, embryogenic tissue appeared to develop normally and retained its competence to produce mature embryos and plantlets. Similar results were obtained with Nem, although the survival percentages were much lower.Abbreviations BA 6-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid - MS Murashige and Skoog (1962) medium     

Somatic embryo formation and germination from immature embryo-derived suspension-cultured cells of Angelica sinensis (Oliv.) Diels

Embryogenic callus was induced from immature embryos of Angelica sinensis cultured on Murashige and Skoog (MS) basal medium. Embryogenic callus growth was more rapid on MS basal medium than on B5 or White medium. Embryogenic callus was used to establish a suspension culture and somatic embryos and germinating embryos developed during the culture. A shaking speed of 80 rpm was found to be optimal for establishing suspension cultures, while 100 rpm produced more somatic embryos and germinating embryos with an initiation cell density of 0.2 ml packed cell volume/25 ml medium. Adding 0.3% agar to the liquid medium also stimulated the formation of somatic and germinating embryos. While no plant growth regulators were needed for culture initiation and plant regeneration, the addition of 0.5–1 mg/l 2,4-dichlorophenoxyacetic acid was needed to maintain the embryogenic suspension culture by preventing embryo germination. Forty percent of the germinating embryos survived after culturing on filter paper moistened with liquid half-strength MS medium containing 3% sucrose. The plants were successfully transferred into soil. Received: 19 March 1997 / Revision received: 21 November 1997 / Accepted: 19 January 1998     

Initiation of embryogenic callus and suspension cultures of eastern white pine (Pinus strobus L.)

Embryogenic callus and suspension cultures of eastern white pine (Pinus strobus) have been obtained. The whole female gametophyte was plated on a medium containing 50 mg/l glutamine, 500 mg/l casein hydrolysate, 3% sucrose, 2 mg/1 2,4-D, 1 mg/1 BA and 0.2% Gelrite as a solidifying agent. Embryogenic calli could be seen as early as 5 days following culture. Histological studies indicate proliferation of pre-existing embryogenic tissue in the corrosion cavity followed by extrusion of embryogenic callus through the micropylar end of the gametophyte. Embryogenic suspension cultures were obtained by placing embryogenic callus into liquid medium. Embryogenic suspension cultures were subcultured weekly and proliferated as early-stage embryos with attached suspensors. Embryo development was obtained following transfer of the embryogenic tissue to an auxin-free medium containing 50 mM glutamine, 38 M abscisic acid, and 6% sucrose. Although embryo development could be consistently obtained, whole plants have not yet been recovered from these somatic embryos.Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid - ABA Abscisic acid - BA 6-Benzyladenine Salaries and research support were provided by State and Federal funds appropriated to OSU/OARDC. Journal Article No. 62–89     

The use of TTC reduction assay for assessment of Gentiana spp. cell suspension viability after cryopreservation

This study was aimed at improving the 2,3,5-triphenyl-tetrazoliumchloride (TTC) reduction test for initial assessment of cell survival after cryopreservation. Experiments were carried out on three embryogenic cell suspensions of different ages: 9-year-old Gentiana tibetica (King ex Hook. F.), 2-year-old G. kurroo (Royle), and 1-year-old G. cruciata (L.). The suspensions were maintained in MS medium supplemented with 1.0 mg 1⁻¹ 3,6-dichloro-o-anisic acid, 0.1 mg 1⁻¹ naphthaleneacetic acid, 2.0 mg l⁻¹ 6-benzylaminopurine, 80.0 mg 1⁻¹ adenine sulphate and 0.09 M sucrose. Four weeks before freezing, part of the tissue was subcultured to the same medium with sucrose concentrations elevated from 0.09 M (3%sMS) to 0.175 M (6%sMS) or 0.26 M (9%sMS). In freezing treatments without cryoprotection, tissue was plunged directly into liquid nitrogen (LN) or cooled gradually. In freezing treatments with cryoprotection, the cells were pretreated with 1 M sucrose, or with 0.4 M sorbitol + 0.25 M proline or + 0.08 M DMSO, or with vitrification solution (PVS2). Encapsulation was another variant. TTC reduction activity was spectrophotometrically assessed immediately, 1, 3, 5, 24 and 48 h after thawing. Cells without cryoprotection were lethally damaged, but TTC reduction activity in those cells ranged from 6.5% (tissue from 3%sMS) to 73 % (tissue from 9%sMS) directly after thawing. Formazan production was reduced to zero after 24 h. The TTC test showed 50% formazan content immediately after thawing of DMSO-protected G. tibetica tissue, but only 22.47% after 24 h and 2.9% after 48 h. Ultrastructural analysis of those cells showed lethal damage in many of them. For the PVS2 treatment, the formazan content was similar in samples analyzed directly after thawing and 24 h later. Cells treated with PVS2 did not show structural disturbances. Encapsulated cell aggregates of G. cruciata treated with concentrations of sucrose increasing up to 1 M produced 2.6 times more formazan. When applied at least 48 h after thawing, the TTC test can reflect cell viability and can be used to compare the effectiveness of cryoprotectant performance and freezing protocols, but it must be carefully evaluated, with appropriate controls.     

Cryopreservation of in vitro sugar beet shoot tips using the encapsulation-dehydration technique: influence of abscisic acid and cold acclimation

It has been previously shown that shoot tips of in vitro plantlets of sugar beet (Beta vulgaris L. clone SES1) can be cryopreserved using the encapsulation-dehydration technique (survival rate of 37% after freezing). This article reports the influence of abscisic acid (ABA) and cold acclimation on survival after cryopreservation. When ABA was added to the multiplication medium of the plants, the survival rate of shoot tips after cryopreservation was not increased (45%). After cold acclimation of the plants, their growth pattern differed (plants became apically dominant) and the survival rate of the shoot tips after cryopreservation clearly increased (70% survival and 50% plant regeneration after freezing). This improved protocol was successfully applied to three other clones. Received: 28 October 1996 / Revision received: 28 January 1997 / Accepted: 15 March 1997     

Enhanced production and germination of somatic embryos by temporary starvation in tissue cultures of Daucus carota

Embryogenic callus was induced from cotyledonary explants of Daucus carota L. cultured on solidified MS medium supplemented with 1 mg l^-1 2,4-D. Following callus initiation somatic embryos were developed from the callus on MS medium without 2,4-dichlorophenoxyacetic acid. To stimulate the production and germination of somatic embryos we cultured the callus under physically and chemically modified conditions during subculture. When the embryogenic callus was cultured on half-strength MS medium or MS medium without sucrose or cultured under conditions of reduced humidity (69.3%), the production of embryos increased 3.4- to 4.5-fold compared to culture on MS medium containing 3% sucrose (control). Embryogenic callus cultured on MS medium after 5 days of starvation (by being placed in empty 12-well tissue culture plates) showed a 20-fold increase in somatic embryo production and enhanced maturation and germination of embryos. An important point is that the germination of somatic embryos with cup-shaped cotyledons, after a period in culture without medium, was remarkably improved (92%) compared to that of the controls (23%).Thus, we were able to show that stress by starvation without medium led to the enhanced production and increased germination of somatic embryos.     

Effects of medium composition and culture duration on in vitro morphogenesis of sweet potato

In vitro morphogenesis of sweet potato (Ipomoea batatas) shoot explants after cultures in callus initiation medium (CIM) with two sucrose contents and plant regeneration medium (PRM) with three growth regulator combinations for different durations was studied. After 4 weeks, explants on 5 % sucrose CIM had significantly more shoots but similar or lower root fresh mass and callus fresh mass than those on 3 % sucrose CIM subsequent to transfer for 6 weeks on all three PRM. Cultures transferred to growth regulator-free PRM after 4 and 12 weeks on 5 % sucrose CIM formed plants through organogenesis and embryogenesis, respectively. Embryogenic cultures from 4 weeks on CIM + 10 weeks on callus proliferation medium when transferred to PRM without growth regulator for 4 and 8 weeks produced multiple embryos in the prior and both embryos and shoot buds in the later.     

Droplet-vitrification and morphohistological studies of cryopreserved shoot tips of cultivated and wild pineapple genotypes

Germplasm conservation of pineapple [Ananas comosus (L.) Mer.] is crucial to preserve the genus’ genetic diversity, to secure material for genetic improvement and to support innovative and new research. Long-term conservation is accomplished through cryopreservation, that is done by storing cells or tissues at ultra-low temperature in liquid nitrogen (−196 °C). Droplet-vitrification, a combination of droplet freezing and solution-based vitrification, was used to establish a protocol for cryopreservation of pineapple genetic resources. This protocol was tested on cultivated and wild pineapple genotypes to establish a long-term germplasm security duplicate as well as to investigate cryo-injuries in the tissues by means of histological techniques. Excised shoot tips (0.5–1 mm with one primordial leaf) of different pineapple genotypes were precultured for 48 h on solid MS medium containing 0.3 M of sucrose. Three PVS2 exposure times (30, 45 and 60 min) were tested. The results showed high post cryopreservation survival for all genotypes evaluated. The best PVS2 exposure time varied according to genotype, although 45 min gave the best survival for the majority of genotypes. The technique was highly efficient in cryopreserving meristem shoot tips of different pineapple genotypes, and was also less laborious than techniques previously reported. This is a first report on application of the droplet-vitrification technique to diverse genotypes of cultivated and wild pineapples and the first report on histological changes occurring in cryopreserved Ananas tissue.

     

`Isubgol' as an alternative gelling agent in plant tissue culture media

`Isubgol', the mucilaginous husk derived from the seeds of Plantago ovata, was successfully used as a gelling agent in tissue culture media for in vitro seed germination, shoot formation and rooting in Syzygium cuminii and anther culture in Datura innoxia. For seed germination, Knop's basal medium supplemented with 1% sucrose was employed, whereas for the development of shoots the epicotyl segments excised from in vitro-developed seedlings were cultured on MS basal medium supplemented with 4% sucrose and 1 mg/l 6-benzyladenine. Shoots that developed from the epicotyl segments were rooted on Knop's medium enriched with 2% sucrose and 1 mg/l indole-3-acetic acid. The anthers of D. innoxia excised at the late uninucleate to early binucleate stages of microspore development were cultured on Nitsch's basal medium containing 2% sucrose. Media were either gelled with 0.9% agar or 3% `Isubgol'. The response on media gelled with `Isubgol' in each of the cases was similar to that on media solidified with agar. Received: 9 October 1996 / Revision received: 22 July 1996 / Accepted: 30 July 1997     

Endogenous hormone levels in habituated nucellar Citrus callus during the initial stages of regeneration

 Embryogenic nucellar callus cultures of different Citrus species and cultivars growing in hormone-free medium were transferred to medium containing either sucrose or glycerol as the only carbohydrate source. Glycerol has been reported to induce further development of Citrus somatic embryos, while in the presence of sucrose they continue to proliferate in an 'undifferentiated' manner. The endogenous hormone levels of the cultures were evaluated after 2 and 5  days to characterise the initial steps of embryo development. In most cases, differences among treatments were observed only after 5 days of culture. Higher cytokinin levels were found in most of the cultures transferred to the glycerol-containing medium. The effect of ageing sweet orange cultures on their endogenous hormone levels was determined by leaving them in the original culture medium without subculturing for 60 days. While no changes were observed in the free indoleacetic acid and gibberellin contents, lower levels of abscisic acid and cytokinins were found in the aged cultures than in those transferred at the normal interval, every 30 days. The endogenous hormone contents of Citrus callus of different genotypes were compared. Significant differences were observed in the levels of all hormones evaluated, even when the in vitro ontogeny of the different genotypes was very similar. Received: 10 February 2000 / Revision received: 25 August 2000 / Accepted: 29 August 2000     

Medium,container and genotype all influence in vitro cold storage of apple germplasm

The goal of this study was to evaluate the in vitro storage of apple germplasm by screening a range of genotypes followed by more comprehensive testing of multiple parameters on two genotypes of differing species, Malus domestica cultivar Grushovka Vernenskaya and wild Malus sieversii selection TM-6. Stored plants were rated on a 6 point scale (0 low to 5 high) for plant appearance at 3 month intervals after storage at 4°C. Combinations of carbon source (sucrose and/or mannitol), nitrate nitrogen content (25, 50 or 100%) and plant growth regulators (ABA, BAP, IBA) were studied in three types of containers (tissue culture bags, test tubes or jars). An initial screen of 16 genotypes stored in tissue culture bags indicated that plantlets could be stored at 4°C for 9–14 months without subculture on standard 3% sucrose Murashige and Skoog (1962) (MS) medium with no plant growth regulators (PGRs). In subsequent in-depth studies on the two genotypes, ANOVA indicated highly significant interactions of medium, container and genotype. ‘Grushovka Vernenskaya’ shoots with no PGRs and 3% sucrose remained viable (ratings of ≥1) for 21 months of storage in bags. Storage on reduced nitrogen (MS with 25% nitrogen), PGRs, and 3% sucrose kept ‘Grushovka Vernenskaya’ shoot condition rated >2 at 21 months. Addition of 0.5 or 1 mg⁻¹ abscisic acid (ABA) also improved plant ratings at 21 months. The longest storage for ‘Grushovka Vernenskaya’ was 33–39 months with PGRs and 3% sucrose in either tubes or jars. Addition of abscisic acid (ABA) to the medium did not improve storage of plantlets in jars and tubes at 15 months. TM-6 stored best in tubes on 3% sucrose with PGRs or in jars on 2% mannitol and 2% sucrose. Overall it appears that cold storage of apple shoot cultures can be successful for 21 months in tissue culture bags with 25% MS nitrate nitrogen, 3% sucrose, and no PGRs or for 33 months in jars or tubes on MS with 3% sucrose and PGRs. Preliminary RAPD analysis found no significant differences between plants stored for 39 months and non-stored controls.     

Cryopreservation of in vitro-grown shoot-tips of hop (Humulus lupulus L.) using encapsulation/dehydration

 A cryopreservation procedure using encapsulation/dehydration was established for shoot-tips obtained from in vitro-grown shoots of hop. After dissection, shoot-tips were encapsulated in medium with alginate and 0.5 M sucrose. Optimal conditions consisted of preculture for 2 days in solid medium with 0.75 M sucrose, or in increasing sucrose concentrations, desiccation for 4 h with silicagel in a flow cabinet (16% water content) followed by rapid freezing and slow thawing. Shoot recovery after freezing 60 min in liquid nitrogen was around 80%. No phenotypical changes were observed in the recovered plants from cryopreserved shoot-tips growing in the field. Received: 20 April 1997 / Revised: 20 February 1998 / Accepted: 1 Dezember 1998     

An efficient regeneration system via somatic embryogenesis in mango ginger (Curcuma amada Roxb.)

A reproducible protocol for somatic embryogenesis was established for mango ginger (Curcuma amada Roxb.)—an important horticultural aromatic rhizomatous plant. Embryogenic callus induction was obtained from leaf sheath explants of in vitro raised plants on Murashige and Skoog (MS) agar medium containing 2.0 mg/L 2,4-dichlorophenoxyacetic acid and 0.5 mg/L 6-benzyladenine (BA). Embryogenic callus proliferation, somatic embryo (SE) formation and subsequent plantlet conversion occurred under optimal culture conditions. The effects of MS medium strength, sucrose and BA on SE formation were also evaluated. Half strength MS liquid medium necessary for SE formation and optimal sucrose concentration was found to be 3.0 %. BA at 0.3 mg/L produced the highest number (84.71 %) of SEs from leaf sheath explants. Secondary somatic embryos originated from primary somatic embryos on the same medium supplemented with 0.4–0.6 mg/L BA. Stereo microscopic and scanning electron microscopic observation revealed that the globular and torpedo shaped somatic embryos resulted in suspension culture during development. Mature somatic embryos germinated readily and developed into normal plantlets after 3 weeks on half strength MS basal agar medium under dark condition. Well rooted plantlets were successfully acclimatized at the survival rate of 70 %.     

Media and genotype effects on the development and conversion of somatic alfalfa (Medicago sativa L.) embryos

Summary Various preconditioning treatments of alfalfa (Medicago sativa L.) somatic embryos to improve embryo quality and conversion were studied. Four different regenerating genotypes were compared. Embryogenic cultures were established in liquid culture. Globular embryos were collected and plated on an embryo development medium until they reached cotyledonary stage. They were then exposed to three treatments: a standard embryo development medium (control), media supplementation with 1 μM abscisic acid (ABA), 50 mM glutamine and 5% sucrose (T), additional supplementation with 50 μM ABA (TT), and additional supplementation followed by desiccation (TTD). Treatments affected embryo conversion, but not uniformly for all genotypes. Embryo conversion was increased (P<0.05) by pretreatment (T), while only one exhibited any response to additional ABA (T vs. TT). Desiccation decreased (P<0.05) conversion of pretreated embryos (TT vs. TTD) of all genotypes. The effect of treatments on plantlet weight was less pronounced and inconsistent across genotypes.     

Induction of somatic embryogenesis and genetic transformation of ohio buckeye (Aesculus glabra willd.)

Summary Mature embryo axes of the Ohio buckeye were germinated on a medium containing 1 mg gibberellic acid (GA) per 1. Three wk following germination, stem, petiole, and leaf blade tissues were excised and placed on media containing either 1 mg (4.5 μM) 2,4-dichlorophenoxy acetic acid (2,4-D) per 1, 1 mg (4.7 μM) kinetin per 1, 1 mg of both 2,4-D (4.5 μM) and kinetin (4.7 μM per 1, or 2 mg of both 2,4-D (9.1 μM) and kinetin (9.3 μM) per 1. Embryogenic tissue was formed only from stem segments after 2–3 mo. of culture on media containing both 2,4-D and kinetin. Embryogenic tissue could be either maintained on solid medium for proliferation of embryogenic callus or placed in liquid medium for proliferation of embryogenic suspension cultures. For transformation of suspension cultures, tissues were inoculated with Agrobacterium EHA105 containing the binary plasmid Vec035, briefly sonicated, and cultured in the presence of 100 μM acetosyringone for 2 d. To eliminate Agrobacterium, tissues were washed and placed in liquid proliferation medium containing either 500 mg Cefotaxime per 1 or 400 mg TimentinŖ per 1. Selection on 20 mg hygromycin per 1 was initiated 2 wk after inoculation, and after an additional 10 wk, hygromycin-resistant tissue was isolated and separately cultured. Although some hygromycinresistant clones were recovered with no sonication treatment, four to five times more clones were obtained following sonication. Putative transformed clones were confirmed to be transgenic via both histochemical β-glucuronidase (GUS) assay and southern hybridization analyses. Development of transgenic embryos occurred on a growth regulator-free medium containing 3% sucrose. After 2 mo. of embryo development, the embryos were transferred to fresh medium for germination.     

Efficient cryopreservation of adventitious shoots of Begonia x erythrophylla using encapsulation–dehydration requires pretreatment with both ABA and proline

Despite the widespread use of tissue culture as a means of propagating begonias and concerns regarding the preservation of germplasm, little information is available on the cryopreservation of these commercially important plants. For this reason studies were conducted to develop an encapsulation–dehydration method for the cryopreservation of adventitious shoots of the rhizomatous begonia, Begonia x erythrophylla. Adventitious shoots of B. x erythrophylla were found to be sensitive to dehydration and very sensitive to freezing. While pre-treatment with 0.75 M sucrose significantly increased the percentage of encapsulated shoots surviving dehydration, pre-treatment with sucrose did not afford cryoprotection without prior dehydration. Addition of abscisic acid and proline to the pre-treatment medium significantly improved the percentage of shoots surviving freezing. Pre-treatment of shoots with a medium containing, 0.75 M sucrose, 3.8 μM abscisic acid and 2.15 mM proline resulted in greater than 50% of shoots surviving freezing.     

High frequency plant regeneration from protoplasts in cotton <Emphasis Type="Italic">via</Emphasis> somatic embryogenesis

A highly reproducible system for efficient plant regeneration from protoplast via somatic embryogenesis was developed in cotton (Gossypium hirsutum L.) cultivar ZDM-3. Embryogenic callus, somatic embryos and suspension culture cells were used as explants. Callus-forming frequency (82.86 %) was obtained in protoplast cultures from suspension culture cells in KM₈P medium with 0.45 μM 2,4-dichlorophenoxyacetic acid (2,4-D), 0.93 μM kinetin (KIN), 1.5 % glucose and 1.5 % maltose. Protocolonies formed in two months with plating efficiency of 14 %. However, the callus-forming efficiencies from other two explants were low. The calli from protoplast culture were transferred to somatic embryo induction medium and 12.7 % of normal plantlets were obtained on medium contained 3 % maltose or 1 % of each sucrose + maltose + glucose, 2.46 μM indole-3-butyric acid (IBA) and 0.93 μM KIN. Over 100 plantlets were obtained from protoplasts derived from three explants. The regenerated plants were transferred to the soil and the highest survival rate (95 %) was observed in transplanting via a new method.     

Sucrose dilution of glycerol from mouse embryos frozen rapidly in liquid nitrogen vapour

The toxic effects of sucrose and the conditions of in-straw glycerol removal after freezing and thawing were studied using Day-3 mouse embryos. At 20 degrees C, exposure to less than or equal to 1.0 M-sucrose for periods up to 30 min had no adverse effects on freshly collected embryos. At 25 and 36 degrees C, however, greater than or equal to 1.0 M-sucrose significantly reduced the developmental potential (P less than 0.001). In the freezing experiments the embryos were placed in 0.5 ml straws containing 40 microliters freezing medium separated by an air bubble from 440 microliters sucrose solution. The straws were frozen rapidly in the vapour about 1 cm above the surface of liquid nitrogen. The post-thaw viability was substantially better after sucrose dilution at 20 degrees C than at 36 degrees C. Mixing the freezing medium with the sucrose diluent immediately after thawing further improved the rate of survival relative to mixing just before freezing (P less than 0.001). The best survival was obtained when the freezing medium contained 3.0 M-glycerol + 0.25 M-sucrose; it was mixed with the diluent after thawing and the glycerol was removed at 20 degrees C. Under such conditions the sucrose concentration in the diluent had no significant effect on the rate of development (0.5 M, 69%; 1.0 M, 73%; 1.5 M, 64%). The results show that during sucrose dilution the temperature should be strictly controlled and suggest that intracellular and extracellular concentrations of glycerol are important in the cryoprotection of embryos.     

Volume contents volume 38 (1985)

Isolated microspores from six cultivars of Brassica napus and one of B. carinata were cultured in modified Nitsch and Nitsch (NN) medium supplemented with 13% (W/V) sucrose, 0.05 mg/l benzyladenine (BA) and 1.00 mg/l nahpthaleneacetic acid (NAA). Embryogenic responses were observed at cultured temperatures ranging from 22 to 32°C. For most genotypes tested, the highest frequency of embryos occurred at 30°C and 7–54 embryos per anther (approx. 17 000 microspores per anther) developed. Although incubation at 30°C produced the highest frequency of embryos, lower culture temperatures induced better quality embryos. A split temperature culture regime of incubation at 32°C for 3 days followed by incubation at 25°C resulted in both high embryo yields and a high percentage of normal embryos. Plantlet development from microspore-derived embryos appeared to be influenced by both genotype and medium.