High frequency somatic embryogenesis with a pampeana-derived genotype of alfalfa (Medicago sativa L.)

Summary Somatic embryos of genotype R11 of the alfalfa variety Pampeana were produced from embryogenic calli derived from leaf sections. They were induced by an auxin shock and its development was attempted on six different media. The best condition for somatic embryo production was inducing callus on MS medium plus 10 M 2,4-D and 4,6 M KIN and transferring them, after the auxin shock, to MS with 10–20 mM NH₄ ⁺ and 30 mM proline. More than 500 somatic embryos per plate were produced. Embryos were grown to plants on MS or half strength MS media and all regenerated plants resembled the original R11 genotype. This technique could be useful in alfalfa Pampeana improvement using genetic modification.     

Heritable tissue culture induced variation in Zinnia marylandica

Summary Adventitious shoots of Zinnia marylandica, an amphidiploid with limited genetic segregation, were regenerated from cotyledonary tissue on Murashige-Skoog (MS) media containing 0.2 or 22.2 M thidiazuron (TDZ) and grown through flowering. Fisher's Test for Equal Variance indicated tissue culture induced plants had more variation than seed-derived control plants. Twelve of 149 (8%) plants derived from 0.2 M TDZ and three of 23 (13%) plants from 22.2 M TDZ had variant characters. Aberrant characteristics in self-pollinated variants included plant height, fertility, flower color and morphology, and were sexually transmitted, indicating genetic change had occurred. Aberrant characteristics not observed in regenerated plants arose in progeny.Abbreviation TDZ thidiazuron     

Effects of salicylic acid on cold preservation and cryopreservation of encapsulated embryonic axes of Persian lilac (Melia azedarach L.)

Embryonic axes of Persian lilac (Melia azedarach L.)encapsulated into calcium alginate beads with sucrose (0.75 M) and salicylicacid (0, 50 M, 200 M) were subjected to cryopreservationtechnique with dehydration and freezing in liquid nitrogen or to coldpreservation by stocking alginate beads in empty petri dishes for 4 monthsat 4 °C. In these two cases 200 M salicylic acid enhancedsignificantly the percentage of viability of encapsulated embryonic axes andthe role of salicylic acid in increasing tolerance to dehydration is discussed.     

Interspecific hybrids of Cucumis metuliferus × C. anguria obtained through embryo culture and somatic embryogenesis

Summary Interspecific crosses between Cucumis metuliferus Naud. and C. anguria L. were obtained through embryo culture. Embryos in the rabbit-ear to advanced fluke-shaped stages were rescued 34–99 days after pollination. Plants were obtained through direct embryo culture, and through somatic embryogenesis from immature embryos. For direct embryo culture, fluke-shaped embryos were stored in sterile water in darkness for three days at 25C prior to transfer on Murashige and Skoog (MS) culture medium plus 1.0 M 6-benzylamino-purine. Multiple plants were obtained from single embryos through somatic embryogenesis of rabbit-ear stages on MS plus 10 M indole-3-acetic acid and 5 M 6-benzylamino-purine. Evidence of hybridization included leaf shape intermediate between the two parents, penduncle shape prior to fertilization which resembled the male parent, low pollen viability and isoelectric focussing of protein bands for acid phosphatase of leaf extracts.     

Plant recovery of cryopreserved apical meristem-tips of Melia azedarach L. using encapsulation/dehydration and assessment of their genetic stability

A cryopreservation process usingencapsulation/dehydration technique was setup for apical meristem-tips of invitro plantlets of `paradise tree' (Melia azedarach L. var. gigantea,clone `El dorado'). Apical meristem-tipswere cultured for one day on MS basalmedium with 2 M BA and 0.5 M IBAand encapsulated with 3% sodium alginate.The highest shoot proliferation rate aftercryopreservation was obtained whenencapsulated apical meristem-tips werepregrown for 3 days in liquid medium with0.5, 0.75 and 1 M of sucrose for 24 hoursprogressively, desiccated for 5 hours withsilicagel followed by rapid or slowcooling. Survival after freezing in liquidnitrogen ranged between 67–83% andshoot proliferation ranged between 43–60%. This cryopreservation treatmentpreserved genetic stability, when it wasevaluated using the electrophoreticpatterns of nine isozyme systems and RAPDprofiles.     

Inhibitory effect of streptomycin on rice tissue cultures and the selection of resistant callus clones

Summary Callus tissues were initiated from seed, radicle and anther cultures of rice (Oryza sativa L.) in order to study the effect of streptomycin on callus growth. Our results showed that the addition of 250 g/ml or more streptomycin to the culture medium caused a significant inhibition of callus proliferation. The degree of inhibition depended upon the genotype, the drug concentration and the tissue source of callus. Selection of resistant cell lines began with seed and immature embryo cultures grown on various levels of streptomycin. The fastest growing sectors of callus were subcultured for additional selection. In this way, a total of 11 comparatively vigorous callus clones were isolated after 7 or 12 subcultures. Some of these clones exhibited a significant increase of resistance index when compared with unselected starting material. After 5 or 6 selection cycles, 79 plantlets were regenerated from resistant callus, but none grew to maturity because all were white (albino).     

Slow-growth conservation of potato microplants: efficacy of ancymidol for long-term storage in vitro

Ancymidol was investigated as an alternative mediumsupplement to mannitol for slow-growth conservation ofpotato microplants in vitro. Differentconcentrations of ancymidol (0, 5, 10, 15, 20, 25, 30,35 and 40 M) were tested in slow-growthmedia based on MS medium supplemented with either 30or 60 gl^-1 sucrose. The cultures were conservedunder a 16-h photoperiod at two temperature regimesi.e. 24 ± 1 ^°C and 6 ± 1 ^°C. Therewere significant interactions between ancymidol andother factors such as sucrose, temperature andgenotype for microplant survival, microshoot heightand overall microplant growth. Ancymidol did have abeneficial effect on culture viability after prolongedmaintenance in vitro. The growth-inhibitingeffect of ancymidol persisted through a 16-monthculture period. Combined effect of ancymidol, sucroseand temperature showed that optimum culture viabilityand desirable microplant growth were obtained when thecultures were grown in MS medium supplemented with 10M ancymidol plus 60 gl^-1 sucrose at6 ± 1 ^°C. Vitrification and flaccidity, whichare very frequently observed in potato microplantcultures during prolonged maintenance in vitrounder osmotic stress (mannitol), were not observedwhen the microplants were conserved in ancymidolmedia. Genetic stability of potato microplantsconserved in ancymidol media was evaluated usingrandomly amplified polymorphic DNA (RAPD)fingerprints. Ancymidol did not induce any detectablegenetic variation in genomic DNA as visualized by theabsence of either any additional RAPD fragment oralterations in RAPD fragment patterns.     

Transient gene expression in transformed banana (Musa cv. Bluggoe) protoplasts and embryogenic cell suspensions

Summary In order to introduce currently-available genes with agronomical value into banana, two genetic transformation protocols have been optimized.Firstly, regenerable protoplasts isolated from embryogenic cell suspensions of the cultivar Bluggoe have been used for the introduction of several chimaeric uidA gene constructs by electroporation. With the inclusion of polyethylene glycol and heat shock, the frequency of transiently expressing protoplasts reached 1.8% as shown by an in situ -glucuronidase assay. A duplicated 35S promoter with an alfalfa mosaic virus leader sequence (pBI-426) induced the highest expression rate among the constructs tested.Embryogenic cell suspensions of cv. Bluggoe have also been bombarded with accelerated particles coated with a high expression uidA gene construct (pEmuGN) using a biolistic gun. After a partial optimization of the procedure, transient GUS assays reproducibly demonstrated the presence of 400 blue foci in 30 l of settled cell volume (approximately 25 mg cells). Selection and characterization of antibiotic-resistant transformed cultures is in progress.Abbreviations AMV alfalfa mosaic virus - GUS -glucuronidase - TGE transient GUS expression - uidA gene for -glucuronidase     

In vitro propagation of Japanese garden iris,Iris ensata Thunb.

Summary Explants of young scapes of Iris ensata were cultured on MS medium with 1 mg/l NAA, 1 mg/l 6-BA, 30 g/l sucrose and 10 g/l agar, and this species was characterized by high variety specificity for callus, shoot and root induction. Among 23 varieties and one wild form tested, Okichidori, Miyukisudare and Meiji-l exhibited a considerable rate of shoot induction, although these induced poorly rooted shoots. In addition, two types of callus induction such as green and white calli were observed, and the induction of green-type calli was significantly correlated with that of shoots. Surprisingly, the only modification, half-strength MS inorganic salts, for the above medium proved to be very effective for shoot induction in the scape culture. For shoots obtained from the scape culture, effects of sucrose concentrations and activated charcoal on root induction were examined by using 1/2 MS with 1 mg/l NAA, 1 mg/l 6-BA, 30 g/l sucrose and 10 g/l agar as the basic medium. The addition of 1% activated charcoal to the media had a marked effect for root induction independent of sucrose concentrations and varieties tested. The in vitro propagation technique of I. ensata is discussed.     

Studies on glucosinolates in clubroot resistant selections and susceptible commercial cultivars of cabbages

Summary Heads of 59 commercial cabbage cultivars, all susceptible to clubroot disease, and of 86 individual clubroot resistant cabbage plants of various breeding selections were analysed for their composition in glucosinolates, determined by quantifying their hydrolytic breakdown products, thiocyanate, goitrin, and volatile isothiocyanates. The mean thiocynate ion content was significantly lower in the breeding selections (120 g/g dry weight) than in the commercial cultivars (204 g/g). In contrast, the mean goitrin content was significantly higher in the breeding selections (193 g/g) than in the commercial cultivars (35 g/g). Similar to goitrin, the range of volatile isothiocyanates and total glucosinolate were higher in the breeding selections, but the mean contents of each were not statistically different between selections and cultivars. Fourteen cultivars and four selections were found to be free of goitrin; three cultivars, but no breeding selection were free of volatile isothiocyanates. The breeding selections will provide germplasm for breeding new clubroot resistant and low glucosinolate cultivars.Contribution No. J-965.     

Interspecific hybridization between Abelia × grandiflora ‘Francis Mason’ and A. schumannii via ovule culture

Ethiopian mustard (Brassica carinata Braun) is a potential oil crop for the Mediterranean area. The objective of this study was to develop an efficient system of mutagenesis using ultraviolet (UV) light irradiation of isolated microspores from Brassica carinata. From the survival curve based on embryo yield after irradiation of the microspores with UV light, the LD50 was estimated to be an exposure of 8 min. Total content of glucosinolates and fatty acid composition were analysed in the seeds of the doubled haploid homozygous plants with the purpose of selecting lines with modified glucosinolate and erucic acid contents. Three groups of doubled haploid lines exhibiting low and high glucosinolate contents, and high erucic acid content have been identified from a population of 270 doubled haploid lines. In eight lines, the content of glucosinolates was reduced from an average of 80.6 mol g^-1 seed to 37.5 mol g^-1 seed, whereas in four lines, the content of glucosinolates was increased up to 99.2 mol g^-1 seed. In six additional lines, the content of erucic acid was increased from 42.8% in the nontreated lines to 49.5% of the totalfatty acid composition in some of the mutant lines. All lines showed stablelevels of erucic acid in two generations, the M₂ and M₃.     

Colchicine, trifluralin, and oryzalin promoted development of somatic embryos in Ilex paraguariensis (Aquifoliaceae)

A protocol for somatic embryogenesis and plant regeneration of Ilexparaguariensis St. Hil. from embryos cultures was developed. Heart stage zygotic embryos were removed from seeds of immature, light green fruit and treated with antimicrotubule agents (0.1; 0.2, and 0.5% colchicine for 24 and 48 h; 1; 10, and 20 M of either trifluralin, - trifluoro- 2,6-dinitro-N,N- dipropyl-p-toluidine, or oryzalin, 3,5-dinitro-N₄, N-dipropylsulphate during 48 h). The embryos were cultured aseptically on quarter-strength Murashige and Skoog medium containing 3% sucrose, 0.65% agar (1/4MS), and 0.46 M zeatin. Cultures were incubated in darkness at 27 ± 2 °C. All thetreatments provoked a diminution of the number of germinated embryos and in some of the treated embryos somatic embryogenesis was induced. Somatic embryo maturation and conversion into whole plants could be achieved by culturing the embryos on 1/4MS lacking hormones and incubated at 27 ± 2 °C, 14 h photoperiod (116 mol m^-2s^-1). Mostof the plants regenerated from somatic embryos appeared morphologically normaland grew under greenhouse conditions. Only 2 plants out of 152 studied contained the tetraploid number of the chromosomes (2n = 4x = 80), meanwhile the rest of the plants had the normal diploid number of chromosomes (2n =2x = 40). Somatic embryos with abnormal morphology were also observed.     

In vitro morphogenetic capacity of Nicotiana tabacum L. var. BEL W3 upon cyclic culture and regeneration

Summary Variation in the capacity to regenerate shoots and morphogenetic potential were investigated by cyclic culture and regeneration of Nicotiana tabacum var. BEL W3. Explants were excised from different shoots formed by the same callus line. Differences in the organogenetic in vitro response among regenerated plantlets was observed in two subsequent regenerate generations. A progressive decrease of the regenerative capacity was observed across callus line generations.     

Genotypic and exogenous factors affecting shoot regeneration from anther callus of linseed (Linum usitatissimum L.)

Summary The objective of this study was to investigate factors affecting the regeneration capacity of linseed anther culture. Four different environmental conditions in a phytotron were tested with regard to their effects on anther donor plants of cv. Hella. Anther response and shoot regeneration from anther callus was maximal when donor plants were grown in a 16 hrs-day at 14°C day/8°C night temperature. Anthers of four linseed genotypes were cultured on different media. Maximum shoot regeneration was achieved when the induced calli were transferred onto a modified N6 medium containing zeatin (1 mg l^-1). Most of the calli regenerated shoots in the second subculture on regeneration media. Shoots were rooted on modified B5 or MS media containing NAA (0.1 mg l^-1). Cytological examinations of incubated anthers and root tips of regenerated plants indicated that the anther calli were derived from microspores.Abbreviations B5 Gamborg's (1975) medium - BAP 6-benzylaminopurine - 2,4D dichlorophenoxyacetic acid - N6 Chu's (1978) medium - NAA -naphthaleneacetic acid - MS Murashige & Skoog's (1962) medium - ZEA zeatin     

The influence of cytokinins and ionic strength of Anderson's medium on shoot establishment and proliferation of evergreen azalea

Shoot-tip explants of evergreen azalea cv. Fuchsia grown on Anderson's medium and containing different cytokinins produced the highest proliferation rate on a medium containing thidiazuron (TDZ). TDZ concentrations ranging from 0.23 to 2.3 M resulted in both good bud-break rate (4 to 5) and shoot quality (> 0.5 cm in length). Adding 2.3 M zeatin to Anderson's medium containing 0.23 or 2.3 M TDZ increased the number of axillary shoots/explant. However, increasing the zeatin concentration to 4.6 M resulted in a reduced shoot proliferation rate. A medium containing 1.15 M TDZ and 2.3 M zeatin resulted in an 18-fold increase for 'Fuchsia' and a 9-fold increase for 'Hino Crimson' after 6 weeks of culture. It was found that explants grown on a half-strength Anderson's medium with 87.6 mM sucrose generally had better shoot proliferation rate and shoot quality than at higher ionic strength.     

In vitro adventitious shoot regeneration for effective maintenance of male sterile niger Guizotia abyssinica (L.f.) Cass.)

A rapid and efficient method of in vitro plant regeneration for large scale propagation of male sterile plants of niger (Guizotia abyssinica) was developed. Leaf segments from mature plants were cultured on Murashige & Skoog's basal medium (MS) supplemented with N⁶-benzyladenine (BA) and kinetin individually and in combination with low concentrations of indole-3-butyric acid (IBA); and -Naphthaleneacetic acid (NAA). Prolific direct adventitious shoot regeneration occurred on most of the media tested. The best response in terms of frequency of shoot regeneration and the number of shoots per leaf explant was observed on medium supplemented with 2.22 M BA. Transfer of shoot bud clusters to fresh medium with same composition promoted further multiplication of the shoot buds, while medium with reduced BA concentration (0.89 M) facilitated shoot elongation. Shoots that were rooted on half-strength MS medium gelled with 0.2 or 0.4% agar and supplemented with 4.9 M IBA survived with a frequency of 61.36% on transfer to ex vitrum conditions. Field evaluation of the regenerants revealed the genetic stability of the plantlets and are being used in breeding of experimental hybrids.     

Selection of a highly-regenerative genotype of white clover (Trifolium repens L.) and plant regeneration from protoplasts derived from this genotype

Summary Callus cultures were induced from hypocotyl sections of 24 varieties of white clover (Trifolium repens L.). The calli did not show any significant difference of growth among the varieties. After the calli has been transferred to three regeneration media, green-spot formation was observed on calli derived from some seedlings. Remarkable intra- and intervarietal variations in the emergence of green spots and some trends between the origin of varieties and the frequency of green spots were observed. In most cases, the green spots turned brown without showing further differentiation, and only two genotypes formed shoots. A callus from a seedling of the Swedish variety Undrom has sustained high levels of plant regeneration throughout 24 months of culture. Protoplasts derived from this selected genotype were divided into cell colonies. 8P (Kao, 1977) medium containing 0.5 mg/1 2,4-D and 0.5 mg/1 kinetin was the most suitable medium for inducing divisions in protoplasts. When subcultured into solid B5 medium, the colonies produced calli, which when transferred to a regeneration medium, formed shoots. This genotype is expected to a useful subject for genetic engineering of white clover.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - 2ip 6-, -dimethylallylamino purine - NAA -naphthaleneacetic acid     

Somaclonal variants of sunflower with modified coumarin expression under stress

Summary Two coumarins, scopoletin and ayapin, have been shown to accumulate in sunflower following insect damage or fungal infection (Tal & Robeson, 1986a, 1986b & Olson, 1989). The technique of shoot regeneration from embryo-derived callus was used to produce somaclonal variants of sunflower with enhanced levels of coumarin expression after an applied stress. Five regenerates (R1–R5) of inbred lines RHA 297 produced viable seeds following self-pollination. One of the S1 families from these plants had significantly higher concentration of scopoletin (12.75 g/g fresh weight) compared to the parental inbred line (5.66 g/g). The increased levels of scopoletin in S1 plants were partially inherited after a second self-fertilization (S2 plants). The ploidy of the regenerates and their S1 and S2 progeny was not examined. No consistent correlation between higher levels of scopoletin in stimulated S1 and S2 plants, and deterrence to feeding of Heliothis virescens (Fab) larvae on unstimulated plants was shown after in vivo assays. From inbred line HA 300, three regenerates (H1–H3) were derived which produced S1 progeny with higher or lower concentrations of ayapin or scopoletin than the parental inbred. The inheritance of increased ayapin in the HA 300-derived lines was not assessed in the S2 generation because of poor seed germination. Feeding deterrence of H. virescens larvae was not consistently correlated with ayapin concentrations in S1 progeny of HA 300 regenerates. However, a correlation (-0.61) between ayapin concentration and daily feeding score was observed in one planting of these unstimulated S1 progeny. Sunflower germplasm was developed with enhanced levels of stress-induced coumarin expression through somaclonal variation and this germplasm may also show increased resistance to some leaf-feeding insects.     

Evaluation of Triticum aestivum germplasm from Croatia and Yugoslavia for aluminium tolerance

Summary Aluminium tolerance of 90 genotypes of Triticum aestivum L. germplasm from the breeding programmes of eight Croatian and Yugoslav institutions was evaluated in nutrient solutions having Al³⁺ activities of 0, 12.5 and 25M. Overall distribution of Al tolerance of wheat genotypes was skewed toward lower tolerance rankings. Average Al tolerance differed among gene pools created at different breeding institutions. Genotypes tolerant enough to be useful in the breeding programmes aimed at selecting cultivars with improved Al tolerance are identified in germplasm from four institutions. No correlation was found between chemical characteristics of soils used over the years by breeding institutions for their field trials and the Al-tolerance ranking of the corresponding germplasm material.Abbreviations HSD Tukey's Honestly Significant Difference - RRL-4 relative root length, in % (25M Al³⁺/0 Al)     

Plant regeneration from in vitro cultures of cotyledon explants and anthers of Sinapis alba and its implications on breeding of crucifers

Summary Protocols for plant regeneration from cotyledon explant and anther cultures of Sinapis alba have been developed for creating doubled-haploids and somaclonal variation. Among the several cultivars tested in this study, only Arda responded well to in vitro plant regeneration both from anther-as well as cotyledoncultures. Multiple shoot formation in cotyledon explants, which always followed a brief callusing phase, was found to be the best on MS medium with ZEA (1.0mg/l) and NAA (0.1mg/l). Regeneration frequency declined sharply in the absence of auxin or presence of other cytokinins and/or auxin. The frequency of shoot regeneration also declined with reduction in the photoperiod to 16h. On MS + BAP (1.0mg/l) + NAA (1.0mg/l) medium, cotyledonary explants showed profuse callusing, which could regenerate shoots on high ZEA + low NAA/IAA medium. However, it declined with progressing time in culture. Anthers, excised from fresh as well as cold pretreated buds, cultured on 10% sucrose containing MS media with different hormonal constitution, developed calli and/or embryos. Initial culture temperature was important with embryogenesis occurring only in anthers cultured at 30°C for 3 weeks. A high temperature (35°C) treatment was lethal for both callus as well as embryo formation. While BAP + NAA and ZEA + NAA/IAA supported embryogenesis, further plant regeneration from anther-or embryo-callus could be achieved in ZEA + NAA/IAA media. Some of the regenerants flowered already in vitro and had small and sterile flowers. Cytological examination of some of the root differentiating calli indicated the presence of haploid as well as diploid cells. Shoots were rooted during prolonged incubation on the same medium or on transfer to MS (reduced)/ B5 + ZEA + NAA media.