Establishment of oil palm cell suspensions and plant regeneration

Primary globular callus from immature zygotic embryos and friable embryogenic tissue derived from mature zygotic embryos were used to establish suspension cultures. Callus cultures were established either on modified Y3 or MS medium containing 475–500 M 2,4-D or 250 M picloram and 0.3% (w/v) activated charcoal. Suspension cultures of both cell lines were established in modified Y3 medium containing 10 M 2,4-D. The establishment of cell suspensions from friable embryogenic tissue took only 2 months, in contrast with suspensions from primary globular callus which took 3–5 months to establish. Embryo differentiation was observed only in cell suspensions derived from the friable embryogenic tissue after plating aliquots on regeneration medium. Germinated embryos were recovered and plantlets were successfully established under greenhouse conditions.Abbreviations CET compact embryogenic tissue - FET friable embryogenic tissue - CIM callus induction medium - PGC primary globular callus - 2,3-D 2,4-dichlorphenoxyacetic acid Y3-Eeuwens' medium - MS Murashige & Skoog medium - PVP-40 polyvinylpyrrolidone - KM Kao & Michayluk vitamins - ABA abscisic acid     

Plant regeneration via somatic embryogenesis in creeping bentgrass (Agrostis palustris Huds.)

We have established a high-frequency plant regeneration system via somatic embryogenesis from mature seeds of creeping bentgrass (Agrostis palustris Huds). The effects of 2,4-dichlorophenoxyacetic acid (2,4-D), 3.6-dichloroo-anisic acid (dicamba) and 6-benzyladenine (BA) on callus formation and embryogenesis were evaluated. Callus produced on the Murashige and Skoog (MS) (1962) medium containing 2,4-D had low embryogenic potency. In the presence of 30 M dicamba, addition of 2.25 to 9 M BA significantly enhanced embryogenic callus formation over dicamba alone. Optimum frequency of somatic embryogenesis was achieved on MS basal medium containing 30 M dicamba and 2.25 M BA. Over 80% of somatic embryos germinated and formed plantlets on half-strength MS basal medium. These plantlets grew normally in the greenhouse.Abbreviations MS Murashige and Skoog medium - 2,4-D 2,4-dichlorophenoxyacetic acid - BA 6-benzyladenine - dicamba 3, 6-dichloro-o-anisic acid     

Influence of photoperiod and medium formulation on peanut somatic embryogenesis

Somatic embryos were produced from peanut (Arachis hypogaea L.) immature zygotic cotyledons. Comparisons were made of the level of -naphthaleneacetic acid during induction, nitrogen formulation of the medium, and photoperiod. Over 70% embryogenesis was obtained regardless of NAA level used. Percent embryogenesis and number of embryos were markedly lower in explants induced on NAA compared to 2,4-D. Embryo production was not greatly affected by either the use of Murashige & Skoog versus Finer & Nagasawa salts or light versus dark culture conditions. However, embryo morphology was noticeably affected by photoperiod. Embryos produced under a 16 h photoperiod were tough, woody and difficult to separate for subsequent germination and conversion. Those produced under a 0-h photoperiod were succulent and pliable.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - MS Murashige & Skoog (1962) - B5 Gamborg et al. (1968) - picloram 4-amino-3,5,6-trichloropicolinic acid - FN Finer & Nagasawa (1988) - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid     

Repetitive somatic embryogenesis in peanut cotyledon cultures by continual exposure to 2,4-d

Somatic embryos from immature cotyledons in peanut (Arachis hypogaea) were initiated on media supplemented with 2,4-dichlorophenoxyacetic acid (2,4-d). Over 90% primary embryogenesis and 41–46% repetitive embryogenesis were obtained 12 weeks after initiation by maintaining embryogenic cultures on medium containing 20 mg 1^-1 2,4-d. Maintenance of cultures on medium with 30 or 40 mg I^-1 2,4-d resulted in lower primary and secondary embryogenesis, and proliferation of nonembryogenic callus. Transfer of embryogenic cultures to a secondary medium with 10 or 20 mg I^-1 2,4-d significantly enhanced secondary embryogenesis compared to basal medium without the growth regulator. The use of Murashige & Skoog versus Finer's media had no significant effect on embryogenesis (85–95%), repetitive embryogenesis (11–37%) or mean embryo number. Secondary embryogenesis was also maintained for over one year by repeated subculture of isolated somatic embryos on medium with 20 mg I^-1 2,4-d.Abbreviations B5 Gamborg et al. medium (Gamborg et al. 1968) - 2,4-d 2,4-dichlorophenoxyacetic acid - FN Finer & Nagasawa medium (Finer & Nagasawa 1968) - MS Murashige & Skoog medium (Murashige & Skoog 1962)     

TDZ-induced somatic embryogenesis in non-responsive caryopses of rice using a short treatment with 2,4-D

Caryopses cultures of rice on an auxin medium (2,4-D, 20 M) formed slow-growing tissues that failed to regenerate. The embryogenic tissue possibly lost its regeneration potential on the auxin medium. Reproducible regeneration, however, could be achieved by a short treatment with 20 M 2,4-D for 3 days. Transfer of these caryopses to the medium containing TDZ or BA at 10 M resulted in regeneration of somatic embryos and shoots in 30% of the cultures within 10–15 days. TDZ was better than BA for inducing shoot regeneration. Short treatment for 3 days at higher concentrations of auxin (40–80 M) and subsequent transfer to TDZ or BA medium resulted in an increased frequency (up to 50%) of regeneration.     

Somatic embryogenesis from immature inflorescences of oil palm

Summary Immature inflorescences of oil palm (Elaeis guineensis) var. Pisifera were inoculated onto modified MS medium containing 0.3% (w/v) activated charcoal and 475 M 2,4-D. After 2—3 months of culture, a hard yellow callus proliferated at the base of the shoot-like structures. The high incidence of phenolic oxidation required the use of increased levels of activated charcoal (0.5% w/v) and 2,4-D (500 M). Development of floral structures from inflorescence expiants was frequently observed during the culture period. After 81 weeks of culture, an embryogenic tissue characterized by compact consistency and pearly white color was observed in tissues derived from very young inflorescences. This compact embryogenic tissue differentiated into normal somatic embryos when transferred onto regeneration medium containing NAA (15 M) and ABA (2 M). Normal plantlets were recovered from these somatic embryos after 8 weeks on regeneration medium.Abbreviations 2, 4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - ABA abscisic acid - PVP-40 polyvinylpyrrolidone     

Stepwise decrease of 2,4-D and addition of BA in subculture medium stimulated shoot regeneration and somatic embryogenesis in buffalograss

Plant regeneration of buffalograss `Texoka' was achieved through both somatic embryogenesis and organogenesis by culturing immature male inflorescences collected from field-grown plants. Three passages of subculture for calluses derived from male `Texoka' on medium containing 2.25, 4.5, or 9 M 2,4-D combined with either 0.44 M or 1.32 M BA led to shoot formation via organogenesis. Higher concentrations of 2,4-D (4.5 or 9 M) resulted in higher percentages of embryogenic callus while 2,4-D at 2.25 M generated shoot-producing callus but with a lower percentage of embryogenic callus. Transfer of calluses from medium containing 4.5 M 2,4-D and 0.44 M BA to the somatic embryo initiation medium containing 0.9 M 2,4-D gelled with either 7 g 1^–1 agar or 3 g 1^–1 Gelrite led to the formation of somatic embryos. Somatic embryo initiation medium gelled with 3 g 1^–1 Gelrite led to significantly higher frequency of somatic embryo formation than in medium gelled with 7 g 1^–1 agar. Callus of a female genotype `315' generated under similar treatments did not produce shoots or somatic embryos.     

Somatic embryogenesis from immature embryos in meadowfoam (Limnanthes alba)

Developing embryos from immature seeds were excised and cultured. Optimal proliferation of differentiated secondary embryos occurred on Murashige-Skoog media containing 7% sucrose, 0.1 M 2,4-D, and 0.1–1.0 M zeatin. Higher levels of auxins inhibited embryo proliferation. Secondary embryos were subcultured to produce more embryos. The results indicate the feasibility of clonal propagation of meadowfoam.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BA 6-Benzyladenine     

Somatic embryogenesis in callus cultures of Rosa hybrida L. cv. Landora

Callus was initiated from immature leaf and stem segments of rose (Rosa hybrida cv. Landora) and subcultured every four weeks on a basal medium of half-strength Murashige & Skoog (1962) salts plus 30 g l^-1 sucrose (1/2 MS) and supplemented with 2.2 M BA, 5.4 M NAA and 2.2–9.0 M 2,4-D. Embryogenic callus and subsequently somatic embryos were obtained from 8-week-old callus culture on 1/2 MS+2.2 M BA+0.05 M NAA+0.3 M GA₃+200–800 mg l^-1 L-proline. Long-term cultures were established and maintained for up to 16 months by repeated subculture of embryogenic callus on L-proline deficient medium. About 12% of cotyledonary stage embryos taken from cultures cold-stored at 8±1°C for 4 days germinated on 1/2 MS+2.2 M BA+0.3 M GA₃+24.7 M adenine sulphate.Abbreviations BA benzyladenine - NAA -naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid     

Regeneration of Cucumis sativus var. sativus and C. sativus var. hardwickii,C. melo,and C. metuliferus from explants through somatic embryogenesis and organogenesis

Regeneration in six inbred lines or F₁ hybrids of Cucumis sativus was achieved on Murashige & Skoog's medium containing various concentrations of 2,4-D/BA, NAA/BA, NAA/Z or NAA/K. The range of regeneration frequency for cotyledon, leaf and petiole explants was 0–38, 0–75 and 14–96%, respectively, after 6–8 weeks in culture. Only one subculture of calli to growth regulator-free medium was required for regeneration. Preincubation of explants in the dark for 2–3 weeks was essential to achieve optimal regeneration. Highest frequency of plantlet formation occurred with petiole explants incubated on NAA/BA (5.0/2.5 M), NAA/Z (5.0/5.0 M) or 2,4-D/BA (5.0/5.0 M). Approximately 80% of these plantlets survived after transplanting to greenhouse soil, and they flowered and set fruit. The F₁ hybrid, Endeavor, gave the highest regeneration frequency of 91% on 2,4-D/BA at 5.0/5.0 M. Formation of somatic embryos was observed on 2,4-D/BA, while organogenesis and embryogenesis both were evident on NAA/BA and NAA/Z. Cotyledonary explants yielded the lowest frequency (ca. 7%) of plantlet formation in this study. Plantlets of C. sativus var. hardwickii and an F₁ hybrid of C. sativus x C. s. var hardwickii were regenerated on NAA/Z and NAA/K at frequencies of 15–65%, predominantly by the formation of somatic embryos. Shoots were obtained from cotyledon and leaf explants of C. metuliferus on IAA/BA (7.5/5.0 M) and from leaf and petiole explants of C. melo on NAA/BA (5.0/2.5 M), but plantlets were recovered only in C. melo.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxy-acetic acid - IAA indoleacetic acid - K kinetin - MS Murashige & Skoog's medium - NAA naphthaleneacetic acid - Z zeatindihydroside     

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     

Control of direct and indirect somatic embryogenesis by exogenous growth regulators in immature zygotic embryo cultures of rose

Immature zygotic embryos of rose (Rosa hybrida L.; cv. Sumpath) did not form somatic embryos or embryogenic calluses when cultured on half-strength Murashige and Skoog's medium supplemented with various con-centrations of 2,4-dichlorophenoxyacetic acid (2,4-D) as the sole growth regulator. However, the zygotic embryos produced somatic embryos without an intervening callus phase at a frequency of 27.3% on medium with 4.44 M 6-benzyladenine (BA) alone. Immature zygotic embryos formed embryogenic calluses at a frequency of 25% on medium with a combination of 1.36 M 2,4-D and 4.44 M BA. Upon transfer to medium without growth regulators, embryogenic calluses produced numerous somatic embryos that subsequently developed into plantlets. Somatic embryos were induced directly from immature zygotic embryos, or indirectly via an intervening callus phase, by manipulating the exogenous growth regulators. Plantlets were successfully transplanted to potting soil and grown to maturity in a greenhouse.     

Somatic embryogenesis in cultured immature kernels of Pistachio,Pistacia vera L.

Embryogenic tissue was produced from kernels of immature fruits of Pistachio (Pistacia vera L.) cultured in liquid Murashige and Skoog media, supplemented with 200 mgl^–1 casein hydrolysate, 114 M 1-ascorbic acid, and benzylaminopurine. Compact embryogenic masses differentiated directly from the fruit explants after culture for 2 weeks in liquid medium with 8.9 M benzylaminopurine. After transfer of the embryogenic masses into the same medium, but with 4.4 M benzylaminopurine, somatic embryos appeared. Several stages of embryogenesis were present in the cultures. Adventive embryos were readily separated from the friable embryogenic masses by shaking. Separated somatic embryos, germinated on solidified Murashige & Skoog medium without growth regulators, developed into plantlets.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BAP benzylaminopurine (N⁶-benzyladenine) - EMS embryogenic mass - MS Murashige and Skoog medium (Sigma M-0404) - NAA -naphthalene acetic acid - PGR plant growth regulator - TDZ thidiazuron (1-phenyl-3-(1,2,3, thiadiazol-5-yl)urea) - WP McCown's Woody plant medium (Sigma M6774) - ABA abscisic acid     

Preliminary studies of micropropagation of Hopea odorata,a dipterocarp tree

Plantlet development from in vitro cultures of Hopea odorato Roxb. is described. Embryos excised from seeds and cultured on Gamborg's B5 or modified Murashige and Skoog (MS) medium with benzyladenine (BA, 2.2–22.2 M) produced axillary shoots at cotyledonary and/or stem nodes. Shoot production was greatest in germinated embryos on modified MS medium with 8.9 M BA. Excised axillary shoots formed few buds when cultured on medium with BA and limited root development occurred on Woody Plant Medium with naphthaleneacetic acid. Nodal explants from aseptically grown plantlets sprouted axillary shoots in modified MS medium with BA.Abbreviations BA 6-benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IBA indolebutyric acid - NAA naphthaleneacetic acid - PVPP polyvinylpolypyrrolidone     

High efficiency adventive embryogenesis on somatic embryos of anther,filament and immature proembryo origin in horse-chestnut (Aesculus hippocastanum L.) tissue culture

Adventive embryogenesis was successfully induced in cultures of zygotic and somatic embryos on MS medium supplemented with BA and NAA. A procedure has been proved successful for the in vitro multiplication of somatic embryos regenerated at low frequencies from filament and callus cultures. The occurrence and rate of adventive embryogenesis did not depend on the origin of the primary embryos (zygotic and somatic), but did depend on the developmental stage. Primary embryos are capable of embryogenesis in each of the different phases of embryogenesis, though the rate is different. BA concentrations of 22–44 M increased the rate of adventive embryogenesis and accelerated the development of embryos. The highest proliferation rate (22–25x/5 weeks) was achieved at hormone concentrations of 44 M BA and 5.4 M NAA.Abbreviations BA benzyladenine - CH casein hydrolysate - CM coconut milk - 2,4-D dichlorophenoxyacetic acid - MS Murashige & Skoog medium - WPM woody plant medium - NAA 1-naphtaleneacetic acid     

Influence of plant growth regulators,basal media and carbohydrate levels on the in vitro development of Pinus ponderosa (Dougl. ex Law.) cotyledon explants

Applications of in vitro screening techniques for Pinus ponderosa resistance to Peridermium harknessii could be beneficial in a tree breeding program. Plant growth regulators, basal media formula and carbohydrate levels were examined to determine the various effects each would have on excised cotyledon growth and development. Proliferating green callus was initiated from cotyledon explants on SH basal medium containing 4.4 M BA:5.4 M NAA and 1% sucrose. Subsequent subculturing onto LS medium supplemented with 44.0 M BA: 5.4 M NAA and 2% sucrose improved callus maintenance. The highest frequency of caulogenesis from cotyledon explants occurred on a modified GD medium containing 44.0 M BA: 0.054 M NAA and 4% glucose. The influence of nitrogen source, osmoticum and medium salt concentrations are discussed relative to callus initiation and caulogenesis.Abbreviations BA 6-benzyladenine - NAA -naphthaleneacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - 2,4-D 2,4-dichlorophenoxyacetic acid - CD Campbell & Durzan - GD Gresshoff & Doy - LP Le Poivre - LS Linsmaier & Skoog - MC McCown - SH Schenk & Hildebrandt     

High frequency somatic embryogenesis from coffee leaves

An improved procedure for the induction, proliferation and regeneration of embryogenic callus from coffee leaf explants has been developed. The optimal culture conditions for callus induction and somatic embryogenesis yielded so-called high frequency embryogenic callus ofCoffea canephora P. ex Fr., Arabusta and Congusta, more rapidly and abundantly than other published procedures.Coffea arabica L. genotypes, however, were less responsive to the procedure. The highest multiplication rate of embryogenic callus in liquid culture, which avoided the differentiation of embryos, was obtained by culture at an inoculum density of 10 g callus 1^-1 in a modified MS medium containing 4.5 M 2,4-dichlorophenoxyacetic acid, under 3 mol m^-2 s^-1 illumination, and subcultured every 7–10 days. The best long-term maintenance of embryogenic potential was obtained by culture of aggregates (250–1000 m in diameter) at an inoculum density of 5 g 1^-1, with medium renewed every 3–4 weeks. Under these conditions, embryogenic potential ofC. canephora callus was maintained for over 2 years. Analysis of nutrients absorbed by the callus cultures demonstrated that half strength MS macro- and micro-salts were not depleted during at least 3 weeks of sustained culture. The highest regeneration of embryogenic callus required the omission of 2,4-D and a reduced culture density of 1 g 1^-1. Under these conditions of culture, 1 g ofC. canephora or Arabusta callus produced 1.2 and 0.9×10⁵ somatic embryos, respectively, after 8–10 weeks in liquid regeneration medium. This was an overall reduction of 4–6 months from explant to regenerant, when compared with other procedures.Abbreviations BA N⁶-benzyladenine - HFSE high frequency somatic embryogenesis - IAA indole-3-acetic acid - IBA indole-3-butyric acid - rpm rotations per minute - LFSE low frequency somatic embryogenesis - MS Murashige & Skoog medium - PPF photosynthetic photon flux - 2,4-D 2,4-dichlorophenoxyacetic acid - 2-iP 2-isopentenyladenine     

Isolation,culture and plantlet regeneration from cotyledon and mesophyll protoplasts of two pickling cucumber (Cucumis sativus L.) genotypes

Optimal protoplast yields from cotyledons (2.0×10⁶ protoplasts/ 0.5 g tissue) and from true leaves (5.0×10⁶ protoplasts/g tissue) of two Cucumis sativus genotypes were obtained following a 16 h digestion with, respectively, 1.25% pectinase+0.5% Cellulysin and 0.5 % pectinase+ 1.0% Cellulysin. Enzyme solutions were prepared in modified MS medium containing half-strength major salts, full complement of minor salts and vitamins, 2% sucrose and 0.25 M mannitol. A plating density of 3.5–4.0× 10⁴ protoplasts/ml or higher was required for sustained division, with first division occurring in 6–7 days, second-third division in 8–9 days, and minicalli formation by day 13. Embedding in 0.4% agarose provided the highest plating efficiency (proportion that formed minicalli) of mesophyll protoplasts, which was 28.3% for genotype 3672 and 15% for genotype 3676. By comparison, liquid culture and droplet culture gave lower plating efficiencies (10–19%). Cotyledon and mesophyll protoplasts of one genotype formed minicalli on MS medium containing 2,4-D/BA at 1.0/2.5 M and 5.0/5.0 M, respectively, within 21 days, while mesophyll protoplasts of the second genotype formed minicalli on MS medium containing NAA/BA at 5.0/5.0 M within 12 days. Shoot buds or somatic embryos were obtained upon subculture of calli to MS medium containing lower concentrations (0.05–0.01 M) of 2,4-D/BA or NAA/BA and a few plantlets, ca.18, were recovered on hormone-free medium.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog medium - NAA naphthaleneacetic acid     

High frequency somatic embryogenesis and plant regeneration in zygotic embryo cultures of Japanese honeysuckle

Japanese honeysuckle plant (Lonicera japonica Thunb.) is rich in iridoid secologanin and is a potentially useful model for the study of secologanin biosynthesis. Culture conditions for high frequency plant regeneration via somatic embryogenesis from zygotic embryo cultures and zygotic embryo-derived embryogenic cell suspension cultures of this species are described. Mature zygotic embryos formed embryogenic calluses at a frequency of 46.7% when cultured on Murashige and Skoog (MS) medium supplemented with 4.52 M 2,4-dichloro-phenoxyacetic acid (2,4-D). Cell suspension cultures were established with embryogenic calluses using liquid MS medium with 4.52 M 2,4-D. Upon plating onto MS basal medium, embryogenic cell suspension cultures produced numerous somatic embryos, which subsequently developed into plantlets at a frequency of 68%. Regenerated plantlets were transplanted to potting soil and grown to maturity in a greenhouse.     

High frequency somatic embryogenesis and plant regeneration in petiole and leaf explant cultures and petiole-derived embryogenic cell suspension cultures of Hylomecon vernalis

Culture conditions are described for high frequency somatic embryogenesis and plant regeneration in petiole and leaf explant cultures and petiole-derived embryogenic cell suspension cultures of Hylomecon vernalis Max. Petiole explants formed embryogenic calluses at a frequency of 53% when cultured on B5 medium supplemented with 13.6 M 2,4-dichlorophenoxyacetic acid (2,4-D) alone. Leaf explants formed embryogenic calluses at a frequency of 21% when cultured at a combination of 4.52 M 2,4-D and 2.22 M 6-benzyladenine. Cell suspension cultures were established with petiole-derived embryogenic calluses using liquid B5 medium with 4.52 M 2,4-D. Upon plating onto B5 basal medium, cell suspension cultures produced numerous somatic embryos, which then developed into plantlets. Regenerated plantlets were transplanted to potting soil and grown to maturity in a greenhouse.