Subculture Frequency Research Articles

Enhancing Protoplast Isolation and Early Cell Division from Cannabis sativa Callus Cultures via Phenylpropanoid Inhibition.

De novo regeneration of Cannabis sativa L. (cannabis) using tissue culture techniques remains unreliable and infrequent. Conventional methods for the regeneration and transformation of cannabis have not achieved the reliability and replicability that need to be integrated into research and breeding programs. Protoplast systems are effective for gene expression studies and transformation and genome-editing technologies and open the possibility of somatic hybridization to create interspecific hybrids. To date, leaf-derived protoplasts have been isolated for transient gene expression studies, but protoplast-to-plant regeneration has not been reported. The present study aims to evaluate the efficacy of using a callus culture system as an abundant tissue source for protoplast isolation and lays the groundwork for a protoplast-to-plant regeneration system. Using hypocotyl-derived callus cultures, which are known to have relatively greater regenerative potential, the efficacy of protoplast isolation and initial cell division were assessed. In this study, the effect of 2-aminoindane-2-phosphonic acid (AIP), a competitive inhibitor of phenylalanine ammonia lyase (PAL), in callus culture media and the effect of subculture frequency on protoplast yield were assessed. This study found that inclusion of AIP at 1 mM resulted in a 334% increase in protoplast yield compared with AIP-free medium, representing the first known use of AIP in cannabis tissue culture. Inclusion of AIP led to a 28% decrease in total soluble phenolics and 52% decrease in tissue browning compared with the control medium. Lastly, a two-phase culture system for protoplast regeneration was tested. At a concentration of 2.0 × 105 protoplasts per mL, cell wall reconstitution and cell division were observed, providing one of the first know reports of cell division from cannabis protoplasts and setting the stage for the future development of a protoplast-to-plant regeneration system.

Coconut Callus Initiation for Cell Suspension Culture

The development of a cell suspension culture system for the scaling up of coconut embryogenic callus (EC) production would drastically improve efforts to achieve the large-scale production of high-quality clonal plantlets. To date, the hard nature of coconut EC appeared to be the main constraint for developing cell suspension cultures. Hence, this study attempted to acquire friable EC through the following approaches: The manipulation of (1) medium type and subculture frequency, (2) a reduced 2,4-dichlorophenoxy acetic acid concentration during subculture, (3) the nitrate level and the ammonium-to-nitrate ratio, and the addition of amino acid mixture, (4) the addition of L-proline, and (5) the reduction of medium nutrients. Unfortunately, none of these culture conditions produced friable coconut EC. Even though friable EC was not achieved via these approaches, some of the conditions were found to influence the formation of compact EC, therefore these results are important for further studies focused on somatic embryogenesis in coconut and other species.

EFFECTS OF LIGHT ILLUMINATION AND SUBCULTURE FREQUENCY ON BIOMASS PRODUCTION IN CELL SUSPENSION CULTURES OF Clinacanthus nutans

Clinacanthus nutans (Burm. f.) Lindau is a medicinal plant rich in polyphenols and possesses multiple pharmacological properties. It is widely used as a side treatment for cancer and to prevent oxidative stress-related diseases. The present study aims to investigate the effects of light illumination and subculture frequency on the growth and proliferation of cells in the suspension culture of C. nutans. Friable callus induced from leaf explants C. nutans were cultured in liquid Murashige & Skoog medium supplemented with 0.25 mg/L 2, 4-dichlorophenoxyacetic acid, and 0.25 mg/L 6-benzylaminopurine to initiate the cell suspension culture. The suspension cultures were incubated under different conditions (16 hr photoperiod and continuous darkness). Subcultures were conducted every two weeks for ten subculture cycles. Results revealed that higher cell biomass was obtained under the exposure of 16 hr photoperiod with the highest biomass production at the fifth subculture cycle (growth index of 11.8) exhibiting stable growth from the eighth to the tenth subculture cycle. The suspension cells showed stable growth rates up to the 10th subculture cycle producing small aggregates and single cells. The present study indicated that light illumination significantly impacts the growth of suspension cultures for C. nutans in the production of valuable secondary metabolites.

Effect of Subculture Frequency Toward Growth And Carotenoid Content from Tomato (Lycopersicon Esculentum Mill.) Callus

Tomatoes (Lycopersicon esculentum Mill.) are a source of carotenoids they are easy to find. These compounds function as precursors of vitamin A, antioxidant, and prevent cancer. The extraction of carotenoid compounds for commercial products usually uses fresh plants, which are less efficient and require a lot of raw materials. The supply of these raw materials can be done through tissue culture. The frequency of subculture or supply of nutrients in tissue culture is very influential on the content of callus carotenoids produced. This study aimed to determine the effect of subculture frequency on growth, development and callus carotenoid content and to find out the right frequency of subculture to produce callus with optimal growth, development and carotenoid content. The design this study was a single Completely Randomized Design (CRD) with 4 treatments of subculture frequency and 5 replications. The data obtained were analyzed by ANOVA at the 95% test level followed by DMRT in case a significant different was found The results showed subculture frequency affected growth, development and carotenoid content of callus Lycopersicon esculentum. Mill. The most optimal treatment to induce growth and production of carotenoids in this study was treatment of thrice subculture while the most optimal treatment in inducing development was the twice subculture treatment.

Increased endogenous indole-3-acetic acid:abscisic acid ratio is a reliable marker of Pinus massoniana rejuvenation

ABSTRACTPinus massoniana is a recalcitrant tree species for rooting in vitro. We rejuvenated 26-year-old P. massoniana trees by successive grafting. Rooting rates of rejuvenated shoots were > 83.1% after rooting induction. We compared endogenous levels of indole-3-acetic acid (IAA), abscisic acid (ABA), gibberellins (GAs) and zeatin-riboside (ZR), and the rhizogenesis ability of axillary shoots of mature and rejuvenated materials in vitro, i.e., somaplants and grafts. Enhancement of the rooting ability of mature materials in vitro following somatic embryogenesis or repeated grafting onto juvenile rootstocks was accompanied by increased IAA and GAs levels, and by decreased ABA levels in scions used as starting material for micropropagation in vitro. Successive subcultures did not influence the rooting ability of shoots from untreated mature material. Rooting ability of shoots in vitro, however, gradually increased with subculture frequency during repeated subculturing in grafting materials. The IAA:ABA ratio in shoots in vitro after grafting five times, and consequently capable of root organogenesis, was higher than in shoots of untreated mature material incapable of root organogenesis in vitro. A high IAA:ABA ratio was detected in scions of somaplants that were capable of rooting in vitro despite subculture times. We found that the endogenous IAA:ABA ratio is a reliable marker for the recovery of root organogenesis in vitro after rejuvenating treatments for mature P. massoniana trees.

Reduction of the subculture frequency in three Mediterranean species during micropropagation

Reduction of the subculture frequency in three Mediterranean species during micropropagation

Genetic Stability of Cassava Plants Regenerated Through Organogenesis Using Microsatellite Markers

Tissue culture technology of cassava (Manihot esculenta Crantz) is a viable alternative to currently adopted techniques for mass propagation, germplasm conservation and genetic improvement. However, somaclonal variation is a common phenomenon in tissue culture which makes it mandatory to monitor the genetic stability of plants. Therefore, the objective of this study was to evaluate the genetic stability of cassava plants regenerated from axillary bud explants through direct organogenesis using simple sequence repeat (SSR) markers. High shoot regeneration (81.2 – 90.0%) occurred in MS medium supplemented with 10 mg/L 6-bnzylaminopurine (BAP) and multiple shoots (2 – 4 shoots per from axillary bud explant) were formed for all the three cultivars (TME14, TMS60444 and Kibandameno) tested. High frequency of rooting (100%) was obtained after transferring the plantlets to cassava basic medium (CBM) and the rooted plants were successfully hardened and acclimatized in the glasshouse with 100% survival rate. Three-month old plants exhibited normal morphological characters comparing with the mother plant. A total of 10 SSR markers were used to validate the genetic homogeneity amongst five randomly selected plants along with the donor mother plants. DNA fingerprints of axillary bud regenerated plants displayed monomorphic bands similar to mother plant, indicating homogeneity among the regenerated plants with donor mother plant. The effect of subculture frequency on genetic stability of axillary bud-derived regenerants and micropropagated plants was also assessed using SSR markers. All the SSR profiles from axillary bud regenerants and micropropagated plants were monomorphic and comparable to mother plants from 1st to 5th subculture, confirming the genetic stability among clones and mother plants. At the 6th subculture, similarity indicators between the progenies and the mother plants ranged from 0.95 to 1.0 and such a similarity indicated a very low polymorphism. The dendrograms generated through Unweighted Pair Group Method with arithmetic mean (UPGMA) analysis of the 6th subculture revealed 96% similarity amongst axillary bud regenerants and micropropagated plants with donor mother plant. This low polymorphism ratio between mother plants, axillary bud regenerants and micro-propagated plants indicates the little effect of somaclonal variations, the high genetic similarity between mother plants and progenies and demonstrates the reliability of this propagation system for cassava. These results suggest that direct organogenesis from the axillary buds is the safest method for regeneration of true-to-type plants and this system can be used for clonal mass propagation, germplasm conservation and genetic transformation of cassava.

Effect of subculture times on genetic fidelity, endogenous hormone level and pharmaceutical potential of Tetrastigma hemsleyanum callus

The effects of subculture frequency on genetic variations of Tetrastigma hemsleyanum callus under a long-term tissue culture were evaluated by means of flow cytometry (FCM), inter simple sequence repeat (ISSR), and sequence-related amplified polymorphism (SRAP) markers to clarify the variation pattern and frequency of somatic variation in the course of the subculture. The effect of subculture frequency on endogenous hormone content was also assessed by Enzyme-linked immunosorbent assay to explore the correlation within the differentiation rate, somaclonal variation frequency of callus and the endogenous hormone content. Moreover, the flavonoid content in the callus of different subculture times was compared and the antitumor activity of them was evaluated by using MTT cell proliferation and cytotoxicity assay. FCM confirmed similar ploidy level in mother plant and all callus, but some changes were observed in the ISSR and SRAP patterns, of which 10.3 and 6.06 % were polymorphic, respectively. The somatic variation frequency tended to increase with the increasing subculture time in the early stage of subculture, with a peak in the 4–6 times and then kept nearly at a constant after 8 times. High IAA content may be one reason for this variation by this period. The flavonoid content and antitumor potential increased and remained at high level during the 2nd and 6th subculture. Though there was a significant decline after the 6th subculture, they still had about the same flavonoid content and antitumor potential as those of mother plant. The results suggested that the in vitro callus of T.hemsleyanum is potentially useful for pharmaceutical uses. For supplying true-to-true seedlings, germplasm conservation and further genetic transformation, subculture frequency of callus should be controlled within 4 times to obtain clonally identical plantlets. However, for the development of germplasm improvement of the species, subculture frequency should be controlled in 6–8 times to make better use of somatic variations.

저온보존기간이 담자균류의 균사생장과 목질분해효소의 활성에 미치는 영향

Subculture is the most common method for preservation fungi, but has a disadvantage of accumulation of sponta- neous mutations during the repeated subculture. To reduce the subculture frequency, the effect of preservation period at 4 o C in a slant culture was examined on the mycelia growth and lignocellulolytic enzyme activities of various basidiomycetes. Mushrooms, including Stereum ostrea, Coprinellus micaeus, Trametes versicolor, Hypholoma fasciculare, Wolfiporia extensa, Pleurotus pulmo- narius, Piptoporus betulinus and Ganoderma applanatum were not affected by the preservation period more than two years, indicating that they can be maintained by subculture every two years. Some other tested fungal strains showed a significant decrease in both viability and enzyme activity when they were maintained for two years, suggesting that they should be subcultured at least once in a year. A little correlation was found between the recovery of mycelial growth and extracellular enzyme activity. In conclusion, mycelial activity and enzyme activity according to storage period is expected to be a way of deciding on subculture times for fungal preservation.

Regeneration and Agrobacterium-mediated transformation of multiple lily cultivars

To pursue genetic improvement of lily, efficiency of both regeneration and transformation from callus cultures induced from different explants were evaluated in multiple cultivars. Thirty-five callus lines induced from filaments or styles and one control callus line derived from bulb scales of in total twenty lily cultivars representing Lilium longiflorum, Oriental × Trumpet and Longiflorum × Asiatic hybrids were maintained on a medium with 8.3 μM picloram (PIC). In this study, they were tested for their regeneration potential by transferring them onto a regeneration medium supplemented with 0.4 μM PIC and 0.044 μM 6-benzyladenine. Regeneration was obtained in all cultivars examined and the percentage varied from zero to 89 % in the 36 callus lines. Regeneration frequency was significantly influenced by the genotype (cultivar). Subculturing the calli every 4 weeks by refreshing the regeneration medium contributed positively to bulblet formation, when compared to an eight week subculture frequency. It was found that the regeneration ability generally decreased with an increasing age of the callus cultures for all cultivars. The origin of the callus (style or filament) did not lead to significant differences in regeneration frequency, but there was an interaction between callus origin and genotype. Calli of eight randomly chosen cultivars were co-cultivated with Agrobacterium tumefaciens strain AGL0 carrying binary vectors with the gus gene as reporter and putative transgenic plants were produced. GUS histochemical assays demonstrated transient and stable expression of the gus gene in both calli and regenerated lily plants. Transient expression frequencies ranged from 0.3 to 20.6 % while stable transformation was much lower, only 1.4 % as the maximum.

OPTIMIZATION OF PROTOCOLS FOR THE IN VITRO MULTIPLICATION AND CONSERVATION OF ACORUS CALAMUS, AN ENDANGERED MEDICINAL PLANT

ISHS IV International Symposium on Acclimatization and Establishment of Micropropagated Plants OPTIMIZATION OF PROTOCOLS FOR THE IN VITRO MULTIPLICATION AND CONSERVATION OF ACORUS CALAMUS, AN ENDANGERED MEDICINAL PLANT

Effect of Plant Growth Regulators and Subculture Frequency on Callus Culture and the Establishment of Melastoma malabathricum Cell Suspension Cultures for the Production of Pigments

Effect of Plant Growth Regulators and Subculture Frequency on Callus Culture and the Establishment of Melastoma malabathricum Cell Suspension Cultures for the Production of Pigments

Karakterisasi secara Morfologi Abnormalitas Embrio Somatik Kelapa Sawit (Elaeis guineensis Jacq) dari Eksplan Daun

<p>Morphological Characterization on Abnormalities of Oilpalm<br />(Elaeis guineensis Jacq) Embryo Somatic Generated<br />from Leaf Explant. Nesti F. Sianipar, Gustav A.<br />Wattimena, Hajrial Aswidinnoor, Maggy Thenawidjaya<br />S., Nurita Toruan-Mathius, and Gale Ginting. Somatic<br />embryogenesis is the development of somatic cells to form a<br />structure alike zygotic embryo direct or indirectly. Somatic<br />embryos from young leaf explants could be induced from<br />primary callus formed surrounding the palm-leaf rib. Embryogenic<br />callus will develop to be somatic embryos which<br />grew nonuniformly. Embryo somatic growth pattern of<br />globular, asymmetric heart shape, and cotyledon ary stage<br />produced different morphological variation. Morphological<br />variability of in vitro somatic embryos could be due to high<br />application of growth regulator 2,4-D at the beginning of<br />initiation, subculture frequency, loaded cells, and polysomic<br />cells from certain tissues. From the three clones used,<br />which were clone 638, 636, and 558, there were different<br />variation at each step of development stages, grouping<br />morphologically into normal and abnormal based on the<br />development of somatic embryos. The percentage of abnormality<br />from the three clone used was clone 27% (638), 30%<br />(636), and 46% (558). The normal somatic embryos at globular<br />stage were round and bipolar shaped; while the abnormal<br />embryos were oval and no bipolar. At heart-shape stage,<br />the normal somatic embryos had symmetrical polarized surface;<br />while the abnormal embryos had asymmetrical polarized<br />surface. At the cotyledon stage, the normal embryos<br />had monocot-tyledon; the abnormal ones were more than<br />one cotyledon.</p>

High subculture frequency, maltose-based and hormone-free medium sustained early development of somatic embryos in maritime pine

Summary Embryonal-suspensor mass (ESM) lines characterized by a spiky morphotype (i.e., developed early embryos escaping from the ESM periphery) were recently shown to produce the best maturation yields in Maritime pine. How to select or preserve such a valuable morphotype during ESM maintenance (prior to maturation treatment) is still unknown. Several maintenance procedures were tested; 2400 ESM from 10 lines were subcultured each 7 or 14 d on maltose- or sucrosecontaining medium without plant growth regulator (PGR) or supplemented with 2,4-dichlorophenoxyacetic acid and 6-benzylaminopurine or a low concentration of abscisic acid. Multiple components analyses based on growth rate, macromorphology data, and micromorphology data, collected for 6 mo., allowed for the association of the spiky morphotype with additional traits (defining the phenotype I) such as whitish aspect, high vigor, high growth rate, and complex cellular organization (resulting from high early embryogenic ability). Since a gradual increase in growth rate as well as a decrease in cellular organization were concomitantly observed during the 6 mo. experiment, we concluded that ESM growth and early somatic embryo development were disconnected. In some lines, the progressive loss of early embryogenic ability (aging process) could be decreased using maltose-based and PGR-free medium. For most lines, the aging effect was minimized using a weekly subculture. An improved procedure for ESM maintenance prior to the maturation step is thus proposed.

Growth profile of ectomycorrhizal fungal mycelium: emphasis on substrate pH influence.

A knowledge of behaviour of ectomycorrhizal fungal isolates to substrate pH would help in identifying candidate fungi for plantation programs. Ectomycorrhizal fungus isolates were studied to determine the pH optima for growth, substrate acidification by the culture and the effect of substrate acidification on culture growth. Of the isolates tested, the members of Agaricales (except Laccaria laccata) and Aphyllophorales favored neutral to near neutral pH, while members of the order Sclerodermatales strictly favored acidic pH. The change in substrate pH (deltapH) was maximum at the optimum growth pH for acidophilic isolates but minimum for neutro/basophilic isolates. The experiments indicate that the substrate pH would not only determine the growth rate of the fungus but also limits further proliferation of the fungus in medium. This gives vital information for determining the subculture frequency and for designing substrate parameters for nursery/plantation programs.

CRESCIMENTO E OXIDAÇÃO DE EXPLANTES DE BANANEIRA PRATA (Musa AAB) IN VITRO: IV. CONCENTRAÇÕES DE SAIS, ÁCIDOS ASCÓRBICOS E FREQÜÊNCIA DE SUBCULTIVOS

Avaliaram-se diferentes concentrações de sais do meio MS (Murashige & Skoog, 1962), freqüência de subcultivos e adição de ácido ascórbico ao meio de cultura, objetivando o controle da oxidação de explantes de bananeira-'Prata' (Musa AAB) na fase de estabelecimento. Os tratamentos constituíram-se das diluições dos sais do meio MS (100%, 50% e 33,33%), subcultivos (a cada 7 dias, a cada 14 dias e a cada 28 dias) e ácido ascórbico (0 e 25 mg L-1). O delineamento experimental foi inteiramente casualizado, cujos tratamentos foram arranjados em um fatorial 3 x 2 x 2. Utilizaram-se 15 repetições. As avaliações relativas à massa da matéria fresca, altura e oxidação foram feitas aos 28 dias após a inoculação. Quando o período de subcultivos foi maior (28 dias), o crescimento em massa da matéria fresca foi reduzido em função da redução da concentração de sais do meio MS. Para meios de cultura menos concentrados e freqüência maiores de subcultivos, não houve necessidade da adição do ácido ascórbico para a redução do escurecimento e houve uma tendência de maior crescimento em massa dos explantes.

Micropropagation of Echinacea angustifolia, E. pallida, and E. purpurea from Stem and Seed Explants

Micropropagation of three Echinacea species, E. angustifolia DC., E. pallida Nutt., and E. purpurea Moench., was investigated as a potential means of germplasm preservation of species faced with overcollection in the wild and rapid clonal propagation of elite individuals with unique medicinal or ornamental properties. Very high contamination rates occurred with shoot-tip explants but not with nodal segments. Contamination rates for seed explants were inversely related to the number of seedcoat layers removed, ranging from 100% contamination from intact seeds to near 0% contamination from excised embryos. Dormancy of seed explants was also eliminated when the pericarp and integument were removed. Addition of benzyladenine (BA) to the culture medium induced shoot multiplication and inhibited root formation in all three species. Shoot multiplication rates were low (1-3 shoots per culture) when seed explants were placed on a medium with BA levels low enough to avoid adventitious shoot formation (0.45 μm). Shoot count was higher on half-strength Murashige and Skoog (MS) minerals, while leaf size was greater on full-strength MS minerals. Cultures did not perform well in Woody Plant Medium. Reducing subculture frequency from 4 to 2 weeks increased shoot multiplication from 1.4 to 1.8 shoots per subculture and total shoots produced per subculture after 12 weeks from 2.8 to 23.9. Rooting occurred readily on shoots isolated from E. purpurea cultures and was not promoted by addition of IBA to the rooting medium. Rooting was low and nil on shoots from cultures of E. angustifolia and E. pallida, respectively. Methods described in this study allow rapid multiplication of three Echinacea species and subsequent rooting of E. purpurea. Future improvements in root induction treatments will allow these methods to be used effectively for micropropagation and maintenance of disease-free germplasm of Echinacea species. Chemical names used: N-(phenylmethyl)-1H-purine-6-amine (BA); 1H-indole-3-butyric acid (IBA).

333 Micropropagation of Echinacea angustifolia, E. pallida, and E. purpurea from Vegetative and Seed Explants

Micropropagation of three Echinacea species, E. angustifolia, E. pallida, and E. purpurea, was investigated as a potential means of germplasm preservation of species faced with overcollection in the wild and rapid clonal propagation of elite individuals with unique medicinal or ornamental properties. Comparison of explant sources indicated vegetative explants resulted in high contamination rates when collected from shoot-tips (100%),but not when collected from nodal explants (11% to 39). Seed coat removal reduced contamination from 100% in intact seeds to near 0% in excised embryos. Removal of seed coats (pericarp and integument layers) also eliminated dormancy requirement for germination. All species responded with shoot multiplication and loss of rooting when BA or thidiazuron was added to culture medium. Medium with thidiazuron resulted in excessive adventitious shoot formation. Shoot multiplication rates were low (one to three shoots/explant) on medium with BA levels low enough to avoid adventitious shoot formation. Medium containing half-strength MS minerals resulted in more shoots with smaller leaves than full-strength MS minerals. Cultures did not perform well on Woody Plant Medium. Increasing subculture frequency from every 4 weeks to every 2 weeks increased shoot multiplication rates from 1.4 to 1.8 shoots per subculture and total shoots produced after 12 weeks of culturing (per initial explant) from 2.8 to 23.9. Rooting occurred readily on shoots isolated from E. purpurea without addition of IBA. Rooting was low or non-existent on shoots from E. angustifolia and E. pallida, respectively, regardless of IBA level, light conditions, or temperature. Methods described in this study allow rapid multiplication of three Echinacea species and subsequent rooting of E. angustifolia and E. purpurea. Future improvements in root induction treatments will allow more effective use of micropropagation for Echinacea germplasm preservation and multiplication. Chemical names used: N-(phenylmethyl)-1H-purine-6-amine (BA), 1H-indole-3-butyric acid (IBA).

Increased root and shoot production during micropropagation of cherry and apple rootstalks: effect of subculture frequency.

Shoot cultures of apple rootstock cv. M9 and cherry rootstock cv. F12/1 were established and then divided into several sub-lines that were subcultured at 28-42-day intervals. Consequently, similarly aged cultures received various numbers of subcultures. Cultures kept at 24 degrees C showed an increase in shoot and root production over time. There were differences in shoot and root production between apple lines, but there were no differences among sub-lines. In cherry, altering the subculture interval affected rooting competence, which increased with time. Cherry cultures maintained at 4 degrees C gave rise to cultures that were as easy to root as cultures kept at 24 degrees C with more frequent subculturing. We conclude that total time in culture is the most important factor bringing about physiological changes in these genotypes of micropropagated apple and cherry.

Soybean somatic embryogenesis: Effects of hormones and culture manipulations

Somatic embryos were induced in cultures of immature soybean (Glycine max (L.) Merr) embryos, or isolated cotyledons on MS modified medium supplemented with NAA and 2,4-D, BAP and ABA. When NAA and 2,4-D were compared at similar concentrations (25 and 23 μM), 2,4-D produced larger number of somatic embryos, however, embryogenesis efficiency was improved in media containing NAA by using higher levels (100–150 μM) of the auxin. Somatic embryo morphology varied with auxin type: NAA-induced embryos more closely resembled zygotic embryos than did 2,4-D-induced embryos. Additions of BAP or ABA to auxin-containing media had either no effect or reduced embryo production, although ABA altered the morphology of 2,4-D-induced embryos. In media containing both NAA and 2,4-D, the latter was dominant in terms of embryo morphology. The effects of subculture frequency and of transfers between 2,4-D and NAA media were investigated: Subculture frequency influenced mainly the frequency of normal embryos, while preculture on 2,4-D increased subsequent embryogenesis efficiency on NAA medium but reduced the frequency of normal embryos.