In vitro culture of Cucumis sativus L.

Summary The ability to regenerate plants from leaf explants has been tested for three highly inbred cucumber lines (B, G, S), their reciprocal hybrids, F₂ and BC₁ generations. The lines differed from each other in their regenerating ability, which was expressed by the percentage of explants regenerating embryoidal callus and mean number of plantlets per plant. Thus, the lines could be classified as frequently (B), intermediately (G) or occasionally regenerating ones (S). There were no reciprocal cross differences in the regeneration. It was found that the intermediately and intensively regenerating lines contain two pairs of dominant genes responsible for plant regeneration, characterized by complementary and probably additive interaction. The frequently regenerating line differed from the intermediately regenerating in the effect of one gene. It is supposed that the above-mentioned genes belong to three different loci. The ability to regenerate plants from leaf expiants had high heritability.     

Effect of silver nitrate on long-term culture and regeneration of callus from Brassica oleracea var. gemmifera

Incorporation of AgNO₃ (1–10 mg1^-1) into the culture medium of Brassica oleracea var. gemmifera callus significantly improved growth and allowed long-term callus culture. In the absence of AgNO₃, callus died shortly after removal from the hypocotyl explants. Regeneration of shoots from callus on low-hormone medium was also enhanced by AgNO₃. Significant differences in shoot production were found between the three genotypes examined. Cv. Aries produced large numbers of shoots even in the absence of AgNO₃. Investigation of callus production from the inbred parent lines of cv. Aries indicated that tissue culturability may be determined genetically.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid     

High-frequency plant regeneration through callus initiation from mature embryos of maize (<Emphasis Type="Italic">Zea Mays</Emphasis> L.)

An efficient maize regeneration system was developed using mature embryos. Embryos were removed from surface-sterilized mature seeds and sliced into halves. They were used as explants to initiate callus on induction medium supplemented with 4.0 mg l^–1 2,4-dichlorophenoxyacetic acid (2,4-D). The induction frequency of primary calli was over 90% for all inbred lines tested. The primary calli were then transferred onto subculture medium supplemented with 2.0 mg l^–1 2,4-D. Following two biweekly subcultures, embryogenic calli were formed. Inclusion of a low concentration (0.2 mg l^–1) of 6-benzylaminopurine (BA) in the subculture medium significantly promoted the formation of embryogenic callus. The addition of silver nitrate (10 mg l^–1) also supported an increased frequency of embryogenesis. The embryogenic callus readily formed plantlets on regeneration medium supplemented with 0.5 mg l^–1 BA. The regenerated plantlets were transferred to half-strength Murashige and Skoog medium supplemented with 0.6 mg l^–1 indole-3-butyric acid to develop healthy roots. The regenerated plantlets were successful on transfer to soil and set seed. Using this system, plantlets were regenerated from seven elite maize inbred lines. The frequency of forming green shoots ranged from 19.8% to 32.4%. This efficient regeneration system provides a solid basis for genetic transformation of maize.Abbreviations BA 6-Benzylaminopurine - 2,4-D 2,4-Dichlorophenoxyacetic acid - IBA Indole-3-butyric acid - KT KinetinCommunicated by M.C. Jordan     

Strategies for overcoming genotypic limitations of in vitro regeneration and determination of genetic components of variability of plant regeneration traits in sorghum

Development of suitable strategy to overcome genotypic limitations of in vitro regeneration in sorghum would help utilize high yielding but poor tissue culture responsive genotypes in genetic manipulation programmes. A factorial experiment was conducted with two explants (immature embryos and inflorescences), eight genotypes (five Sorghum sudanense and three Sorghum bicolor genotypes), three levels of 2,4-D (1 mg l⁻¹, 3 mg l⁻¹, and 5 mg l⁻¹), and two levels of kinetin (0.0 mg l⁻¹ and 0.5 mg l⁻¹). The induced callus was transferred to the regeneration media with factorial combinations of IAA (1.0 mg l⁻¹ and 2.0 mg l⁻¹) and kinetin (0.5 mg l⁻¹ and 1.0 mg l⁻¹). S. sudanense regenerated at significantly higher frequency (38.91%) and produced shoots more intensely (2.2 shoots/callus) than S. bicolor (26.93%, 1.26 shoots/callus). Immature inflorescences regenerated at a much higher frequency (46.48%) and produced significantly more number of shoots (2.71 shoots/callus) than immature embryos (22.35%, 0.99 shoots/callus). Moreover, differences for plant regeneration between genotypes of the same species were minimal when using immature inflorescences. Increase in the 2,4-D concentration in callus induction media exhibited inhibitory effect on callus induction, growth, shoot induction and number of shoots/callus but inclusion of kinetin in callus induction media improved these responses. Use of immature inflorescence explant and inclusion of kinetin in callus induction media could overcome genotypic limitations of plant regeneration to a large extent. The extent of variability, heritability and expected genetic advance was more in plant regeneration traits than in callus induction traits. This indicated that the variability in respect of these attributes in the genotypes may be due to the additive gene action and selection of genotypes for these characters would be rewarding.     

Regeneration of zoysia grass (Zoysia matrella L. Merr.) cv. Konhee from young inflorescences and stem nodes

We have optimized conditions for efficient regeneration of the vegetatively propagated zoysia grass (Zoysia matrella L. Merr) cultivar “Konhee”. Two explants, young inflorescences, and stem nodes, were used and they displayed different responses to combinations and concentrations of plant growth regulators in callusing, embryogenic callus formation, and regeneration. The highest callus initiation rate from young inflorescences was obtained on medium supplemented with 4.5 to 9.0 μM 2,4-dicholorophenoxy acetic acid (2,4-D) and 0.44 μM 6-benzyl amino purine (BA). When the BA concentration was lowered to 0.044 μM, the highest percent embryogenic callus induction from young inflorescences was achieved. The highest callus initiation rate from stem nodes was obtained, when young inflorescences were cultured on MS medium supplemented with 4.5 to 9.0 μM 2,4-D, 0.44 μM BA, and 0.037 μM abscisic acid (ABA). But embryogenic callus formation from the stem node was highest in the presence of 4.5 to 9.0 μM 2,4-D, 0.044 μM BA, and 0.037 μM ABA. Addition of ABA significantly increased embryogenic callus formation from stem nodes, but not from young inflorescences. Regeneration percentage was variable in response to BA level, and inclusion of α-naphthalene acetic acid (NAA) and gibberellic acid (GA₃) further increased the regeneration percentage. The highest regeneration percentages obtained from the young inflorescences and stem nodes were 82% and 67%, respectively. This is the first report showing that plants can be regenerated from young inflorescences and stem nodes of vegetatively propagated zoysia grass.     

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     

The identification of QTLs associated with the <Emphasis Type="Italic">in vitro</Emphasis> response of rye (<Emphasis Type="Italic">Secale cereale</Emphasis> L.)

This study was conducted in order to identify quantitative trait loci (QTLs) for the in vitro culture response of winter rye (Secale cereale L.) immature embryos and immature inflorescences. A genetic linkage map comprising 67 SSRs, 9 ISSRs, 13 SAMPLs, 7 RAPDs, 2 SCARs and one EST marker was created based on the analyses of 102 recombinant inbred lines from the cross between lines L318 (which has a good response in tissue cultures) and L9 (which is unable to regenerate plants from somatic tissues and anthers). The map spans 979.2 cM, and the average distance between markers is 9.9 cM. Two characteristics were evaluated: callus induction (CI) and somatic embryogenesis ability (SE). They were expressed as the percentage of immature embryos/inflorescences producing callus (designated ECI/ICI) and the percentage of explants producing somatic embryos (ESE/ISE). All the analysed traits showed continuous variation in the mapping population but a non-normal frequency distribution. We identified nine putative QTLs controlling the tissue culture response of rye, explaining up to 41.6% of the total phenotypic variation: two QTLs for ECI — eci-1, eci-2; 4 for ESE — ece-1, ese-2, ese-3, ese-4; 2 for ICI — ici-1, ici2; and 1 for ISE — ise-1. They were detected on chromosomes 1R, 4R, 5R, 6R and 7R.     

Genetic analysis of leaf explant regenerability inSolanum chacoense

An F₁ population consisting of 51 genotypes, derived from two unresponsive parental lines ofSolanum chacoense Bitt., was examined for shoot regeneration from leaf explants. Fourteen genotypes failed to respond whereas, among the responsive genotypes, four produced multiple shoots on over 90% of the explants. Estimates of broad-sense heritability were high for both frequency of responsive explants (83%) and the number of shoots per responsive explant (82%). The segregation of the F₁ hybrid progeny was in agreement with the theoretical ratios of a genetic model for tissue culture responsiveness involving three unlinked genes. This study confirms earlier findings concerning the genetic control ofin vitro shoot regeneration from leaf explants inS. chacoense.     

Effects of explants and growth regulators in garlic callus formation and plant regeneration

We intended to evaluate the effects of different explants and growth regulators on callus induction and plant regeneration in garlic (Allium sativum L.). Furthermore, we intended to differentiate among different morphological types of callus by light microscopy and to relate them with their abilities to regenerate plants in the red-garlic cultivar 069. A factorial design with BDS—basal Dunstan and Short (1977)—medium, as a control and supplemented with 0.042, 0.42 and 4.24 μM picloram or with 0.045, 0.45 and 4.5 μM 2,4-D, in both cases with and without 4.43 μM N⁶-benzylaminopurine (BAP), was used. The cultures were grown in darkness at 25 ± 2°C and they were subcultured over a 6-month period. Basal plates and meristems were highly responsive explants, while immature umbels and root-tips were less responsive ones, as indicated by percentage of induced callus, growing callus and regenerating callus. The best response was 41% regenerating callus with 0.045 μM 2,4-D and BAP from basal plates while 57, 56 and 20% regenerating callus were obtained with 0.45 μM 2,4-D from meristems, root-tips and immature umbels, respectively. Also, these treatments showed a higher percentage of nodular and embryogenic callus (type I). Thus, it can be concluded that the use of meristems and 2,4-D will enhance callus production and quality, increase plant regeneration and allows to develop a protocol suitable for further transformation experiments in garlic.     

The influence of genotype and medium on rye (Secale cereale L.) anther culture

Anthers of three rye inbred lines - L9, L318, Dw28, one synthetic hybrid - F₁(5) and one variety, Dakowskie Zote (DZ), were cultured on two media based on N6, and two based on P2 components. The induction rate significantly depended on genotype - the best results were obtained for line L318 and the lowest percentage of responding anthers was noticed in line L9. There was no universal medium for all tested genotypes. The highest induction rate (IR) for lines L318, L9 and hybrid F₁(5) was obtained on medium CI (11.94, 0.71 and 1.75 respectively). For DZ, the P2I medium was better than the others while for Dw28 CIP turned to be as suitable as CI. A highly significant interaction between genotype and medium was proved. Single anthers of DZ, L318 and L9 produced embryos on CI, CIP and P2I media. They did not develop into plants but after transferring them to CS1.7 medium, a secondary, embryogenic callus was obtained. Such a reaction has not been described in rye until now. In spite of a relatively high IR, plant regeneration was rather poor. An elaboration of this step in haploid production is needed. Most of the analyzed calluses and microspore derived regenerants proved to be haploids, according to flow cytometry analysis. Plants treated with colchicine were doubled haploids.     

Improvement of embryogenic callus induction and shoot regeneration of buffalograss by silver nitrate

Addition of the ethylene antagonist, silver nitrate (AgNO₃), into callus induction medium significantly enhanced embryogenic callus production (both induction frequency and callus growth) of field-collected male immature inflorescence cultures of buffalograss NE84-45-3 and 'Texoka'. No stimulatory effect of AgNO₃ was observed on embryogenic callus induction for female immature inflorescence culture of a female genotype `609' and `Texoka'. Calli initiated on AgNO₃-containing media had more shoot-regenerating calli than those initiated on AgNO₃-free media, when they were transferred to the regeneration media. Benzyladenine at 2.2 μM gave the best response for regeneration, regardless of the callus source. Although average number of shoots regenerated per callus was lower for calli initiated on AgNO₃-containing media, total number of shoots regenerated was higher. The stimulatory effect, however, was environment and genotype dependent. While the addition of AgNO₃ significantly stimulated embryogenic callus induction of NE84-45-3 immature inflorescences collected in Fall 1995 and May 1997, it only slightly increased the embryogenic callus induction frequencies in May 1996 when rainy conditions occurred. For male inflorescences of `Texoka' collected in early May, AgNO₃ significantly enhanced embryogenic callus production consistently over the two-year period (1996, 1997). Published as Journal Series No. 1351, Agricultural Research Division, University of Nebraska. This revised version was published online in June 2006 with corrections to the Cover Date.     

Plant regeneration of Solanum lycopersicoides Dun. from stem explants,callus and suspension cultures

Protocols were established for achieving plant regeneration from stem internode, callus, and cell suspension cultures of Solanum lycopersicoides Dun. Two accessions of S. lycopersicoides exhibited different responses as to callus formation on various media, requirement of gibberellic acid for shoot regeneration, and ability to grow in suspension culture. The optimum medium for initiation and maintenance of cell suspension cultures was Murashige and Skoog [9] medium with 15 mg l^– NAA. For shoot regeneration, of three cytokinins tested, zeatin was found most effective relative to number, rapidity of response and overall quality of shoots. Shoot regeneration from stem explants, callus and suspension cultures was optimum on MS + 3.0 mg l^–1 zeatin + 0.1 mg l^–1 gibberellic acid.Michigan Agricultural Experiment Station Journal Article No. 11589.     

Effect of growth regulators and ethylmethane sulphonate on growth,and chlorophyll,sugar and proline contents in <Emphasis Type="Italic">Dracaena sanderiana</Emphasis> cultured <Emphasis Type="Italic">in vitro</Emphasis>

A high efficient four step protocol (callus initiation, regeneration, shoot elongation and rooting) for in vitro propagation of Dracaena sanderiana Sander ex Mast was developed. Callusing was achieved from nodal stem segment explants treated with various concentrations of ethylmethane sulphonate (EMS) on MS medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D; 1.5 g m⁻³). A significant increase in callus induction percentage and biomass production was noticed from lower EMS treated lines (ET₁ and ET₂) comparatively to control and other (ET₃, ET₄ and ET₅) lines. Calli of ET₁ line showed high regeneration potential on MS medium with N ⁶-benzylaminopurine (BAP; 1.75 g m⁻³). Length of microshoots, which was reduced by EMS, restored by addition of gibberellic acid (GA₃; 0.4 g m⁻³). A marked increase in rooting with increasing EMS concentration was noticed on MS medium fortified with 3-indolebutyric acid (IBA; 1.5 g m⁻³).     

Somatic embryogenesis from mesocotyl and leaf-base segments of <Emphasis Type="Italic">Paspalum scrobiculatum</Emphasis> L., a minor millet

Summary Somatic embryos could be induced from embryogenic callus originating from mesocotyl as well as leaf-base segments of Paspalum scrobiculatum on Murashige and Skoog (MS) or Chu et al. (N₆) medium supplemented with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D; 4.5, 9.0, 18.0, and 22.5 μM). N₆ medium was better than MS, for both explants, for high-frequency somatic embryogenesis. Also, mesocotyl tissues were relatively more totipotent than leaf-base segments. The somatic embryos ‘germinated’ and formed plantlets on transfer of embryogenic calluses to hormone-free MS or N₆ regeneration medium. Embryogenic cultures could be maintained on low hormone medium which readily regenerated to form plantlets on hormone-free medium. A higher frequency of plantlet formation occurred on MS than on N₆ medium. In vitro-formed plantlets were gradually acclimatized in the culture room and on transfer to soil flowered and set seed. Somatic embryogenesis and plantlet regeneration from mesocotyl and leaf-base segments are potentially simpler systems than regeneration from ‘embryonic’ explants such as immature embryos and unemerged inflorescences.     

Genetic analysis of the anther-culture response of three spring wheat crosses

Summary Anther-culture response was examined among three spring wheat (Triticum aestivum L.) cultivars to evaluate the genetic component of response and to determine whether androgenetic performance could be improved by selection. The three lines, the three possible F₁'s among the three lines, their F₂'s, and the backcrosses to the parents were evaluated for callus production and regeneration capacity. Significant variation was observed among the generations of the three crosses for callus formation. Genetic variation for regenerability was nonsignificant. Callus production was negatively correlated (-0.24) with regeneration capacity. The random variation in the study was too great to determine whether major-gene differences for antherculture response exist among the three lines by examining population distributions. When the material was evaluated for quantitative gene effects, the estimates for the additive gene effects were generally greater than the estimates for the dominance gene effects for callus formation. Only the Pavon x Chris cross, however, exhibited a significant narrow sense heritability estimate for callusing response (0.94). Due to the large component of random variation and the varying selection potential among crosses for androgenetic performance, improving anther-culture response in wheat by selection could prove difficult unless the anther-culture process itself selects for response traits at the gametic level.     

In vitro propagation of Agave fourcroydes Lem. (Henequen)

In vitro plant regeneration of Agave fourcroydes Lem. (Agavaceae) is described. Results suggest that the NO₃ ^-:NH₄ ⁺ balance in the culture medium is a key factor controlling callus growth and organogenesis in rhizome cultures. Stem callus showed limited organogenic capacity, but high cytokinin concentrations induced adventitious shoot formation on stem explants. When these shoots were excised and subcultured, new callus formed at their base from which new shoots arose. The shoots from stem explants and rhizome callus formed extensive root systems in vitro and were transferred to pot culture with a 90% survival rate.     

The effects of genotype,inflorescence developmental stage and induction medium on callus induction and plant regeneration in two <Emphasis Type="Italic">Miscanthus</Emphasis> species

Several grass species of the genus Miscanthus are considered to be outstanding candidates for a sustainable production of biomass to generate renewable energy. The purpose of this study was to investigate the effects of genotype, the developmental stage of the explant donor inflorescence and the induction medium on the success rate of micropropagation. The experiments were conducted on three genotypes of M. sinensis and one of M. x giganteus. Explants from the youngest inflorescences (0.1–2.5 cm in length) showed a significantly higher callus induction rate than those from more developed inflorescences (2.6–5 cm in length). In addition, cultures initiated from explants from the youngest inflorescences showed significantly the highest rates of callus regeneration and the highest shoot regeneration rate. Three out of the four genotypes tested showed the best shoot regeneration from calli initiated from the youngest inflorescences when cultured on the Murashige and Skoog basal medium (MS) with 5 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.1 mg l⁻¹ 6-benzyladenine (BA). The percentages of calli from those genotypes showing regeneration ranged from 45 to 76.7%, and the corresponding shoot regeneration rates ranged from 1.85 to 6.33 shoots/callus. This demonstrates that, with some adjustments, efficient micropropagation of Miscanthus sp. is feasible.     

Reciprocal effects in anther cultures of wheat hybrids

This study was conducted to determine the reciprocal effects for anther culture response in wheat (Triticum aestivum L.) using a set of 4 × 4 full diallel crosses. Both reciprocal and nuclear genetic effects were highly significant for anther culture response and useful for selection and breeding purposes. General combining ability (GCA) effects were predominant for all investigated anther culture traits. Also, significant differences for specific combining ability (SCA) effects were detected between reciprocal crosses. Although significant reciprocal differences for responding anther, callus number and green plant regeneration were recorded in some reciprocal crosses, there were no significant reciprocal differences for albino plant regeneration. The use of one parent as male or female could lead to change at the production of green plants from the F₁ hybrids and screening of inbred lines for response to anther culture, without reciprocal effects, could decrease the utilization of breeding material.