The production of callus capable of plant regeneration from immature embryos of numerous Zea mays genotypes

In the summer of 1983, immature embryos from 101 selfed inbred lines and germplasm stocks of Zea mays L. were examined for their ability to produce callus cultures capable of plant regeneration (regenerable cultures) using a medium with which some limited success had previously been obtained. Forty-nine of the genotypes (49%) produced callus which visually appeared similar to callus previously cultured and shown to be capable of plant regeneration. After five months, 38 of these genotypes were alive in culture and plants were subsequently regenerated from 35 (92%) of them. No correlation was observed between plant regeneration and callus growth rate, the vivipary mutation (genes vp1, 2, 5, 7, 8 and 9), or published vigor ratings based on K⁺ uptake by roots. When F₁ hybrid embryos were cultured, 97% of the hybrids having at least one regenerable parent also produced callus capable of plant regeneration. No regenerable cultures were obtained from any hybrid lacking a parent capable of producing a regenerable callus culture.In the summer of 1984, immature embryos from 218 additional inbred lines and germplasm stocks were plated and examined for their ability to produce regenerable callus cultures on media containing altered micronutrient concentrations, 3,6-dichloro-o-anisic acid (dicamba), glucose, and elevated levels of vitamin-free casamino acids and thiamine. Of these genotypes 199 (91%) produced callus that was regenerable in appearance. In the 1984 study, plant regeneration was noted in many commercially important inbreds, including B73, Mo17, B84, A632, A634, Ms71, W117, H99³H95 and Cm105. Thus tissue-culture techniques are now available to obtain callus cultures capable of plant regeneration from immature embryos of most maize genotypes.Abbreviations trade names 2,4-D 2,4-dichlorophenoxyacetic acid - dicamba 3,6-dichloro-o-anisic acid     

Structural analysis of colonies derived from sunflower (Helianthus annuus L.) protoplasts cultured in liquid and semi-solid media

Summary Colonies have been generated from cotyledon protoplasts of sunflower (Helianthus annuus L.) and transferred to solid regeneration medium. The development of these protoplasts was strikingly dependent on their physical environment (liquid or solidified medium) and the nature of the gelling agent (agarose, alginate). In particular, extremely compact multicellular structures developed in agarose solidified medium, similar to those observed in cultures of hypocotyl- and petiole-derived sunflower protoplasts (termed proembryoids). A detailed study at the histological level, however, indicates that these structures are degenerative, rather than embryonic in nature. The quality of the colonies, with regard to their regeneration potential, appears to be best in alginate solidified medium.Abbreviations MS medium of Murashige and Skoog (1962) - NAA 1-naphthaleneacetic acid - BAP 6-benzylaminopurine     

Protoplasts from cotyledon and hypocotyl of sunflower (Helianthus annuus L.): shoot regeneration and seed production

Summary Cotyledon and hypocotyl protoplasts of Helianthus annuus inbred line 47 302 bcd were embedded in alginate and plated on L4 medium (Lenée and Chupeau 1986). After one month, the calli were transferred on MSSH regeneration medium (Murashige and Skoog 1962; Schenk and Hildebrandt 1972) where they regenerated shoots (overall efficiency 10^–2%). The shoots were elongated on B5 (Gamborg et al. 1968) medium first without hormones, then supplemented with GA3 and BAP (both 0.05 mg/l). In order to overcome the difficulty to induce rooting by classical methods, the elongated shoots were grafted on a sunflower rootstock. The grafted shoots produced flowers and seeds. Different factors have been shown to have an important influence on the capacity to regenerate shoots: the genotype, the physical culture conditions at the callus regeneration step (e.g. protoplasts embedded in alginate), and the media composition.Abbreviations BAP 6-benzylaminopurine - GA3 gibberellic acid - IBA indole-3-butanoic acid - IAA indole acetic acid - MES 2-N-morpholinoethane sulfonic acid - NAA 1-naphthalene acetic acid - 2,4D 2,4 dichlorophenoxyacetic acid     

Effects of growth regulator concentrations and explant size on shoot organogenesis from callus derived from zygotic embryos of sunflower (Helianthus annuus L.)

Effects of medium growth regulator composition and embryo size on shoot organogenesis of callus derived from globular- to torpedo-shaped zygotic embryos of five sunflower (Helianthus annuus L.) genotypes were examined. Forty growth regulator combinations composed of 0 to 5 mgl^-1 naphthaleneacetic acid (NAA) and 0 to 1 mgl^-1 6-benzylaminopurine (BA) were tested. The frequency of zygotic embryos forming shoot-regenerating callus was analysed according to categorical data modelling using a maximum-likelihood approach. Both NAA and BA must be present to induce the formation of morphogenic callus from zygotic embryos, but each growth regulator effect varied with the genotype. For four genotypes, NAA and BA effects were neither linear nor quadratic; whereas, they were linear for the fifth one. Most effective concentrations across genotypes were 0.1 mgl^-1 NAA and 0.5 mgl^-1 or 0.2 mgl^-1 BA. However, the optimal growth regulator combination depended on the genotype and an interaction between the two growth regulators. The frequency of shoot-regenerating callus also varied with the size of the embryo explant. For all five genotypes, 0.4 to 1.2 mm long heart-shaped zygotic embryos formed morphogenic callus more frequently than smaller less-developed ones.     

Somatic embryogenesis in sisal (Agave sisalana Perr. ex. Engelm)

A protocol has been developed for somatic embryogenesis and plant regeneration of sisal (Agave sisalana Perr. ex. Engelm). Embryogenic callus cultures were initiated from young shoots raised in vitro from the stem portion of the bulbil on medium supplemented with 1–2 mg l^-1 kinetin (KN) and 0.2–0.5 mg l^-1 -naphthaleneacetic acid plus KN or 1–1.5 mg l^-1 benzylaminopurine (BAP) or 0.25–0.5 mg l^-1 2,4-dichlorophenoxyacetic acid plus BAP or 0.5–1.0 mg l^-1 KN. Embryos at various developmental stages (globular-, heart- or torpedo-shaped) produced mature and germinating embryos on being transferred to a new medium containing 0–0.25 mg l^-1 KN. After 28 days, a maximum of 76% germinated embryos was obtained on a medium supplemented with 0.1 mg l^-1 KN. The capacity for embryogenesis remained constant in the callus upon subculturing on the same medium for more than 48 months. Histological observations showed a distinct multicellular origin for most of the somatic embryos as they developed from epidermal, sub-epidermal and inside callus cells, while a few of them originated from a superficial callus cell. Plantlets regenerated from embryos were transferred to the field where their survival rate was 100%.Abbreviations BAP Benzylaminopurine - 2,4-D 2,4-Dichlorophenoxyacetic acid - IAA Indoleacetic acid - KN Kinetin - NAA -Naphthaleneacetic acidCommunicated by W. Barz     

High frequency plant regeneration from anther-derived cell suspension cultures via somatic embryogenesis in Catharanthus roseus

Summary A system for high frequency plant regeneration from cell suspension cultures in Catharanthus roseus is described. Calli were obtained from anthers cultured on Murashige and Skoog's medium supplemented with 1 mgl^-1 -naphthaleneacetic acid and 0.1 mgl^-1 kinetin. After the second subculture on solid medium, embryogenic callus was identified and transferred to liquid medium to initiate suspension cultures. Cells dispersed finely in the medium were subcultured at 14-day intervals. Upon plating onto the basal medium, yellowish compact colonies proliferated from the cells and more than 80% of them gave rise to somatic embryos. Subsequently, plantlets developed from the embryos. Both the plantlets and the source plants showed the normal somatic chromosome number of 2n=2x=16.Abbreviations MS Murashige and Skoog - MSNK MS medium + 1 mgl^-1 NAA + 0.1 mgl^-1 kinetin - NAA -naphthaleneacetic acid     

Tissue culture response of Beta germplasm: callus induction and plant regeneration

Callus cultures of 18 sugarbeet (Beta vulgaris) lines, two accessions of B. maritima and a B. macrocarpa accession were initiated from aseptically germinated seeds. Plant regeneration through organogenesis was obtained either on MS or B5 medium containing various concentrations and combinations of naphthaleneacetic acid (NAA), 6-benzylaminopurine (BAP), 2,3,5-triiodobenzoic acid (TIBA) and abscisic acid (ABA). Genotypes differed in their abilities of callus formation and regeneration: seven out of 18 sugarbeet lines, and an accession of B. maritima were capable of regenerating plantlets. Our data also indicated that 2 M TIBA promoted morphogenesis from callus culture in the presence of 5 M BAP.     

Screening of diploid Medicago sativa germplasm for somatic embryogenesis

Nineteen accessions of diploid Medicago sativa L. belonging to the four subspecies sativa, caerula, falcata and xvaria were screened for their ability to produce somatic embryos on hypocotyl-derived callus. Two medium protocols were used in this study, a three-step sequence with exposure of the callus cultures to a high 2,4-D concentration and a two-step sequence without exposure to a high 2,4-D concentration. Considerable variation for callus proliferation was observed. In general, the diploid M. sativa accessions showed poor regenerability and it was not possible to correlate high regeneration frequencies with a particular germplasm source. It was, however, possible to identify regenerable genotypes in all four subspecies. One falcata accession produced somatic embryos on the callus induction media at high frequencies. This response was also obtained with a few genotypes from one xvaria accession. All regenerable plants were maintained as shoot cultures and were able to form somatic embryos on petiole-derived calli.Abbreviations BA 6-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - 2iP iso-pentyladenine - NAA -naphthaleneacetic acid Contribution No. 772 Ottawa Research Station     

High frequency somatic embryogenesis and plant regeneration from zygotic embryo-derived callus cultures of three Allium species

The plant regeneration ability of zygotic embryo-derived callus cultures was studied for 12 A. cepa varieties and accessions, two A. fistulosum varieties, one A. fistulosum x A. cepa interspecific hybrid and two A. porrum varieties. Compact embryogenic callus was induced on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid. The embryogenic calluses of all three Allium species were similar in appearance. For all accessions tested plants could be regenerated at a high frequency from this compact callus through somatic embryogenesis, when using kinetin supplemented MS medium (regeneration medium). Addition of abscisic acid to the regeneration medium stimulated the formation of both somatic embryos and shoots for a number of varieties. Concerning shoot regeneration from callus cultures, significant differences existed between genotypes of all accessions except one.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - VDH Van Der Have Seed company     

Somatic embryogenesis from immature zygotic embryos of oil palm

Immature zygotic embryos at different developmental stages were used for callus induction and regeneration studies. Immature embryos excised from fruits 77, 91, 100, 114, 128, 140 and 193 days after pollination and mature embryos were cultured on modified Y3 medium containing 500 mgl^–1 cysteine, 0.5% (w/v) PVP-40, 500 M 2,4-d and 0.3% (w/v) charcoal. Compact embryogenic tissue began differentiating directly from embryo explants after 2 weeks of culture. The percentage of embryos forming compact embryogenic tissue ranged from 28.6% for 91-day-old embryos to 0% for 140-day-old and older embryos. Friable embryogenic tissue was observed in callus cultures derived from 100-day-old embryos. Although both compact and friable embryogenic tissues were successfully isolated, normal embryo and plantlet development was observed only from friable embryogenic tissue.Abbreviations ABA abscisic acid - 2,4-d 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - PVP polyvinylpyrollidone     

Antioxidants enhance in vitro plant regeneration by inhibiting the accumulation of peroxidase in Virginia pine (<Emphasis Type="Italic">Pinus virginiana</Emphasis> Mill.)

Plant tissue necrosis and subsequent cell death are usually observed during in vitro regeneration in conifers, especially in plant regeneration via somatic organogenesis in pine species. Cell death is correlated with the elevated levels of peroxides. In this investigation, the effects of antioxidants on in vitro regeneration of Virginia pine (Pinus virginiana Mill.) were evaluated. Antioxidants, polyvinylpolypyrrolidone (PVPP) and 1,4-dithio-dl-threitol (DTT), were found to improve callus formation, shoot differentiation and growth, and shoot rooting by inhibiting tissue necrosis during the initiation of cultures and subculture of shoots. These treatments enabled the recovery and regeneration plants at high frequency through somatic organogenesis. Compared to the control, the frequencies of callus formation, shoot growth, and shoot rooting increased 15, 26, and 19%, respectively, by addition of 5 g/l PVPP and 2 g/l DTT. Higher peroxidase activity of tissue cultures during subculture from callus proliferation medium to shoot differentiation medium and to rooting medium was observed. The addition of antioxidants reduces and inhibits browning by reducing the accumulation of peroxidase.Abbreviations BA 6-Benzylaminopurine - 2,4-D 2,4-Dichlorophenoxyacetic acid - DTT 1,4-Dithio-dl-threitol - IBA Indole butyric acid - NAA -Naphthaleneacetic acid - PVPP Polyvinylpolypyrrolidone     

Plant regeneration from callus cultures derived from mature zygotic embryos in white pine (Pinus strobus L.)

Plant regeneration via adventitious shoot organogenesis from callus cultures initiated from mature embryos in white pine (Pinus strobus L.) was achieved in this study. Callus cultures were induced from mature embryos cultured on PS medium supplemented with 2,4-dichlorophenoxyacetic acid, -naphthaleneacetic acid, or indole-3-acetic acid. Adventitious shoot regeneration from callus cultures was induced on medium containing 2 M indole-3-butyric acid (IBA) and 3–12 M N₆-benzylaminopurine, thidiazuron (TDZ), or 6-(,-dimethylallylamino) purine. Sucrose was the most suitable sugar for adventitious shoot organogenesis in white pine. Shoot organogenesis was improved by treatment at 4°C for 6 weeks. The frequency of adventitious shoot formation increased when 0.1 mM putrescine was added to basal medium supplemented with 6 M TDZ and 2 M IBA. Putrescine improved adventitious shoot organogenesis by decreasing lipid peroxidation. These findings provide useful information on adventitious shoot organogenesis and may be valuable to genetic transformation in white pine.     

Involvement of ethylene on growth and plant regeneration in callus cultures of Heliconia psittacorum L.f.

The level of ethylene accumulated in morphogenic callus cultures of Heliconia psittacorum L.f. was only one quarter that of non-morphogenic cultures. The rate of ethylene production in the morphogenic callus cultures during early stages of differentiation of protocorm-like bodies leading to plantlet regeneration was 10-fold higher than that during callus proliferation. In cultures sealed with gastight serum caps, fresh weight gain was reduced 2-to 3-fold compared to those that were closed with Kaputs. Treatment with 1-aminocyclopropane-1-carboxylic acid ( 100 M) caused complete inhibition of plant regeneration from the morphogenic callus on subsequent culture under inductive conditions. Silver nitrate and aminoethoxyvinylglycine also reduced plant regeneration. These results indicate that while high levels of ethylene were inhibitory, a low level of endogenous ethylene production may be necessary during the plant regeneration phase in callus cultures of Heliconia.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - AC activated charcoal - ACC 1-aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglycine - BM basal medium - CH casein hydrolysate - DM development medium - MM maintenance medium - PLB protocorm-like body     

Organogenesis and somatic embryogenesis from callus cultures in Muscari armeniacum Leichtl. ex Bak.

Summary Establishment of fast-growing, highly regenerable callus cultures was examined in Muscari armeniacum Leichtl. ex Bak. in order to develop an efficient genetic transformation system. High-frequency callus formation was obtained from leaf explants of cv. Blue Pearl on media containing 2,4-dichlorophenoxyacetic acid (2,4-D), α-naphthaleneacetic acid (NAA) or 4-amino-3,5,6-trichloropicolinic acid (picloram, PIC). Fast-growing, yellowish nodular callus lines and white friable callus lines containing a few somatic embryos were established on initiation medium supplemented with 4.5 μM 2,4-D and with 54 μM NAA, respectively. The yellowish nodular calluses vigorously produced shoot buds after transfer to media containing 0.44–44 μM 6-benzyladenine (BA), whereas the white friable calluses produced numerous somatic embryos upon transfer to plant growth regulator-free (PGR-F) medium. Histological observation of shoot buds and somatic embryos indicated that the former consisted of an apparent shoot meristem and several leaf primordia, and the latter had two distinct meristematic regions, corresponding to shoot and root meristems. Both shoot buds and somatic embryos developed into complete plantlets on PGR-F medium. Regenerated plants showed no observable morphological alterations. High proliferation and regeneration ability of these calluses, were maintained for over 2 yr.     

Callus growth and plant regeneration in diverse cultivars of cucumber (Cucumis sativus L.)

The growth and differentiation of callus tissues derived from cotyledons of ten cultivars ofCucumis sativus L. were investigated. Cotyledonary explants from all ten cultivars formed callus tissue on Murashige and Skoog (MS) medium supplemented with 0.5 M 2,4-dichlorophenoxyacetic acid and 5 M 6-benzylaminopurine. Fresh weight of the callus tissues averaged 1 to 8 g per flask after five weeks of culture. Shoot development was achieved in three cultivars, Hukchinju, Manchoonchoungjang and Seoul, on MS medium supplemented with 0.5 M -naphthaleneacetic acid and 5 M 6-benzylaminopurine. Reducing the 6-benzylaminopurine concentration to 0.01 M resulted in root formation on callus tissues and on shoots transferred to this medium. All cultivars gave the same response in tests of root formation, but shoot regeneration from callus culture of cucumber cotyledons was dependent on genotype with cultivar Manchoonchoungjang exhibiting the best shoot differentiation capability among the genotypes examined. Examination of mitotic metaphase from the regenerants revealed that all were tetraploid.     

Agrobacterium-mediated transformation of sunflower (Helianthus annuus L.) shoot apices: transformation patterns

Apical segments of embryonic axes of sunflower (Helianthus annuus L.) embryos were submitted to co-culture experiments with a disarmed strain of Agrobacterium tumefaciens, harbouring a plasmid coding for the marker enzyme -glucuronidase. The expression patterns of this marker were analysed at different developmental stages of the regenerated shoots. The results are consistent with the hypothesis that transformed shoots originate from transformation events that have occurred within the existing meristems. Two of the resulting chimaeric plants have been analysed in detail, and some representative gene integration patterns are presented.     

Plant regeneration in Kentucky bluegrass (Poa pratensis L.) via coleoptile tissue cultures

Summary Plant regeneration in Kentucky bluegrass (Poa pratensis L. cv. Touchdown) via culture of seedling tissues was investigated. When coleoptile, leaf, and stem sections of dark-germinated seedlings were cultured on Murashige and Skoog (MS) medium, different types of callus were produced, depending on the expiant source and growth regulator combinations. Only compact-friable callus (type 3) and moderately compact, friable callus (type 2) produced shoots upon subculture. The nonstructured watery callus (type 4) produced roots without shoots. Shoot differentiation from callus tissues was highest when the culture medium contained 0.2 mgL^–1 picloram + 0.01 mgL^–1 -naphthaleneacetic acid (NAA). Calli grown from coleoptiles had higher shoot regeneration frequency (32%) than that obtained from either stem sections (12%) or young leaf tissues (2%) of the same seedlings. Some organogenic callus lines produced exclusively green plants, while others produced albino shoots or a mixture of green and albino shoots. The green plants were multiplied in a medium containing 0.1 mgL^–1 BAP plus either 0.2 mgL^–1 picloram or 0.1 mgL^–1 indole-3-acetic acid (IAA). Over 90% of the cultures in the shoot proliferation medium produced roots in 4 weeks. The rooted plants were successfully established in soil medium and grown in the greenhouse.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - MS Murashige and Skoog (1962) medium - NAA -naphthaleneacetic acid - picloram 4-amino-3,5,6-trichloropicolinic acid - TDZ thidiazuron     

Effect of genotype on somatic embryogenesis from axes of mature peanut embryos

Genotypes representing the three botanical varieties of peanut (Arachis hypogaea L.) were assessed for somatic embryogenesis and subsequent plant conversion from mature zygotic embryo axes. Explants were initially cultured on Murashige and Skoog medium supplemented with 12.42 M 4-amino-3,5,6-trichloropicolinic acid. Individual somatic embryos wer isolated from explant tissue and used to initiate repetitive liquid cultures. There were significant differences among genotypes and varieties for somatic embryo formation and plant regeneration using a single media sequence. Botanical variety fastigiata had a lower embryogenic frequency and produced significantly fewer embryos than either hypogaea or vulgaris, which were similar in response.Abbreviations EA zygotic embryo axes - MS Murashige and Skoog (1962) medium - picloram 4-amino-3,5 - 6 trichloropicolinic acid     

Tissue culture and long-term regeneration of Phragmites australis (Cav.) Trin. ex Steud.

Phragmites australis tissue cultures were initiated from mature seeds on MS medium supplemented with 1 mgl^-1 each of 2,4-D and IAA. Cultures displayed typical embryogenic callus that was compact and bright yellow. Selection for embryogenic callus established long-term regenerable cultures. Removal of auxin from the basal medium allowed numerous complete plants to be recovered from the cultures. Histological study indicated both the presence of embryogenic-type cells and the bipolar development of regenerated plants.     

Production of solamargine by in vitro cultures of Solanum paludosum

Callus cultures of Solanum paludosum were established from roots, hypocotyles, cotyledons and leaf limbs of plantlets cultivated in sterile conditions on a Murashige and Skoog's modified medium. Non organogenous calluses were obtained with addition of BA or kinetin (10^-5M to 10^-6M) as the cytokinin and 2,4-d or NAA (10^-5M to 10^-6M) as the auxin. These calluses permitted the establishment of a cell suspension culture with BA (10-6M) and 2,4-d (10^-6M). Zeatin (10^-6M) with IAA (10^-6M) gave rise to organogenous calluses. These organogenous callus cultures developed multiple shoots which either proliferated if they were cultivated on a medium containing zeatin with IAA or IBA or were able to regenerate into whole plants when zeatin was used as the only hormone. The different plant material produced solamargine, the main steroidal glycoalkaloid present in the unripe fruits. The best production was obtained with the fruits of regenerated plants from organogenous callus cultures after reintroduction of these plants in their brasilian biotope. The solamargine content of the two types of plant materials was about 0.06% and 2.5% (dry weight) respectively for the callus cultures and the fruits from in vitro plants. The fruits were harvested a year after the beginning of the plantlet regeneration step.Abbreviations HPTLC high performance thin layer chromatography - HPLC high performance liquid chromatography - 2,4-d 2,4-dichlorophenoxyacetic acid - BA benzylaminopurine - IAA 3-indolebutyric acid - NAA -naphthaleneacetic acid - IBA 3-indolebutyric acid - IPA isopentenyladenine