Embryogenic callus induction and plant regeneration media for bentgrasses and annual bluegrass

Summary Embryogenic callus induction and plant regeneration systems have long been established for creeping bentgrass (Agrostis palustris Huds.), but little research has been reported on optimal medium for embryogenic callus induction and plant regeneration in velvet bentgrass (Agrostis canina L.), colonial bentgrass (Agrostis capillaries L.), and annual bluegrass (Poa annua L.). The present study compared 14 callus induction media and eight regeneration media for their efficacies on embryogenic callus induction and plant regeneration in these four species. The embryogenic callus initiation media contained the Murashige and Skoog inorganic salts and vitamins supplemented with 2,4-dichlorophenoxyacetic acid or 3,6-dichloro-anisic acid and 6-benzyladenine. l-Proline or casein hydrolyzate was included in some media to stimulate embryogenic callus formation and plant regeneration. The frequencies of embryogenic callus formation ranged from 0% to 38% and exhibited medium differences within each of the four species. Callus induction media, plant regeneration media, and genotypes affected plant regeneration rates, which varied between 0% and 100%. The embryogenic callus induced on Murashige and Skoog medium supplemented with 500 mgl⁻¹ casein hydrolyzate, 6.63 mg l⁻¹ (30 μM) 3,6-dichloro-anisic acid and 0.5–2.0 mg l⁻¹ (2–9 μM) 6-benzyladenine had much higher regeneration rates than those formed on other callus induction media. Embryogenic callus of annual bluegrass had higher regeneration rates than those of bentgrass species. MSA2D, a media containing 2 mgl⁻¹ (8 μM) 2,4-dichlorophenoxyacetic acid, 100 mgl⁻¹ myo-inositol, and 150 mgl⁻¹ asparagine, was effective in promoting embryogenic callus formation in creeping bentgrass but not in colonial and velvet bentgrasses and annual bluegrass.     

Plant regeneration from cotyledon tissues of common buckwheat (Fagopyrum esculentum Moench)

Summary Plants were regenerated from cotyledon tissue of greenhouse grown seedlings of common buckwheat (Fagopyrum esculentum Moench.). Maximum callus regeneration was induced on Murashige and Skoog (MS) medium containing 2,4-D (2.0 mg l⁻¹) and kinetin (KIN) (0.2 mg l⁻¹) and either 3 or 6% sucrose. Friable callus was transferred to MS media containing KIN and benzylaminopurine (BAP) at varied concentrations for embryogenic callus induction. The optimum medium for embryogenic callus induction was found to be MS medium supplemented with 0.2 mg l⁻¹ KIN, 2.0 mg l⁻¹ BAP and 3% (w/v) sucrose. Variation of sucrose from 3 to 6% did not show any significant effect on callus induction or embryogenesis. Regeneration of embryonic callus varied from 13 to 32%. Whole plants were obtained at high frequencies when the embryogenic calluses with somatic embryos and organized shoot primordia were transferred to half-strength MS media with 3% sucrose. Regenerated plants after acclimation were transferred to greenhouse conditions, and both vegetative and floral characteristics were observed for variation. This regeneration system may be valuable for genetic transformation and cell selection in common buckwheat.     

The effect of auxin type and cytokinin concentration on callus induction and plant regeneration frequency from immature inflorescence segments of seashore paspalum (<Emphasis Type="Italic">Paspalum vaginatum</Emphasis> Swartz)

Seashore paspalum (Paspalum vaginatum Swartz) is a salt tolerant, fine textured turfgrass used on golf courses in coastal, tropical, and subtropical regions. A callus induction and plant regeneration protocol for this commercially important turfgrass species has been developed. Induction of highly regenerable callus with approximately 400 shoots per cultured immature inflorescence (1 cm in length) was achieved by culturing 0.2 cm segments on media with 3 mg l⁻¹ 3,6-dichloro-2-methoxybenzoic acid (dicamba) and 0.1 or 1.0 mg l⁻¹ benzylaminopurine (BA). A multifactorial experiment demonstrated the combination of 3 mg l⁻¹ dicamba and 1.0 mg l⁻¹ BA for induction of callus resulted in 12 times higher plant regeneration frequency compared to 3 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) alone or ten times higher plant regeneration frequency than the combination of 3 mg l⁻¹ 2,4-D and 1.0 mg l⁻¹ BA. These results are expected to support the development of a genetic transformation protocol for seashore paspalum.     

Somatic embryogenesis and plant regeneration in centipedegrass (Eremochloa ophiuroides [Munro] Hack.)

Centipedegrass (Eremochloa ophiuroides [Munro] Hack.) is an important warm-season turfgrass and pasture grass. To explore the potential use of biotechnical tools in breeding of centipedegrass, we established an efficient plant regeneration system for this species. Four basal media and 24 combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzyladenine (BAP) were examined for their effects on callus induction from mature seed explants. Twenty combinations of naphthaleneacetic acid (NAA) and BAP were tested for their effect on plant regeneration. Results indicated that Murashige and Skoog basal medium supplemented with 4.5 mg l⁻¹ 2,4-D and 1 mg l⁻¹ BAP was the best medium for callus induction, while the combination of 2 mg l⁻¹ BAP and 1 mg l⁻¹ NAA induced the highest rate of regeneration and development of shoots and roots. This work provides a basis for the breeding of centipedegrass through somaclonal variation and genetic transformation.     

Efficient plant regeneration from shoot apices of sorghum

An efficient and rapid regeneration protocol was developed using shoot apices from germinating seedlings of two cultivars of sorghum, SPV-462 and M35-1, as explants. A vertical slit given from the base of each dissected apex enhanced the efficiency of callusing response by two fold. MS medium containing 0.5 mg dm⁻³ each of 2,4-D and kinetin was most effective in producing friable and embryogenic calli. Scanning electron microscopy of these calli detected somatic embryogenesis. Calli thus induced gave rise to approximately 42 green shoots per callus in both the genotypes when transferred to regeneration medium containing 1.5 mg dm⁻³ kinetin.     

<Emphasis Type="Italic">In vitro</Emphasis> plant regeneration through somatic embryogenesis and direct shoot organogenesis in <Emphasis Type="Italic">Pennisetum glaucum</Emphasis> (L.) R. Br.

An efficient in vitro plant regeneration protocol through somatic embryogenesis and direct shoot organogenesis has been developed for pearl millet (Pennisetum glaucum). Efficient plant regeneration is a prerequisite for a complete genetic transformation protocol. Shoot tips, immature inflorescences, and seeds of two genotypes (843B and 7042-DMR) of pearl millet formed callus when cultured on Murashige and Skoog (MS) medium supplemented with varying levels of 2,4-dichlorophenoxyacetic acid (2,4-D; 4.5, 9, 13.5, and 18 μM). The level of 2,4-D, the type of explant, and the genotype significantly effected callus induction. Calli from each of the three explant types developed somatic embryos on MS medium containing 2.22 μM 6-benzyladenine (BA) and either 1.13, 2.25, or 4.5 μM of 2,4-D. Somatic embryos developed from all three explants and generated shoots on MS medium containing high levels of BA (4.4, 8.8, or 13.2 μM) combined with 0.56 μM 2,4-D. The calli from the immature inflorescences exhibited the highest percentage of somatic embryogenesis and shoot regeneration. Moreover, these calli yielded the maximum number of differentiated shoots per callus. An efficient and direct shoot organogenesis protocol, without a visible, intervening callus stage, was successfully developed from shoot tip explants of both genotypes of pearl millet. Multiple shoots were induced on MS medium containing either BA or kinetin (4.4, 8.8, 17.6, or 26.4 μM). The number of shoots formed per shoot tip was significantly influenced by the level of cytokinin (BA/kinetin) and genotype. Maximum rooting was induced in 1/2 strength MS with 0.8% activated charcoal. The regenerated plants were transferred to soil in pots, where they exhibited normal growth.     

Rapid and efficient regeneration in soybean [<Emphasis Type="Italic">Glycine max</Emphasis> (L.) Merrill] from whole cotyledonary node explants

A rapid and efficient regeneration protocol was established for soybeans [Glycine max (L.) Merrill]. Whole cotyledonary node explants were collected from aseptic seedlings cultured on MSB₅ medium containing 0.4 mg l⁻¹ N⁶-benzyladenine (BA). The effects of the plant growth regulators BA, kinetin (KT), indole-3-butyric acid (IBA) as well as the explant genotype on shoot regeneration were evaluated by the orthogonal design [L₁₆(4⁵)]. The process of shoot development was also observed. The regenerated shoots were elongated on MSB₅ medium and sufficiently elongated shoots were rooted on MSB₅ medium containing 0.5 mg l⁻¹ IBA. The results showed that all three of the plant growth regulators significantly affected shoot regeneration, with BA exhibiting the greatest benefit. The best combination for shoot regeneration was MSB₅ medium supplemented with 3.0 mg l⁻¹ BA , 0.2 mg l⁻¹ IBA and 0.5 mg l⁻¹ KT on Hefeng 25 genotype. Under these most favorable conditions, one explant could regenerate 30–35 shoots. Plantlets could be obtained within 2 months. The result of histocytological analysis indicated that protein accumulated gradually and reached to peak at late shoot bud formation.     

Callus induction and plant regeneration of U.S. rice genotypes as affected by medium constituents

Summary This study was conducted to establish and optimize a regeneration system for adapted U.S. rice genotypes including three commercial rice cultivars (LaGrue, Katy, and Alan) and two Arkansas breeding lines. Factors evaluated in the study were genotype, sugar type, and phytohormone concentration. The system consisted of two phases, callus induction and plant regeneration. In the callus induction phase, mature caryopses were cultured on MS medium containing either 1% sucrose combined with 3% sorbitol or 4% sucrose alone, and 0.5 to 4 mg·L⁻¹ (2.26 to 18.10 μM) 2,4-D with or without 0.5mg·L⁻¹) (2.32 μM) kinetin. In the plant regeneration phase, callus was transferred to 2,4-D-free MS medium containing 0 or 2 mg·L⁻¹ (9.29 μM) kinetin combined with 0 or 0.1 mg·L⁻¹ (0.54 μM) NAA. Callus induction commenced within a week, independent of the treatments. Callus growth and plant regeneration, however, were significantly influenced by interactions among experimental factors. Generally, the greatest callus growth and plant regeneration were obtained with 0.5 mg·L⁻¹ (2.26 μM) 2,4-D and decreased with increasing 2,4-D concentrations. Kinetin enhanced callus growth only when combined with 0.5 mg·L⁻¹ (2.26 μM) 2,4-D, and 4% sucrose. Inducing callus on kinetin-containing medium generally enhanced regeneration capacity in the presence of sucrose but not with a sucrose/sorbitol combination. Media containing sucrose alone generally supported more callus proliferation, but the sucrose/sorbitol combination improved regeneration of some cultivars. NAA and kinetin had little effect on regeneration.     

Callus induction and shoot regeneration of <Emphasis Type="Italic">Phaseolus lunatus</Emphasis> L. cv. Wonder Bush and cv. Pole Sieva

We report the first protocol for callus induction and shoot regeneration for Phaseolus lunatus L. cv. Wonder Bush and cv. Pole Sieva. We used different explants viz., epicotyls, cotyledons and hypocotyls. The medium used was MS basal medium with thidiazuron (0.5 mg l⁻¹) and IAA (0.05 mg l⁻¹) for the induction of callus followed by BAP (1.0 mg l⁻¹) for the induction of shoots. Epicotyl explants showed the fastest response and the highest percentage of shoot regeneration. This protocol opens new biotechnological strategies to transfer economically important genes to this important crop species.     

Plant regeneration from leaf-derived callus cultures of the tropical pasture legume Stylosanthes scabra Vog.

Callus cultures of 5 genotypes of S. scabra Vog. were optimally established from leaf tissue on Murashige and Skoog (MS) basal medium supplemented with 0.5–2.0 mg l^-1 2, 4-Dichlorophenoxy acetic acid (2, 4-D) and 1.0–2.0 mg l^-1 6-benzylaminopurine (BAP). On media containing 2, 4-D only, calli were soft, and rhizogenesis occurred on calli of 4 genotypes. Calli formed on media containing BAP only, but not with kinetin only. In the presence of 2, 4-D, BAP inhibited rhizogenesis and promoted better callus growth than kinetin. High frequency shoot induction was achieved for 3 genotypes on MS +2.0 mg l^-1 BAP. Roots formed on shoots when sub-cultured on half-strenght MS without growth regulators. The form of cytokinin used in the callus induction media appeared to affect subsequent shoot organogenesis. Genotypic differences were observed for shoot organogenesis. There was some morphological variation evident among regenerants.     

Callus induction and plantlet regeneration in <Emphasis Type="Italic">Withania somnifera</Emphasis> (L.) dunal

Summary Callus induction was observed from hypocotyl, root, and cotyledonary leaf segments, grown on Murashige and Skoog (MS) medium supplemented with various concentrations and combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin (KN). Maximum callusing (100%) was obtained from root and cotyledonary leaf segments grown on MS medium supplemented with a combination of 2 mg l⁻¹ (9.1 μM) 2,4-D and 0.2 mg l⁻¹ (0.9 μM) KN. The calluses, when subcultured in the same medium, showed profuse callusing. However, these calluses remained recalcitrant to regenerate regardless of the quality and combinations of plant growth regulators in the nutrient pool. When hypocotyl segments were used as explants, callus induction was noticed in 91% of cultures which showed shoot regeneration on MS medium supplemented with 2 mg l⁻¹ 2,4-D and 0.2 mg l⁻¹ KN. These shoots were transferred to fresh medium containing various concentrations and combinations of 6-benzyladenine (BA) and N⁶-(2-isopentenyl)adenosine (2-iP). Maximum shoot multiplication was observed after 60 d of the second subculture on MS medium containing 2 mg l⁻¹ (8.9 μM) BA. These shoots were rooted best (87%) on MS medium containing 2 mg l⁻¹ (9.9 μM) indole-3-butyric acid (IBA). The plantlets were transferred to the field after acclimatization and showed 60% survival.     

In vitro differentiation and plant regeneration of Albizia chinesis (Osb.) Merr

Summary Petiolar and distal cotyledonary segments (PCS and DCS) of Albizia chinensis were cultured on Murashige and Skoog's (MS; 1962) medium and induced to form adventitious shoot buds in the presence of either cytokinins 6-benzylamino purine (BAP), kinetin (KN) or thidiazuron (TDZ). Superiority of BAP in inducing shoot bud and differentiation was observed. PCS was more morphogenic to shoot bud differentiation than DCS. TDZ was highly effective in inducing shoot buds, but arrested shoot growth, while KN produced more callus during differentiation of shoots. Rapid and high rate of shoot multiplication per explant was achieved through subculture in MS medium containing BAP (1.0 mg l⁻¹) and indole-3-acetic acid (IAA) (0.5 mg l⁻¹). BAP at low concentration was required to enhance shoot multiplication and elongation. Successful rooting of regenerated shoots was carried out in a two-step culture procedure in MS media with indole-3-butyric acid (IBA) (2.0 mg l⁻¹) and subsequent subculture in IBA-free medium.     

Plant regeneration from phyllode explants of Acacia crassicarpa via organogenesis

In this paper we report the establishment of Acacia crassicarpa regeneration through organogenesis. We used phyllode (leaf) explants excised from 60-day-old in vitro seedlings for green compact nodule induction and, tested Murashige and Skoog (MS) media supplemented with various concentrations of 1-phenyl-3-(thiadiazol-5-yl) urea (thidiazuron) (TDZ) and α-naphthaleneacetic acid (NAA). Under the optimized condition, green compact nodules and adventitious shoots were induced in 10 and 40 days, respectively, on the medium containing a combination of 0.5 mg l⁻¹ TDZ and 0.5 mg l⁻¹ NAA. This medium also yielded the highest rate (56%) of adventitious shoots forming from the nodules. Efficient shoot elongation was achieved by transferring the clusters of adventitious shoots to medium containing 0.1 mg l⁻¹ TDZ within 2 months. The elongated adventitious shoots were rooted at a rate of 96.5% on half-strength MS medium with 0.5 mg l⁻¹ 3-indolebutyric acid (IBA) in 1 month. Rooted plantlets were hardened and successfully established in soil with an 80% survival rate. To our knowledge, this is the first report describing a detailed protocol for regeneration through organogenesis using phyllodes as explants for A. crassicarpa.     

Comparison of callus induction and plant regeneration from different explants in triploid and tetraploid turf-type bermudagrasses

The turf-type bermudagrasses are genetically variable and do not respond uniformly to tissue culture and plant regeneration protocols. We evaluated the callus induction response of two explant types, young inflorescences and nodes, from multiple genotypes including triploid TifSport, TifEagle, and Tift97-4 and tetraploid Tift93-132, Tift93-135, Tift93-156 and Tift93-157 on MS medium supplemented with 1–1.5 mg l⁻¹ 2,4-D + 0.01 mg l⁻¹ BA + 1.16 g l⁻¹ proline. Four types of callus were observed. Type I was fluffy, soft, and white non-embryogenic callus, common to all cultures. Type II was globular, transparent, and hard, but sticky callus, which was pre-embryogenic and could be selected for subculture. Type III callus was transparent, compact, and embryogenic. Type IV callus was opaque white and compact. Both Type III and Type IV calluses were embryogenic and regenerative. A combination of gelling agents in the medium (2 g l⁻¹ Gelrite and 5 g l⁻¹ agar) improved callus quality and increased the rate of compact callus formation during subculture. Embryogenesis from compact callus was observed in TifEagle, TifSport and Tift93-132, and shoots were regenerated on MS medium with 0.1 mg l⁻¹ 2,4-D + 0.5–4.0 mg l⁻¹ BA. Low intensity light treatment (30 μmol m⁻2 s⁻¹ of cool white fluorescence) to callus before regeneration greatly enhanced regeneration frequency from 6.7% to 40% in recalcitrant TifSport.     

High frequency somatic embryogenesis and regeneration in different genotypes of <Emphasis Type="Italic">Sorghum bicolor</Emphasis> (L.) Moench from immature inflorescence explants

This study describes a protocol for the induction of high frequency somatic embryogenesis directly from immature inflorescence explants in three sorghum genotypes (SPV-462, SPV-839, and M35-1). The effect of various growth regulators on somatic embryogenesis was investigated. High frequency somatic embrogenesis was obtained on Murashige and Skoog (MS) medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D), and addition of 0.5 mg l⁻¹ kinetin (KN) in the medium further improved the formation of somatic embryos per explant in all genotypes. The presence of 1.5 mg l⁻¹ 6-benzylaminopurine plus 1.0 mg l⁻¹ KN in MS medium was most efficient for maturation and germination of somatic embryos. The genotype SPV-462 performed better than SPV-839 and M35-1 in terms of induction and germination of somatic embryos. Organogenesis also occurred in callus of all genotypes at the frequency of 20–25%. Regenerated plants from somatic embryos were successfully acclimatized in soil in the greenhouse where plants were grown to maturity, flowered, and set seeds. Regenerated plants appeared normal like that of the seed-raised plants.     

Efficient regeneration system from wheat leaf base segments

Efficient plant regeneration system from leaf base segments of wheat (Triticum aestivum L.) was developed. The factors affecting the callus formation and regeneration capacity of leaf segments of two genotypes; Bobwhite and Pavon 76, were investigated. The highest number of somatic embryos (SE) was obtained on Murashige and Skoog medium supplemented with 2 mg dm⁻³ 2,4-dichlorophenoxyacetic acid + 1 mg dm⁻³ naphthalenacetic acid (14.7 SE per segment). Highest frequency of embryogenic callus (96 %) and somatic embryo formation (24.3 SE per segment) were achieved in the first segments. The highest plantlet regeneration was obtained after transfer of embryogenic calli to regeneration medium supplemented with 1 mg dm⁻³ kinetin (6.3 plantlets per segment).     

Regeneration through organogenesis in rattan

Organogenic cultures were induced from zygotic embryo and megagametophyte explants of the Central American cycad species, Dioon edule. Plant growth medium consisted of B5 major salts, Murashige and Skoog minor salts and organics, 400 mg l⁻¹ glutamine, 100 mg l⁻¹ arginine, 100 mg l⁻¹ asparagine, 60 g l⁻¹ sucrose, 8 g l⁻¹ Difco Bacto agar and was supplemented with kinetin (0 – 13.94 μM) and 2,4-dichlorophenoxyacetic acid (2,4-D) (0 – 9.05 μM) arranged as a 5×4 factorial in a randomized block design. Callus initiation occurred on a wide range of medium formulations from megagametophyte explants; however, shoot formation occurred only on medium supplemented with 2.26 μM 2,4-D. In comparison, callus initiation from explanted zygotic embryos occurred on fewer medium formulations, and adventitious shoot induction occurred from callus on formulations with 9.29–13.94 μM kinetin + 0.45–9.05 μM 2,4-D. Rooted shoots, derived from megagametophyte and zygotic embryo cultures, have been regenerated. This revised version was published online in June 2006 with corrections to the Cover Date.     

High-efficiency plant regeneration from leaf explants of male himalayan poplar (Populus ciliata wall.)

Summary This study reports a protocol for high-efficiency plant regeneration from leaf explants of male Himalayan poplar (Populus ciliata Wall.). Shoots were regenerated at high frequencies from explants grown on Murashige and Skoog (MS) medium supplemented with 0.5 mg l⁻¹ kinetin and 0.2 mg l⁻¹ indole-3-acetic acid (IAA). Regenerated shoots developed roots in MS medium supplemented with 0.1 mg l⁻¹ IAA. Himalayan poplar plantlets could be produced within 2 mo. after acclimatization in a sterile mixture of sand and soil.     

The effects of genotype,inflorescence developmental stage and induction medium on callus induction and plant regeneration in two <Emphasis Type="Italic">Miscanthus</Emphasis> species

Several grass species of the genus Miscanthus are considered to be outstanding candidates for a sustainable production of biomass to generate renewable energy. The purpose of this study was to investigate the effects of genotype, the developmental stage of the explant donor inflorescence and the induction medium on the success rate of micropropagation. The experiments were conducted on three genotypes of M. sinensis and one of M. x giganteus. Explants from the youngest inflorescences (0.1–2.5 cm in length) showed a significantly higher callus induction rate than those from more developed inflorescences (2.6–5 cm in length). In addition, cultures initiated from explants from the youngest inflorescences showed significantly the highest rates of callus regeneration and the highest shoot regeneration rate. Three out of the four genotypes tested showed the best shoot regeneration from calli initiated from the youngest inflorescences when cultured on the Murashige and Skoog basal medium (MS) with 5 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.1 mg l⁻¹ 6-benzyladenine (BA). The percentages of calli from those genotypes showing regeneration ranged from 45 to 76.7%, and the corresponding shoot regeneration rates ranged from 1.85 to 6.33 shoots/callus. This demonstrates that, with some adjustments, efficient micropropagation of Miscanthus sp. is feasible.     

The effect of several factors on somatic embryogenesis and plant regeneration in protoplast cultures of <Emphasis Type="Italic">Gentiana kurroo</Emphasis> (Royle)

Protoplasts were isolated from cell suspensions derived from cotyledon and hypocotyl Gentiana kurroo (Royle). Cell walls were digested with an enzyme cocktail containing cellulase, macerozyme, driselase, hemicellulase and pectolyase in CPW solution. Protoplast viability ranged from 88 to 96%. Three techniques of culture and six media were evaluated in terms of their efficiency in producing viable cultures and regenerating whole plants. With liquid culture, cell division occurred in only a low number of the protoplasts isolated, and no plant regeneration was successful. Cell division occurred within 2 or 3 days in case of agarose solidified media. After 10 days of culture, the number of dividing cells was the highest with modified MS medium in which NH₄NO₃ was replaced with 3.0 g l⁻¹ glutamine. The best results were obtained with agarose bead cultures: plating efficiency was 68.7% and 58.1% for protoplasts isolated from cotyledon and hypocotyl derived suspensions, respectively. The results were achieved with using medium containing 0.5 mg l⁻¹ 2,4-D + 1.0 mg l⁻¹ kinetin or 2.0 mg l⁻¹ BAP + 1.0 mg l⁻¹ dicamba + 0.1 mg l⁻¹ NAA + 80 mg l⁻¹ adenine sulfate. Protocalluses transferred on the following composition of plant growth regulators: 0.5 mg l⁻¹ 2,4-D + 1.0 mg l⁻¹ kinetin or 1.0 mg l⁻¹ kinetin + 0.5 mg l⁻¹ GA₃ + 80.0 mg l⁻¹ adenine sulfate developed in embryogenic cultures. However, the best embryo production occurred with the first one. Later embryos were transferred to half-strength MS mineral salts to promote plants formation. Flow cytometry studies revealed increased amounts of DNA in about one third of the regenerants.