Long-duration,high-frequency plant regeneration from cereal tissue cultures

By visual examination of calli derived from germinating seeds of wheat, oats, rice, proso millet, and pearl millet it has been possible to visually select embryogenic (E) callus which, on transfer to a regeneration medium, forms plants an average of 33 times more frequently than non-embryogenic (NE) callus of equal mass. Embryogenic callus consists of small isodiametric cells averaging 31 m in diameter; NE callus consists of long tubular cells averaging 52 m in width and 355 m in length. Production of E callus is in many cases promoted by media containing 2,4-di- or 2,4,5-trichlorophenoxyacetic acid (2,4-D or 2,4,5-T) plus indole-3-acetic acid or tryptophan+kinetin. Production on NE callus is promoted by media containing 2,4-D or 2,4,5-T alone. As a result of initial experiments to optimize both media for E callus production and media for plant regeneration, callus derived in six passages from an average of 26 seeds could produce about 1,000 regenerated plants.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid - IAA indole-3-acetic acid - Kin kinetin - Trp L-tryptophan - E embryogenic - NE non-embryogenic     

Plant regeneration in vitro from embryogenic cultures of spring- and winter-type barley (Hordeum vulgare L.) varieties

Summary Immature embryos of 41 lines of barley were screened in vitro for callus induction and somatic embryogenesis on different media to establish totipotent cultures. The use of modified MS and CC media, both supplemented with 1 g/l casein hydrolysate, and the substitution of agarose for agar resulted in the highest frequencies of somatic embryo induction. Embryogenic callus was induced and plants regenerated from 23 of the lines tested. The auxins 2,4-D, dicamba, picloram and 2,4,5-T were suitable for embryogenic callus induction. High frequencies of somatic embryo germination occurred on CC medium supplemented with 1 mg/l IAA and 0.05 mg/l zeatin. A strong genotypic effect on the capacity and frequency of embryogenic callus formation was found. Cultivar Golden Promise always gave the best results. Experiments with field grown material in 3 consecutive years showed that environmental factors also strongly influenced the induction of somatic embryogenesis and plant regeneration.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid - dicamba 3,6-dichloro-o-anisic acid - picloram 4-amino-3,6,6-trichloropicolinic acid - NAA naphtaleneacetic acid - IAA indole-3-acetic acid - ABA abscisic acid - BAP 6-benzyl amino purine - 2iP 6-(3-methyl-2 butenyl 1-amino)purine - GA₃ gibberellic acid     

In vitro propagation of Iris pallida

Plantlets were regenerated from callus of Iris pallida, an important perfume plant. Only the leaf base attached to the rhizome had the ability to generate yellow-colored callus on LS medium supplemented with 1 mg/l 2,4-D and 0.1 mg/l KT in the dark. Yellow calli grew with partial differentiation into white tissue, probably embryogenic, during subculture on the same medium with a 16-h photoperiod. Only yellow-colored calli with the white tissue could differentiate into plantlets after transfer to kinetin- or gibberellin- supplemented LS medium. Regenerated plantlets which grew on the medium without growth regulators were transferred to the soil. After 2 years of cultivation in soil, the regenerated plants flowered and formed rhizomes. The components of the essential oil in the rhizome of regenerated plants were essentially the same as those in natural plants.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - KT kinetin - NAA alpha-naphthaleneacetic acid - LS Linsmaier and Skoog (1965) medium     

Plant regeneration via somatic embryogenesis in chick pea (Cicer arietinum L.)

Five genotypes of chickpea (Cicer arietinum L.) PG1, PG5, PG12, N59 and C235 were evaluated for induction of somatic embryogenesis. Somatic embryogenesis was induced from immature cotyledons of genotypes PG12 and C235 and immature embryo axes of genotypes PG5, PG12 and C235. Genotypes N59 and PG1 showed no response. The maximum frequency of globular embryo formation occurred in cotyledonary segments on MS medium with 3.0 mg/l 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). Further embryo development was achieved only in somatic embryos derived from cotyledonary segments of genotype PG12. Globular-stage embryos derived from immature embryo axes of PG5, C235, PG12, and cotyledonary segments of C235 dedifferentiated and formed callus. The cotyledonary stage embryos of genotype PG12 germinated on half-strength MS medium supplemented with 1 mg/l zeatin. The regenerated plants were transferred to soil and grown to maturity.Abbreviations ABA abscisic acid - BAP 6-benzylamino purine - CM coconut milk - Dicamba 3,6-Dichloro-2-methoxybenzoic acid - 2,4-D 2,4-dichlorophenoxy-acetic acid - GA₃ gibberellic acid - IAA indoleacetic acid - MS Murashige and Skoog medium (1962) - NAA 1-napthaleneacetic acid - Picloram 4 amino-3,5,6-trichloropicolinic acid - 2,4,5-T 2,4,5-trichlorophenoxy-acetic acid - zeatin (6-[4-Hydroxy-3-methyl-2-butenylamino] purine)     

Plant regeneration and initiation of cell suspensions from root-tip derived callus of Oryza sativa L. (rice)

Root-tip derived suspended callus of Oryza sativa cv. Thaipei showed the capacity for plant regeneration via organogenesis. Cell cultures were induced in liquid Murashige-Skoog medium containing 2 mg/l 2.4-dichlorophenoxyacetic acid. Dicamba or Picloram were effective for induction of organogenesis. Shoots and roots differentiated following subculture on medium lacking auxins but containing kinetin. At 1 and 4 mg/l Dicamba and 1 mg/l Picloram normal green plants were regenerated whereas with 7 mg/l Dicamba in the medium only albino plantlets were obtained. Regenerated plantlets were grown to maturity and set seed. Cell suspension cultures, initiated from the root-tip derived calli, provided suitable material for protoplast isolation.Abbreviations BM Basic medium - 2.4 -D 2,4-dichlorophenoxyacetic acid - Dicamba 3,6-dichloro-2-methoxy benzoic acid - Picloram 4-amino-3,5,6-trichloropicolinic acid     

Plant regeneration through somatic embryogenesis from mature seed and young inflorescence of wild rice (Oryza perennis Moench)

Somatic embryogenesis in the wild rice species (Oryza perennis) was induced from cultured mature seeds and young inflorescences. Murashige and Skoog's (MS) medium supplemented with 2 mg/l 2,4-D and 0.2 mg/l BAP was used for induction of a compact, white nodular callus and somatic embryos. Plant regeneration occurred with the tranfer of the nodular callus to MS basal medium containing 0.5 mg/l IAA, 0.5 mg/l NAA, 4 mg/l BAP and 500 mg/l casein hydrolysate. The embryogenic nature of the callus from both explants was maintained over 10 subcultures for about 12 months. Plant regeneration with respect to the number of calli plated from the 6th to 10th passage varied from 80% to 60% for young inflorescence derived callus and from 75% to 69.8% for seed-derived callus.Abbreviations MS Murashige and Skoog medium - BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - NAA naphthalene acetic acid - CH casein hydrolysate     

Anther culture of some perennial triticeae

Three field grown Agropyron spp. (crested wheatgrasses) and two Thinopyrum spp. (intermediate and tall wheatgrasses) were evaluated for anther culture response. Hormonally modified potato extract and 85D12 media induced pollen embryogenesis. Modified Murashige and Skoog media were tested for their effects on callus proliferation and plantlet regeneration. Callus induction frequency and plantlet production were highest (25.0% and 45.8%, respectively) for Thinopyrum ponticum (2N=70) (tall wheatgrass). One-hundred and nine albino plantlets were produced from T. ponticum Jose both by direct regeneration on 85D12 medium and through a callus phase from potato extract media. This is the first report of plantlet production from anther culture of a Triticeae perennial forage grass. Further experimentation with environmental and cultural conditions may result in the production of green plantlets.Abbreviations MS Murashige and Skoog (1962) medium - NAA naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - 2-ip 2-isopentenyladenosine - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid Cooperative investigations of the USDA-Agricultural Experiment Station and the Utah Agricultural Experiment Station, Logan, UT 84322. Approved as Journal Paper No. 3596     

Production,maintenance and plant regeneration from cell suspension cultures of Stevia rebaudiana (Bert.) Bertoni

A method is described for producing and maintaining Stevia rebaudiana suspensions and regeneration of plants from calli derived from cell suspensions. Suspension cultures composed of isolated cells (ca. 10%) and cellular aggregates (5–100 cells) were obtained in 20–30 days by using friable callus as the initial inoculum in liquid medi with BA (0.5 mg/l)+2,4-D (1.0 mg/l), and periodic filtering (100–500 m sieves) with 6–7 days interval between subcultures. Cultures derived from actively growing calli are mainly diploid (2n=22) whereas those derived from senescent calli showed a wide variation in chromosome number (55–200). Stock cell suspensions which had been maintained for 3 years were plated on basal LS agar medium with BA (0.5 mg/l)+2,4D (0.5 mg/l) to form callus. Calli originating from predominantly 2n cell suspensions when transferred to medium with K (2.0 mg/l)+NAA (0.02 mg/l) were able to form buds. Shoot elongation and further rooting of isolated shoots was better on LS medium devoid of growth regulators. Variation in rooting capacity, plant vigour, morphological characters and chromosome number was found amongst regenerated plants.Abbreviations BA Benzylaminopurine - 2,4-D 2,4 - Dichlorophenoxyacetic acid - GA₃ Gibberellic acid - IAA Indoleacetic acid - IBA Indolebutyric acid - K Kinetin - LS Linsmaier & Skoog     

Somatic embryogenesis and plant regeneration in inflorescence and seed derived callus cultures of Poa pratensis L. (Kentucky bluegrass)

Callus induction and plant regeneration were studied in 15 cultivars of the facultative apomictic species Poa pratensis L. (Kentucky bluegrass).The tissue culture responses of mature seeds and immature inflorescences were compared. Murashige and Skoog's (MS) medium, supplemented with 2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) was used for callus induction and maintenance. Plants could be regenerated from compact and friable callus on MS medium devoid of 2,4-D. Plants were recovered from 14 cultivars at a high frequency (up to 79% of the callus cultures) when young inflorescences were used as the explant material and from only 3 cultivars, at a low frequency (up to 3%), with seeds. Somatic embryos were observed in callus cultures of many cultivars. Fully developed germinating somatic embryos were occasionally observed. Plant regeneration appeared to take place both via somatic embryogenesis and organogenesis. Plants were generally green but albino shoots developed at a low frequency from friable callus.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog's (1962) medium - IAA indole-3-acetic acid - N₆ medium of Chu et al. (1975)     

Regeneration of plantlets from the callus of stem segments of adult plants of Ficus religiosa L.

Stem segments of adult plants of Ficus religiosa L. cultured on MS medium containing 1.0 mg/l 2,4-D produced callus. Shoots were regenerated when the induced calli were transferred to medium supplemented with 0.05 to 2.0 mg/l BAP. Callus derived shoots produced roots and developed into plantlets when transferred to medium supplemented with 1.0 mg/l NAA.Abbreviations MS Murashige and Skoog (1962) - BAP 6-benzylaminopurine - NAA naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid     

Influence of primary callus induction conditions on the establishment of barley cell suspensions yielding regenerable protoplasts

Summary With the aim of the development of a culture method for efficient plant regeneration from barley (Hordeum vulgare L.) protoplasts, we examined several culture conditions for primary calli from immature embryos of cvs. Dissa and Igri, which were used for initiation of cell suspensions. Among the primary callus culture conditions tested, growth condition of donor plants had a great impact on these efficiencies; Igri protoplasts derived from embryos of plants grown in a greenhouse gave rise to albino plants and few green shoots while several cell lines originating from embryos of plants grown in a growth chamber (16h light, 12°C) yielded protoplasts developing into green plants. In contrast, cell suspensions were produced at higher frequencies from calli derived from embryos of greenhouse-grown Dissa plants. In Igri, increased levels of 2,4-dichlorophenoxyaceticacid (2,4-D) significantly reduced the efficiency of cell suspension establishment and plant regeneration from protoplasts was achieved only with suspension cells derived from calli induced at the lowest level (2.5 mg/l), while the effect of the 2,4-D concentration was not clear in Dissa. The developmental stage of immature embryos also affected the efficiency of cell suspension establishment, and the optimal embryo size was determined to be approximately 1mm in diameter. These results demonstrate the importance of callus induction conditions for successful barley protoplast culture.     

Plant regeneration from shoot apex explants of foxtail millet

Green and etiolated shoot apices of foxtail millet (Setaria italica L.) cv. Nese 2A were cultured on Murashige and Skoog medium with four concentrations of 2,4-dichlorophenoxyacetic acid or 2,4,5-trichlorophenoxyacetic acid. In all treatments, embryogenic calli capable of plant regeneration were induced after ten weeks in culture. Calli induced on 2 mg l^-1 of 2,4-d from green apices gave a higher rate of plant regeneration in comparison with etiolated apices on the other treatments. Plant regeneration was obtained from one year-old cultures. Regenerated plants were successfully established in soil, reached maturity and produced seeds.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - EC embryogenic calli - NE nonembryogenic calli - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid     

The effect of auxins, time exposure to auxin and genotypes on somatic embryogenesis from mature embryos of wheat

The effects of different factors on the embryogenesis and plant regeneration from mature embryos of Russian spring and winter genotypes were studied. Embryogenic callus induction was achieved on MS medium supplemented with different concentrations of 2,4-D (2,4-dichlorophenoxyacetic acid), 2,4,5-T (2,4,5-trichlorophenoxyacetic acid) or Dicamba (3,6-dichloro-o-anisic acid). Although all auxins were able to induce callus from explants with high frequency (98–100%), Dicamba was more effective for the induction of embryogenic callus (21.8–38.3%). Maximum embryogenic callus formation and high number of regenerated plants were observed at 12 mg l⁻¹ of Dicamba. The time exposure to Dicamba (7, 14, 21 and 28 days) had a significant effect on efficiency of somatic embryogenesis. When contact of explants with callus induction medium was increased from 7 to 21 days the rate of somatic embryogenesis and number of regenerated plants per embryogenic callus gradually increased from 13.0 to 38.4% and 3.6 to 8.0%, respectively. Supplement of additional auxins (indoleacetic acid (IAA), indolebutyric acid (IBA), and naphthaleneacetic acid (NAA)) to callus induction medium with Dicamba had a positive effect on the rate of embryogenic callus formation, while the average number of regenerated shoots was not affected. The best rate of somatic embryogenesis was observed at the addition of 0.5 mg l⁻¹ IAA with Dicamba (61.0%). The optimum combination of Dicamba and IAA increased the efficiency of somatic embryogenesis and plant regeneration from seven spring and winter wheat genotypes, thought overall morphogenic capacity was still genotype dependent.     

Callus production,regeneration and evaluation of plants from cultured inflorescences of tall fescue (Festuca arundinacea Schreb.)

Twenty genotypes (individual plants) of Kentucky 31 tall fescue (Festuca arundinacea Schreb., 2n=6x=42) were evaluated to determine regeneration response, and meiotic and isozyme changes in the regenerants. Six genotypes (K8, K16, K25, K27, P3 and P13) were selected for study of efficiency in producing calli and regenerating plants. Panicle pieces (3569) were plated on Schenk & Hildebrandt medium with one of three auxins, 2,4-D (2.0 mg 1^-1), pCPA (3.8 mg 1^-1) or 2,4,5-T (2.5 mg 1^-1). Square-root transformed data were analysed as a completely randomized 6×3 factorial with genotype and auxin as the main effects. F-protected means were compared by the Waller-Duncan K-ratio T test. Genotype K8 produced significantly more calli per panicle piece (88/877), whereas genotype K25 regenerated significantly more plantlets per panicle piece (58/214) than the other genotypes. Callus production was significantly higher using 2,4-D (102/1244 calli per piece) than pCPA or 2,4,5-T. The type of auxin did not have a statistically significant effect on plant regeneration, which suggested that genotype was the most important variable. Twelve of the 210 regenerants were albino. Cytological evaluation of 95 green regenerants showed that all plants had 21 bivalents and pollen stainability ranged from 43.0–98.4%, which suggested all plants were male fertile. Zymograms of these 95 regenerants for ACPH, ADH, GOT, MDH, 6-PGD, PGI and SOD showed no differences from that of the parental genotype, which suggested that the green plantlets regenerated from somatic tissue.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - pCPA parachlorophenoxyacetic acid - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid - ACPH acid phosphatase - ADH alcohol dehydrogenase - GOT glutamate oxaloacetate transaminase - MDH malate dehydrogenase - 6-PGD 6-phosphogluconate dehydrogenase - PGI phosphoglucoisomerase - SOD sulfoxide dismutase     

Organogenesis and Plantlet Formation from Organ- and Seedling-Derived Calli of Rice (Oryza sativa)

Callus was induced in different somatic organs of Oryza sativa L. Specific minimum 2,4-dichlorophenoxyacetic acid (2,4-D) concentrations in the medium were necessary for the induction of callus from different organs while high levels of 2,4-D (6–10 mg/l) induced callus formation in each organ tested. The optimum 2,4-D concentration for callus induction and growth for root-derived calli was 2 mg/l and for leaf-derived 6 mg/l. Root and shoot organogenesis were induced in both root- and leaf-derived calli by sub-culturing to a medium lacking 2,4-D. Root organogenesis occurred at a higher frequency than shoot organogenesis. Shoot organogenesis rarely occurred in calli without differentiated roots. Increased age of callus cultures almost completely inhibited shoot development. The addition of the cytokinin 6-γ,γ-dimethylallyl-amino purine partially restored the potential for shoot organogenesis. Whole plants were easily recovered from the calli and grown to maturity with some plants exhibiting phenotypic abnormalities.     

Callus induction and plant regeneration from maize mature embryos

Calli were induced from mature embryos of maize (Zea mays L.) inbred lines A632, B73 and Mol7 on MS medium supplemented with 1–2 mg/1 2,4-dichlorophenoxyacetic acid. Callus induction frequency ranged from 23–100%, with Mol7 having the highest frequency. Plants were regenerated from 4–5% of the B73 and Mol7 explants. Embryogenic and organogenic calli of B73 were maintained for more than two and one half years without losing regenerability. Of 95 regenerated plants, only one R₀ plant with abnormal pollen was detected, and no morphological variants were observed in the R₁ progeny.Abbreviations Dicamba 3,6-Dichloro-o-anisic acid - IAA 3-indoleacetic acid - NAA 1-naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - Ze zeatin     

An efficient and reproducible method for regeneration of whole plants from mature seeds of a high yielding Indica rice (Oryza sativa L.) variety PAU 201

Tissue culture is one of the tools necessary for genetic engineering and many other breeding programs. Moreover, selection of high regenerating rice varieties is a pre-requisite for success in rice biotechnology. In this report we established a reproducible plant regeneration system through somatic embryogenesis. The explants used for regeneration were embryogenic calli derived from mature seeds cultured on callus induction media. For callus induction mature seeds were cultured on MS medium containing 30 g/l sucrose combined with 560 mg/l proline and 1.5-3.5 mg/l 2,4-D and 0.5-1.5 mg/l Kin. For plant regeneration, embryogenic calli were transferred to MS medium containing 30 g/l sucrose, supplemented with 1.0-3.0 mg/l BAP, 0.5-1.5 mg/l Kin and 0.5-1.5 mg/l NAA. The highest frequency of callus induction (44.4%) was observed on the MS medium supplemented with 2.5 mg/l 2,4-D, 0.5 mg/l Kin, 560 mg/l proline and 30 g/l sucrose. The highest frequency of shoot regeneration (42.5%) was observed on the MS medium supplemented with 2.0 mg/l BAP, 0.5 mg/l NAA and 0.5 mg/l Kin. The plantlets were hardened and transferred to soil in earthen pots. The developed method was highly reproducible. The in vitro developed plants showed normal growth and flowering under glasshouse conditions.     

Stimulation of shoot regeneration in Triticum aestivum and Nicotiana plumbaginifolia Viv. tissue cultures using the ethylene inhibitor AgNO3

Silver nitrate effectively promoted shoot regeneration in wheat (Triticum aestivum L.) callus cultures derived from immature embryos. This effect could be observed in both weakly and strongly regenerating cultivars, and in using material from both field and greenhouse grown plants. The role of silver ions as an inhibitor of ethylene action was supported by a reversal of the inhibitory effects of 2,4-D and ethylene on morphogenesis in wheat callus cultures.Enhancement of shoot regeneration by silver nitrate was also observed in callus cultures of non-regenerating or weakly regenerating mutants of Nicotiana plumbaginifolia Viv. derived from cell cultures.Abbreviations BA 6-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - Ethrel 2-chlorethylphosphonic acid - NAA 1-naphthaleneacetic acid - NR^– nitrate reductase deficient     

Embryogenesis and Regeneration of Green Plantlets from Wheat (Triticum aestivum) Leaf Base

A short-term regeneration system from leaf-base-derived callus of wheat (Triticum aestivum L.) was developed. Embryogenic callus formation and shoot regeneration were achieved from the first basal segments of 3–4-day-old seedlings. Callus formation frequency as well as plantlet regeneration frequency was dependent on the composition of basal medium and the concentration of 2,4-dichlorophenoxyacetic acid (2,4-D). MS medium with 2,4-D 4.5–9.0 mol l^–1 was optimal for the culture of wheat leaf base. Effects of different combinations of plant growth regulators, which were added in either callus induction medium or shoot regeneration medium, were tested. Adding of BAP in callus induction medium shortened the time of shoot emergence but could not improve the producing of embryogenic calli and green plantlets. Optimal ratio of 2,4-D, BAP and NAA gave similar regeneration frequency to control. Existence of cytokinins in regeneration medium had no effect on increasing the regeneration frequency. The regenerants could grow to normal, fertile plants after they were transferred into soil.     

Meiotic and isozymic characterization of plants regenerated from euploid and selfed monosomic tall fescue embryos

Summary Tissue culture of tall fescue (Festuca arundinacea Schreb., 2n=6x=42) would be enhanced by improving the callus induction and plant regeneration efficiency, and evaluating the meiotic and isozymic variation induced by culture. Mature embryos were cultured from four lines of Kenhy tall fescue and from the progeny of three selfed monosomics. Evaluation of six media-auxin combinations showed callus initiation was greatest on SH medium with 2.5 mg/l 2,4,5-T or 7.4 mg/l pCPA, while plant regeneration was greatest on SH medium with 0.5 mg/l 2,4-D. Cytological analyses of 27 plants derived from euploid parents showed a high frequency of aneuploidy (15/27). Chromosome numbers of aneuploids ranged from 36 to 41, with one plant having 80 chromosomes and two plants being asynaptic. Two of ten monosomic-derived plants were euploid, five were monosomic, one was monosomic with a fragment and two were double monosomic. Zymograms of the parents and regenerants were obtained for the enzymes ACPH, ADH, GOT, 6-PGD and PGI. Isozyme variation was observed for two groups of plants derived from the same Kenhy embryos. One group of four monosomic-derived plants differed for the enzymes GOT and ACPH, and all four plants had a PGI pattern. different from that of the parental monosomic plant. This indicated loss of a PGI allele, probably as a result of callus culture.Contribution No. 89-3-141 of the Kentucky Agricultural Experiment Station in cooperation with the USDA-ARS. Part of thesis research for senior author's M. S. degree