Cytological Study of Callus and Regenerated Plants of Sunflower (Helianthus annuus L.)

In vitro culture of shoot apical meristems of Helianthus annuus was studied to determine the cytological condition of calli and regenerated shoots. A broad chromosome mosaicism (aneusomary) in bath callus and regenerated shoots was found, reflecting the cytological conditions of the explain. During in vitro plant development, a diplontic selection took place that was more rapid when very precocious in vitro flowering occurred.     

Genetic variability in plants regenerated from in vitro culture of sunflower (Helianthus annum L.)

In vitro regenerated sunflower (Helianthus annuus L.) plants (R₁) were self-fertilized and the R₂ generation was evaluated for qualitative traits. A broad range of phenotypic variation was observed and mutation frequencies were calculated. Some in vitro induced variant phenotypes were similar to known spontaneous or induced mutations in sunflower, while others were new. Chlorophyll and carotenoid deficiencies, chimaerical variegation, fasciated stem and capitulum, abnormal shoot development, and other morphological variations, were noted. Substitution of anthers with petaloid structures in a disk-floret mutant indicates a possible homeotic mutation induced by in vitro tissue culture.     

Somaclonal variation of regenerated plants in chili pepper (Capsicum annuum L.)

The morphological and genetic variations in somaclones of chili pepper (Capsicumannuum L.) derived from tissue culture were evaluated. Cotyledonary node explants of cultivars, Shishitou and Takanotsume, were cultured on Murashige and Skoog (MS) medium supplemented with benzyladenine (BA)5 mg/l for shoots regeneration and regenerated shoots were rooted on MS medium supplemented with naphthalene acetic acid(NAA) 0.1 mg/l and indol-3-butyric acid(IBA) 0.05 mg/l. The regenerated plants(R₀) were selfed to obtain seeds for next generation (R₁ lines). Qualitative characters were studied in R₀generation and both qualitative and quantitative characters were studied in R₁ generation. In R₀ generation, variations were noticed in plant growth habit, stem color, flower color and color of unripe fruits, and expression of anthocyanin in unripe fruits. Comparison among the R₁ lines and their parents were made for morphological and agronomic characters. Significant variation among R₁ lines and differences between R₁ lines and their parents were observed. Genetic variations among three somaclones were revealed by random amplified polymorphic DNA (RAPD) analysis. Variation, such as early flowering and increase of yield components, is an indication of the response to selection for any specific character. Occurrence of productive variants among somaclones of established cultivars, like Shishitou and Takanotsume, indicates the possibility of their improvement through somaclonal variation. This revised version was published online in July 2006 with corrections to the Cover Date.     

Application of in vivo and in vitro mutation techniques for crop improvement

Summary Conventional mutation techniques have often been used to improve yield, quality, disease and pest resistance in crops, or to increase the attractiveness of flowers and ornamental plants. More than 1700 mutant varieties involving 154 plant species have been officially released. In some economically important crops, e.g. barley, durum wheat and cotton, mutant varieties occupy the majority of cultivated areas in many countries. Mutation techniques have become one of the major tools in the breeding of ornamentals such as alstroemeria, begonia, chrysanthemum, carnation, dahlia and streptocarpus. The use of in vitro techniques such as anther culture, shoot organogenesis, somatic embryogenesis and protoplast fusion can overcome some of the limitations in the application of mutation techniques in both seed and vegetatively propagated crops. In vitro culture in combination with induced mutations can speed up breeding programmes, from the generation of variability, through selection, to multiplication of the desired genotypes. The expression of induced mutations in the pure homozygote obtained through microspore, anther or ovary culture, can enhance the rapid recovery of the desired traits. In some vegetatively propagated species, mutations in combination with in vitro culture technique, may be the only method of improving an existing cultivar. Currently, many molecular studies rely on the induction and identification of mutants in model species for construction and subsequent saturation of genetic maps, understanding of developmental genetics and elucidation of biochemical pathways. Once identified and isolated, the genes that encode agronomically-important features can be either introduced directly into crop plants or used as probes to search for similar genes in crop species. It seems most likely that the recent developments based on these technologies will soon provide improved methods for selection of desired mutants.     

Molecular mapping of major genes and quantitative trait loci determining flowering time in response to photoperiod in barley

Two major genes (eam8 and eam10) and two quantitative trait loci (QTL) determining flowering time in barley were associated with restriction fragment length polymorphism markers. The loci eam8 and eam10 were found to map in regions of chromosomes 1HL and 3HL, respectively, already estimated from previous classical linkage analyses. While investigating doubled haploid lines of a spring habit barley mapping population, two QTL for flowering time were detected on chromosomes 1HL and 7HS, respectively, when the material was grown under long photoperiod conditions. When growing the same lines under short photoperiod, no QTL were discernible. Allelic and homoeologous relationships with flowering time loci described earlier in barley and other Triticeae species 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.     

Genetic mapping of quantitative trait loci in rye (Secale cereale L.)

Using the marker information of 275 F₂ plants quantitative traits determining morphological and yield characters were studied analyzing F₃progenies grown in four different experiments at three sites. The map constructed contains 113 markers including the major dwarfing gene Ddw1 with an average distance of about 10 cM between adjacent markers. Of the 21 QTLs detected ten were found to map on chromosome 5RL in the region of Ddw1. Beside the expected effects on plant height and peduncle length that are most probably due to the presence of the major dwarfing gene, additional effects on yield characters and flowering time were discovered in that region which may be caused by pleiotropic effects of Ddw1. An additional supposed gene cluster consisting of four QTLs controlling flowering time and yield components was discovered in the centromere region of chromosome 2R. Further loci are distributed on chromosomes 1R (1), 4R (1) 6R (3) and 7R (1). The map positions of the quantitative trait loci detected in rye are discussed in relation to major genes or QTLs determining agronomically important traits in other cereals. This revised version was published online in July 2006 with corrections to the Cover Date.     

Dissection of genetic variance of fibre quality in advanced generations from an interspecific cross of Gossypium hirsutum and G. barbadense

The interspecific genetic introgression approach has been shown to facilitate the detection and dissection of quantitative trait loci (QTL). A population consisting of 121 F₆ recombinant inbred lines was developed by crossing Gossypium hirsutum cv. ‘Handan 208’ and G. barbadense cv. ‘Pima 90’ via single‐seed descent. Genotyping indicated that the mean ‘Pima 90’ allele frequency at each locus was 21%. Phenotyping and phenotypic distribution indicated a trend of return of individual lines’ characters to ‘Handan 208’ coupled with a wide variance for each trait. Significant loci influencing fibre quality were detected by one‐way analysis of variance (anova; P < 0.005) and association analysis [?log₁₀(P) ≥ 3]: five and three markers for fibre length, four and one marker(s) for uniformity, two and one marker(s) for micronaire, 13 and 15 markers for strength, six and 10 markers for elongation, respectively. Two‐way anova based on genotypes of all marker loci combination showed that 807 two‐locus combinations were significant, and two‐way anova based on marker genotypes of QTL markers combination showed five significant digenic interactions (P < 0.01).     

Effects of loci determining photoperiod sensitivity (Ppd-H1) and vernalization response (Sh2) on agronomic traits in the 'Dicktoo'×'Morex' barley mapping population

The objectives of this research were to determine the individual and interaction effects of the Ppd-H1 and Sh2 loci on agronomic traits under short- and long-photoperiod regimes. Nineteen doubled haploid (DH) lines from the ‘Dicktoo’בMorex’ mapping population, which represented the four genotypes at the Ppd-H1 and Sh2 loci, were pheno-typed in controlled environment photoperiods. Both Ppd-H1 and Sh2 had significant effects on several agronomic traits, in addition to their role in determining first node appearance and flowering time. The magnitude of these effects depended on daylight. Under long-day conditions (18 h) Ppd-H1, and under short-day conditions (12 h) Sh2 was a significant determinant of most characters. The interactions between these two loci were significant for several characters, particularly for yield components, under both long- and short-photoperiod regimes. Under the long-day treatment, Ppd-H1 influenced plant height through the determination of node number. There was an epistatic association between the two loci for both 1000-kernel weight and tillering. The combination of photoperiod insensitivity and vernalization requirement caused a significant increase in tillering. This was paralleled by a decrease in 1000-kernel weight. Under the long-day treatment, neither Ppd-H1 nor Sh2 influenced plant yield. Under short-day conditions, the combination of photoperiod insensitivity and vernalization requirement had a pronounced negative effect on plant yield.     

Breeding the common bean (Phaseolus vulgaris L.) for resistance to bean common mosaic virus: alternatives to backcrossing

A series of field experiments was undertaken in order to determine whether resistance to bean common mosaic virus (BCMV) could be incorporated into genotypes of the common bean (Phaseolus vulgaris L.) suitable for cultivation in Zimbabwe without recourse to backcrossing. Six inbred genotypes carrying the resistance-conferring alleles at the loci I and Bc-3 were crossed with five locally-adapted inbred genotypes. The first experiment comprised F₃ progeny rows, each derived from a single unselected F₂ plant, the second, F₃ bulks selected for resistance, and the third, a comparison of selected and unselected F₂-derived F₄ lines. The number of days to flowering and to maturity, the incidence of mosaic and necrosis symptoms, seed yield and seed size were recorded. There was evidence that late flowering and maturity were associated with BCMV resistance in some crosses, though not strongly enough to present an obstacle to plant breeding. The incidence of virus symptoms and seed yield were influenced by genetic factors additional to the major resistance genes, and variation in seed yield was present not only between bulk populations of crosses, but also between single-row plots of lines within crosses. This indicates that early-generation selection for yield in the presence of BCMV, even among progeny selected for BCMV-resistace, is likely to be effective. However, the variation in yield among F₄ lines was least in the highest-yielding crosses, which may represent a limit to successful selection for yield. Seed size was partly under additive genetic control, but there was also evidence of non-allelic interactions. There was no association between large seed size, preferred by consumers, and susceptibility to BCMV in the progeny, indicating that the association between these characters in the parent lines is fortuitous and will not present an obstacle to plant breeding. It is noted that a considerable amount of useful genetic information can be obtained without recourse to elaborate crossing schemes, provided that unselected progeny are included in experiments as controls. The evidence presented indicates that resistance to BCMV can be combined with appropriate values of maturity date, yield and seed size without the need for backcrossing.     

An assessment of morphogenic and transformation efficiency in a range of varieties of potato (Solanum tuberosum L.)

Summary To provide a truly genotype-independent transformation system, it is necessary to be able to transform a wide range of potato genotypes. The ability to regenerate shoots in vitro was determined for 34 potato varieties using tuber disc explants. Following a culture regime used extensively in previous studies with the variety Desiree, half of the varieties could be regenerated from tuber discs and half could not. From a sample of varieties that could be regenerated from tuber discs, all but one variety gave transgenic plants. Twelve varieties were evaluated for the capacity to regenerate shoots from leaf and internode explants excised from in vitro grown plants. All of the varieties tested regenerated adventitious shoots. Leaf and internode explants from 5 varieties were subsequently used for transformation, and transgenic plants were produced from two potato varieties that did not give transgenic plants from tuber disc explants. Some varieties could not be transformed by either method, and will require modification of the in vitro regeneration and transformation system to be successful.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid     

Genetic basis of resistance against two Tospovirus species in tomato (Lycopersicon esculentum)

Summary A study was conducted to determine if the chlorsulfuron tolerance existing in barley could be improved with the use of in vitro tissue culture. Differential herbicide concentration led to variable culture response for callus culture.Plants regenerated from callus culture were evaluated for chlorsulfuron tolerance in the greenhouse. Regenerants showed improved tolerance when compared to the control or unselected tissue culture-derived lines.     

In vitro screening of strawberry plants for cold resistance

Formation of embryo autonomy of strawberry, plant regeneration fro membryo components, plant freezing conditions in vitro and the possibility to differentiate objectively genotypes by freezing them in vitro and in vivo were studied to create strawberry screening technology in vitro for cold resistance. It was established that autonomy of strawberry embryos manifests itself not earlier than on 14–16^th day after pollination and full autonomy is reached on 20–22^nd day. Plants regenerated from 26 days old embryos grew most intensively. At the highest rate strawberry plants regenerated from an isolated embryo axis on MS medium without phytohormones, and from rescued cotyledons x on the medium with 1.0 BA and 0.5 NAA. The temperature interval, at which genotypes differentiated according to cold resistance in vitro, was -8 to 12 ^°C. Differentiation of strawberry genotypes according to this character conformed to their differentiation in vivo, provided hardening proceeded not less than 21 days. The correlation between cold resistance in vitro and in vivo reached 0.93. Domination of cold resistance manifested itself in strawberry seedlings from various crossing combinations. This revised version was published online in August 2006 with corrections to the Cover Date.     

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.     

Identification of quantitative trait loci for flowering-related traits in the D genome of synthetic hexaploid wheat lines

The gene pool of Aegilops tauschii, the D-genome donor of common wheat (Triticum aestivum L.), can be easily accessed in wheat breeding, but remains largely unexplored. In our previous studies, many synthetic hexaploid wheat lines were produced through interspecific crosses between the tetraploid wheat cultivar Langdon and various A. tauschii accessions. The synthetic hexaploid wheat lines showed wide variation in many characteristics. To elucidate the genetic basis of variation in flowering-related traits, we analyzed quantitative trait loci (QTL) affecting time to heading, flowering and maturity, and the grain-filling period using four different F₂ populations of synthetic hexaploid wheat lines. In total, 10 QTLs located on six D-genome chromosomes (all except 4D) were detected for the analyzed traits. The QTL on 1DL controlling heading time appeared to correspond to a flowering time QTL, previously considered to be an ortholog of Eps-A ^m 1 which is related to the narrow-sense earliness in einkorn wheat. The 5D QTL for heading time might be a novel locus associated with wheat flowering, while the 2DS QTL appears to be an allelic variant of the photoperiod response locus Ppd-D1. Some of the identified QTLs seemed to be novel loci regulating wheat flowering and maturation, including a QTL controlling the grain filling period on chromosome 3D. The exercise demonstrates that synthetic wheat lines can be useful for the identification of new, agriculturally important loci that can be transferred to, and used for the modification of flowering and grain maturation in hexaploid wheat.     

In vitro selection for oleic and linoleic acid content in segregating populations of microspore derived embryos of Brassica napus

Microspore derived embryos (MDEs) in Brassica napuscontain large amounts of storage lipids which show a genotype specific fatty acid composition (FAC). One cotyledon of regenerating emblyos can be dissected at an early stage during the in vitro culture and used for fatty acid analysis. Thus, in breeding programmes to modify oil quality, only MDEs having the desired FAC need to be regenerated to plantlets and transferred to the greenhouse. In the present study the applicability of this method for the selection of a high oleic acid content and a low linoleic acid content in the seed oil has been tested by crossing a Brassica napus mutant line having a high oleic acid (C18:1) content in the seed oil (75%) with a wild type doubled haploid line with 62% C18:1 in the seed oil. Microspore culture was applied to the F1 plants. In total 59 MDEs were obtained, from which 31 were cultured with and 28 without 15μM abscisic acid for 3 weeksin vitro. One cotyledon was dissected under aspetic conditions and used for fatty acid analysis. The remaining part of the embryos were further regenerated to plantlets and transferred to the greenhouse to obtain seeds after self pollination. Seeds harvested from the doubled haploid lines in the greenhouse were used for fatty acid analysis and also for growing in the field. The abscisic acid treatment of the MDEs generally improved the correlations for linoleic and oleic acid between the MDEs and the seeds harvested in the greenhouse and the field. The correlations ranged from 0.68** to 0.81**.This indicates that selection for high oleic acid can be started already during an early stage of the in vitro culture. This revised version was published online in July 2006 with corrections to the Cover Date.     

Flowering behaviour,male sterility and berry setting in tetraploid Solanum tuberosum germplasm

Summary Six hundred and seventy six accessions of cultivated potato (Solanum tuberosum ssp. tuberosum) from 25 countries, were studied for flowering and fruiting behaviour under long days (12–14 h). Flowering intensity ranged from dropping of floral buds just after initiation to profuse blooming. The majority (58.3%) of the accessions bloomed profusely, though 20.4% of the accessions did not bloom at all. Weeks to flowering ranged from 6 to 15 and the majority (66.5%) of the flowering accessions bloomed within 8 to 9 weeks after planting. Duration of flowering ranged from 1 to 10 weeks and the majority (68.1%) of the flowering accessions bloomed for 1 to 4 weeks only. Twentythree per cent of the flowering accessions were completely male sterile. Maximum male fertility was 90% only. No berry setting was observed in 31.8% of the flowering accessions. Only 54.3 per cent of the accessions were found to be fertile in all respects and could be used both as male and female parents. Premature bud abscission was the major cause of sterility. Peru was the best source of profuse-flowering genotypes, Poland was the best source of early flowering genotypes and Mexico was the best source of long duration flowering and good berry setting genotypes. The results suggested that flower bud formation; the growth and development of mature flowers; weeks to flowering and duration of flowering are independent characters controlled by different genes of quantitative nature. Berry setting and duration of flowering were closely associated (r=0.95). Genetic as well as environmental factors interfered with the developmental process leading to flower production and berry setting at different times in different genotypes. The practical implications of these results for true potato seed production are discussed.Publication No. 1298, CPRI, Shimla.     

Epistasis among the three major flowering time genes in rice: coordinate changes of photoperiod sensitivity,basic vegetative growth and optimum photoperiod

Flowering time is affected not only by photoperiod sensitivity (PS) but also by basic vegetative growth (BVG) and optimum photoperiod (OP), although their developmental and genetic relationships are not well understood. The present study was carried out in rice to examine to what extent these three developmental components are modified by the three flowering time genes, Se1 (= Hd1), Ef1 and e1 (= m-Ef1), which are known to contribute to flowering time in temperate and tropical regions of rice cultivation. Photoperiodic response curves were estimated under controlled conditions of different growth regimes, using eight near-isogenic lines possessing different combinations of the alleles at the three loci. The results showed that each of the components is greatly affected by the main effect of the genes, temperature and their epistasis, indicating that none of the three genes controls flowering time by altering any single component in PS, BVG or OP. Epistasis was detected more frequently among the three genes than reported before, suggesting that epistasis contributes to flowering time by changing PS, BVG and OP differently. The comparison of the nucleotide sequences suggested that Ef1 is the same as Early heading date 1 (Ehd1). Since the two genes Se1 (= Hd1) and Ef1 (= Ehd1) are known to up-regulate the rice homolog of Arabidopsis FT, it is suggested that the detected epistasis may respond to diverse environments by modulating the CO/FT system conserved in flowering plants.     

Detection of an earliness per se quantitative trait locus in the proximal region of wheat chromosome 5AL

A homoeologous quantitative trait locus to that of eps5L on barley chromosome 5H was identified in a syntenic region of wheat chromosome 5A. Wheat single chromosome recombinant lines (SCRs) were developed from a cross between ‘Chinese Spring’(‘Cappelle-Desprez’ 5A) and ‘Chinese Spring’(Triticum spelta 5A), these were grown together with the parental controls under different vernalization and photoperiod regimes. The variation for ear emergence time accelerated heading induced by the T. spelta segment indicated an effect associated with the Xcdo412-Xbcd9 interval. Since no differences between the SCRs and controls in responses to vernalization and photoperiod treatments were detected, this effect was identified as an earliness per se gene, Q Eetocs-5 A.2, which may be homoeologous to the eps5L quantitative trait locus of barley. Xbcd926 has been found to be closely linked to the rice flowering time quantitative trait loci, QHd9a or FLTQ2, on chromosome 9, suggesting possible relationships among the quantitative trait loci across wheat, barley and rice genomes.     

Efficient production of doubled haploid wheat plants by in vitro treatment of microspores with trifluralin or APM

Isolated microspore cultures from two doubled haploid (DH) lines of wheat, Triticum aestivum L., were used to develop an in vitro chromosome-doubling protocol. During the initial 24 h or 48 h of culture the microspores were treated with either of the two antimicrotubule herbicides trifluralin or amiprophos-methyl (APM) in concentrations ranging from 0.1 μM to 10μM. Untreated control cultures yielded 209 embryos per 100000 microspores, which is the equivalent of one spike. Among the regenerated plantlets 67% were green, and 15% of the flowering plants were spontaneously chromosome doubled. Treatments with both the herbicides had a significant effect on chromosome doubling, measured as the percentage of fertile regenerants. With the best combination of treatment duration (48 h) and herbicide concentration (10/μM) the percentage of fertile plants among regenerants could be increased up to 74% with APM and up to 65% with trifluralin. The largest numbers of DH plants per spike could be obtained with herbicide concentrations at 1–3 μM. Treatments with either herbicide at these concentrations resulted in an estimated average between the two genotypes of 27 DH plants per 100 000 microspores. These results demonstrate the high potential of APM and trifluralin as chromosome-doubling agents in isolated microspore cultures. The in vitro treatment integrated into tissue culture procedures will constitute an efficient method for chromosome doubling in future wheat breeding