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.     

Genetic factors influencing regeneration ability in rye (Secale cereale L.). I. Immature inflorescences

Immature inflorescences of ten rye inbred lines (inbred degree S10 and S11) were cultured on solidified MS medium supplemented with 3.0 mg/dm³ of 2,4-D. According to their capability for callus production explants were classified into two groups : responsive (giving weak or intensive callus production) and non responsive (lack of callus formation). After transferring responsive material into hormone-free medium the regeneration of roots or shoots from the intensive growing callus was observed. Consistent differences between lines in the portion of explants with a certain response were found. They were divided into five groups reacting in the same way. Lines with different in-vitro response were crossed in an incomplete diallel. F₁, F₂ and F₃ generations were analyzed and the following conclusions drawn: the ability for plant regeneration from immature inflorescences in rye is determined by numerous loci, has a recessive character, and both callus production and regeneration suppression may be controlled by complementary genes.     

A genetic analysis of cell culture traits in tomato

Summary Tomato genotypes superior in regenerating plants from protoplast and callus cultures were obtained by transferring regeneration capacity from Lycopersicon peruvianum into L. esculentum by classical breeding. The genetics of regeneration and callus growth have been studied in selfed and backcross progenies of a selected plant (MsK93) which has 25% L. peruvianum in its ancestry. Segregation data showed that the favourable cell culture traits of L. peruvianum are dominant. Regeneration capacity from established callus cultures was controlled by two dominant genes. Callus growth on primary expiants, callus growth of established cultures and shoot regeneration from explants had high heritabilities (0.47, 0.78, 0.87, respectively). Callus growth and regeneration capacity were not correlated within the populations studied.     

Characterisation of <Emphasis Type="Italic">Solanum lycopersicoides</Emphasis> Dun. root primordia culture with plant recovery

A liquid meristematic root primordia culture (RPC) of Solanum lycopersicoides Dun. based on persistent rhizogenesis in a modified Murashige and Skoog (1962) medium supplemented with NAA (15 mg·l⁻¹) or 2,4-D (1 mg·l⁻¹) was described. The meristematic clumps (2–3 mm in diameter) originating from NAA supplemented medium were capable of regenerating plants through the callus stage (up to 70 %). Efficient direct plant regeneration (up to 21 %) was possible from numerous single globular-shaped root primordia (RP) structures liberated from the parental aggregates in 2,4-D supplemented proliferation medium without NH₄NO₃ and with a 2.5 fold increase in KNO₃. The RP converted into plantlets (artificial seedlings) on solid or liquid media without growth growth regulators through the unipolar followed by the mace-shaped bipolar structure stages. The use of apical shoot bud, root apices or root segments as a primary explants brought about RPC induction and plant regeneration. The plants derived from 2 years old culture were phenotypically identical to their parental S. lycopersicoides plants and possessed the same ploidy.     

The effects of cold-pretreatment,auxins and carbon source on anther culture of rice

The effects of a cold pretreatment, the concentration of different auxins (2,4-D, NAA and IAA) and the type of carbon source (maltose and sucrose) on the induction of callus from anthers of three parental lines and four rice F₁ hybrids (Japonica × Indica, Indica × Japonica) were studied. The results indicated that a cold pretreatment was essential for the induction of callus from anthers of the parental lines and the F₁ hybrids. These effects were genotype dependent. Auxins were essential for the induction of callus, and the type and concentration of auxins also influenced this process, as well as the type of carbon source. The greatest induction of callus was by the hybrid Morelos A92 × Koshihikari after a cold pretreatment of 8 days using 10.74 M –napthaleneacetic acid and 30 g l^–1 maltose.     

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.     

The effect of gamma radiation and N-ethyl-N-nitrosourea on cultured maize callus growth and plant regeneration

Regenerable maize calli of two inbred lines were exposed to 0 to 100 Gy of gamma rays or treated with 0 to 30 mM of N-ethyl-N-nitrosourea (ENU) to determine their effect on growth and plant regeneration capability. Both growth and plant regeneration capacity decreased with increasing levels of either gamma radiation or ENU; however, plant regeneration capacity was more sensitive to either agent than growth. The 50% inhibition dose (I₅₀) for callus growth (fresh-weight gain) was approximately 100 Gy of gamma radiation and 30 mM ENU. The I₅₀ for plant regeneration capacity of treated callus was approximately 25 Gy of gamma radiation and 2.5 mM ENU. The decrease in plant regeneration capacity correlated with a change in tissue composition of the treated callus from a hard, yellow and opaque tissue to a soft, grayish-yellow and translucent tissue. This change was quantified by measuring the reduction of MnO₄ ^- to MnO₂ (PR assay) by the callus. These results suggest that the effect of gamma radiation or ENU on plant regeneration capacity must be taken into consideration if these potentially mutagenic agents are to be used on maize callus cultures, for the purpose of producing useful mutations at a whole plant level. The data also suggest that the PR assay may be useful for predicting the actual plant regeneration capacity of maize callus.Abbreviations g f.w. gram fresh weight - ENU N-ethyl-N-nitrosourea - PR assay permanganate reduction assay - I₅₀ 50% inhibition dose     

Inheritance of somatic embryogenesis in red clover (Trifolium pratense L.)

 Red clover genotypes capable of regenerating plantlets in vitro from non-meristematic tissue-derived callus are rare. Selection for genotypes capable of somatic embryogenesis identified a clone comprised of a group of plantlets regenerated from a hypocotyl-derived callus culture on L2-based media and another group of plantlets originating from crown divisions of the epicotyl-derived plant. The callus-derived plants of this clone were highly regenerative when reintroduced to callus culture, but the epicotyl-derived plants produced nonregenerative callus cultures. F₁, F₂ and BC₁ populations were evaluated to determine the mode of inheritance of the regeneration trait. Reciprocal crosses did not differ, indicating a lack of maternal effects. Results were compatible with genetic control of regeneration by two complementary genes. We propose the genotype Rn1-Rn2- for regenerative plants. Three petiole segment explants were sufficient to evaluate regenerative ability in seedlings. Regenerative ability was often associated with abnormal leaf morphology in a few to several leaves. Received: 17 February 1998 / Accepted: 7 April 1998     

Improved plant regeneration from maize callus cultures using 6-benzylaminopurine

A new protocol for regenerating plants from cultured type I callus of the maize (Zea mays L.) inbred Pa91 includes growing the callus on medium containing 3.5 mg/l (15.5 M) of the cytokinin 6-benzylaminopurine (6BA) for 3 to 6 d and then moving the callus to medium containing no growth regulators (H medium) for an additional 15 to 21 d, where the plants actually develop. The number of plants regenerated from the 6BA treated callus was 113% to 148% greater than the number of plants produced from callus placed directly on H medium. This increased plant regeneration induced by 6BA seemed to maximize the number of plants regenerated from a gram of callus and was slightly affected by callus age or prior treatment of callus with AgNO₃. Exposure to 6BA for 9 d greatly reduced shoot and root development, and longer exposures totally prevented root formation. This inhibition of root formation could be reversed only slightly by naphthaleneacetic acid. The data indicate that high concentrations of 6BA are effective for increasing plant regeneration from maize callus cultures when short exposure times are used. This procedure has also been effective for regenerating many plants from the inbreds H99 and Mo17.Abbreviations 6BA 6-Benzylaminopurine - IAA indole-3-acetic acid - NAA Naphthaleneacetic Acid - 2,4-D 2,4-dichlorophenoxyacetic acid - dicamba 3,6-dichloro-o-anisic acid - GA₃ Gibberellic acid - gfw gram fresh weight     

Synergistic promotion of gibberellin and cytokinin on direct regeneration of floral buds from in vitro cultures of sepal segments in sinningia speciosa hiern

Summary This study reports the direct regeneration of flower buds from cultured sepal segments of Sinningia speciosa Hiern. Two types of floral bud regeneration were observed: regeneration of floral buds only (designed as B^F) and regeneration of both floral and vegetative buds (designed as B^F+V). The capacity of B^F regeneration was closely related to the location of sepal segments and the concentration of exogenous gibberellin (GA₃) and cytokinin in the medium. On the medium containing 1.0mgl^&#x2212;1 GA₃, the addition of 6-benzyladenine (BA) significantly increased the frequency of total flower bud (B^F+B^F+V) formation, with the frequency up to 91.5% in the presence of 0.4mgl^&#x2212;1 BA. On the medium containing 0.1mgl^&#x2212;1 BA, the addition of GA₃ also increased the frequency of total flower bud regeneration, with the frequency up to 74.3% in the presence of 1.0mgl^&#x2212;1 GA₃, but no further increase in regeneration was observed when the GA₃ concentration was higher than 1.0mgl^&#x2212;1. The capacity of B^F regeneration from different locations of sepal segments was differential. The adaxial part of sepal segments gave rise to the highest frequency of 56.7 and 84.3% for B^F and B^F+B^F+V, respectively.     

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.     

Effect of silver nitrate on multiple shoot formation of Virginia-type peanut through shoot tip culture

Summary A protocol for micropropagation of Virginia-type peanut plants, an ancient crop of the New World, is reported. This study was conducted to explore the effect of silver nitrate (AgNO₃), alone or in combination with growth regulators, on multiple shoot formation from shoot tip culture. Incorporation of AgNO₃ into the medium, without growth regulators, induced regeneration of the explants (which did not develop at all in the AgNO₃-free medium), and stimulated the emergence of axillary shoots. When AgNO₃ was added in combination with cytokinins and α-naphthaleneacetic acid (NAA), maximum average shoot number per regenerating explant was recorded (6.3) in Murashige and Skoog (MS) medium containing 33 μM 6-benzyladenine, 5.3 μM NAA, and 23.54 μM AgNO₃. Moreover, AgNO₃ showed a positive and marked effect on both shoot elongation and the reduction of callus proliferation from the basal ends of shoot tips. Following a period of elongation, the shoots were rooted in hormone-free Ms medium, showing no residual effects due to the long-term culture in AgNO₃-containing media. Acclimatization was easily obtained after plantlets were transferred to pots under greenhouse conditions, with 90% survival.     

Influence of nitrogen and phosphorus on induction embryogenic callus of sorghum

Crown and leaf slices of in vitro plantlets of a non-flowering Vetiveria zizanioides from Java were used to induce compact calli and to regenerate plantlets. The influence of different growth regulators (2,4-dichlorophenoxy acetic acid, 6-benzylaminopurine), sucrose concentrations (10–100 g l⁻¹), cultivation in light or dark, and cultivation time on callus induction medium (6 or 12 weeks), on the induction of compact callus and the subsequent regeneration of plantlets was studied. Up to 75% of crown slices cultured on modified Murashige and Skoog medium supplemented with 2.26 μM 2,4-dichlorophenoxy acetic acid, 2.22 μM 6-benzylaminopurine and 75 g l⁻¹ sucrose developed compact callus. For subsequent regeneration of plantlets, callus induction in the light for 6 weeks on the callus induction medium containing 10 g l⁻¹sucrose, and subsequent transfer to the regeneration medium, was the best procedure, regenerating plantlets on around 60% of the crown or leaf slices, with up to 100 plantlets per slice. We have compared the efficiency of the above mentioned procedure with several other methods to regenerate plantlets. Our findings indicate that the procedure developed in this study was best in regenerating plantlets for the used vetiver variant. This revised version was published online in June 2006 with corrections to the Cover Date.     

Factors affecting plant regeneration from immature inflorescence of two winter wheat cultivars

Inflorescence explants of two winter wheat cultivars, Triticum durum cv. Kızıltan-91 and T. aestivum cv. Bezostaja-01, were used to evaluate the effects of vernalization period of donor plants, callus age and medium composition on regeneration capacity. Donor plants were grown for 7 d and they were exposed to 4 °C for 1, 2, 3, 4, and 5 weeks. The maximum inflorescence formation was observed as 79 % at 4 weeks and 73 % at 5 weeks of vernalization period for Kızıltan-91 and Bezostaja-01, respectively. Among 6 different callus induction and regeneration mediums, I₁-R₁ and I₃-R₃ have to be the best responding mediums for Kızıltan-91 and Bezostaja-01, respectively. In Kızıltan-91, calli induced from donor plants, vernalized for 3 weeks, showed a significantly lower regeneration capacity than counterparts vernalized for 4 and 5 weeks. The highest regeneration capacity of 69 % was obtained from 6-week-old calli produced from 4 weeks vernalized Kızıltan-91 donor plants. In contrast to Kızıltan-91 cultures, the effects of vernalization period and callus age on regeneration capacity were not significant in Bezostaja-01 cultures. The maximum numbers of tillers were obtained from 6-and 15-week-old calli for Bezostaja-01 and Kızıltan-91, respectively. In contrast to vernalization period of donor plants, callus age had no effect on seed number.     

Fertile somatic hybrids between Petunia hybrida and a wild species,Petunia variabilis

Somatic hybrid plants were regenerated following electrofusion between leaf mesophyll protoplasts of P. hybrida (2n = 14) and a wild sexually incompatible species, P. variabilis (2n = 18). The selection of hybrids was based on the hybrid vigour, expressed both in the growth of the callus and at the shoot formation stage, resulting from the combination of parental genomes. Calli exhibiting vigorous growth were selected, and upon transfer to regeneration medium gave rise to shoots. Four regenerated plants from three calli had morphological characteristics intermediate between those of the parents. The hybrid nature of these plants was confirmed by chromosome counts as well as isozyme and DNA analyses. They had amphidiploid chromosome numbers (2n = 32) and were fertile. Following self-pollination and backcrossing with P. variabilis, large numbers of F₂ and BC₁ seedlings were obtained.     

A revised scheme for mass propagation of Easter Lily

Lilium longiflorum Thunb., commonly known as Easter Lily is widely propagated by vegetative means for its high ornamental value as a pot plant. Following in vitro technique, mass propagation has been achieved through direct production of bulblets from the explant as well as regeneration from callus. The chromosome analysis of the progeny derived from callus even from long term culture, did not reveal any marked variability in chromosome morphology. The stable nature of callus maintained in modified MS medium in long term culture has been confirmed. Along with rapid growth, the regenerating capacity of calli has been maintained for 3 years of culture in the above medium. Following shake culture, large number of bulblets could be obtained from such differentiated calli within 3–4 weeks. The shake culture technique of calli is ideally suited for securing stable regenerants on a mass scale in this species.Abbreviations MS Murashige & Skoog's medium - NAA -napthaleneacetic acid - IAA indole-3-acetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - BA 6-benzyladenine     

Inheritance of competencies for leaf disc regeneration,anther culture,and protoplast culture in Solanum phureja and corrleations among them

Competence for leaf disc regeneration, anther culture, and protoplast culture was examined in the parental, F₁, and F₂ generations of a population of the diploid, cultivated, primitive potato, S. phureja (2n=2x=24). The parental pair consisted of AM3-8, an anther culture derived homozygous diploid, and NBP2, a heterozygous, field selected line. AM3-8 produced embryos in anther culture, and shoots on cultured leaf discs, but its cells did not divide after protoplast isolation. Cells of NBP2 divided to form calli and shoots in protoplast culture, but the clone did not respond to anther culture or leaf disc regeneration. All the individual plants in the F₁ generation were responsive to both anther and protoplast culture; however, there was segregation for the ability to regenerate shoots from leaf discs. The F₂ population, the result of a sib-cross, segregated for all three tissue culture competencies. Segregation data fit a one gene model for anther culture competence with the homozygous dominant genotype expressing the highest response, the heterozygote resulting in a marginal response, and the homozygous recessive resulting in no response. A two-gene model applied to the protoplast culture data, with a dominant allele at both loci required for division to occur after protoplast isolation. Leaf disc regeneration data could only be explained by a two gene model with recessive alleles at each locus required for the highest response, a dominant allele at either of the loci resulting in a marginal response, and dominant alleles at both loci resulting in no response. No significant correlation was found among these traits, implying three separate genetic mechanisms which segregate independently.Abbreviations BA N⁶-benzyladenine - GA₃ gibberellic acid - IAA indole-3-acetic acid - NAA -naphthaleneacetic acid     

Anther culture response of Hordeum spontaneum-derived winter barley lines

The anther culture response of 18 Hordeum spontaneum derived winter barley lines, as well as that of the F₂ and/or BC₁ progeny of the cross between nine of these lines and the winter barley cultivar Triton was assessed. While the majority of lines showed a poor regeneration capacity (4%), two lines, RS 170-A13 × Sonja and RS 120-27 × Sonja demonstrated an outstanding response, producing 26 and 8 green regenerants per 100 anthers plated, respectively. Five further lines, RS 70-30 × Dura, RS L74-11 × Dura, RS 122-19 × Sonja. RS 142-29 × Dura and RS 170-A51 × Sonja were identified as highly responsive in the F₂ generation. An average production of 4 green regenerants per 100 anthers plated was observed both in the parental and F₂ generation.Abbreviations BA benzyladenine - IAA indole-3-acetic acid     

Cold storage effects on maternal and progeny quality of Gonatocerus ashmeadi Girault (Hymenoptera: Mymaridae)

This study determined the effects of cold storage on the survival, development and reproduction of the mymarid wasp, Gonatocerus ashmeadi Girault (Hymenoptera: Mymaridae). Following storage of the immature parasitoids within host eggs of the glassy-winged sharpshooter, Homalodisca vitripennis (Germar) (Hemiptera: Cicadellidae) under a daily fluctuating temperature for up to 50 d, the quality of the parental and F₁ generations was assessed by examining several reproductive and developmental parameters indicating fitness. Immature wasps were stored for 20 d within the host without reducing their subsequent survival, development or progeny fitness parameters. After 30 d of storage, survival declined, post-storage developmental time was extended, and the fecundity of the adult females decreased. Storage for 40 d severely damaged G. ashmeadi, because it not only yielded a 12% survival rate, 44% female sterility and increased the proportion of progeny males by 155%, but it also reduced parasitism and fecundity by 70% and 73%, respectively. No wasps emerged after 50 d of storage. Cold storage affected the emergence pattern of the parental but not the F₁ and F₂ generations. Parental emergence was extended and the pattern displayed two additional peaks after the initial onset. Analysis of several demographic parameters for the parental and F₁ generations further confirmed that the quality of the adult parents declined after they had been stored as immatures for 30 d. The detrimental effects caused by cold storage of the parental generation do not extend to the F₁ generation. Our results indicate that short-term cold storage of G. ashmeadi within its host could be used for maintaining and accumulating these parasitoids during mass propagation for release in a control program.