Double-phase in vitro culture using sorbitol increases shoot proliferation and reduces hyperhydricity in Japanese pear

This study aimed to improve in vitro shoot proliferation efficiency without inducing hyperhydricity in Japanese pear. The shoot number increased at 2.5–10.0 mg l⁻¹ benzyladenine (BA), while shoot fresh mass increased at 1.0 and 2.5 mg l⁻¹ BA. Different macroelement formulation did not affect shoot proliferation, but adding activated charcoal (AC) to the medium inhibited markedly the production of axillary shoots and biomass and many shoots were hyperhydric. Different carbon sources (CS) significantly increased the shoot number and fresh mass, with the best results for shoot proliferation at 20–30 g l⁻¹ sorbitol. With gelling agents, the shoot number increased at 0.4 and 0.6% agar and 0.3% gellan gum, while fresh mass increased at 0.4% agar. The hyperhydric explants were more than 30% at 0.4–0.6% agar and at any concentration of gellan gum. The improved culture (woody plant (WP) supplemented with 20 g l⁻¹ sorbitol, 0.1 mg l⁻¹ 3-indolyl-butyric acid (IBA), 2.5 mg l⁻¹ BA and 0.8% agar) and double-phase culture (the same medium using a double-phase liquid-gelling agent solidified culture system) produced a higher number of axillary shoots than the conventional culture (1/2MS supplemented with 0.1 mg l⁻¹ IBA, 1.0 mg l⁻¹ BA, 30 g l⁻¹ sucrose and 0.8% agar), moreover, double-phase culture had a higher fresh mass than the other cultures.     

Micropropagation of the strawberry tree,Arbutus unedo L.

Actively growing shoots of potted greenhouse-grown strawberry tree (Arbutus unedo L.) were initially sterilised and established in basal woody plant medium containing 11.1 μM BA. Optimum shoot proliferation was achieved on a basal WPM containing MS vitamins, sucrose, agar and 22.2 μM BA. Microshoots rooted successfully in basal in vitro medium containing 10 μM IBA or IAA, but their survival rate during acclimatisation was low. Addition of a mixture 1 part peat:4 parts perlite in the basal in vitro rooting medium (1:1 v/v) containing 10 μM IAA resulted in high rooting percentage and plantlets with branched roots. These plantlets were successfully acclimatised. This novel rooting medium can be exploited further due to its potential in commercial applications.     

Factors affecting tissue culture of Damask rose (Rosa damascena Mill.)

The present study was conducted to evaluate the regeneration ability of Damask rose. Single-node explants were surface sterilised with 10% chlorox for 15 min and cultured on Murashige and Skoog (MS) medium supplemented with various concentrations of N⁶-benzyladenine (BA) or kinetin (Kin) separately or in combination with different concentrations of indole-3-butyric acid (IBA). Combination of 2.5–3 mg/l BA and 0.1 mg/l IBA was the most suitable treatment for proliferation. In vitro derived shoots were subcultured four times on the fresh medium within a 4-week period. Other treatments such as explant orientation (horizontal, vertical and oblique) and explant wounding were also examined but did not affect shoot multiplication rate significantly. Several experiments were carried out to stimulate in vitro rooting of Damask rose. Application of different media such as MS, 1/2 MS, 1/3 MS and 1/4 MS with different concentrations of indole-3-acetic acid (IAA), IBA and naphthaleneacetic acid (NAA) did not produce satisfactory results. Quick-dip method using sterilised 0–2000 mg/l IAA, IBA and NAA solutions was also studied. Other treatments such as using various concentrations of abscisic acid (ABA) in combination with various concentrations of IAA, IBA and NAA, and using various concentrations of sucrose and agar did not produce any roots. The best treatment for rooting of shoots was 2.5 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) for 2 weeks in MS medium and then transferring the explants to MS medium without any growth regulator. Plantlets were acclimatised in a soil mixture consisting of peat moss and sand 1:1 (v/v) and successfully transferred to the greenhouse after 3 weeks.     

Factors affecting the shoot multiplication of Persian walnut (Juglans regia L.)

A number of experiments were conducted to identify suitable procedure for in vitro shoot multiplication of Persian walnut (Juglans regia L.). Three different nutrient media (DKW, MS and WPM) and three different gelling agents (Phytagel, Difco Bacto agar and a mixture of Phytagel and Difco Bacto agar) were studied in the first experiment. Driver and Kuniyuki walnut (DKW) medium solidified with 2.2 g l⁻¹ Phytagel was found optimum. Performance of explants was better on DKW medium than on MS and WPM. The DKW and MS media were not significantly different from each other, but both of them were significantly better than WPM, which was a very poor medium for this species. Phytagel alone was significantly better than Difco Bacto agar or Phytagel combined with Difco Bacto agar. In another experiment different concentrations of BA were studied. Medium containing 1.0 mg l⁻¹ BA and 0.01 mg l⁻¹ IBA was the best, although medium containing 0.6 and 0.8 mg l⁻¹ BA were also successful, and utilisation of 0.4 mg l⁻¹ BA and 0.01 mg l⁻¹ IBA was optimum for shoot elongation. Application of different kinds of auxins (IAA, IBA and NAA at 0.01 or 0.1 mg l⁻¹) with 1.0 mg l⁻¹ BA were also studied. Media containing IBA were significantly better than media containing IAA for shoot fresh weight, but neither of them was significantly different from media containing NAA. Application of 0.01 mg l⁻¹ or 0.1 mg l⁻¹ auxin, with 1.0 mg l⁻¹ BA, was not significantly different for shoot multiplication of Persian walnut. The morphology of shoots on media containing 0.01 mg l⁻¹ IBA was the best.     

In vitro production of Prunus necrotic ringspot virus-free begonias through chemo- and thermotherapy

Prunus necrotic ringspot virus (PNRSV)-free Begonia spp. plants were raised from petioles of virus-infected plants using in vitro techniques. The petioles were grown on MS medium supplemented with 0.2 mg/l NAA and 0.2 mg/l BAP (pH 5.8). For rooting, half-strength MS medium without any plant growth regulators was used. On rooting medium, shoots were subjected to chemotherapy (virazole, 2-thiouracil or 6-azauracil) and thermotherapy (38 °C for 16 h light period and 22 °C for 8 h dark period) separately or in combination. Regenerated plants (treated with chemo- and thermotherapy) were indexed for PNRSV by DAS-ELISA and RT-PCR. An amplified product of 785 bp was obtained by RT-PCR in PNRSV-infected plants. Virazole at a concentration of 20 mg/l was found to be more effective (30 and 20% of PNRSV-free plants as indexed by ELISA and RT-PCR, respectively) in comparison to the other chemicals. Thermotherapy for 25 days gave 35 and 25% PNRSV-free plants as indexed by DAS-ELISA and RT-PCR, respectively. A combination of both treatments gave a good number of PNRSV-free plants (67.5 and 57.5% as indexed by DAS-ELISA and RT-PCR, respectively). At higher concentrations all three chemicals were found to be toxic. Thermotherapy for more than 25 days caused browning of leaves and shoots died.     

Rapid in vitro multiplication and conservation of Garcinia indica: A tropical medicinal tree species

A simple and efficient method has been developed for rapid regeneration of plantlets via adventitious bud differentiation on mature seeds of Garcinia indica (Thouars) Choisy, a medicinally important facultative apomictic tropical tree species. High frequency direct shoot proliferation was induced in seed segments cultured on Murashige and Skoog's medium supplemented with cytokinins (BAP, kinetin and TDZ) alone and in combination with auxin (NAA). Amongst the various combinations used, BAP proved to be the most effective. Multiple shoots formed within 4–5 weeks of culture. The shoot forming capacity of the seeds was influenced by the BAP concentration tested (5–50 μM) and optimal response was observed at different concentrations (12.5–50 μM) in different genotypes investigated. Significant differences were recorded in terms of percent response (27.78–100%) as well as average number of shoots per explant (3.49–57.67) among the four genotypes investigated. Elongation of the induced shoots was achieved on MS basal medium containing 0.2% activated charcoal. The induction medium had a profound effect on rate of bud elongation with shoots induced on lower concentrations of BAP showing as much as four-fold elongation within 4 weeks. Proliferating shoot cultures were established by repeatedly subculturing the shoot nodes on MS medium supplemented with 5 μM BAP. Maximum rooting (91.66%) occurred in shoots cultured on half-strength MS medium supplemented with 10 μM IBA. The plantlets were successfully transferred to soil after hardening; the time period from initiation of shoot buds to transplantation being 15–17 weeks. For in vitro conservation, the shoot cultures were maintained on medium supplemented with 0.5 μM BAP and the subculture duration could be enhanced up to maximum of 11 months.     

Effect of phytohormones on in vitro shoot multiplication and rooting of lime Citrus aurantifolia (Christm.) Swing

Multiple shoots were produced from node explants of lime (Citrus aurantifolia (Christm.) Swing) on MS medium supplemented with 6-benzylaminopurine (BAP), 6-furfurylaminopurine (kinetin) and α-naphthaleneacetic acid (NAA). The highest number of shoots, nine shoots per node, were produced on a medium containing 2 mg l⁻¹ BAP (8.8 μM), 1 mg l⁻¹ kinetin (4.6 μM) and 1 mg l⁻¹ NAA (5.4 μM). Depending on the concentration of BAP and kinetin, NAA either inhibited, stimulated or did not affect shoot multiplication, which also depended on the cytokinin level. Maximum shoot length was obtained from treatments containing 0.5 mg l⁻¹ BAP (2.2 μM) combined with 1 mg l⁻¹ kinetin (4.6 μM) and 0.5 mg l⁻¹ NAA (2.7 μM). The largest leaves of resultant shoots were produced on a medium containing 0.5 mg l⁻¹ each of kinetin (2.3 μM) and NAA (2.7 μM). Transferring in vitro shoots to rooting media containing indole-3-butyric acid (IBA) and NAA produced complete plantlets. The highest rooting percentage was obtained on a medium containing either 1 mg l⁻¹ NAA (5.4 μM) alone or 0.5 mg l⁻¹ NAA (2.7 μM) combined with 2 mg l⁻¹ IBA (9.6 μM), whereas the highest number of roots were produced on a treatment containing both 2 mg l⁻¹ NAA (10.8 μM) and 2 mg l⁻¹ IBA (9.6 μM). Roots elongated most on treatments containing 0.5 mg l⁻¹ of either NAA (2.7 μM) or IBA (2.4 μM). Shoot growth associated with the rooting phase was the highest in response to 2 mg l⁻¹ IBA (9.6 μM) or 0.5 mg l⁻¹ NAA (2.7 μM). Plantlets that survived acclimatization, 82%, exhibited normal growth in soil under greenhouse conditions.     

The effect of mycorrhizal symbiosis on the development of micropropagated artichokes

In this work, microrosettes of Cynara cardunculus L. var. scolymus Fiori of the “catanese” type were subcultured in a medium supplemented with 6-benzylaminopurine (BAP) (0.05 mg l⁻¹). For root induction, indoleacetic acid (IAA), α-naphthalene acetic acid (NAA) and indole-3-butyrric acid (IBA) were used at three concentrations: 2, 5 and 10 mg l⁻¹. The highest percentage of rooted shoots was aided by the presence of 10 mg l⁻¹ IAA.Once transplanted in pots, the plantlets were inoculated with 10 g Glomus viscosum strain A6 (AM fungus). Acclimatisation was clearly facilitated by the addition of the AM fungus. Indeed, the mycorrhizal plantlets registered a survival of between 90 and 95% for the rooting shoots and 60% for the non-rooting shoots.The botanical characterization of the material produced was carried out in field and was based on several morphological and productive parameters. Data collected confirm the characteristics of the original cultivar, the efficiency of the in vitro propagation material and the possibility of using this technique in early types of artichoke.     

Establishment and plant regeneration of somatic embryogenic cell suspension cultures of the Zingiber officinale Rosc.

Somatic embryogenic cell suspension cultures of four ginger cultivars were established. Somatic embryogenic calli were induced from ginger shoot tips on MS agar medium supplemented with 1.0 mg l⁻¹ 2,4-D and 0.2 mg l⁻¹ Kn, which contained only half concentration of NH₄NO₃. Rapid-growing and well-dispersed suspension cultures were established by subculturing this kind of callus in the same liquid MSN medium. The suspension cultures (about 1–2 mm in diameter) were placed on the MSN agar medium for callus proliferation. Thereafter embryogenic callus (1.5 cm²) was transferred to solid media (MS + 0.2 mg l⁻¹ 2,4-D + 5.0 mg l⁻¹ BA + 3% sucrose + 0.7% agar). Somatic embryos produced shoots and roots, and shoots developed into complete plantlets on solid MS medium supplemented with 3.0 mg l⁻¹ BA and 0.1 mg l⁻¹ NAA. The relationship between the DW of suspension cultures and pH changes in medium is also discussed. The suspension cultures still kept their vitalities after subculture for 8 months.     

Shoot induction and plant regeneration from receptacle tissues of Lilium longiflorum

An efficient system has been developed for the in vitro plant regeneration of Lilium longiflorum Thunb. by culturing receptacle sections from flower buds. The sections were cultured on one-half MS medium plus 30 g l⁻¹ sucrose, 8 g l⁻¹ agar, 5.4 μM NAA or 4.9 μM IBA plus 2.2 μM BAP. A section size of 3–4 mm was found to be optimal. After 60 days an average of 41 shoots were formed per explant. More vigorous shoots were obtained by subculturing on hormone-free medium with 20 g l⁻¹ sucrose. Rooting occurred on one-half MS medium with 1.1 μM NAA. Rooted plants were hardened-off in a greenhouse for two months, and normal flowering plants were produced.     

Micropropagation of Vitis thunbergii Sieb. et Zucc., a medicinal herb,through high-frequency shoot tip culture

This study describes a protocol for rapid and large-scale in vitro propagation of the valuable medicinal herb Vitis thunbergii Sieb. et Zucc.. Culture conditions influencing shoot proliferation and rooting of the two clones (three- and five-lobed) were examined. Three medium formulations, Murashige-Skoog (MS), Woody Plant Medium (WPM) and Nitsch and Nitsch (NN) medium, were tested for growth of shoot tip culture, and WPM was found to have a superior proliferation rate. The chlorophyll content of leaves was highest in those cultured on NN, followed by those on MS and WPM medium. WPM medium supplemented with 0.5 mg l⁻¹ 6-benzyladenine (BA) displayed the highest proliferation rate (15–19 nodes or 3–4 shoots per explant). The rooting was optimized using MS medium supplemented with 0.5 mg l⁻¹ napthaleneacetic (NAA) with eight roots, 3 cm long after 1 month of culture. High frequency callus formation was observed in the basal end of explants cultured on NAA-containing medium. Following acclimatization, rooting plantlets were transferred to the plastic house with a 95% survival rate.     

An investigation on the effect of different plant growth regulating compounds in in vitro shoot tip and node culture of lemon seedlings

The possible application of some less commonly used in vitro growth regulating compounds is outlined. A number of treatments were applied to determine the best way of inducing in vitro shoot proliferation and rooting on a modified Driver–Kuniyuki [HortScience 19 (1984) 507] basal medium of lemon (Citrus limon (L.) Burm, f. cv. Interdonato) seedlings. 6-Benzyladenine (BA) alone (1, 2 and 4 mg l⁻¹) and in combination with either orange juice (10%, v/v), silver nitrate (3 mg l⁻¹), gibberellic acid (GA₃) (0.1 mg l⁻¹ at the establishment stage and 0.5 mg l⁻¹ at all combinations during the proliferation stage) or abscisic acid (ABA) (0.2 mg l⁻¹ only at the establishment stage) were used to stimulate shoot formation during the establishment and the proliferation stage. The combination of BA with ABA gave a high rate of shoot formation, while GA₃ and silver nitrate enhanced shoot elongation. When these shoots were transferred to the rooting stage, the effect of application of two different auxins (indole-3-butyric acid (IBA) and α-napthaleneacetic acid) was examined, as well as different methods of application (auxin added to the basal medium and auxin application by dipping the base of the explant in auxin solution). Dipping the base of the explants in a 50% ethanol solution of IBA at 1000 mg l⁻¹ for 5 s resulted in 80% rooting with subsequent 90% survival of these explants, during acclimatization under mist.     

Improvement of in vitro micropropagation of Lilium oriental hybrid ‘Casablanca’ by the formation of shoots with abnormally swollen basal plates

Bulb scales of Lilium oriental hybrid ‘Casablanca’ were cultured on MS medium supplemented with cytokinins (kinetin, BA (benzyladenine), TDZ (thidiazuron)). The basal part of bulb scales swelled and formed adventitious shoots with foliage small, leafy bulb scales and abnormally swollen basal plates on the media with cytokinins. Shoots were formed rapidly from the basal parts of bulb scales and became shoot clusters. The medium containing 2.2 μM BA was most favorable in the shoot formation from bulb scales. Cutting shoots into small segments (7–8 mm wide × 12–15 mm length) were cultured on media containing BA and IAA (indole acetic acid) and the segments regenerated many new shoots and formed shoot clusters. The shoot section to shoot cluster cycle method improved adventitious shoot proliferation. The media supplemented with 4.4 μM BA and 5.7 μM IAA. or 8.9 μM BA and 0.6–2.9 μM IAA were effective in allowing the proliferation of shoots from shoot segments under light condition. Sucrose and activated charcoal (AC) improved bulblet growth. Bulblet growth was effectively performed on MS medium containing 60 g/L sucrose. Bulblet growth was also improved by the supplement of AC. The medium with 2.0 g/L AC was most effective for bulblet growth. Normal bulblet growth was stimulated more by the culture of shoots than that of bulb scales. Bulblet weight from shoots reached to an average of over 1100 mg of a bulblet after 3 months in culture on MS medium containing 60 g/L sucrose and 2 g/L AC. Most of the bulblets produced by this method generated stems with several leaves in the green house, after cold treatment at 5 °C for 3 months.     

Production of high quality Ardisia plants by stem tip cuttings

To produce commercially acceptable Ardisia plants, stem tip cuttings from mature plants were rooted and forced in greenhouses. Ten centimeter long cuttings were either treated with 200 ppm 1-naphthalene acetic acid (NAA) for 2 h, 2000 ppm indole-3-yl-butyric acid (IBA) for 10 s, or 0.5 and 1.0% IBA powder prior to sticking them in the rooting medium. Rooting percentage at 45 days exceeded 76% with 2000 ppm IBA treatment which was a significant increase over non-treated control. Rooted cuttings developed into three types of plants: those forming only vegetative shoots without flowers, those forming reproductive shoots with flowers, and those forming both vegetative and reproductive shoots. The ideal plant produced only vegetative shoots when rooted cuttings were transplanted into pots. About 50% rooted cuttings were forced to finish, producing 31 or 40% of high quality plants when rooted cuttings with vegetative shoots were grown in a greenhouse (GH) at temperatures higher than 21/19 °C (day/night) in 1995 or 21/18 °C GH in 1997, respectively. This method shortened the total production time to less than 2 years as compared to 4 years when starting from seeds.     

Inhibitory effects of riboflavin (Vitamin B2) on the in vitro rooting and nutrient concentration of explants of peach rootstock GF 677 (Prunus amygdalus × P. persica)

The effect of vitamin riboflavin (B₂) on in vitro rooting of the almond × peach hybrid clone GF 667 was studied. Riboflavin was added in five concentrations: 0 (control), 0.5, 1.0, 1.5 and 2.0 mg l⁻¹. After 4 weeks in culture, riboflavin did not stimulate adventitious rooting of the explants and rooting was very low in comparison to the control treatment. A high percentage of shoots on the rooting media containing the two highest concentrations of Vitamin B₂ had shown symptoms of chlorosis and apex necrosis.     

Shoot regeneration via direct organogenesis from in vitro derived leaves of mulberry using thidiazuron and 6-benzylaminopurine

A brief culture of mulberry leaves for 8–10 days on MS medium with 18.17 μM TDZ followed by transfer to 8.88 μM BAP supplemented medium triggered high frequency shoot organogenesis (77.6–89.2%) and favoured shoot elongation in Morus spp. Shoot proliferation was highest in the presence of 2.22 μM BAP with induction of 9.4–10.6 shoots per culture. High frequency of root induction (76.0–86.6%) was observed on medium supplemented with 0.49 μM IBA whereas increase in the level of IBA (4.92 μM) resulted in induction of roots along with development of callus from the base of the shoots. The regenerated plants established in soil at higher frequency in rainy season compared to winter and summer.     

Direct shoot organogenesis on hypocotyl explants from in vitro germinated seedlings of Psidium guajava L. cv. Allahabad Safeda

Multiple shoots were generated via direct organogenesis on hypocotyl segments excised from in vitro germinated seedlings (45-day-old) of Psidium guajava L. cv. Allahabad Safeda. Modified basal Murashige and Skoog (MMS) medium supplemented with 6-benzylaminopurine (BAP), zeatin or thidiazuron with or without α-naphthalene acetic acid (NAA) were tried. Thidiazuron (0.1 μM) along with 0.54 μM NAA gave the highest response (44.6%) with the regeneration of 3.6 shoots per original explants. These shoots upon subculture gave rise to about 5.0 shootlets per explant in shoot proliferation medium, i.e. MMS supplemented with 2.22 μM BAP + 2.32 μM kinetin. The regenerated microshoots were elongated using a quick dip of gibberellic acid (GA₃; 1.44 mM) prior to culture on MMS medium supplemented with 0.88 μM BAP and adenine sulphate (54.29 μM) for 2 weeks. Rooting of microshoots was achieved best on half strength MMS medium supplemented with 4.90 μM indole-3-butyric acid along with 100 mg l⁻¹ activated charcoal.     

Protocol for rapid propagation,and to overcome delayed rhizome formation in field established in vitro derived plantlets of Kaempferia galanga L.

Single medium based efficient protocol for rapid propagation, and to overcome the delayed rhizome formation in field established in vitro derived plantlets of Kaempferia galanga L. through in vitro rhizome induction was achieved. MS medium with combination of 8.87 μM N⁶-benzyladenine (BA), and 2.46 μM indole-3-butyric acid (IBA) induced a mean of 6.2 shoots per explant. Addition of 11.7 μM silver nitrate to 8.87 μM BA and 2.46 μM IBA supplemented medium facilitated the highest number of shoots (mean of 8.3 shoots) as well as roots within 60 days. Subculture of isolated shoots on medium with the same concentration of BA, IBA and silver nitrate increased the number to a mean of 12.1 shoots. Silver nitrate enriched medium developed rhizome at the base of shoots. Increase of sucrose concentration (6–8%) in medium with BA, IBA and silver nitrate favoured the best rhizome development. Ninety five per cent of the plantlets survived in field conditions. The plantlets established in field without in vitro developed rhizome (from medium with BA and IBA) did not form rhizome even at 7 months after transplantation. Instead, they developed tuberous roots only. The plantlets with in vitro developed rhizome (on medium having BA, IBA, silver nitrate, and 6–8% sucrose), and that established from conventional way (through splitting of old rhizome) showed no difference in growth of the rhizome. The present study emphasizes the efficacy of silver nitrate and sucrose to develop rhizome in vitro, which enabled to overcome the delayed development of rhizome, and reduced yield of plantlets established in field without in vitro developed rhizome.     

Factors controlling high efficiency adventitious bud formation and plant regeneration from in vitro leaf explants of roses (Rosa hybrida L.)

Studies were conducted to improve adventitious bud regeneration in roses (Rosa hybrida L.), specifically to extend the protocol to different genotypes and to initiate production of multiple shoots per explant. The best results were obtained by using a two-stage procedure where excised leaflets were incubated on Murashige and Skoog (MS) (1962) induction medium with 6.8 μM TDZ plus 0.49 μM IBA in the dark for 7 days and subsequently transferred to an MS-based regeneration medium with 2.22 μM BA plus 0.049 μM IBA exposed to a PPFD of 15 μmol m⁻² s⁻¹ PAR. Bud formation capacity was also significantly affected by the genotype and the environment, such as the use of bottom cooling creating a lower RH in the vessel. The addition of silver nitrate to the induction medium also significantly improved the percentage of regeneration in three genotypes tested. Regenerated shoots failed to elongate when transferred to MS proliferation medium containing 0.5 mg l⁻¹ BA, however maximum bud development and elongation were achieved when kinetin in the range 1–2 mg l⁻¹ was used. Elongated shoots were excised and rooted best on zero growth regulator half-strength MS modified medium. Rooted plantlets were acclimatized under greenhouse conditions for evaluation of somaclonal variation.     

Embryo induction via anther culture in papaya and sex analysis of the derived plantlets

We investigated the embryo induction of papaya by anther culture, and identified the sex of plantlets derived from embryos using a sex-diagnostic PCR. Anthers, containing approximately 80% uninucleate pollen, were collected from 10 to 14 mm long male flower buds. They were pre-treated on agar (0.8%) or in liquid medium for 1–5 days at 25 or 35 °C, then transferred to agar medium with 0.1 mg l⁻¹ BA and 0.1 mg l⁻¹ NAA. Agar and liquid media used for the pre-treatment contained water only or MS nutrients with or without sucrose (2.0%). On the agar medium, no embryos were induced at any pre-treatment temperature. In the liquid medium at 25 °C, embryos were induced at about 1.0% (rate of anthers forming embryos) in MS medium with sucrose for 3 or 5 days. At 35 °C, embryo induction rate tended to increase up to about 4.0% when anthers were treated in water for 1 day or MS medium with sucrose for 3 or 5 days. The sex of plantlets established through anther culture was analyzed using a sex-diagnostic PCR. All plantlets were determined as female. From these results, we suggest that all plantlets established through anther culture were of microspore origin, and that the anther culture technique is useful for the breeding of female papaya.