Long-term culture of embryogenic sugarcane callus

Embryogenic calluses of sugarcane capable of regenerating green plants after long-term culture were sought. The largest quantities of embryogenic calluses were produced on Murashige & Skoog medium, but cultures maintained on Chu N6 medium remained embryogenic and totipotent longer. Both media contained 4.5 M 2,4-dichlorophenoxyacetic acid (2,4-d). The effect of supplements on somatic embryogenesis was examined. Kinetin (0.5 M) and 10% (v/v) coconut water in callus initiation medium were inhibitory to subsequent embryogenesis. Embryogenic calluses on N6 medium increased in fresh weight with proline concentration up to 90 mM. Maximum fresh weight was achieved with 5% sucrose. Although genotypic differences were observed, embryogenesis occurred in all 17 sugarcane clones tested. Embryogenic calluses of one cultivar regenerated green plants after 16 months, but suspensions were totipotent for only 8 months. Total number of regenerated plants decreased with time in culture, while the number of pale green plants increased starting after 5 months in culture.Published as Paper No. 785 in the journal series of the Experiment Station, HSPA     

Sucrose accumulation and enzyme activities in callus culture of sugarcane

The activities of sucrose phosphate synthase (SPS), sucrose synthase (SUSY), neutral invertase (NI) and soluble acid invertase (SAI) were measured in callus cultures of four Mexican sugarcane cultivars (Saccharum spp.) with a different capacity to accumulate sucrose in stem parenchyma cells. The results indicated that sucrose accumulation in callus was positively correlated to the activity of SPS and SUSY and negatively to the activity of SAI and NI while SPS explained most of the variation found for sucrose accumulation and NI least.The research was funded by the department of Biotechnology and Bioengineering CINVESTAV Mexico City, and F. G.-M. received grant-aided support from CONACyT, Mexico.     

Pigment and isozyme variation in aspen shoots regenerated from callus culture

Pigment as well as isozyme variations were observed among aspen (Populus tremuloides Michx.) plants regenerated from callus cultures. Out of more than 600 plantlets, two chimeric plants (one with green base and two albino shoots and the other with an albino shoot) were produced. Callus derived from albino shoots produced albino as well as chimeric plants when transferred to shoot inducing medium. Isozyme patterns of 119 plants were examined by starch gel electrophoresis. Thirty plants showed variation in shikimic dehydrogenase isozyme and 41 in isocitric dehydrogenase. Variation was also observed in malate dehydrogenase and phosphoglucose isomerase. No variation was seen in 6-phosphogluconate dehydrogenase. Pigment variation was not associated with any isozyme changes.Abbreviations BA 6-benzyladenine - IBA indole-3-butyric acid - GD Gresshoff & Doy medium - WPM woody plant medium - SKD shikimic dehydrogenase - IDH isocitric dehydrogenase - MDH malate dehydrogenase - PGI phosphoglucose isomerase - 6-PGD 6-phosphogluconate dehydrogenase     

Ploidy levels of plants regenerated from mixed ploidy maize callus cultures

Summary Haploid and diploid anther-derivedZea mays callus lines were treated with the antimicrotubule herbicide pronamide to produce mixed ploidy callus as determined by flow cytometry. The ploidy levels of the plants regenerated from the callus were determined by counting the leaf epidermal guard cell chloroplast numbers. The proportion of diploid regenerated plants was somewhat lower than the proportion of diploid cells of the callus. The diploid plants regenerated somewhat faster than the haploids. The proportion of tetraploids regenerated from the pronamide treated diploid callus, which originated by spontaneous chromosome doubling, was much lower than the proportion of cells indicating that tetraploid cells survive or regenerate plants at a lower frequency than diploid cells.     

Field performance of temporary immersion bioreactor-derived sugarcane plants

Summary The temporary immersion bioreactor has been found to be an important tool for sugarcane micropropagation, allowing higher shoot formation rates and cost reduction. This research was conducted to demonstrate the agricultural value of temporary immersion bioreactor-derived sugarcane plants. The experiment was carried out for about 2 yr to study the field performance of these plants. Two control treatments were also evaluated representing the conventional forms of micro- and macropropagation. Growth of sugarcane stools, first ratoon and the use of micropropagated plants for macropropagation were recorded. Some botanical and chemical characteristics were evaluated. Differences among propagation systems were only found in the first 6 mo. of field growth, regarding the stem length and diameter. Such differences disappeared with the course of the experiment.     

Sugarcane mosaic virus in plantlets regenerated from diseased leaf tissue

Plantlets produced from sugarcane leaf tissue were examined to determine the effect of propagation on the frequency of occurrence of sugarcane mosaic virus (SCMV).Explants from immature leaf tissues of the sugarcane variety CP 72-356 (Saccharum interspecific hybrid), healthy or SCMV-infected, were cultured on Murashige-Skoog medium to which a combination of cytokinin and auxin had been added. Plantlets developed on healthy and infected leaf tissue within 6 weeks. The juice from plantlets was assayed for SCMV on Rio sorghum (Sorghum bicolor (L.) Moench, var. Rio) seedlings and on sugarcane varieties CP 31-294 and CO 31-588 for SCMV-strain identification. Results indicated that SCMV strain H was transmitted from the donor tissue to the regenerated plantlets. Observation on plantlets reared in the greenhouse showed that 23% had symptoms of SCMV. In a second replicated experiment, the leaf tissue from plants of POJ 234 free of mosaic or infected with SCMV strain A, B, D, H, or I was cultured. Each of the five strains was transmitted from donor to plantlet as indicated by assays on sorghum and sugarcane varieties. From 11 to 88% of the plantlets had mosaic symptoms, depending on the strain infecting the donor plant. In this experiment, SCMV-strain M was transmitted from an unidentified donor variety to 23% of the regenerated plantlets.Portions of this paper have been presented to the American Society of Sugar Cane Technologists, at the meeting in Clearwater, Florida in June, 1984.     

Field performance of artificial seed-derived sugarcane plants

This study shows the behaviour of sugarcane plants cv. CP-5243 derived from artificial seed compared with traditional and isolated bud methods. Artificial seed-acclimatised plants were planted in field conditions simultaneously with two-control treatments previously germinated: macropropagated plants derived from stems of three buds and axillary buds isolated from field-grown plants. Plants from artificial seed were taller and had a smaller diameter at 8 months, but these differences disappeared at 12 months. With respect to sugar analysis and yield, no differences in all parameters evaluated were found between artificial seed-derived plants and plants derived from the two other methods.     

Identification of somaclonal variants of sugarcane (Saccharum spp.) resistant to sugarcane mosaic virus via RAPD markers

Somaclonal variants resistant to sugarcane mosaic virus (SCMV) were obtained from susceptible sugarcane cv PR62258 through somatic embryogenesis by increasing the number of subcultures of the embryogenic callus tissue in MS medium with 3 mg/L 2,4-dichlorophenoxyacetic acid. Transfers were made at 30-day intervals for 1, 2 or 3 subcultures. Two somaclones, namely AT626 and BT627, were selected by their resistance to SCMV. These subclones have maintained the resistance trait over seven years of testing in the field. In this report we identified the somaclonal SCMV resistant variants from the maternal line and the nonresistant somaclones, using the RAPD technique.     

Isolation and culture of protoplasts from callus tissue of Hibiscus syriacus L.

Protoplasts were isolated from callus tissue of Hibiscus syriacus L. using a solution of 3% Onozuka cellulase, 1% Onozuka macerozyme, and 0.5% hemicellulase. Highest yields of viable protoplasts were obtained from friable, white or yellow callus 8–9 days after subculture on Murashige & Skoog medium with 0.5 mg l^-1 2,4-dichlorophenoxyacetic acid and 0.1 mg l^-1 kinetin. Protoplasts cultured in thin liquid layers of this medium with mannitol continued dividing for longer than those cultured in droplets or in an agar medium. Cultures were maintained until protoplasts had divided to form groups of more than ten cells. Cell groups developed into callus and continued to grow on an agar medium, but failed to differentiate on a regeneration medium with 2 mg l^-1 naphthalene acetic acid and 1 mg l^-1 benzylaminopurine.     

Karyotypic variability in plants of Solanum melongena regenerated from callus grown in presence of culture filtrate of Verticillium dahliae

Summary Plantlets were regenerated from calli derived from leaf expiants of three genotypes of Solanum melongena (two parental genotypes and their hybrid). The cytological analysis showed that a) plants regenerated were all mixoploid, b) toxic medium (basal medium added with filtrate culture of Verticillium dahliae) was able to evidence karyotypic differences between genotypes not displayed by plants regenerated from callus grown on control medium, c) chromosomal mosaicism persists up to plant maturity and also in the selfed progeny. The results are discussed in terms of a selective process involving genes controlling chromosome number and/or a direct effect of toxic medium on the activity of the same genes.This research is supported by a grant from ERSO (Ente per la Ricerca e Sperimentazione in Ortoflorifrutticoltura e Sementi) — Regione Emilia Romagna     

Regeneration of plants from Antirrhinum majus L. callus

Somaclone production in Antirrhinum majus plants by regeneration of plants from callus cultures has been achieved using three types of explant tissue. Regeneration from mature stem internode-derived callus was extremely poor. Callus derived from seedling shoot tips could be induced to form new shoots in six of seven cultivars tested. Regeneration was achieved in all seven cultivars when callus was produced from segments of hypocotyl and was most effective using agar-solidified medium containing 0.25 mgl^-1 naphthoxyacetic acid + 10% coconut milk. In this case, five of the cultivars produced shoots directly, one produced leaves from the petioles of which new shoots emerged, and one regenerated plants chiefly through the production of embryoids.     

Thidiazuron stimulates shoot regeneration of sugarcane embryogenic callus

Summary Efficient shoot regeneration of sugarcane (Saccharum spp. hybrid cv. CP84-1198) from embryogenic callus cultures has been obtained using thidiazuron (TDZ). Callus was placed on modified Murashige and Skoog (MS) medium containing 2.3 μM 2,4-dichlorophenoxyacetic acid (2,4-D), or 9.3 μM kinetin and 22.3 μM naphthaleneacetic acid (NAA) and compared with the same MS medium supplemented with 0.5, 1.0, 2.5, 5.0 or 10.0 μMTDZ, A11 TDZ treatments resulted in faster shoot regeneration than the kinetin/NAA treatment, and more shoot production than either the 2,4-D or kinetin/NAA treatments. Maximum response, as determined by total number of shoots (26 per explant) and number of shoots greater than 1 cm (4 per explant) 4 wk after initiation, was obtained with 1.0 μM TDZ. The shoots rooted efficiently on MS medium supplemented with 19.7 μM indole-3-butyric acid (IBA). These results indicate that TDZ effectively stimulates sugarcane plant regeneration from embryogenic callus, and may be suitable to use in genetic transformation studies to enhance regeneration of transgenic plants.     

Association and heritability of sugarcane smut resistance to races A and B in Hawaii

Summary This study was conducted to estimate the degree of association of smut (Ustilago scitaminea Syd.) resistance in sugarcane (Saccharum spp.) between races A and B in Hawaii and to estimate the heritability of resistance to both races. The estimated degree of association was 0.18. Although statistically significant, the degree of association of resistance between races A and B was near zero and therefore of no practical importance. Selection for resistance to one race would have little effect on the frequency of resistance to the other race in the following sexual generation. Heritabilities in the broad sense estimated from the parent population on a plot mean basis were 0.96 and 0.91 for races A and B, respectively. Selection for smut resistance should be very effective between populations of asexual generations. Heritabilities in the narrow sense estimated from parent-progeny regression analysis on a family mean basis were 0.51 and 0.47 for races A and B, respectively. Selecting and breeding for resistance should result in a fairly rapid increase in the frequency of resistance in the progeny population.Contribution from Department of Genetics and Pathology, Hawaiian Sugar Planters' Assn. Exp. Stn., P.O. Box 1057, Aiea, HI 96701. Published with the approval of the Director as paper no. 652 in the Journal Series of the Exp. Stn., Hawaiian Sugar Planters' Assn. Research funded in part by USDA/ARS Cooperative Agreement No. 58-9AHZ-0-492     

Regeneration of Coronilla varia L. (crownvetch) plants from callus culture

Coronilla varia L. (crownvetch) plants were regenerated from callus cultures through somatic embryogenesis. Callus cultures were initiated using hypocotyls excised from sterile seedlings. Cultures were then transferred from a modified Gamborg's B5 medium containing 2,4-D to a medium containing no plant growth regulators (basal B5). Formation of embryos was evident in 12 of 32 callus lines after transfer of callus to BOi2Y (modified Blayde medium supplemented with 100 mg inositol and 2 g yeast extract/L). Basal B5 supplemented with 10 mM asparagine or 20 mM NH₄Cl could be substituted for BOi2Y. Embryos subsequently transferred to basal B5 developed roots and shoots. Plants thus formed were first transferred to vermiculite and then to soil.Contribution No. 8219 of the U.S. Regional Pasture Reasearch Laboratory, USDA-ARS, University Park, PA, U.S.A.     

Regeneration of whole plants from callus culture of diverse genetic lines of Pisum sativum L.

Sixteen genetic lines of peas were screened for their ability to regenerate whole plants from callus cultures. Epicotyl sections from germinating seeds were placed on callus-inducing medium; the resulting callus was subcultured monthly and was tested every other month for its regeneration ability. Six lines were found that would regenerate after 2 months' growth as callus. Four of these continued to regenerate after 4 months and, of these, two after 6 months. The cultivars Frosty and Alaska were among the lines that would not regenerate at all.Michigan Agricultural Experiment Station Journal Article No. 8932     

The initiation of callus and regeneration from callus culture ofTulipa gesneriana

Intergeneric Fragaria vesca x Potentilla fruticosa hybrids were produced using in vitro culture. Hybrid plants were not obtained by direct embryo rescue, but were regenerated from cotyledon-derived callus. Experiments with F. vesca indicated that using cotyledon halves was not more productive than using entire cotyledons. A polarity was observed in cotyledons and in cotyledon halves, with callus and regenerated shoots produced more frequently from proximal ends. Cotyledons from 17% of hybrid embryos produced callus and regenerated mature plants. The technique enabled rapid multiplication of some embryos, with the production of more than one hybrid plant. In some cases more than 100 shoots were obtained from one embryo, demonstrating the potential usefulness of this technique for the production of intergeneric hybrids.Abbreviations BA 6-benzylaminopurine - IAA indole-3-acetic acid - NAA -naphthaleneacetic acid     

Plant regeneration from a cryopreserved embryogenic cell suspension of a commercial sugarcane hybrid (Saccharum sp.)

Efficient plant regeneration was obtained from a cryopreserved embryogenic cell suspension of sugarcane established from leaf derived callus. Pregrowing the cells for three days in MS basal medium supplemented with 0.33 M sorbitol was essential to the process. The cells were cooled at a rate of 0.5°C/min to –40°C and then stored in liquid nitrogen. Thawing was carried out rapidly in water at +40°C, and the cells were then plated without washing onto filter paper discs placed on a semi-solid regeneration medium (MS basal + 3% sucrose + 0.13 mg/1 2,4-D +0.25 mg/1 BAP + 0.25 mg/1 kinetin + 0.25 mg/1 zeatin). The filter paper discs, along with the cells, were transferred to the same, fresh medium after five hours. After 24 hours the cells were scraped off, placed on fresh semi-solid medium and incubated at 28°C in the dark for two weeks before transfer to light. A regeneration efficiency of 92% was obtained (regenerated plants, expressed as a percent of unfrozen control). Plants regenerated from cryopreserved cells, and grown to maturity in the greenhouse, were morphologically identical to regenerated control plants.Abbreviations DMSO dimethyl sulfoxide - PEG polyethylene glycol - 2,4-D 2,4-dichlorophenoxyacetic acid - BAP benzyl aminopurine - TTC 2,3,5-triphenyl tetrazolium chloride     

Transgenic tobacco plants regenerated from leaf disks can be periclinal chimeras

Amongst rolC transgenic tobacco plants regenerated from leaf disks 6.5% are periclinal chimeras, i.e. plants with genetically different cell populations in different cell layers. The expression of the rolC gene of Agrobacterium rhizogenes causes a reduction in pigment content in leaves. The chimeric composition of the regenerated plants becomes thus apparent as light green leaf tissue in the transgenic region, tissue flanked by dark green wild-type sectors. Southern and northern blot analysis confirmed the chimeric nature of such plants. Investigation of selfed progeny of chimeric plants on selective media indicates that layer invasion in reproductive tissues can occur in tobacco early during the formation of the flower buds. The results show (1) that tobacco plants regenerated from leaf disks and grown on selective media have not necessarily the same clonal origin and (2) that they can give rise to non-transgenic offspring. The chimeric plants provide insight on the effect of rolC gene expression on microsporogenesis.     

Plant regeneration from leaf base callus of turmeric and random amplified polymorphic DNA analysis of regenerated plants

Callus cultures were initiated from leaf bases of turmeric on Murashige and Skoog's basal medium (MS) supplemented with dicamba, picloram (2 mg l⁻¹) or 1-naphthaleneacetic acid (NAA) (5 mg l⁻¹) in combination with benzyladenine (BA) (0.5 mg l⁻¹). On transfer of callus cultures to medium supplemented with benzyladenine (BA) (5 mg l⁻¹) in combination with triiodebenzoic acid (TIBA) or 2,4-dichlorophenoxyacetic acid (2,4-D) (0.1 mg l⁻¹), green shoot primordia were seen to differentiate from the surface of the callus. On transfer of regenerating cultures to half MS media supplemented with Kn, shoot primordia developed into well developed shoots. When shoots were transferred to medium devoid of phytohormones, complete rooted plants were obtained. Ninety percent of the plants survived to maturity on transfer to soil. Random Amplified Polymorphic DNA (RAPD) analysis of eight regenerated plants using 14 primers when separated on non-denaturing polyacrylamide gels showed 38 novel bands. About 51 bands present in the control were absent in the regenerants. The result indicates that variation at DNA level has occurred during in vitro culture. This revised version was published online in June 2006 with corrections to the Cover Date.