Heritable tissue culture induced variation in Zinnia marylandica

Summary Adventitious shoots of Zinnia marylandica, an amphidiploid with limited genetic segregation, were regenerated from cotyledonary tissue on Murashige-Skoog (MS) media containing 0.2 or 22.2 M thidiazuron (TDZ) and grown through flowering. Fisher's Test for Equal Variance indicated tissue culture induced plants had more variation than seed-derived control plants. Twelve of 149 (8%) plants derived from 0.2 M TDZ and three of 23 (13%) plants from 22.2 M TDZ had variant characters. Aberrant characteristics in self-pollinated variants included plant height, fertility, flower color and morphology, and were sexually transmitted, indicating genetic change had occurred. Aberrant characteristics not observed in regenerated plants arose in progeny.Abbreviation TDZ thidiazuron     

利用花药培养选育烤烟抗黑胫病突变体

首先在接种有不同浓度黑胫病菌发酵液活性组分的选择培养基上,获得高钾烤烟GK2的花药培养苗,并采用离体叶片法对这些花药培养苗进行黑胫病抗性鉴定,结果花药培养植株表现出不同程度的抗性;然后选其中6株抗性较高且农艺性状与GK2相近的单株,经秋水仙素加倍后收获种子,在黑胫病病圃及大田条件下于2016年对其R-1代、2017年对3个抗病性表现较好的R-2代植株进行黑胫病抗性及农艺性状测试,2年试验结果一致表明：在染色体加倍以后,所选植株的后代在基因型上已完全纯合,黑胫病抗性较GK2有较大提高且均可稳定遗传,是不同于原GK2的烤烟新品系,在生产上具有广泛应用前景。     

Regeneration in sugarcane via somatic embryogenesis and genomic instability in regenerated plants

In the present study, embryogenic calli of sugarcane variety BL4 were induced using 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin in different concentrations and combinations. In contrast to earlier studies, embryogenic callus sectors were identified and isolated microscopically within 1–2 weeks. Subsequently, 51 media formulations were used for regeneration of proliferated embryogenic callus, using MS medium supplemented with three different cytokinins [kinetin, 6-Benzylamino purine (BAP), and thidiazuron (TDZ)] and auxins (IAA/NAA and IBA) in different combination and concentrations. After acclimatization, the genomic DNA of regenerated plants was studied to explore the insertion polymorphism of transposable elements in order to ascertain the variation among somaclones. Though low concentration of kinetin with 2,4-D was found supportive to embryogenic callus development, the highest number of regenerated plantlets was observed using BAP (1 μM), however the plantlets had very low fresh weight (2.2 g). Conversely, TDZ alone supported a significant increase in the number of plantlets regenerated (38–40) with higher fresh weight. The somaclones generated during this study showed considerable positional polymorphism of activator-like transposable elements possibly due to the stress associated with in vitro culture. This study provides a procedure to produce regenerated sugarcane plants from embryogenic callus in a relatively short time.     

An allotriploid derived from a amphidiploid × diploid

A fully fertile interspecific hybrid (Cucumis hytivus Chen and Kirkbride, 2n =4x =38) between Cucumis hystrix Chakr. (2n= 2x =24) and C. sativus L. (2n = 2x = 14) was previously produced by means of F₁ (2n = 19) embryo rescue and subsequent chromosome doubling. This amphidiploid, a new synthetic species, may serveas a genetic bridge in Cucumis, and thus is a source for broadening the genetic base of C. sativus. The identification and characterization of fertile progeny possessing lower ploidy levels would facilitate bridging among Cucumis species. Putative allotriploids (2n = 26) were recovered from C. hytivus × C. sativus matings by means of embryo culture, and experiments were designed to confirm their genetic constitution, describe their morphology, and establish an efficient protocol for their micropropagation. Apical and axillary buds of these putative allotriploid plants were used as explants to establish a micropropagation system for subsequent verification and characterization of ploidy. Of the array of micropropagation media tested, then ability to be most effective for the induction of adventitious buds (desginated Stage II) was a Murashige and Skoog (MS)growth media containing 13.3μM BA + 1.1μM NAA or containing10 μm BA only. The mean number of adventitious buds per explant in the two media was 6.8 and 6.5, respectively. Shoots resulting from adventitious buds produced roots (Stage III) in relative abundance (39 of 42, 92.8%) on half-strength MS medium containing 1.0 μm IBA. The survivorship of rooted plantlets after acclimatization as assessed by relative production of leaves in plantlets (designated Stage IV) was 91.4% (148 of 162). The chromosome number in putative allotriploid plants as determined in mitotic root tip figures in all plants was 2n = 26, the number expected for allotriploids derived from such a mating. An examination of pollen viability in five samples of each plant by cytochemical staining revealed stainability to be < %.Compared to their parents, the allotriploid genotypes possess a high degree of parthenocarpy (84.8%) as measured by setting fruit in pollen-free conditions. While allotriploid fruit are black-spined and similar to the maternal parent C. hytivus, the dark green leaves typical of allotriploid plants mirrors that of the paternal C. sativus parent. This revised version was published online in July 2006 with corrections to the Cover Date.     

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

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

冬小麦对除草剂莠去津反应敏感性及其遗传控制

以２４个冬小麦品种（或品系）为试材,研究测定了冬小麦对莠去津反应的敏感性。结果表明,在苗前土壤喷３０００ｍＬ／ｈｍ＾２莠去津的条件下,出苗后３０ｄ有６个品种表现不敏感（Ｔ）,７个品种表现中感（ＭＳ）,４个品种表现中抗（ＭＴ）;小麦根部受莠去津的为害重于地上部。观察了不同反应型小麦品种之间杂种Ｆ１、Ｆ２和ＢＣ１的敏感和不敏感植株分离比例,小麦对莠去津反应受细胞核基因控制,与细胞质基因无关。该基因属单基因遗传,敏感性为隐性,不敏感性为显性。研究了莠去津对小麦幼胚培养的影响,低浓度莠去津对小麦幼胚愈伤组织诱导率没有明显影响,高浓度莠去津降低了愈伤组织的诱导率。无论培养基中的浓度高低,莠去津对继代培养中愈伤组织的生长状态都有影响。     

转阿特拉津氯水解酶基因烟草的获得及其生物降解能力分析

阿特拉津氯水解酶(AtzA)能有效催化有毒的阿特拉津脱氯生成无毒的羟基阿特拉津。本文将来自假单胞菌ADP的atzA-ADP和来自节杆菌AD1的atzA-NK分别插入Ti载体pBin438, 构建了atzA植物表达载体pBin438-atzA- ADP和pBin438-atzA-NK。并通过农杆菌介导法将其转入烟草, 经基因组PCR筛选及RT-PCR分析, 获得16株转atzA-ADP烟草株系和11株转atzA-NK烟草株系。在含150 mg L^-1阿特拉津的1/2MS培养基上生长50 d时, 株系401的降解能力最高, 达79.26%, 远高于野生型烟草的0.47%, 说明转基因烟草可用于建立阿特拉津残留环境的植物修复系统。     

Antimicrotubule herbicides for in vitro chromosome doubling in Beta vulgaris L. ovule culture

In vitro chromosome doubling during ovule culture of sugar and fodder beets (Beta vulgaris L.) was studied with four anti-microtubule herbicides: amiprophos-methyl (APM), oryzalin, pronamide, and trifluralin at concentrations of 0–300 μM. Best chromosome doubling results were obtained by treatment of the ovules with 100 μM APM which produced 4.7 diploid plants per 100 ovules. Highest chromosome doubling was found with oryzalin using 1 μM, with trifluralin at 10 μM, and with pronamide at 10 μM producing 2.8, 2.0, and 2.0 diploid plants per 100 ovules, respectively. The APM treatments showed relatively low toxicity on embryo formation which in combination with a high chromosome doubling effect, resulted in up to 89 diploids per 100 plants regenerated. Oryzalin and trifluralin had more severe toxic effects, which reduced embryo formation, thereby lower percentages of chromosome doubled plants were obtained from these treatments. Pronamide had no significant toxic effect but it induced chromosome doubling at lower frequencies. Compared to colchicine, APM seems to be as efficient for chromosome doubling during beet ovule culture, but at molar concentrations 100 times lower than those used for chromosome doubling with colchicine. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

Variants of Digitalis obscura from irradiated shoot tips

Shoot tips excised from genotype T4 of Digitalis obscura were exposed to gamma rays (20–100 Gy). Radiosensitivity was assessed and LD₅₀ determined was about 60 Gy. The effect of the herbicide amitrole on shoot-tip culture was investigated, efficient bleaching was obtained with 10 mg/l amitrole. Shoot tips mutagenized (20–40 Gy) were cultured on several selective amitrole-containing media, but none of them permitted prolonged survival of the green shoots formed. Ploidy level of the plantlets developed was analysed by flow cytometry 8 and 18 months after culture establishment. Variations detected corresponded to aneuploid changes and increased in parallel with the gamma radiation dose. Plantlets developed from irradiated shoot tips presented a high variability in their cardenolide production (878 to 3291 μg/g d.w.), including variants with similar or even higher productivities than the native T4 plant. This revised version was published online in August 2006 with corrections to the Cover Date.     

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.     

Boron deficiency induced male sterility in wheat (Triticum aestivum L.) and implications for plant breeding

Boron (B) deficiency causes grain set in wheat to fail. A wide range of genotypic variation in the response to low B has been observed. Genotypes were screened in low B in soil and sand culture, and classified into five groups, namely, very sensitive, sensitive, moderately sensitive, moderately tolerant and tolerant. At very low levels of B, the very sensitive to sensitive genotypes were completely male sterile and set only a few or no grain, while the tolerant genotypes set grain normally. Natural outcrossing was detected in these male sterile plants when a tolerant genotype was growing nearby. Grain set by cross fertilisation was markedly enhanced by a B application directly on the ear of the male sterile plants. Three practical implications are suggested. Firstly, genotypes that are tolerant to low B can provide a solution for grain set failure caused by B deficiency. Secondly, the potential for outcrossing in male sterile B deficient wheat has to be taken into account in the maintenance of pure lines in low B soils even though wheat is normally self pollinated. Thirdly, a simple and novel method for hybridization is suggested, in which B deficiency is used as fertility selective medium and male sterile female parents and fertile male parents are provided by genotypic variation in the response to low B. This revised version was published online in July 2006 with corrections to the Cover Date.     

Monitoring genetic fidelity vs somaclonal variation in Norway spruce (Picea abies) somatic embryogenesis by RAPD analysis

Summary Somaclonal variation, which is a welcome source of genetic variation for crop breeding, is unwanted when direct regenerants have to be used in tissue culture mass propagation (eg. in many forest trees), or in the regeneration of genetically transformed plants. Random amplified polymorphic DNA (RAPD) was used to analyse somatic embryos and plants regenerated from embryogenic cell lines in Norway spruce, Picea abies (L.) Karst. RAPD facilitated the identification of clones, as material from the same cell lines shared identical patterns of amplified fragments, whereas regenerants from different cell lines were easily distinguishable by their respective patterns. For comparisons with explant donor genotypes, cell lines were initiated from cotyledons. Some of the seedlings that had parts of their cotyledons removed were grown on as control plants. Somatic embryos regenerated from cotyledon cell lines showed no aberrations in RAPD banding patterns with respect to donor plants. We conclude that gross somaclonal variation is absent in our plant regeneration system.Abbreviations ESM embryogenic suspensor mass - RAPD random amplified polymorphic DNA - RFLP restriction fragment length polymorphism - (2,4-dichlorophenoxy)acetic acid 2,4-D - 1-naphthaleneacetic acid NAA     

Influence of genotype and culture conditions on the production of embryos from anthers of tetraploid wheat (Triticum turgidum)

Summary Anther culture of 10 tetraploid wheat (Triticum turgidum) genotypes and two backcross lines representing a wide range of genetic variation was studied in a randomized block design with three replications. Each replication consisted of 2 pots with 3 plants. The day length was 16h and temperature 25° C/15°C for day/night in a controlled greenhouse where the anther donor plants were grown. Two different treatments were used for anther culture. The first one was potato 2 medium (Chuang et al., 1978) modified by adding 0.5 mg/l glutamine and solidified by gelrite (4g/l) (Henry & De Buyser, 1981). Cultures were incubated in light (15 E m^–2 S^–1) at 26°C at 16h day length. The second medium was described by Fadel & Wenzel (1990), differing from the first by the nature of the sugar (maltose) and consistency of the medium (semiliquid by ficoll). Anther cultures were incubated in the dark at 28°C. The study of about 1300 anthers per genotype and treatment showed that both genotype and treatment affected embryo formation of tetraploid wheat. The backcross lines exhibited significant differences for androgenic abilities when compared to their common parent. Most of the genotypes were medium dependent for androgenesis and revealed significant interactions with the two treatments. Five green plantlets were regenerated and fertile doubled haploid plants were obtained from three out of the 12 studied genotypes.     

Nutrient culture media with agar is effective for early and rapid screening of iron toxicity tolerance in rice

The breeding for iron toxicity tolerant rice needs an effective, efficient, and reliable screening method. The study was aimed to evaluate the best method for screening iron tolerant genotypes at the seedling stage in the greenhouse. Two rice genotypes, Mahsuri (tolerant) and IR64 (sensitive) were grown in three modified media solutions namely, Yoshida-conventional solution (YCS), Yoshida with etylenediamintetraacetic acid (1:2) (YES), and Yoshida with 0.2% agar (YAS). Three levels of iron were tested to observe the severity of their leaf bronzing score (LBS). The optimized solution in the greenhouse was then evaluated using 24 rice genotypes. Using the same genotypes interrelationship, the LBS in the greenhouse with grain yield and its attributes was validated under acute and moderate Fe toxicity in the field. The results showed that the optimized media culture was YAS with 400 mg L^-1 of FeSO₄. This media had more stable pH and redox potential, it could maintain sufficient Fe²⁺ supply over 10 days, and it could discriminate of LBS between tolerant and sensitive genotypes. Evaluation using the optimized media solution showed that there was a significant variation among genotypes in shoot dry weight and a significant correlation of relative reduction of shoot dry weight with LBS. The LBS in the greenhouse was correlated with LBS in acute iron stress in the field (r=0.673**) and the grain yield (r= -0.618**). This study has proven that YAS culture media can be used as early identification of iron toxicity tolerant genotypes for supporting breeding programs.     

Genetic stability at nuclear and plastid DNA level in regenerated plants of <Emphasis Type="Italic">Solanum</Emphasis> species and hybrids

In this work we detected the extent of variability at nuclear and cytoplasmic DNA level of regenerated plants belonging to Solanum genotypes with a different genetic background and somatic chromosome number. As for the nuclear characterization, a total of 66 (18.5%) polymorphic bands were scored using 13 ISSR primers on 45 randomly selected regenerants. Our results show that the regenerants obtained from clone cmm 1T and, at lower level, those from cph 1C are unstable under in vitro conditions or rather more prone to in vitro-induced stress leading to somaclonal variation than the other genotypes used. Two types of changes were observed: disappearance of parental ISSR fragments, termed “loss”; appearance of novel ISSR fragments, termed “gain”. The most frequent event occurring in the regenerants was the loss of fragments (41 bands). Regenerated plants were analyzed with seven plastid universal primers to determine the cytoplasmic composition at chloroplast level. All cpDNA primer pairs tested produced amplicons of the same size in all genotypes analyzed and no polymorphic fragments were observed with any universal primers used. Our results show that under in vitro culture conditions genotype affects the integrity of the genome. In addition, the absence of polymorphism at plastid level confirms the greater genetic stability of cytoplasmic DNA.     

Genotypic and exogenous factors affecting shoot regeneration from anther callus of linseed (Linum usitatissimum L.)

Summary The objective of this study was to investigate factors affecting the regeneration capacity of linseed anther culture. Four different environmental conditions in a phytotron were tested with regard to their effects on anther donor plants of cv. Hella. Anther response and shoot regeneration from anther callus was maximal when donor plants were grown in a 16 hrs-day at 14°C day/8°C night temperature. Anthers of four linseed genotypes were cultured on different media. Maximum shoot regeneration was achieved when the induced calli were transferred onto a modified N6 medium containing zeatin (1 mg l^-1). Most of the calli regenerated shoots in the second subculture on regeneration media. Shoots were rooted on modified B5 or MS media containing NAA (0.1 mg l^-1). Cytological examinations of incubated anthers and root tips of regenerated plants indicated that the anther calli were derived from microspores.Abbreviations B5 Gamborg's (1975) medium - BAP 6-benzylaminopurine - 2,4D dichlorophenoxyacetic acid - N6 Chu's (1978) medium - NAA -naphthaleneacetic acid - MS Murashige & Skoog's (1962) medium - ZEA zeatin     

In vitro selection and plant regeneration of copper-tolerant plants from leaf explants of Nicotiana tabacum L. cv. 'Xanthi'

Copper tolerance of Nicotiana tabacum L. var. Xanthi in vitro was achieved through plant regeneration from leaf explants on Murashige and Skoog's (MS) medium supplemented with 0.5 mg/l BA, 0.1–0.25 mg/l IAA and 60  μ m Cu. Tolerant organogenic calli showed more vigorous growth in medium containing 60  μ m Cu than the non-tolerant calli. Standard growth parameters such as fresh and dry weight of organogenic callus, growth tolerance index (GTI), enzyme activity (peroxidase and catalase) and copper accumulation were used as indicators of copper tolerance. The activities of peroxidase and catalase as well as estimation of protein, total amino acid and chlorophyll were greater in tolerant calli than non-tolerant ones. The GTI in the 4 weeks after the beginning of treatments yielded significant differences among the tolerant and non-tolerant organogenic callus cultures. The accumulation of copper in the tolerant calli increased significantly with an increase in copper concentration in the medium. Shoot bud regeneration was achieved in both tolerant and non-tolerant organogenic calli on MS medium containing 0.5 mg/l BA and 0.1 mg/l IAA. The tolerant regenerated shoots were rooted on half-strength basal MS medium with 60  μ m Cu for selection of tolerant clones. This study may help in the selection and characterization of Cu-tolerant lines of N. tabacum cv. 'Xanthi' for building conservation strategies and also for phytoremediation programmes.     

Suspension culture performance in commercial varieties of perennial ryegrass (Lolium perenne L.)

Summary Suspension culture performance in commercial varieties of perennial ryegrass was studied to assess the effect of variety on suspension culture response and plant regeneration. 179 suspension cultures were established from embryos of mature seeds of 21 varieties and one breeding population. Of these, 123 suspensions were morphogenic (21 varieties) and 66 suspensions (18 varieties) regenerated green plants. A number of suspension lines, originating from two different suspensions, retained the capacity for green plant formation for almost four years. Replicates performed with seed lots of different ages indicated that suspensions initiated from young seeds (1 year) were of better quality than suspensions initiated from older seeds (2–4 years). Varieties differed in their capacity to form morphogenic suspensions and suspensions capable of regenerating green plants, although the effect of variety was relatively small. It was concluded that responsive genotypes can be found within most varieties of Lolium perenne.     

Variation for tolerance to high concentration of ferrous iron (Fe<Superscript>2+</Superscript>) in Australian hexaploid wheat

High concentration of reduced iron (Fe²⁺) in waterlogged acid soils is a constraint for growing wheat in high rainfall (waterlogged-prone) areas of Western Australia. Growing crop genotypes tolerant to high Fe²⁺ concentrations may be desirable in such situations, but there is no knowledge about the extent of variability in Fe²⁺ tolerance in the wheat germplasm. A bioassay for tolerance to high concentrations of iron in wheat was developed and optimised using Siete Cerros (Fe-tolerant) and BH1146 (Fe-intolerant) as control genotypes and a range of FeSO₄ concentrations (36, 313, 625, 1250, 1875, 2500 and 3125 μM Fe²⁺) in nutrient solution in a controlled-temperature environment. Increasing external concentration of iron decreased both shoot and root dry weight, increased shoot iron concentration and intensified the development of toxicity symptoms to a greater degree in intolerant BH1146 as compared to tolerant Siete Cerros. Increased iron supply negatively affected uptake of Ca (r = −0.41) and Mg (r = −0.40). The tolerant genotype Siete Cerros showed an improved avoidance/exclusion of high external concentration of Fe²⁺ compared with intolerant BH1146. The genotypic discrimination based on relative root dry weight and the development of toxicity symptoms was most pronounced at 625 μM Fe²⁺. This concentration was chosen for screening of 20 bread wheat and one durum genotype chosen from a preliminary screening of 94 Australian wheat genotypes. A relatively narrow but significant variation (22–38%) in terms of relative root dry weight under Fe²⁺ toxicity was observed among Australian advanced breeding lines and varieties. The presence of genotypic variation for Fe²⁺ tolerance across and within the Australian breeding programs could be exploited in a deliberate selection process to enhance Fe²⁺ tolerance in wheat. Durum wheat (Arrivato) and several Australian wheat varieties and advanced lines in this study were as tolerant to Fe²⁺ toxicity as Siete Cerros, a variety representing common parentage of iron-tolerant genotypes.