Chromosomal intervals responsible for tissue culture response of wheat immature embryos

To study the genetic mechanism underlying the tissue culture response (TCR) of immature embryos, callus induction and regeneration were performed in two separate trials using the recombinant inbred line (RIL) derived from a cross of Nanda2419 with Wangshuibai. In the first trial, immature embryos were collected from plants grown in the greenhouse in the winter of 2005; while in the second trial, immature embryos were collected from donor plants grown in the field during the growing season. Through whole genome screening, seven chromosome regions conditioning percent embryos forming embryogenic callus (PEFEC) and one conditioning percent callus pieces regenerating plantlets (PCRP) were detected. These QTLs were distributed on chromosomes of homoeologous groups 2, 3, 5 and 7. Among all, QPefec.nau-3B.2, QPefec.nau-7D, and QPcrp.nau-3A were consistently identified. The relationship of these identified wheat TCR QTLs with those of other cereal crops has been evaluated. PCR markers linked to TCR QTLs would facilitate germplasm identification, marker-assisted evaluation and utilization of these QTLs.     

The effect of rye chromosomes on callus induction and regeneration in callus cultures of immature embryos of wheat-rye substitution lines, Triticum aestivum L. cultivar Saratovskaia 29/Secale cereale L. cultivar Onokhoiskaia

The effect of individual rye chromosomes on the induction of callus and the character of its regenerating capacity was studied with cultured immature embryos of wheat-rye (Triticum aestivum L. cv. Saratovskaya 29-Secale cereale L. cv. Onokhoiskaya) substitution lines. The genotypic diversity of the substitution lines proved to significantly affect variation of parameters characterizing the major types of callus cultures, that is, frequencies of embryogenic calli, which are capable of shoot regeneration, and of morphogenic calli, which produce root structures. Functioning in the genotypic background of common wheat cultivar Saratovskaya, chromosomes 2R and 3R of rye cultivar Onokhoiskaya stimulated significantly the induction of embryogenic callus highly capable of shoot regeneration. Rye chromosome 2R present in place of chromosome 2D in the common wheat genome suppressed the induction of callus producing root structures. Rye chromosomes 1R and 6R suppressed the induction of embryogenic callus capable of shoot regeneration.     

The Effect of Rye Chromosomes on Callus Induction and Regeneration in Callus Cultures of Immature Embryos of Wheat–Rye Substitution Lines,Triticum aestivum L. Cultivar Saratovskaya 29–Secale cereale L. Cultivar Onokhoiskaya

The effect of individual rye chromosomes on the induction of callus and the character of its regenerating capacity was studied with cultured immature embryos of wheat–rye (Triticum aestivum L. cv. Saratovskaya 29–Secale cereale L. cv. Onokhoiskaya) substitution lines. The genotypic diversity of the substitution lines proved to significantly affect variation of parameters characterizing the major types of callus cultures, that is, frequencies of embryogenic calli, which are capable of shoot regeneration, and of morphogenic calli, which produce root structures. Functioning in the genotypic background of common wheat cultivar Saratovskaya 29, chromosomes 2R and 3R of rye cultivar Onokhoiskaya stimulated significantly the induction of embryogenic callus highly capable of shoot regeneration. Rye chromosome 2R present in place of chromosome 2D in the common wheat genome suppressed the induction of callus producing root structures. Rye chromosomes 1R and 6R suppressed the induction of embryogenic callus capable of shoot regeneration.     

Efficient callus induction and plant regeneration from mature embryo culture of winter wheat (Triticum aestivum L.) genotypes

Immature and mature embryos of 12 common winter wheat (Triticum aestivum) genotypes were cultured in vitro to develop an efficient method of callus formation and plant regeneration from mature embryo culture, and to compare the responses of both embryo cultures. Fifteen days after anthesis, immature embryos were aseptically dissected from seeds and placed with the scutellum upwards on a solid agar medium containing the inorganic components of Murashige and Skoog (MS) and 2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D). Mature embryos were moved slightly in the imbibed seeds. The seeds with moved embryos were placed furrow downwards in dishes containing 8 mg/l 2,4-D for callus induction. The developed calli and regenerated plants were maintained on 2,4-D-free MS medium. Plants regenerated from both embryo cultures were vernalized and grown to maturity in soil. Regenerated plantlets all maintained the hexaploid chromosome number. A strong genotypic effect on the culture responses was found for both explant cultures. Callus induction rate, regeneration capacity of callus and number of plants regenerated were independent of each other. Mature embryos had a high frequency of callus induction and regeneration capacity, and therefore, being available throughout the year, can be used as an effective explant source in wheat tissue culture. Received: 4 February 1997 / Revision received: 1 April 1997 / Accepted: 5 May 1997     

QTL mapping for some grain traits in bread wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

Grain traits are important agronomic attributes with the market value as well as milling yield of bread wheat. In the present study, quantitative trait loci (QTL) regulating grain traits in wheat were identified. Data for grain area size (GAS), grain width (GWid), factor form density (FFD), grain length-width ratio (GLWR), thousand grain weight (TGW), grain perimeter length (GPL) and grain length (GL) were recorded on a recombinant inbred line derived from the cross of NW1014?×?HUW468 at Meerut and Varanasi locations. A linkage map of 55 simple sequence repeat markers for 8 wheat chromosomes was used for QTL analysis by Composite interval mapping. Eighteen QTLs distributed on 8 chromosomes were identified for seven grain traits. Of these, five QTLs for GLWR were found on chromosomes 1A, 6A, 2B, and 7B, three QTLs for GPL were located on chromosomes 4A, 5A and 7B and three QTLs for GAS were mapped on 5D and 7D. Two QTLs were identified on chromosomes 4A and 5A for GL and two QTLs for GWid were identified on chromosomes 7D and 6A. Similarly, two QTLs for FFD were found on chromosomes 1A and 5D. A solitary QTL for TGW was identified on chromosome 2B. For several traits, QTLs were also co-localized on chromosomes 2B, 4A, 5A, 6A, 5D, 7B and 7D. The QTLs detected in the present study may be validated for specific crosses and then used for marker-assisted selection to improve grain quality in bread wheat.     

Control of tissue culture response in wheat (Triticum aestivum L.)

Summary The ability of immature embryos of wheat (Triticum aestivum L.) to respond to tissue culture has been shown to involve the group 2 chromosomes. The available group 2 ditelosomic and nullisomic-tetrasomic lines of Chinese Spring wheat were used to determine the chromosome arm location and chromosome dosage effect associated with the expression of tissue culture response (TCR). Significant differences were found between the aneuploid lines and the euploid control for the expression of both regenerable callus formation and callus growth rate. A model is proposed suggesting that a major TCR gene is located on 2DL and that 2AL and 2BS possess minor TCR genes. Furthermore, a major regulatory gene controlling the expression of TCR genes may be located on chromosome 2BL.     

Identification of QTLs associated with tissue culture response through sequencing-based genotyping of RILs derived from 93-11 × Nipponbare in rice (Oryza sativa)

Key message

The performance of callus induction and callus differentiation was evaluated by 9 indices for 140 RILs; 2 major QTLs associated with plant regeneration were identified.

Abstract

In order to investigate the genetic mechanisms of tissue culture response, 140 recombinant inbred lines (RILs) derived from 93-11 (Oryza sativa ssp. indica) × Nipponbare (Oryza sativa ssp. japonica) and a high quality genetic map based on the SNPs generated from deep sequencing of the RIL genomes, were used to identify the quantitative trait loci (QTLs) associated with in vitro tissue culture response (TCR) from mature seed in rice. The performance of callus induction was evaluated by indices of induced-callus color (ICC), induced-callus size (ICS), induced-callus friability (ICF) and callus induction rate (CIR), respectively, and the performance of callus differentiation was evaluated by indices of callus proliferation ability (CPA), callus browning tendency (CBT), callus greening ability (CGA), the average number of regenerated shoots per callus (NRS) and regeneration rate (%, RR), respectively. A total of 25 QTLs, 2 each for ICC, ICS, ICF, CIR and CBA, 3 for CPA, 4 each for CGA, NRS and RR, respectively, were detected and located on 8 rice chromosomes. Significant correlations were observed among the traits of CGA, NRS and RR, and QTLs identified for these three indices were co-located on chromosomes 3 and 7, and the additive effects came from both Nipponbare and 93-11, respectively. The results obtained from this study provide guidance for further fine mapping and gene cloning of the major QTL of TCR and the knowledge of the genes underlying the traits investigated would be very helpful for revealing the molecular bases of tissue culture response.     

小麦成熟胚愈伤组织诱导及分化研究

以2个小麦品种成熟胚为外植体进行离体培养,研究了不同预处理、不同2,4-D浓度及与KT组合、不同蔗糖浓度等因素对愈伤组织诱导及分化的影响。结果表明:4℃低温预处理可提高愈伤组织的出愈率及再生苗率,2个材料的出愈率及再生苗率均达到90%和30%以上;在不同预处理条件下,2,4-D浓度对出愈率及再生苗率的影响与基因型有关,2,4-D浓度为1~2 mg/L更有利于愈伤组织诱导及分化;附加KT能缓解高浓度2,4-D对再生苗率的抑制作用,而对于在1、2 mg/L 2,4-D的培养基中附加KT则不表现这种作用;蔗糖浓度则在30 g/L条件下更有利于愈伤组织诱导。因此通过4℃低温预处理,在MS基本培养基中附加1~2mg/L 2,4-D及30 g/L蔗糖亦可促进小麦成熟胚愈伤组织的诱导和分化。     

Plant regeneration through callus initiation from thin mature embryo fragments of wheat

A wheat regeneration system was developed using mature embryos. Embryos were removed from surface-sterilised mature caryopses (winter wheat Odeon cultivar and spring wheat Minaret cultivar) and ground to pieces through a sterile nylon mesh. The fragments were characterised by means of the image analysis technique. They were 500 M mean diameter and most of them were elongated. They were used as explants to initiate embryogenic calli on solid medium supplemented with 10 M 2,4-dichlorophenoxyacetic acid. The morphogenic pathway of the initiated calli was followed for a 40-day culture period. Active cellular division occurred within 24 hours of cultivation. Several hundred calli were produced from 100 fragmented embryos within 3 days. A 90% callus induction rate was achieved and proembryos appeared by the 8th day of culture. The highest embryogenic calli induction rate of 47% was obtained when 2,4-dichlorophenoxyacetic acid was suppressed after a 3–4 week induction period. Two regeneration methods were finally compared. A total of 513 plantlets were produced. The optimal protocol produced 25–30 plants per 100 embryos. This regeneration method may be suitable for transformation applications.     

Mapping QTLs controlling kernel dimensions in a wheat inter-varietal RIL mapping population

Key message

Seven kernel dimension QTLs were identified in wheat, and kernel thickness was found to be the most important dimension for grain weight improvement.

Abstract

Kernel morphology and weight of wheat (Triticum aestivum L.) affect both yield and quality; however, the genetic basis of these traits and their interactions has not been fully understood. In this study, to investigate the genetic factors affecting kernel morphology and the association of kernel morphology traits with kernel weight, kernel length (KL), width (KW) and thickness (KT) were evaluated, together with hundred-grain weight (HGW), in a recombinant inbred line population derived from Nanda2419?×?Wangshuibai, with data from five trials (two different locations over 3 years). The results showed that HGW was more closely correlated with KT and KW than with KL. A whole genome scan revealed four QTLs for KL, one for KW and two for KT, distributed on five different chromosomes. Of them, QKl.nau-2D for KL, and QKt.nau-4B and QKt.nau-5A for KT were newly identified major QTLs for the respective traits, explaining up to 32.6 and 41.5% of the phenotypic variations, respectively. Increase of KW and KT and reduction of KL/KT and KW/KT ratios always resulted in significant higher grain weight. Lines combining the Nanda 2419 alleles of the 4B and 5A intervals had wider, thicker, rounder kernels and a 14% higher grain weight in the genotype-based analysis. A strong, negative linear relationship of the KW/KT ratio with grain weight was observed. It thus appears that kernel thickness is the most important kernel dimension factor in wheat improvement for higher yield. Mapping and marker identification of the kernel dimension-related QTLs definitely help realize the breeding goals.

     

QTL mapping of flag leaf-related traits in wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

Key message

QTL controlling flag leaf length, flag leaf width, flag leaf area and flag leaf angle were mapped in wheat.

Abstract

This study aimed to advance our understanding of the genetic mechanisms underlying morphological traits of the flag leaves of wheat (Triticum aestivum L.). A recombinant inbred line (RIL) population derived from ND3331 and the Tibetan semi-wild wheat Zang1817 was used to identify quantitative trait loci (QTLs) controlling flag leaf length (FLL), flag leaf width (FLW), flag leaf area (FLA), and flag leaf angle (FLANG). Using an available simple sequence repeat genetic linkage map, 23 putative QTLs for FLL, FLW, FLA, and FLANG were detected on chromosomes 1B, 2B, 3A, 3D, 4B, 5A, 6B, 7B, and 7D. Individual QTL explained 4.3–68.52% of the phenotypic variance in different environments. Four QTLs for FLL, two for FLW, four for FLA, and five for FLANG were detected in at least two environments. Positive alleles of 17 QTLs for flag leaf-related traits originated from ND3331 and 6 originated from Zang1817. QTLs with pleiotropic effects or multiple linked QTL were also identified on chromosomes 1B, 4B, and 5A; these are potential target regions for fine-mapping and marker-assisted selection in wheat breeding programs.

     

Improvement of rice callus regeneration in the presence of NaCl

The effects of abscisic acid (37.8 μM), polyethylene glycol (5%), proline (10 mM), tryptophan (490 μM) and indoleacetic acid (5.7 μM) on rice callus regeneration were studied at various doses of NaCl (0, 50 and 100 mM) on three month-old mature embryo-derived callus of two japonica (I Kong Pao and Aiwu) and two indica (IR 2153 and Nona Bokra) rice cultivars differing in salinity tolerance. NaCl strongly decreased the regeneration frequency of all cultivars but slightly increased the survival of regenerated plantlets. Tryptophan stimulated regeneration and increased subsequent survival rates of regenerated plantlets in all cultivars at all NaCl doses. Abscisic acid and polyethylene glycol, though not affecting the final regeneration percentages, delayed regeneration and reduced the mean number of plantlets produced per regenerating callus in all cultivars, as well as rooting ability and survival of regenerated plantlets in indica genotypes. Proline had no marked effect on regeneration, whatever the NaCl dose or cultivar, while indoleacetic acid reduced shoot regeneration and increased root regeneration. This revised version was published online in June 2006 with corrections to the Cover Date.     

The effects of level of 2,4-D and time in culture on regeneration rate and chromosome numbers of regenerants from calli of the hybrid Triticum aestivum cv. Chinese Spring ph1b x Thinopyrum ponticum (2n = 10x = 70).

Thinopyrum ponticum (Podp.) Barkworth &D. R. Dewey (2n = 10x = 70) has excellent resistance for both leaf and stem rusts. Long-term callus cultures were established from the immature embryos of a hybrid between Triticum aestivum L. (2n = 6x = 42) x Th. ponticum. They were maintained in culture for over 2 years and continued to grow and have organogenetic capacity. With increasing time on a maintenance medium, the plant regeneration rates of the hybrid calli decreased when transferred to regeneration media containing 0.1, 0.2, or 0.5 mg/L 2,4-D, but the rate of decrease was much higher at 0.5 mg/L than at either 0.1 or 0.2 mg/L 2,4-D. After 3 months of subculture, the highest plant regeneration rate was obtained on the medium containing 0.5 mg/L 2,4-D (1.11 plantlets/callus), while on the 24th month of subculture the highest plant regeneration rate was obtained on the medium containing 0.1 mg/L 2,4-D (0.20 plantlets/callus). Thus, it was shown that as the calli aged it was important to reduce the level of 2,4-D in the regeneration medium. Over 2 years, a total of 667 regenerants were successfully transferred and grown to maturity. Chromosome numbers in root-tip cells were determined for 539 regenerants and ranged from 36 to 70. Telocentric chromosomes were frequent. A fertile plant was found among the regenerants after 15 months of subculture. It had 56 chromosomes with 2.15 (1-6) univalents, 22.76 (17-26) closed bivalents, 3.55 (1-9) open bivalents, and 0.41 (0-3) trivalents and was highly resistant to stem rust race 15B-1. Callus culture of wide hybrids can be used to introgress characters from alien species into wheat.     

A new,endosperm-supported callus induction method for wheat (Triticum aestivum L.)

A new, endosperm-supported callus induetion method was developed using mesocotyls of mature wheat embryos. After seed germination under aseptic condition, most of the germ tissues were cut off and only a few mm of the mesocotyl tissue with the scutellum was used for callus induction. The seeds were placed furrow downwards in 2,4-D solution (6–8 mg l^-1). Proliferating callus tissues were already observed on the cut surface of the mesocotyls on the 2nd day after inoculation. On the MS nutrient medium, callus formation from the isolated scutella with attached mesocotyls was negligible even after 6 days. For shoot and root regeneration, the calli produced up to 10 days were removed from the seeds and transferred onto a hormone-free MS medium. As shown by histological methods, the plantlets regenerated via organogenesis.     

Callus Induction and Plant Regeneration in Alstroemeria

Callus was induced from mature embryos of Alstroemeria cv. ‘Butterfly’cultured on MS medium supplemented with 2·0 or 4·0mg dm^–3 2,4–D or picloram and incubated at 25°Cin the dark. The effect of auxin concentration and precultureof embryos was studied. Callus was capable of regeneration aftertransfer to MS medium containing 4.0 mg dm°3 BAP. Shootsand whole plantlets were regenerated. The effect of growth regulators,used in the callus induction medium and the regeneration medium,on plant regeneration was studied Key words: Alstroemeria, callus, plant regeneration.     

The effect of auxin on plant regeneration of wheat,barley and triticale

One of the basic components of a medium influencing somatic embryogenesis of cereals from immature embryos is the type of auxin. According to some researchers, phytohormones can also play an important role during Agrobacterium-mediated transformation. In this first part of research, the influence of three types of auxins used alone or in combination of two on somatic embryogenesis and plant regeneration in three cereal species has been tested. Eight cultivars of barley, five cultivars of wheat and three cultivars of triticale have been used. Efficiency of plant development on two regeneration media, with and without growth regulators has been compared. Efficiency of regeneration characterized by frequency of explants that form embryogenic callus ranged from 25% for wheat cultivar Torka to 100% for two barley cultivars. Mean number of plantlets regenerating per explant differed significantly (from 2 to 58) depending on the type of auxin in inducing media, the type of regenerating media as well as cultivar. The biggest differences in regeneration efficiency were observed between barley cultivars, however regeneration of plants occurred in all combinations tested. The best regeneration coefficients for most barley cultivars were obtained after culture on dicamba or dicamba with 2,4-D. However, in the case of highly regenerating cv Scarlett, the most effective culture media contained picloram or 2,4-D alone. The highest values of regeneration coefficients for two triticale cultivars (Wanad and Kargo) were obtained on picloram (26.1 and 21.4, respectively) and for `Gabo' on picloram with dicamba (12.6). The range of mean number of regenerated plantlets was from 12 to 30. Dicamba alone or lower concentrations of picloram with 2,4-D were the best media influencing embryogenic callus formation in five wheat cultivars. However, the highest values of regeneration coefficients ranging from 10.6 to 26.8 were obtained at lower concentrations of picloram with 2,4-D or picloram with dicamba. R2 regeneration medium containing growth regulators was significantly better for plantlet development in several combinations (cultivar and induction medium) than the one without growth regulators. Generally, regeneration coefficients for all tested cultivars of three cereal species on the best media were high, ranging from 5.5 for barley cultivar Rodion to 51.6 for another barley cultivar Scarlett. Plantlets developed normally, flowering and setting seed.     

Mapping quantitative trait loci for tissue culture response in VCS3M-DH population of Brassica rapa

Key message

Quantitative trait loci (QTL) controlling callus induction and plant regeneration were identified in the VCS3M-DH population of Brassica rapa.

Abstract

Quantitative trait loci (QTL) controlling callus induction and plant regeneration were identified in the VCS3M-DH population of Brassica rapa. The VCS3M-DH population showed wide and continuous variation in callus induction and shoot regeneration. Significant coefficient correlations were detected between these two parameters. Broad-sense heritability (h ²) for the two traits was around 0.7, indicating genetic regulation of regeneration ability in this population. In the composite interval mapping analysis, two QTLs for callus induction ability, qCi2 and qCi7, were mapped on chromosome A02 and A07, explaining 28.6 % of phenotypic variation. For plant regeneration, four QTLs, qPr6-1 qPr6-2, qPr7, and qPr9 were identified on chromosome A06, A07, and A09, which in total explained 50.1 % of phenotypic variation. Furthermore, 15 putative candidate genes were found on the interval of the six QTLs, which were related to various plant hormones, MADS-box genes, and serine/threonine related genes. These results provide important information to identify genes related to tissue culture ability in B. rapa.     

Increased Copper Content of the Medium Improves Plant Regeneration from Immature Embryo Derived Callus of Wheat (Triticum aestivum)

The effect of various concentrations of CuSO₄ on the induction and regeneration of embryogenic callus from immature embryos of wheat was investigated. Immature embryos of wheat cvs C-306 and R-3777 were cultured on MS medium supplemented with 2,4-D (11.3 µM) and different levels of cupric sulphate, i.e. 0, 0.1 (MS level), 0.5, 1 and 5 µM. Relatively high induction frequency of callus was obtained on MS medium supplemented with 2,4-D (11.3 µM) and 0.5 µM CuSO₄. The compact, nodular, embryogenic callus was maintained on the medium having 2,4-D (11.3 µM) and proline (86.8 µM) by regular subculturing. Plant regeneration from the embryogenic callus occurred on MS medium supplemented with NAA (1.07 µM) and BAP (44.4 µM). Regenerated plantlets were rooted on MSmedium supplemented with IAA (2.85 µM). The average number of regenerated plantlets produced from primary callus induced on 2,4-D (11.3 µM) and 5x CuSO₄ was significantly higher.     

In Vitro Regeneration of Onion through Repetitive Somatic Embryogenesis

A reliable protocol for the regeneration of onion through repetitive somatic embryogenesis was established. Embryogenic callus was derived from mature seeds on Murashige and Skoog (MS) medium supplemented with 2 mg dm-3 2,4-dichlorophenoxyacetic acid (2,4-D). Somatic embryos aroused on the surface of calli cultures and formed plantlets after the removal of 2,4-D or its substitution with 1 mg dm-3 kinetin (Kin). Reculturing the somatic embryos on 2,4-D containing medium led to secondary embryos formation. The embryogenic cultures which were preserved for five months on maintenance medium containing 2 mg dm-3 2,4-D + 0.5 mg dm-3 Kin have retained their ability for regeneration, while those kept on 2,4-D only, failed to form plantlets. Electrophoretic analysis of total soluble proteins revealed that the competence for successful conversion of somatic embryos into plantlets is associated with the expression of new set of proteins (112, 58 and 30 kD). The regenerated plants were successfully transferred to the soil.     

玉米优良自交系成熟胚再生体系的建立

选用生产上广泛应用的10个玉米优良自交系,用幼胚通过组织培养研究其再生特性,结果表明:玉米自交系基因型间的培养能力有较大的差异,自交系178的再生率高达78%。在此基础上以其中的178玉米优良自交系为材料,研究了影响玉米成熟胚再生的各种因素,结果表明:高浓度的2,4-二氯苯氧乙酸(2,4-D)(4.0 mg/L)是诱导愈伤组织必须的;在继代培养基中添加适量的2,4-D(2.0 mg/L)、6-苄基嘌呤(6-BA)(0.2 mg/L)和硝酸银(10 mg/L)显著增加胚性愈伤组织的形成;在分化培养基中添加0.5 mg/L 6-BA有利于提高愈伤组织的分化频率。该再生体系的建立,为以成熟胚为受体系统的遗传转化体系的建立奠定了基础。