In vitro techniques for selecting wheat (Triticum aestivum L.) for Fusarium-resistance. II. Culture filtrate technique and inheritance of Fusarium-resistance in the somaclones

Summary Calli of resistant, intermediary and susceptible wheat (Triticum aestivum L.) varieties were selected using culture filtrates of Fusarium graminearum and F. culmorum and the regenerants were evaluated for resistance up to R₃. Czapek-Dox broth medium was inoculated with mycelia of Fusarium isolates and incubated for 2–6 weeks. Filtrates were added to MS callus growing medium, then 5 weeks-old calli were transferred onto this medium (MST) for 4–5 weeks. MST containing 30% filtrate was found to be suitable for selection. Resistant calli were transferred again to fresh MST for further two selection cycles. The surviving calli produced less fertile regenerated lines (R₀) than the non-selected ones. Among 18 R₁ lines tested for Fusarium-resistance in the seedling stage by artificial inoculation in the greenhouse, two (11.1%) were significantly more resistant, one (5.6%) was more susceptible than the original cultivar and the rest (83.3%) behaved similarly to the donor plants. Among unselected R₃ lines of three varieties, practically the same number of resistant plants were found as among the related selected ones. When the R₃ selfed generations obtained through double-layer and culture filtrate selection techniques were tested for Fusarium-resistance, 35.7% of the lines were found to be more resistant than the original cultivars, none was more susceptible and 64.3% had a reaction similar to that of the source materials. Thus, inheritance of the disease reaction was not stable in all cases. Success of in vitro selection for Fusarium-resistance depended also on the genotype, and toxin analysis showed that although being effective, the selective media contained deoxynivalenol only exceptionally. In selecting wheat for Fusarium-resistance in vitro, the culture filtrate technique proved better than the double-layer procedure.     

In vitro selection of wheat callus tolerant to high levels of salt and plant regeneration

Summary Embryogenic calli isolated from immature embryos of four wheat cultivars were subjected to three in vitro selection methods for salt tolerance. The effect of NaCl on the selected and unselected cell lines has been investigated. The results indicated that the relative growth rate of callus decreased as the concentration of NaCl increased in both callus lines. The selected callus line gave a higher growth weight in the presence of NaCl in the medium and was highly significant as compared with unselected callus line across medium protocols in all wheat cultivars. The dry weight of both kinds of callus lines of all wheat cultivars increased markedly with increasing NaCl concentration in most cases. The Na⁺ and Cl^- contents of both callus lines were increased with increasing salinity levels while K⁺ content was decreased. The selected callus line of each cultivar at the same salinity level produced significant amounts of Na⁺, K⁺ and Cl^- higher than the unselected callus line in most salinity levels. However, the unselected callus lines of the cultivars Giza-157 and Sakha-90 at the same salinity level produced significant amounts of K⁺ higher than the selected callus line in most salinity levels. The proline content of both kinds of callus lines for all wheat cultivars was increased with increasing salinity level. However, the selected callus line gave a significantly higher proline content than the unselected callus line in all wheat cultivars at the same Salinity level. Results from the in vitro selection for NaCl tolerance showed that the stepwise method of increasing NaCl in the medium was more effective for plant regeneration than other methods.     

Genetic factors influencing the regeneration ability of rye (Secale cereale L.). II. Immature embryos

Summary Immature embryos of seven rye inbred lines were cultured on modified MS medium containing 3 mg/dm^–3 2,4-D. According to thein vitro response lines were divided into four groups: (1) those producing non-embryogenic callus (NEC) from above 30% of the embryos and having a high rate of non-responding (NR) embryos, (2) those producing non-embryogenic callus (NEC) from the majority of embryos, (3) those producing NEC by the majority of embryos with a high percentage of calli regenerating roots, (4) those producing embryogenic callus (EC) and regenerating plants by above 50% of the embryos. The inheritance of these response types was analysed in F₁, F₂, and F₃ generations of crosses of some lines. The results obtained indicate that EC production and both plant and root regeneration are determined by recessive genes whereas the reduced ability for NEC production most probably by dominant genes. The lack of response is controlled by at least two interacting genes.     

Utilization of Asymmetric Somatic Hybridization for the Transfer of Disease Resistance from Brassica nigra to Brassica napus

Asymmetric somatic hybrid calli were produced between Brassica napus and a transgenic (Hyg ^R) line of B. nigra using a donor recipient fusion method for the production of cybrids. The transgenic line of B. nigra used as a donor also possessed genetic resistance to the pathogenic fungi Phoma lingam and Plasmodiophora brassicae. Using hygromycin for selection, 332 hybrid calli were obtained from which 30 produced shoots (1—-20 per callus) which were rooted on a hormone-free culture medium. The rooted shoots were transferred into soil and cultivated in a growth chamber where the plants were tested for resistance against the two pathogens. Out of 129 hybrid plants tested for resistance against P. brassicae, 30 (23.3 %) plants proved to be resistant and from 78 plants tested for resistance against P. lingam, 41 (52.6 %) plants remained disease-free after infection.     

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.     

Culture of unpollinated ovules,ovaries, and flower buds in some species of the genus Allium and haploid induction via gynogenesis in onion (Allium cepa L.)

Summary Induction of haploid plants is of great importance for breeding purposes because of the possibility to obtain from haploids homozygous material by artificial chromosome doubling in relatively short times. The present study reports the first evidence of successfull haploid induction in onion. Isolated ovules, ovaries, or whole flower buds of different Allium species were cultured on BDS agar medium. Testa browning in the ovules and an extensive growth of the latter were observed. In cultures of ovaries and flower buds, development of callus and subsequent regeneration of plantlets from the region of the nectaries were observed. In leek, sometimes supernumerary flower organs like ovules were formed in this callus. In onion (Allium cepa L.), plantlets developed from the ovules in all culture methods. Chromosome numbers of these plantlets were counted in root tip squash preparations. They were found to be haploid. Haploid plants were significantly smaller than diploid ones. They were transferred to soil and developed until bulb formation. Because of their importance for breeding, haploid plants obtained by gynogenesis are further stored in vitro.     

High frequency regeneration and heritable somaclonal variation in Brassica juncea

Summary Multiple shoot formation in cotyledonary callus of Indian mustard (Brassica juncea cv. Prakash) was induced on modified MS media supplemented with high cytokinin (kinetin or zeatin) and low IAA concentrations. Complete plants were obtained on prolonged incubation of shoots on the same medium. 6-Benzyladenine alone or in combination with IAA or NAA did not support plantlet regeneration. A total of 71 plants were transferred to greenhouse. The seed, however, could be collected from 37 plants only. The seed was sown in the field to evaluate the material for somaclonal variation in R₁ generation. Data were recorded for yield, plant height, number of primary branches, siliqua number, 1,000 seed weight and oil content. Somaclonal lines showed tremendous amount of variation for all the characters studied. A number of plants in this generation showed significantly higher yield and/or other improved agriculturally important characteristics as compared to the control. A line with dwarf plant type was also identified. A number of plants were selected from this generation and carried forward to R₂ generation. Most of these lines bred true in R₂ generation. The material seems to be very promising for future breeding programmes.     

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.     

In vitro screening and field evaluation of tissue-culture-regenerated sorghum (Sorghum bicolor (L.) Moench) for soil stress tolerance

Summary Sorghum bicolor (L.) Moench is generally quite sensitive to salt and acid (high aluminium) soil stresses, but quite tolerant of drought stress. As with any stress phenomenon, intra-specific variability exists within the genus. In vitro cell selection and somaclonal variation offer an alternative to traditional breeding methodology for generating improved breeding lines for hybrid development. A field selection protocol was developed for the three soil stresses and inter-stress evaluations were conducted in an effort to find multiple, stress-tolerant genotypes. The acid soil-drought stress, super-tolerant selections were located by the R₇ generation when exposed to a combined aluminium-drought stress field environment and when the regeneration population (number of regenerated lines from one callus source) was maintained at 15,000 plants or higher. A variant frequency of 0.1 to 0.2% for stress tolerance and acceptable agronomic traits among the surviving somaclones, provided an adequate number of phenotypes with desirable agronomic characteristics and a high level of soil stress tolerance. Subsequent research verified that the stress-tolerant regenerants had superior acid soil and drought stress tolerance to that of the donor parents, that their yield capabilities under stress were superior to their parents, and that their stress tolerance attributes were transferred in hybrid combinations. In vitro selection was not effective in increasing the number of field stress survivors. In fact, superior germplasms were developed from non-stressed callus or salt-stressed callus. In vitro selection reduced regeneration frequency and subsequent survival of plants under field stress. In vitro-stressed regenerants should be subjected only to non-stressed environments to maintain population numbers for field selection and thereafter should be subjected to stress environments during later (R₅₊) generations. The optimal strategy for the exploitation of somaclonal variation may be through short-term cell culture (< 12 months) with no attempt at in vitro selection.     

Production of intergeneric hybrid plants between <Emphasis Type="Italic">Sandersonia aurantiaca</Emphasis> and <Emphasis Type="Italic">Gloriosa rothschildiana</Emphasis> via ovule culture (Colchicaceae)

Intergeneric hybrid plants between Colchicaceous ornamental plants, Sandersonia aurantiaca and Gloriosa rothschildiana, have successfully been produced via ovule culture. After 5 days of reciprocal cross-pollination, a few pollen tubes were observed in the ovary. Although seeds were obtained in both reciprocal cross-combinations, they did not germinate under ex vitro conditions. Ovules with placental tissues isolated 14 days after cross-pollination of S. aurantiaca × G. rothschildiana were cultured on a medium containing 0.01 mg l^–1 each of -naphthaleneacetic acid (NAA) and 6-benzyladenine (BA), on which 41.5% of ovules swollen and produced callus-like structures within 10 weeks. When such swollen ovules were transferred to a medium containing 0.1 mg l^–1 each of NAA and BA, 7.5% of the initially cultured ovules produced rhizome-like structures within 6 weeks. Among the rhizome-like structures, those derived from two independent ovules (3.7% of the initially cultured ovules) produced multiple shoots following transfer to a medium containing 0.25 mg l^–1 NAA and 2.5 mg l^–1 BA. Multiple shoot-derived plantlets were established on a plant growth regulator-free medium, and they were successfully transplanted to pots. Early verification of their hybridity was accomplished by flow cytometry (FCM) analysis, chromosome observation and rDNA analysis.     

Progress made in FEC transformation of cassava

In cassava friable embryogenic callus (FEC) has been used to obtain transgenic plants using particle bombardment, electroporation, and Agrobacterium tumefaciens. FEC cultures have been obtained in 6 of the10 tested genotypes. In all genotypes FEC could be regenerated into plants,however the efficiency differed between the genotypes. Almost all plants regenerated from 6 months old FEC cultures of TMS604444, Adira 4,Thai 5 and M7 were morphological similar to control plants. However, in R60 and R90 a large number of plants were not identical to control plants. Older FEC lines of TMS60444 have a reduced ability to regenerate plants and the plants show somaclonal variation. Somaclonal variation is observed in the same extend in transgenic and non-transgenic plants. The origin of this variation is both genetic and epigenetic. Luciferase based selection is less efficient in producing transgenic lines than chemical selection. Furthermore Agrobacterium tumefaciens mediated transformation is much more efficient than particle bombardment with respect to the production of transgenic lines. A tentative model is introduced which best describes the effect of different selection regimes on the time period required to produce transgenic plants. Kanamycin and stringent luciferase selection required a shorter period of time than selection based on hygromycin, phosphinothricin or non-stringent luciferase. However, a more significant reduction of time was obtained if young instead of old FEC lines of genotype TMS60444 were used for genetic modification. In accordance to the model these young FEC lines of TMS60444 produced transgenic plants within 4 months with both Agrobacterium tumefaciens combined with kanamycin selection and particle bombardment combined with stringent luciferase selection. This revised version was published online in July 2006 with corrections to the Cover Date.     

Green haploid plants from unpollinated ovary culture in tetraploid wheat (Triticum durum Defs.)

In Triticum durum Defs., gynogenesis was investigated on six genotypes, Cocorit, Isly, Jori, Oued Zénati, Sarif, issued from Morocco,and Cham1 from Syria. The experiments described here were made during two seasons (spring and summer) in which batches of 1,036 and 3,750 unpollinated ovaries were used. A 4 ^°C pretreatment was applied, during 7, 11 or 15 days and the A (modified Jähne's et al.,1991) and B (modifed San N?um's, 1976) induction media were compared. The spikes were harvested when microspores were at bi- or trinucleate stage. After the 4 ^°C cold pretreatment, they were sterilized, and the excised ovaries underwent the following phases and sequence of successive media. The A or B ‘induction’ media, induced cell divisions in the female gametophytes, and after 4 to 9 weeks in dark conditions, the swelling ovaries burst. The growing calluses were excised and transferred to 16 h. light onto a differentiation medium (dif); there generated shoots were then placed on a development (dev), followed by a rooting medium (r). The comparison of the two experiments showed that summer was the best season, for in vitro unpollinated ovary culture; this period corresponds to a growth of mother-plants during spring, while those developing in winter gave rise to lower scores. In Experiment 1 the best level of regenerated plants per 100 ovaries were obtained for Isly with 14.3% and for Oued Zénati with 10.1%. In Experiment 2, the best values were those of Isly with 21.8%, after a 7 day cold pretreatment and use of induction medium B, and of Cocorit with 18.1%. Jori, exhibited good results, in three define situations, with 17.1%, 16%, and 9.3%.Only Cham1 and Sarif showed very low scores or no plants. Jori remaining aside, for each genotype, the best values rose up from a specific treatment combination, as a very strong interaction was expressed between genotype, induction medium and cold pretreatment duration. After this ‘first phase’ of regeneration by ovary culture 150 haploid plants were obtained. When combining these results with other attempts, a total number of 191 green plants was regenerated, 90 for Isly, 74 for Jori, 19 for Cocorit, 7 for Oued Zénati and 1 for Cham1. In a ‘second phase’, permanent regenerating callus lines were obtained through subculture on dif medium. Depending on genotype, 2 to 9 transfers (up to one year for Jori) were performed, giving 507 regenerated plants. From direct regeneration and subcultures, a total of 698 plants were produced, all green and mostly haploid. Fertile doubled haploid plants were obtained, either spontaneously or by colchicine treatment, for all these genotypes. These results are of interest for breeders, who need pure lines for genotypes evaluation and for the creation of improved homozygous varieties.     

Factors affecting in vitro flowering and fruiting of green pea (Pisum sativum L.)

Multiple shoots were efficiently regenerated from cotyledonary node and shoot tip explants of Pisum sativum within 15 days on MS medium containing B₅ vitamins and supplelmented with 2.0 mgl^-1 6-benzylaminopurine. The elongated shoots produced on the same medium were excised and transferred to MS medium containing half strength ammonium nitrate (8.25 gml^-1) and supplemented with auxins (indole-3-butyric acid or naphthalene acetic acid) either alone or in combinations with gibberellic acid. Rooting and flowering were observed on the 7th and 15th day after their transfer to rooting medium. The flowers self-fertilised in vitro and produced mature pods within 25 days of rooting. These seeds were germinable both in vitro and in vivo. In vitro seeds sown in pots under field conditions developed into flowering plants, and subsequently produced pods with viable seeds. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

Towards development of Al-toxicity tolerant lines in indica rice by exploiting somaclonal variation

Somaclonal variations, induced in vitro, were used to enhance tolerance to aluminium (Al) toxicity in rice. Tolerant plants were developed through in vitro screening of embryogenic calli. The calli were derived from mature seed embryos and cultured on medium stressed with different concentrations of Al₂(SO₄)₃⋅18H₂O. Seed germination, callus induction, plantlet regeneration and callus health declined with increased concentration of Al. At higher Al concentrations, callus health deteriorated drastically with partial to total necrosis. Plantlet regeneration varied largely among varieties and treatments. The variety IR72 produced maximum plantlets among all genotypes tested. An amount of 60 ppm or more Al was highly toxic, which greatly reduced plantlet regeneration from callus. R₀ plantlets were grown under glasshouse. Based on the appearance of bronzing symptoms on leaves, the tolerant R₁ plants were selected. R₁ and R₂ lines derived from putative tolerant somaclones, were evaluated in fiberglass tanks filled with Al-toxic soil. R₃ population was evaluated in the field. A few lines derived from IR72 showed high yield and good plant type. The progenies at R₃ showed normal root growth under stressed environment in sand culture. The study revealed that in vitro screening would be an appropriate alternative to conventional breeding in evolving Al-tolerant lines as observed in case of other abiotic stresses. The technique was useful in creating de novo synthesized Al-tolerance character in rice.     

Monohaploid plants from anthers of a dihaploid genotype of Solanum tuberosum L.

Summary Monohaploid Solanum tuberosum plants were produced from the anthers of a dihaploid genotype. From another dihaploid genotype plants containing 36 chromosomes were obtained. For plantlet production anthers containing pollen at the uninucleate microspore stage were inoculated on a Linsmaier and Skoog-based medium supplemented with 1 mg/1 indole-3-acetic acid and 1 mg/1 benzyl aminopurine.Anthers from donor plants grown either in the summer or in the winter responded similarly on a range of media. Anther response in the form of callus induction and root formation was at least partly dependent on the genotype of the donor dihaploid plant.     

Effect of inbreeding and growth regulators on the in vitro androgenesis of wheatgrass, Agropyron glaucum

The capacity of haploid production by anther culture has been analysed in 42 crossbred genotypes (wild type) and in 32 genotypes from inbreeding generations (I₂-I₃) of wheatgrass, Agropyron glaucum. On potato-2 medium, about 50% of the genotypes investigated were capable of embryoid and callus induction. For the regeneration of haploid plants, five media containing various growth regulators were used. The crossbred genotypes were superior to the inbred lines in all the androgenesis parameters examined.     

Segregation of 12 isozyme genes among doubled haploid lines derived from a japonica x indica cross of rice (Oryza sativa L.)

Summary The segregation of 12 heterozygous isozyme markers was analyzed among F₂ plants and 51 anther culture (AC)-derived lines obtained from the japonica × indica cross of rice, IRAT 177 × Apura. All the lines except two were homozygous products of recombination of the two parental phenotypes. Doubled haploid (DH) lines derived from plants regenerated from the same callus were identical, confirming previously obtained results in rice. Surprisingly, some lines derived from different calli were also identical, suggesting a phenomenon of early callus fragmentation. All these observations at the isozyme level were confirmed by field evaluation. Deviations of segregations from the expected 1 : 1 ratio were observed at 4 loci among the DH lines. Among these, two were also noted among the F₂ plants. The two other distortions, both in favor of the japonica allele, were observed specifically in the AC-derived materials.Although this concerns a small proportion of the genes under study, it suggests that the embryogenic microsporal population does not represent a random gametic array. On the other hand, evaluation of recombination between isozyme genes located on chromosome 6 appears consistent with F₂ data and data previously recorded on the other japonica × indica crosses. The potential use of isozymes in breeding doubled haploids derived from remote crosses in rice is discussed.Abbreviations MCPA = 2-methyl-4-chlorophenoxyacetic acid - IAA = indolacetic acid - AC plant or line = anther culture-derived plant or line - DH line = doubled haploid line     

深山草莓花瓣离体培养诱导变异植株及性状稳定变异株系的品种特性

应用均匀设计法研究了影响深山草莓花瓣愈伤组织诱导及愈伤组织再分化变异植株的主要因子和水平。深山草莓花瓣愈伤组织诱导率超过65.0%以上的适宜培养基共有11个,经过组合优化后可知,培养基LS+TDZ2.06 mg?L-1+2,4-D0.60 mg?L-1对深山草莓花瓣愈伤组织诱导效果最好,诱导率为98.0%;其中,以培养基LS+TDZ2.20 mg?L-1 +2,4-D0.40 mg?L-1诱导形成的愈伤组织在10个适宜的分化培养基中培养均分化出再生芽苗,优化后的最佳再分化培养基为1/2LS+TDZ 2.30 mg?L-1+KT 1.38 mg?L-1,分化率为96.3%,田间试验发现变异率高达2.77%。对10个含有不同激素和浓度配比及1个优化后的培养基诱导形成的再生芽苗生根后经5年的田间栽培观察可知,有6个性状稳定的优良变异株系。这说明深山草莓花瓣在适当激素和浓度配比的条件下可产生变异植株,并能趋向稳定性状变异。     

Anther culture response of wild Oryza species

Thirteen different wild species of the genus Oryza were investigated for their response to anther culture and plant regeneration. Callus induction from microspores of anthers was found to be strongly dependent on the species. Although large numbers of anthers from wild Oryza species, including O. barthii, O. latifolia, O. australiensis, O. minuta, O. nivara, O. paraguagensis and O. eichingeri, were cultured, no calli could be obtained. However, calli were produced from anthers of O. punctata, O. perennis, O. alta, O. ridleyi and O. rufipogon, although the frequency of callus induction was different. Similar species-dependence was observed in plant regeneration from microspore-derived calli. In total, 62 plants were derived from anther culture, including 47 albino and 15 green plants (of which 26.7% were haploids) from O. perennis; for the first time, six albino plants were obtained from O. ridleyi. Phytohormone combinations in the callus induction medium were found to influence callus induction and different wild Oryza species exhibited their own preferred phytohormone combinations for anther culture. In general, NK medium containing suitable concentrations of auxin and cytokinin may be successfully applied for anther culture of selected wild Oryza species.