Genetic variation and control of plant regeneration in leek (Allium ampeloprasum L.)

Zygotic embryos of leek (Allium ampeloprasum L.) were isolated from mature seeds of different cultivars, selfings and full-sib families. The embryos were cultured on callus induction and shoot regeneration medium and employed to study several parameters: percentage of embryos forming calluses, percentage of embryos forming compact calluses, callus weight, percentage of regenerating calluses, numbers of shoot primordia and numbers of regenerated shoots. Differences between cultivars and selfings were found for most parameters studied. For all cultivars all parameters, except callus weight, decreased after one generation of selfing. Compact callus types enhanced primordia formation and shoot regeneration. Genetic characteristics of callus development and plant regeneration were studied in a 4 × 4 diallel cross. Analysis of variance revealed significant differences between full-sib families. The diallel analysis showed that additive gene effects were significant for all parameters. The predominance of additive gene effects indicated high narrow-sense heritability. Breeding for an increased number of regenerated shoots was successful.     

Linamarin content and genetic stability of cassava plants derived by somatic embryogenesis

A protocol was established for high frequency cyclic somatic embryogenesis for different varieties of cassava. An efficient plant regeneration system was developed for the high cyanogenic variety PRC60a. Linamarin content and linamarase activity were determined in various tissues of secondary somatic embryos and regenerated plants of PRC 60a. Both linamarin and linamarase activity were not detected in embryogenic callus, roots induced from callus and somatic embryo tissues. The stems and leaves of regenerated plants (in vitro) and storage roots and leaves of mature plants (in vivo), however, contained variable amounts of linamarin and linamarase activity whereas in the non storage root tissues (in vitro) only linamarin was detected. The present study suggested that the linamarin biosynthetic pathway may be absent or not switched on in the embryogenic callus and somatic embryos. The ploidy level and somatic chromosome number of the regenerated plants were found to be same as the source plants. The availability of this regeneration system would be useful not only for investigating cyanogenesis but also for genetic manipulation in cassava. This revised version was published online in July 2006 with corrections to the Cover Date.     

Somatic Embryogenesis and Plant Regeneration from Tritordeum

Regeneration of plants by somatic embryogenesis from immature embryos of hexaploid tritordeum (AABBH^chH^ch, amphiploid Hordeum chilense×Triticum turgidum conv. durum) and durum wheat (Triticum tergidum) was induced on MS medium supplemented with different 2.4-D concentrations. Well-defined embryoids were formed with a high frequency on the scutellar callus from 1 or 2 weeks onwards and plantlets were developed from them. In the best cases from one single explant more than 100 plants could be obtained. Plants were also regenerated by somatic embryogenesis from inflorescences of Hordeum chilense×Triticum turgiditm conv. durum hybrid and its respective hexa-amphiploid. With regard to callus induction and regenerative ability, evident differences between hexa- and octoploid (H. chilense×T. aestivum) tritordeum were found, the latter showing a very low response.     

Somatic embryogenesis and plant regeneration of Tripsacum dactyloides L.

Summary This article reports the culture and plant regeneration of Tripsacum dactyloides. Mature embryos of Tripsacum dactyloides dactyloides were used to obtain embryogenic callus cultures. Currently, 180 normal plants have been regenerated from these cultures. Callus was initiated on MS medium supplemented with dicamba (10 mol or 20 mol) and sucrose (3% or 6%), and plants were regenerated on hormone free MS medium containing 2% sucrose. No significant differences were found in callus initiation frequency or in embryogenic response of cultures on the four combinations of sucrose and dicamba tested. The embryogenic cultures have been maintained for 9 months (12 subcultures) and have retained regeneration capacity. Plants regenerated from tissue culture of maize-by-Tripsacum hybrids could be useful in maize improvement.     

Somatic hybrids between Gossypium hirsutum L. (4×) and G. davidsonii Kellog (2×) produced by protoplast fusion

Somatic hybrids were obtained from electrofused protoplasts derived from embryogenic suspension cultures of tetraploid cotton (G. hirsutum L. cv. Coker 201) and embryogenic callus of diploid wild cotton G. davidsonii. The regenerants were initially identified as hybrids by RAPD (random amplified polymorphic DNA) analysis. Subsequently, observation on chromosome counting, morphology and SSR (simple sequence repeat) confirmed the hybrid status. Cytological investigation of the metaphase root-tip cells of the regenerated plants revealed there were 74 to 84 chromosomes in the plants, close to the expected 78 chromosomes. SSR analysis revealed the regenerated plants contained specific genomic fragments from both fusion partners, further confirmed their hybridity. The morphology of the plants was intermediate between the two fusion partners. The regenerants were difficult to develop into mature plants because their roots browned and they wilted from the stem apex before forming 3 to 5 true leaves. The hybrid plants were transferred to soil by grafting in vitro onto rootstocks.     

Callus induction and plant regeneration from immature and mature embryos of winter durum wheat genotypes

Seven genotypes of winter durum wheat (Triticum durum Desf.) were cultured to establish an efficient method of callus formation and plant regeneration from mature embryo culture, and to compare the responses of immature and mature embryo cultures. Immature embryos were aseptically dissected from seeds and placed, with the scutellum upwards, in dishes containing Murashige and Skoog's (MS) mineral salts and 2mg 2,4- dichlorophenoxyacetic acid (2,4-D) per litre. Calli and regenerated plants were maintained on 2,4-D-free medium. Mature embryos were moved slightly on the imbibed seeds. For callus formation, the seeds with moved embryos were placed, furrow downwards, in dishes containing 8 mg 2,4-D per litre. The developed calli and regenerated plants were maintained on the MS medium. Plants regenerated from both embryo cultures were vernalized and grown to maturity in soil. Variability was observed among the wheat genotypes tested for various culture responses in both explant cultures. Callus induction rate and regeneration capacity of callus were independent of each other. Mature embryos have a low frequency of callus induction but a high regeneration capacity. Considering availability, rapidity and reliability, this form of mature embryo culture can be used as an alternative method for immature embryo culture.     

Analysis of protoplast-derived plants of Saintpaulia ionantha H. Wendl

Of 3272 plants regenerated from protoplasts of 10 Saintpaulia ionantha genotypes, 98.4% survived transfer to the greenhouse. The frequency of regenerants with chlorophyll deficiencies, i.e. variegated leaves or albinos, was low (1.5%). There was a higher number of polyploid, in most cases tetraploid plants, regenerated from protoplasts (16%) which were identified by their altered morphology. Measurements of stomatal length and counting the number of chloroplasts per guard cell also allowed a clear differentiation between diploid and polyploid plants. The classification was confirmed by DNA content determination using flow cytometry. Mechanisms leading to polyploidization included spontaneous protoplast fusion as well as chromosome doubling during callus growth and shoot regeneration. Two genotypes with instabilities in flower colour showed completely altered flower colours in plants regenerated from protoplasts as well as in plants regenerated on leaf explants in vitro.     

Plants regenerated from embryo cultures of an apomictic clone of Kentucky bluegrass (Poa pratensis L. ‘Baron’) are not apomictic in origin

Plants were regenerated from tissue cultures of embryos dissected from seeds that were harvested from a self-pollinated clonal selection of Kentucky bluegrass (Poa pratensis L.) ‘Baron’, an apomictic cultivar. Plants were regenerated from 35 embryo-derived callus cultures of the 3280 embryos that were plated. Flow-cytometric (FCM) and RAPD-marker analyses were performed to determine if regenerants were or were not apomictic in origin. Fifteen regenerants with a 3c DNA content were classified as arising from 2n + n aberrant embryos, which was a higher frequency than expected, based on a chi-square analysis. Of the remaining 20 regenerants with a 2c DNA content, a chi-square test showed that all could have arisen from n + n sexually-derived embryos, based on the observed segregation of n + n regenerants, which fit the expected 3:1 ratio of dominant:recessive RAPD-marker phenotypes. The apparent lack of regenerants of apomictic origin, and implications for genetic transformation and breeding of Kentucky bluegrass are discussed.     

Plant regeneration through callus initiation from mature embryo of Triticum

The behaviour of diverse Triticum genotypes in the tissue culture response of mature embryo callus was compared, and factors affecting tissue culture response were studied in this paper. Significant differences were detected in callus induction, embryogenic callus differentiation, plantlet regeneration and culture efficiency when mature embryos of 31 plants of different Triticum species were compared. These were the main wheat cultivars of the Chinese northern winter‐type wheat region and breeding lines (Triticum aestivum L.), durum wheat (Triticum durum Desf.), cultivable emmer wheat (Triticumdicoccum Schuble) and the common wheat progenitors Triticum dicoccoides and Triticum aegilopides. The genotype dependency was particularly high in tissue culture of mature embryos of these Triticum genotypes. The efficiency of induction, differentiation and regeneration of mature embryos callus was high in genotypes selected out. Mature embryo‐derived callus of HB341, TS021, SN2618, T. dicoccum, HB188, and T9817 showed better tissue culture response than the other genotypes. Plantlets can be regenerated from mature embryo‐derived callus of 31 genotypes, saving on growth facility resources and time required for the collection of other explants, and providing a solid basis for the genetic transformation and molecular plant breeding of Triticum plants.     

In vitro tetraploid induction via colchicine treatment from diploid somatic embryos in grapevine (Vitis vinifera L.)

A protocol for in vitro induction of tetraploids via colchicine-treated somatic embryos from immature zygotic embryos of diploid grapevine (Vitis vinifera L.) is reported. Embryogenic callus was initiated from immature zygotic embryos cultured on Nitsch and Nitsch (NN) medium supplemented with 1.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D). The callus was transferred to NN medium containing 1.0 mg/l α-naphthalene acetic acid (NAA) and 0.5 mg/l benzyladenine (BA) to establish somatic embryogenesis. The vigorously growing globular embryos were selected and treated by 0, 10 or 20 mg/l colchicine for 1, 2 or 3 days, and then immediately transferred to NN medium supplemented with 0.03 mg/l NAA and 0.5 mg/l BA, for somatic embryo conversion and plant regeneration. The number of surviving embryos and regenerated plantlets following colchicine treatment decreased with increasing colchicine concentration and treatment time. Among 29 randomly investigated plantlets regenerated from colchicine-treated somatic embryos, five solid tetraploids (2n = 4× = 76) were identified by chromosome counting analysis; all others were diploid (2n = 2× = 38). Ploidy level of plant regenerated was also determined from leaves using flow cytometry. No chimeras with both 2C and 4C nuclei was produced from colchicine-treated somatic embryos. Significant differences in leaf stomata parameters were observed between diploid and induced tetraploid plantlets.     

Somatic Embryogenesis in Maize and Comparison of Genetic Variability Induced by Gamma Radiation and Tissue Culture Techniques

Sixteen inbred lines and one hybrid of manse were tested for their capability of somatic embryogenesis, and fully developed plants could be regenerated, from ten inbred, lines. The highest frequency of plant regeneration was expressed in the inbred line CHI 31, and when this line was crossed with a recalcitrant, non-regenerating line, the F₁ and BC hybrids were regenerable. The results of reciprocal crosses demonstrated that dominant nuclear genes and cytoplasmic factors are primarily responsible for the heritable determination of embryogenic callus proliferation and in vitro regeneration of maize plants. Somaclonal and radiation-induced variability was studied in maize to assess their nature and potential contribution to plant breeding., The inbred line CHI 31 possessing a high in vitro capacity of somatic embryo formation was used as experiments.] material. CHI 31 plants were selfed and twelve-day old zygotic embryos irradiated with ⁶⁰Co gamma radiation in situ. Mature caryopses were harvested and assigned as M₁ material. In another series, immature zygotic embryos (size 1.2—1.5 mm) were cultured in vitro on N-6 medium supplemented with 2,4-D (2.5 μM), and somatic embryos regenerated into plants; these were transplanted into soil and self-pollinated. Regenerants from non-irradiated cultures were grown as R₁ generation, while regenerants from irradiated explants were considered as M₁R₁ generation. The genetic variability was evaluated in the M₂, R₂ and M₂R₂ generations, respectively, and compared with a non-treated seed control. Irradiation induced a variety of chlorophyll and morphological variants segregating in the M; generation; however, the frequency of deviant types obtained in the R: generation (somaclonal variation) was significantly exceeding the one derived from the M₂ populations. The combination of expert irradiation and in vitro regeneration was most effective for the manifestation of chlorophyll and morphological o if types in the M₂R₂ generation, and increased drastically the frequency of early flowering variants. Differences in the segregation patterns of mutant phenotypes amonsister somaclones in the R₃ and M₃R₃ generations indicate a different genetic basis, of plants originating from the same explant. This phenomenon suggests a mutational sectoring of the callus during culture. Radiation induced and somaclonal variation exerted a similar spectrum of chlorophyll and morphological deviants.     

The effects of kinetin on callus characters in alfalfa (Medicago sativa L.)

Summary Three callus initiation media, B2-k, B2, and 7951, were used to study the effects of kinetin on callus initiation, morphology, histology, and regenerability in alfalfa (Medicago sativa L.). The presence of kinetin in callus initiation media retarded callus initiation, but enhanced division and differentiation of callus cells. Calluses induced on kinetin-containing media (B2 and 7951) had many compact cell aggregations, which were considered meristematic regions that might differentiate to plantlets on a regeneration medium. Visually, these calluses were compact and had many nodular structures. In contrast, most calluses induced on a kinetin-free medium were composed of large, individual cells and had friable structures without nodules. After transfer to a hormone-free medium, calluses induced on kinetin-containing media regenerated more frequently than those induced on a kinetin-free medium, but cytokinin (kinetin) autotrophism also occurred. Autotrophism was sexually transmissable and especially affected by the female parent.     

Plant regeneration from embryo-callus culture in barley

Summary Plants were regenerated from callus cultures initiated from immature embryos of barley, Hordeum vulgare L. Immature embryos from seven diverse genotypes were cultured on modified Murashige and Skoog (MS) medium supplemented with 1.5 mg 2,4-D and 6.5 mg IAA/l. Of the 249 embryos cultured, 30% initiated callus within 8 days. Subculture of callus for 80 to 100 days on half-MS medium supplemented with 0.5 mg/l 2,4-D and 1.0 mg/l zeatin resulted in organogenesis. Culture of organogenic calli for 30 days on half-MS medium without growth regulators produced plants which originated mostly via multiple shoot formation. Callusing response of the tested genotypes ranged from zero to 44%; however, only 23% of the calli were regenerative. Regenerated plants included variants for chlorophyll deficiency, plant height, stem thickness, spike shape, pollen fertility, seed set and ploidy.     

Genotypic differences in callus formation and regeneration of somatic embryos in Cyclamen persicum Mill

This study was performed in order to check the applicability of a protocol for somatic embryogenesis in Cyclamen persicum and to compare the in vitro response of cultivars belonging to different cyclamen growth types and flower colours obtained from two breeders. One hundred ovules per plant were prepared from 32 tested F₁ hybrid cultivars represented by five plants each. Callus induction monitored after 8 weeks varied between 42 and 85%. Variation occurred between replications and single plants of a cultivar. Consistency and colour of the developing calluses were diverse. Only in 1 cultivar out of the 32 tested no regeneration of somatic embryos was obtained. The regeneration rates varied from 0 to 65% among cultivars. In the group of midi-type cultivars the regeneration response was lower than that in mini and giganteum types. High regeneration rates were observed in cultivars with flamed flowers.     

In vitro techniques for selecting wheat (Triticum aestivum L.) for Fusarium-resistance. I. Double-layer culture technique

Summary Calluses of spring and winter wheats (Triticum aestivum L.) were selected for Fusarium resistance in vitro, using the double-layer culture technique. Potato-dextrose agar medium in vials was inoculated with mycelia of Fusarium graminearum and F. culmorum. After one week, fungal cells were killed by autoclaving and the agar medium containing the thermostable toxic metabolites was overlayered with MS callus-growing medium. Later, wheat calluses were placed on the upper medium for 4–5 weeks, and from the surviving calluses plants were regenerated. R₂ seedling populations from self-fertilized R₁ plants of 4 varieties were tested for Fusarium resistance by artificial infections in the greenhouse, and 3% of the regenerated R₂ plants have been found to be more resistant than the original cultivars.     

Embryo rescue and plant regeneration following interspecific crosses in the genus <Emphasis Type="Italic">Hylocereus</Emphasis> (Cactaceae)

The aim of this study was to develop an efficient methodology to rescue embryos following interspecific crosses in the genus Hylocereus. Crosses between the diploids Hylocereus polyrhizus and H. undatus in both directions were performed. Fertilized ovules carrying embryos at very early pro-embryonic stages were excised from ovaries 5 days after pollination (DAP) and placed on half-strength basal MS medium containing 680 μM glutamine, 0.55 μM α-naphthaleneacetic acid (NAA), 0.45 μM thidiazuron (TDZ) and various concentrations of sucrose. After 30 days in culture, ovules were isolated from the surrounding tissue and transferred to the same fresh medium. Significant differences were found between the main effects (cross and sucrose concentration) in ovule response, i.e., increased ovule size and callus formation. The best responses were obtained in the cross: H. polyrhizus × H. undatus; and sucrose concentration of 0.09 M. In terms of embryo conversion, polyembryony and number of regenerated plants, the highest responses were observed on the culture medium supplemented with 0.17 M sucrose in both interspecific crosses. All tested plants were found to be diploid by flow cytometric analyses. Fluorescent amplified—fragment length polymorphism (fAFLP) confirmed the hybrid origin of the regenerated plants. This study reports on the success of a three-step embryo rescue procedure for Hylocereus species. The procedure developed here provides the means for producing plants from very-early embryo stage, thus expanding the prospects for vine-cactus breeding programs.     

Effect of Mutagenic Treatments on Somaclonal Variation in Wheat (Triticum aestivum L.)

Different wheat genotypes were treated with gamma-rays, sodium azide (SA) and EMS before tissue culture and immature embryos from M₁ plants or plants shortly after exposure to gamma-rays were used to initiate callus culture. Thousands of plants were regenerated and used to investigate the effect of mutagenic treatments on the regenerated plants and somaclonal variation in the M₃R₂ and M₂R₂ generations. The results showed that mutagen-induced damage in terms of reduction in plant height, fertility and spike length were not outstanding in the regenerated plants as compared with the untreated control. In the M₃R₂ generation, only SA treatment had significantly higher frequencies of somaclonal variations than the control. Increases in the variation frequencies were observed when explant embryos were irradiated with 2.5 and 5 gy gamma-rays and the highest frequency appeared when embryos were exposed to 5 gy gamma-rays on the 5th day after anthesis. Increased variation spectra also resulted from mutagenic treatments and most of the variants recovered were unsuitable for plant improvement.     

Production and molecular characterization of Citrus intergeneric somatic hybrids between red tangerine and citrange

Somatic hybridization has been an effective and successful technique for plant improvement. In this paper, embryogenic callus protoplasts of red tangerine (Citrus reticulata Blanco) were electrically fused with mesophyll protoplasts from citrange (C. sinensis × P. trifoliata, a Chinese local strain) in an effort to produce complementary tetraploid citrus rootstocks. Regenerated embryoids grew slowly and were vulnerable to browning. Twelve plants were finally regenerated, rooted and transplanted into a greenhouse. Root‐tip chromosome counting of five randomly‐selected plants revealed most cells were tetraploid (2n = 4x = 36), but aneuploid cells also existed. Flow cytometry analysis further confirmed their tetraploid nature. Nuclear simple sequence repeat (SSR) analysis verified their hybridity. Further mitochondrial genome analysis by restriction fragment length polymorphism and cleaved amplified polymorphic sequence revealed their mtDNA banding pattern was identical to that of red tangerine, the embryogenic callus parent; while their chloroplast DNA inheritance was random as revealed by chloroplast SSR analysis, in addition to cpDNA co‐existence detected in one plant. Cytological and molecular analysis indicated that somatic hybrid plants between red tangerine and citrange had been successfully obtained.     

The Response of Anther Culture in a Genetically Wide Material of Winter Wheat (Triticum aestivum L.)

In a screening experiment with 215 different winter cultivars of Triticum aestivum L. for response in anther culture, pollen embryos or callus were obtained from 200 different cultivars and green plants from 93 different cultivars. On average, from the whole material, 1.3 green plants were obtained per 100 anthers cultured. Variance components estimated from replicates with selected parts of the plant material indicated that for the formation of pollen embryos from anthers, interactions between genotypes and replications were dominating, accounting for 45 to 50 per cent of the variation. Main effects from genotypes were less prominent, accounting for 25 to 30 per cent of the variation, and the replications showed relatively little effect, accounting, for about 4 to 12 per cent of total variation. Regeneration of plants from pollen embryos or callus was not significantly influenced by either genotype or replicates. The frequencies of plants regenerated being green, however, were influenced from both genotype and environments contributing 42.5 and 34.9 per cent of the total variation respectively, while only minor interactions between genotype and environments were indicated for this character.     

Grafting in vitro Regenerated Pea (Pisum sativum L.) Shoots as a Method for Obtaining Mature Plants

A method of grafting pea plants is proposed to allow rapid availability of a large number of mature plants from in vitro regenerated shoots. Plant regeneration was obtained from macerated vegetative apices and immature embryos, but regenerated shoots rooted with low frequency (ca. 10 %). When those shoots were grafted onto seedling stocks of the same cultivar, 80 to 85 % of the grafts survived, grew to maturity and produced seed.