Amphidiploids between Cyclamen persicum Mill. and C. purpurascens Mill. induced by treating ovules with colchicine in vitro and sesquidiploids between the amphidiploid and the parental species induced by conventional crosses

Summary We cultured colchicine-treated hybrid ovules in vitro to produce fertile amphidiploids of C. persicum (2n=2x=48. referred to as AA) × C. purpurascens (2n=2x=34, referred to as BB). Seedlings and mature plants were obtained from the ovules without colchicine and those exposed to 50 mg/l colchicine for 5, 10 and 15 days, whereas they were not obtained from the ovules exposed to 50 mg/l colchicine for 20 days and 500 mg/l for 5, 10, 15 and 20 days. Although 8 mature hybrids derived from the ovules without colchicine produced a few fertile pollen grains, they failed to produce viable seeds by self-fertilization. The hybrids had 41 somatic chromosomes. Four and 3 mature plants were derived from ovules exposed to 50 mg/l colchicine for 10 and 15 days, respectively. One each among 4 and 3 mature plants showed a high frequency of pollen grain fertility, produced several seeds by self-fertilization, and had 82 somatic chromosomes which is twice the number of hybrid chromosomes (2n=41, AB). These findings indicated that these plants are amphidiploids (2n=82, AABB) between C. persicum and C. purpurascens. Three and 2 viable seeds were derived by the conventional crosses of diploid C. persicum × the amphidiploid and the amphidiploid × C. purpurascens, respectively. Flowering plants that developed from the seeds of diploid C. persicum × the amphidiploid were barely fertile and had 65 somatic chromosomes (2n=65, AAB), whereas those that developed from the seeds of the amphidiploid × C. purpurascens were barely fertile and had 58 somatic chromosomes (2n=58, ABB). The somatic chromosomes indicated that these plants are probably sesquidiploids between the amphidiploid and either C. persicum or C. purpurascens. The interspecific cross-breeding of cyclamen using the amphidiploids and the sesquidiploids is discussed.     

Spontaneous and induced chromosome doubling in gynogenic lines of onion (Allium cepa L.)

The presence of 2,4-dichlorophenoxyacetic acid (2,4-D) or naphthaleneacetic acid (NAA) and 6-benzyladenine (6—BA) in the medium is required to induce an acceptable yield of gynogenic embryos from unfertilized ovary and flower cultures in onion. Four different exposure times of ovary and flower cultures to exogenous growth regulators (15, 30, and 45 days, and the entire culture period) were assayed. The objective was to ascertain the effect of these substances and of their period of application on the formation of gynogenic embryos and on the yield of haploids. An exposure of 15 days was sufficient for ovaries and flowers to be stimulated towards the gynogenic response, whereas, for the remaining period of 30—80 days, the pro-embryos could easily grow on a growth-regulator-free medium. In the gynogenic material obtained, phenotypic differences were visible between genetically independent lines but not between plants within each line, even when they had a different ploidy level (n or 2n). Almost all lines obtained by gynogenesis were sterile. Only a small percentage (1%) became fertile through spontaneous chromosome doubling, and these produced 2—20 seeds each, giving normal plants. The recovery of fertility occurred more often during the generations of bulbils. To exploit this natural propensity towards diploidization in this phase, different amounts and numbers of applications of colchicine were evaluated in two experiments. The treatments corresponding to 10 and 100 mg/1 of colchicine applied for 24h gave the highest number of diploid cells in root tips but no diploidization occurred in the shoot apices. Three days of colchicine treatment at 10 mg/1 produced 46% of plants completely diploid in the shoot apex. The flower fertility of these doubled haploid plants is being evaluated.     

In vitro chromosome doubling with colchicine during microspore culture in wheat (Triticum aestivum L.)

Isolated microspores of two DH lines of wheat were treated with 8 different colchicine concentrations up to 3 mM for either 24 h or 48 h during microspore culture. Untreated control cultures produced on average 220 embryos per spike (100,000 microspores), 68% of the regenerated plantlets were green, and 15% of the flowering plants were fertile. The colchicine treatments had a significant effect on chromosome doubling as measured by the percentage of fertile regenerants. Using colchicine concentrations around 1 mM the percentage of fertile plants among the regenerants was increased up to 53%. The highest number of embryos and regeneration rates were observed after 24 h colchicine treatment, while the highest frequencies of green plants and fertile plants were obtained with 48 h colchicine treatments. The highest number of DH plants per spike was found after treatment with colchicine concentrations of 300 to 1000 μM. Such treatments resulted in an estimated average between the two genotypes of 23 doubled haploid plants per spike. This revised version was published online in July 2006 with corrections to the Cover Date.     

Durum Wheat under Mediterranean Conditions as Affected by Seed Size

This study was carried out in order to determine the effect of seed size on the growth and yield of durum wheat. Four field experiments, composed of six durum wheat (Triticum turgidum L. var. durum) varieties and three seeding sizes were conducted in north‐east Spain in 2000 and 2001 in randomized complete block designs. The growth of seedlings was dramatically affected by seed size. Large seeds produced greater plot stands, but the plants had fewer tillers, leaves and spikes and less green area and dry weight than plots from small seeds. Grain yield was 16 % greater in plots from large seeds, which resulted in greater biomass, green area index, spikes per m² and heavier kernels than in plots from small seeds. Kernel weight was the yield component most related to grain yield in the three seed sizes. The percentage of yield variation explained by kernel weight increased as the weight of kernels increased. This was a consequence of the use of larger seeds, the same effect being observed when the comparison was made between varieties with different kernel weights. Selection for heavy kernel varieties may help to improve the yield of durum wheat in Mediterranean environments similar to that prevailing in north‐east Spain.     

Dwarf-twisted: An induced mutation in Triticum durum Desf

Summary A mutant phenotype, mainly characterized by undulated leaf surface, dwarfism and sterility, has been repeatedly induced by physical (X-rays; thermal and fast neutrons) and chemical (diethylsulphate and ethylmethanesulphonate) mutagenic agents in different varieties of durum wheat. This phenotype is easily detectable at the seedling stage. Neutron treatments are the most effective method to induce this phenotype and a maximum of 0.6% mutations per M₁ spike-progeny was induced after the highest dose of thermal neutrons. On the average 0.23 mutations per 100 M₁ spike progenies and 0.55 mutant seedlings per 1000 M₂ plants were found after all treatments.Genetic analysis of different dwarf-twisted phenotypes shows that this character is conditioned by one recessive gene with an average segregation ratio of 22.9%. As the dwarf-twisted phenotypes segregate in highly fertile M₁ spikes and no evident chromosome changes have been detected in this caryotype, the hypothesis of a point mutation has been proposed.Contribution no. 171 from the Laboratorio Applicazioni in Agricoltura del C.N.E.N., C.S.N. Casaccia, S. Maria di Galeria, Roma, Italy.     

Chemical induction of apomictic seed formation in maize

Summary Silks of 18 maize (Zea may L.) F₁ hybrids were treated with different combinations of 9 growth regulators, colchicine, and dimethyl sulfoxide (DMSO) for the purpose to induce apomixis (agamospermy) in 1988 and 1989. Hybrid K301 × K303 gave the highest (0.36%) average frequency of seed induction among the hybrids. The most effective treatments were DMSO, gibberellic acid plus 6-benzyl aminopurine (6-BA), and DMSO plus methanesulfonic acid. Individually, the highest frequency of seed induction was 1.4% for hybrid K731×K306 when treated with -naphthalene acetic acid (NAA)-zeatin mixture. The frequency of seed induction seemed to depend partially on the interaction between chemicals and hybrids. Cytological observation of root-tip cells indicated that the majority of the seeds obtained were diploid, some were mixoploid, and a few were haploid. Diploid plants from induced seeds from the same parent were morphologically uniform and resembled the parent. Variations in plant and ear heights were comparable to those of the hybrid parent. Cytological and morphological investigations suggested that the chemically induced seeds originated mainly from somatic tissue but occasionally came from reduced cells in the embryo sac, leading to haploids. The results showed that chemical induction of adventitious embryony in maize hybrids is possible, but the more effective chemicals, their concentrations, and ways of application for increasing the frequency of seed induction need to be explored for practical use.     

Application of in vitro organ culture in wide-cross breeding of rapeseed

Oil radish (Raphanus sativus var. raphanistroides Makino) is resistant to drought and low temperature. In order to breed more resistant cultivars of rapeseed, the wide cross between rapeseed (Brassica napus L.) and oil radish was made. Rapeseed was not compatible with oil radish, and the frequency of hybrid plants (F₁) was very low. Moreover, the hybrid plants were sterile. In order to recover the intergeneric hybrids (F₁), the in vitro organ culture technique was applied in our experiments. The frequency of hybrid plants (F₁) was increased up to 25.55% by means of in vitro culture of pollinated ovaries. Some fertile amphidiploid hybrid plants were obtained by means of colchicine treatment of small buds obtained from cultured flower receptacle segments of hybrid plants (F₁). It is suggested that the technique of in vitro culture of pollinated ovaries and flower receptacle segments is useful in the wide-cross breeding of rapeseed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effects of Agrobacterium tumefaciens-mediated transformation and tissue culture on storage lipids in Brassica napus

The variation obtained in storage fatty acids induced by the procedures of tissue culture and transformation with Agrobacterium tumefaciens was investigated and compared in rapeseed, Brassica napus, cv. Hanna. An increased variation in the fatty acid profiles was noted after tissue culture and transformation compared with plants derived directly from seeds. In the second generation of rapeseed transformants, T₂, the content of oleic acid ranged from 39–72%, 12–31% for linoleic acid and 7–16% for linolenic acid. This could be compared with the oleic acid content in the T₂ generation of tissue culture-derived plants which ranged between 47–76% and in seed-derived material where oleic acid ranged between 55–69%.In the T₃ generation the ranges in transgenic seeds were decreased but still larger than in the seed derived plants. The range in transgenic plants was 49–64% for oleic acid, 20–28% for linoleic acid and 9–18% for linolenic acid. The most extreme individuals, both highest and lowest in the common fatty acids, were found in the group of transformed plants independent of generation. The total lipid content was also affected by the two treatments and seeds with the lowest and highest lipid content were both found among the transformed plants. In conclusion, care should be taken to use proper controls when performing transformation experiments in order to distinguish variation in the fatty acid profiles induced by the transformation procedure and tissue culture treatments from the changes due to transgenic expression. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effects of recurrent selection for resistance to scab (Gibberella zeae) in wheat

Summary Scab caused by Gibberella zeae Petch., in common wheat, is one of the most severe diseases in China. A source population C0, bred for scab resistance, was developed through three cycles of multiple-parent crossing and intercrossing by means of the dominant male-sterile gene Ta1 (Ms2), according to Wu's scheme. Phenotypic recurrent selection methods for increasing the resistance to scab-infection of spikelets and seeds with the male-sterile plants were carried out simultaneously in Nanjing and Shanghai and at Jianyang, Fujian Province, for three cycles. The generations from C0 to C3 and two check cultivars were evaluated, using a randomized block design, under conditions of an artificially induced epidemic of scab during 1988–1990. The results indicate that there were significant differences in the resistance to scab between these generations. On average, the percentages of diseased spikelets and seeds of the male-fertile plants were reduced by 9% and 10%, respectively. The frequency of resistant plants was distinctly enhanced by recurrent selection. Analysis of variance showed that no significant differences existed between cycles of recurrent selection in agronomic characters such as plant height, spikes per plant, spike length, numbers of spikelets and seeds per spike, weight of seeds per spike and 100-kernel weight, days to heading and to maturity. Except for plant height, most of these traits tended to be slightly improved with improvement of resistance in the gene pool. The variance for resistance in the generations was decreased under selection. Recurrent selection for scab resistance using the dominant male-sterile gene Ta1 (Ms2) was both an effective and feasible breeding method for producing this character in wheat.     

Improved application of tribenuron-methyl as a chemical hybridizing agent with forchlorfenuron for rapeseed hybrid breeding

Chemicals or agents that can induce plant male sterility are considered chemical hybridizing agents (CHAs) and are quite useful for plant hybrid breeding. Tribenuron-methyl (TBM) can effectively induce male sterility in rapeseed plants. However, applications of TBM in rapeseed breeding practices have not been reported. Here, treatment with the CHA referred to as CHA-TBM, which contained 0.1–0.9 mg/L TBM, induced male sterility in all 22 tested rapeseed inbred lines. However, the highest induced male sterile rates in the tested rapeseed lines were only approximately 54–57% together with poisoned rates of approximately 36–39%, which were resulted from two treatments of CHA-TBM containing 0.5 mg/L TBM at an interval of 9 days beginning at the floral bud stage (FBS) of 2.0–3.5 mm. Such male sterile rates and poisoned rates caused by CHA-TBM indicated that TBM is not an ideal CHA for rapeseed hybrid breeding. The results of many screening experiments showed that forchlorfenuron can alleviate the toxic effects of TBM on rapeseed plants. Two treatments with the CHA referred to as CHA-TBM-FCF, which contained 0.6 mg/L TBM and 2.1 mg/L forchlorfenuron, at an interval of 9 days beginning at the FBS of 2.0–3.5 mm induced male sterility in approximately 96–98% of plants of all the 22 tested rapeseed inbred lines and poisoned only approximately 2–4% of the tested plants. The CHA-TBM-FCF treatment did not affect pistil fertility of the rapeseed plants, and had no carry-over influence on stamen fertility of the rapeseed hybrids produced with this CHA. Some rapeseed hybrids were successfully produced using CHA-TBM-FCF (e.g., Qinrong 1, Qinrong 2, Qinyou 19, Qinyou 33, Qinzayou 4 and Qinzayou 5). The hybridity values of the F1 seeds of the rapeseed hybrids produced using CHA-TBM-FCF were determined to be approximately 93–98% with the simple sequence repeat (SSR) marker techniques. Together, the results indicate that CHA-TBM-FCF containing TBM and forchlorfenuron at suitable concentrations is an ideal CHA for rapeseed hybrid breeding.     

Waterlogging differentially affects yield and its components in wheat,barley, rapeseed and field pea depending on the timing of occurrence

Waterlogging on croplands is increasing in various areas of the world. This study evaluated the yield penalty by early and late waterlogging on wheat (Triticum aestivum L.), barley (Hordeum vulgare L.), rapeseed (Brassica napus L.) and field pea (Pisum sativum L.). Plants cultivated outdoors were exposed to a 14-day waterlogging during vegetative (at 65 days after sowing (DAS)) or reproductive (at 85/87 DAS) stages, followed by drained conditions until maturity. Yield (seed weight per plant) and its components (number of spikes/siliques/pods per plant, number of grains per spike/silique/pod and 1,000 grain weight) were assessed at maturity, along with morphological (number of tillers/branches) and shoot and root dry weight responses after waterlogging and during recovery. Wheat was the most tolerant species achieving 86% and 71% of controls in yield with early and late waterlogging, related to fewer grains per spike. Barley and rapeseed tolerated early waterlogging (yields 85% and 79% of controls) as compared to late waterlogging (32% and 26% of controls), mainly due to fewer spikes per plant (barley) or reductions in seeds per silique (rapeseed). Field pea was greatly affected by waterlogging at both timings, attaining a yield of only 6% of controls on average due to much fewer pods and fewer seeds per pod. So, wheat could be an option for areas facing either winter or spring transient waterlogging (i.e. early or late stages); barley and rapeseed are recommended only with if water excess occurs in early stages and field pea is intolerant to waterlogging.     

Aneuploids of perennial ryegrass (Lolium perenne)

Summary Aneuploid plants of perennial ryegrass (Lolium perenne L.) with 2n=15 to 30 chromosomes were obtained by crossing a near-triploid (2n=3x+1=22) with a diploid or on open-pollination with diploids and tetraploids. Aneuploids occurred with a frequency of 83% in near triploid × diploid progeny and 92% on open-pollination with diploid and tetraploid plants. Aneuploid plants with 15 to 18 chromosomes resembled diploids in morphology and those with 19 to 30 chromosomes were akin to tetraploids. Meiotic studies suggested that most aneuploid plants resulted from transmission of aneuploid egg cells (n=8 to 23). Aneuploid plants with 2n=27 to 30 chromosomes in the progeny of 22×14 cross originated from unreduced egg cells. Plants with 19 to 21 chromosomes were recovered only by immature seed culture. Aneuploid plants with 26 to 30 chromosomes and triploids (2n=21) had higher pollen fertility and bigger seeds than plants with 15 to 22 chromosomes.     

Use of herbicide-resistant genic male sterility in hybrid rice seed production

One of the difficult problems in hybrid rice seed production is the low outcrossing frequency and requirement for much labor to produce hybrid seeds. In order to simplify the process of hybrid rice seed production, herbicide-resistant photoperiod sensitive genic male sterile (HRPGMS) rice was utilized in this study. The herbicide resistance gene bar was transferred into the photoperiod sensitive genic male sterile (PGMS) rice 920S by Agrobacterium-mediated transformation and the HRPGMS line YA3530ms with good agronomic characteristics was bred by applying conventional pedigree breeding technique. The seeds of HRPGMS and pollen parent were mixed with the ratio of 4:1 in weight, and were sowed in seedling box. The mixed seedlings of HRPGMS and pollen parent grown for 30 days were transplanted by the small transplanting machine in the field. The herbicide glufosinate ammonium was sprayed at 7 days after flowering to kill all the plants of pollen parent, whereas hybrid seeds were harvested from the survived HRPGMS parent at maturity. The outcrossing frequency of HRPGMS line from two combinations in 2002 and from five combinations in 2004 were compared with a control cultivated by the conventional 2-line system. As the result, the mean outcrossing frequency in HRPGMS of the treatments were 10.6–24.5% compared with 5.5% in PGMS of the control in 2002, and that were 24.7–32.0% compared with 7.5% in the control in 2004. Consequently, using HRPGMS in two-line system was proved to be a new method that would simplify the process of hybrid rice seed production and to increase outcrossing frequency without any artificial supplementary pollination processes.     

Hybridization, Through Culture of Embryos and Immature Seeds, of a Range of Tomato Cultivars with a Tomato Somatic Hybrid [Lycopersicon esculentum (+) L. peruvianum]: Emergence of a Possible New Marker Gene for Tomato Breeding

Two hexaploid somatic hybrids [Sh; L. esculentum (+) L. Peruvianum] accessions 6 and 18 were back-crossed with two diploid L. Esculentum cultivars ‘Moneymaker’ (mm) and ‘pusa Ruby’ (pr). Twenty-two plants of the bc2 generation were produced by backcrossing 7 bcl plants (mm x sh, 6, 18) with five tomato cultivars. Fourteen of the bc2 plants were self-fertile, five produced anther cones with anthocyanin pigmentation not present in the parents. A bc3 generation was developed by crossing the four cultivars as female parent with three bc2 generation plants. The bc3 progeny derived from one pollen parent plant were produced without the need to culture immature seeds. They segregated with respect to pigmented anther cones and were self-fertile. The anther cone pigmentation of the pollen parent plant was associated with increased seed set, greater fruit size and an orange-red fruit colour. These features were transmitted to the fertile bc3 generation. Conversely, bc3 offspring involving the other two parent plants were only recovered by culture of immature seeds. The recovery of diploid plants in BCl and self-fertility in BC2 resulted in almost total recovery of the tomato cultivar characteristics (fruit size, colour and number of seeds) by BC3.     

The effect of colchicine treatment on Solanum acaule and S. bulbocastanum; A complete analysis of ploidy chimeras in S. bulbocastanum

Summary Seeds of tetraploid Solanum acaule (2n=48) and diploid S. bulbocastanum (2n=24) were germinated in petri-dishes on filter paper soaked in 0.3% colchicine. An additional treatment with 0.3% colchicine was applied one month after sowing at four successive days in the axils of the cotyledons of the seedlings. S. acaule appeared much more sensitive to colchicine (14 surviving seedlings from 500 seeds) than S. bulbocastanum (109 surviving seedlings from 450 seeds). Six S. acaule plants with 2n=96 chromosomes were obtained against 38 S. bulbocastanum plants with 2n=48 chromosomes.The ploidy level in each of the three germ layers L₁, L₂, L₃ was determined in 113 plants of S. bulbocastanum and the following results were obtained. Four of the eight possible ploidy types were detected, viz 2x-2x-2x (72 plants), 4x-2x-2x (3 plants), 2x-4x-4x (9 plants) and 4x-4x-4x (29 plants). Doubling the number of chromosomes resulted in a highly significant increase of the number of chloroplasts in the guard cells of stomata and a greatly significant decrease in the proportion of trimerous pollen, male fertility and leaf index. The variability for all characters studied, except for leaf index, was clearly lowest in the 2x-2x-2x group. All plants with a 2x-L₂ were highly male fertile and self-incompatible, also in the three bud stages tested. Male fertility of the plants with 4x-L₂ varied greatly: 12 plants had more than 90% stainability, 5 plants must be considered male sterile. All non-sterile plants with 4x-L₂ were found to be self-compatible, pointing to a gametophytic system of incompatibility in S. bulbocastanum.     

Callus induction and plant regeneration from anther and inflorescence culture of Sorghum

Summary Twenty-five inbred lines, including grain and forage types from the USA and China, two hybrids, one Sorghum almum, and one Parasorghum (S. versicolor) were tested for their response to anther culture. Three nutrient media were effective in inducing anther calli from six cultivars (Xin White, TX 403-TSB, DDY Sommer Milo, TX 2779, Brawley, and Spur Federal) and one was effective for plant regeneration for one cultivar, Xin White. Averaged over media, callus induction frequency (number of calli per 100 anthers) was highest in cultivars Xin White and TX 403-TSB (6.7 and 3.9%, respectively). The means of cultivars for media C17-2 and Ms-t-z-2, 4.3 and 3.2%, respectively, were superior to that for medium 85D3-2 (0.1%). Expressed as an average of the six cultivars and three media the mean calli induction frequency was 2.6%; however, differential responses of genotype and medium were noted. Among the 10 regeneration media tested, medium MS-d-4 containing Murashige and Skoog basal components plus 2.0 mg/l indole-3-acetic acid (IAA) and 2.5 mg/l kinetin was the most effective for plant regeneration. Numbers of albino plants and calli developing only roots increased directly with callus-induction time, whereas the frequency of plant regeneration decreased. Regenerated plants had varied numbers of chromosomes in root tip cells: 10, 15, 20, 40, and 60. The 29 regenerated plants that reached maturity, however, were highly fertile and contained only 10 bivalents in pollen mother cells. Normal chromosome number and behavior for the regenerated plants suggest that induced calli originated from cells other than microspores. However, spontaneous chromosome doubling in microspore-derived haploids may occur. The appearance of albinos also implies that haploids may have been produced from anther culture.Joint contribution of the Dept. of Agronomy and USDA-ARS, Kansas Agricultural Experiment Station, Manhattan, KS 66506-5501, USA. Contribution no. 88-566-J.     

Cytogenetical studies on African rice,Oryza glaberrima Steud. 3. Primary trisomics produced by pollinating autotriploid with diploid

Summary In order to promote cytogenetical studies on Oryza glaberrima (2n=2x=24), I produced primary trisomics in the progeny of the autotriploid. When the autotriploid was pollinated with the diploid, 136F1 seeds (5.8% of the number of spikelets pollinated) were obtained. The numbers of chromosomes of 31 surviving progeny varied from 24 to 28, with plants having 2n=25 occurring at the highest frequency (34.4%). The primary trisomics differed from one another as well as from the normal disomics in many aspects. The differences were attributed to a single extra chromosome in each trisomic type. Nine primary trisomics were classified into 8 types on the basis of morphological and reproductive features. Seeds were obtained from all primary trisomics except for Type D by self pollination, open pollination or pollination with the disomics. I was able to maintain 7 types of primary trisomics by seed propagation. The primary trisomics which are capable of expressing subtle genetic dissimilarities should be useful for studying the relationship between the two cultivated species of rice at the level of individual chromosomes.     

Medium, Explant and Genotype Factors Influencing Shoot Regeneration in Oilseed Brassica spp.

The effects of culture media, explants and genotypes on shoot regeneration in oilseed Brassica species were examined in this study. The maximum shoot regeneration frequency was obtained in Murashige and Skoog medium supplemented with 3 mg l^?1 6‐benzylaminopurine and 0.15 mg l^?1 1‐naphthaleneacetic acid. The addition of 2.5 mg l^?1 AgNO₃ was very beneficial to shoot regeneration in B. napus and Ag₂S₂O₃ (10 mg l^?1) was even superior to AgNO₃ (2.5 mg l^?1). Explant age, explant type and carbon source also significantly affected shoot regeneration. Four‐day‐old seedlings of cotyledonary explants showed the maximum shoot regeneration frequency and number of shoots per explant. Of the four explants – peduncles, hypocotyls, cotyledons and leaf petioles – cotyledons produced the highest shoot regeneration frequency (56.67 %). Four carbon sources – glucose, maltose, starch and sucrose – were compared for their respective effects on shoot regeneration from cotyledonary explants. Sucrose appeared to be the best carbon source for shoot regeneration with the highest shoot regeneration frequency (76.00 %). Considerable variation in shoot regeneration from cotyledonary explants was observed both between and within Brassica species. The shoot regeneration frequency ranged from 10.00 % for cv. R5 (B. rapa) to 83.61 % for cv. N1 (B. napus). Two B. napus, one B. carinata and one B. juncea cultivars exhibited shoot regeneration frequency higher than 70 %. In terms of the number of shoots produced per explant, B. rapa showed the highest variation, ranging from 5.64 for cv. R3 to 1.33 for cv. R5. Normal plantlets were regenerated from all induced shoots and developed normally. The regenerated plants were fertile and identical with the source plants.     

Transgenic fertile soybean plants derived from somatic embryos transformed via the combined DNA-free particle bombardment and <Emphasis Type="Italic">Agrobacterium</Emphasis> system

An Agrobacterium-mediated transformation procedure for soybean [Glycine max L. Merrill] proliferating somatic embryos is here described. The Agrobacterium tumefaciens LBA4404 strain harboring pTOK233, pCAMBIA1390-olp or pH7WG2Dwrky plasmids was used to mediate gene transfer into the plant genome. Prior to Agrobacterium inoculation, proliferative soybean embryogenic clusters were microwounded by DNA-free tungsten particle bombardment. Three independent transformation experiments were performed. In Experiment I, 26 transgenic plants were obtained from a unique clone of cv Bragg, while 580 plants were recovered from 105 clones of cv IAS5. In Experiment II, a single hygromycin-resistant clone of cv BRSMG68 Vencedora was recovered and gave rise to five plants. In Experiment III, 19 plants of cv Bragg and 48 plants of IAS5 were recovered, representing five and 14 independent transformation events, respectively. PCR and Southern analyses confirmed the transgenes’ integration into plant genomes. Transgenic plants were fertile. They flowered, set pods and seeds. Transgene segregation in two T₁ progenies fits the Mendelian pattern (3:1 transgenic:non-transgenic plants). This is the first report of transgenic fertile soybean plants obtained from somatic embryogenic tissues transformed by the system that combines DNA-free particle bombardment and Agrobacterium.     

Regeneration of fertile doubled haploid plants from colchicine-supplemented media in wheat anther culture

The effect of colchicine added to induction medium for the production of fertile doubled haploid plants after in‐vitro anther culture was studied in wheat, Triticum aestivum L. For this, one winter and two spring wheat varieties were used. Anther cultures of the three genotypes were treated with 0.03% colchicine for 3 days at the beginning of microspore induction. Colchicine had no significant effect on anther response and embryoid production of the genotypes examined. However, in the winter wheat genotype ‘Mv Szigma’, colchicine caused a significant reduction in microspore‐derived structures. A significant decrease was also observed in plant regeneration ability of two genotypes (‘Vergina’ and ‘Acheloos’) after colchicine treatment. In addition, a significant reduction of the albinos produced was observed in all genotypes after olchicine treatment. In contrast, the regenerants obtained from the colchicine‐supplemented induction media produced significantly higher percentages of fertile plants in all genotypes. However, the level of fertility, was significantly different among the fertile plants obtained. This, together with the observation that in the case of the winter wheat variety the colchicine treatment resulted in 100% completely fertile plants with a high seed‐setting ability indicate that there is space for further improvement of the method when it is applied to spring cultivars. Finally, the increased number of seeds per 100 plated anthers obtained from all three genotypes after colchicine treatment, clearly demonstrates that the addition of colchicine to induction medium was superior to the conventional anther culture method and it could therefore be introduced into wheat breeding programmes.