The use of molecular markers to study patterns of genotypic diversity in some invasive alien Fallopia spp. (Polygonaceae)

Random amplified polymorphic DNA (RAPDs) and inter-simple sequence repeats (inter-SSRs) have been used to study clonal growth and hybridization in some non-native, gynodioecious, invasive weeds from the genus Fallopia (Polygonaceae). At the study site (the River Kelvin, Glasgow, UK) a single genotype of Japanese knotweed ( Fallopia japonica ) was detected, consistent with all the individuals sampled being ramets of a single clone. Two genotypes of giant knotweed ( F. sachalinensis ) were detected, with one genotype accounting for all but one of the samples, again indicative of widespread clonal growth. Five genotypes of the hybrid between Japanese and giant knotweed ( F. × bohemica ) were recovered. F. × bohemica is the only male-fertile taxon present at the site and it seems probable that at least some of this genetic variation is attributable to hybrid fertility. A single plant identified using morphological methods as a backcross between F. japonica and F. × bohemica was analysed, and the molecular data were consistent with this theory. A comparison of RAPDs and inter-SSRs showed that the two techniques gave data that are broadly congruent, and both techniques showed a similar sensitivity in the number of genotypes detected.     

Taxa distribution and RAPD markers indicate different origin and regional differentiation of hybrids in the invasive Fallopia complex in central-western Europe

Interspecific hybridization can be a driving force for evolutionary processes during plant invasions, by increasing genetic variation and creating novel gene combinations, thereby promoting genetic differentiation among populations of invasive species in the introduced range. We examined regional genetic structure in the invasive Fallopia complex, consisting of F. japonica var. japonica , F.   sachalinensis and their hybrid F.  ×  bohemica , in seven regions in Germany and Switzerland using RAPD analysis and flow cytometry. All individuals identified as F. japonica var . japonica had the same RAPD phenotype, while F. sachalinensis (11 RAPD phenotypes for 11 sampled individuals) and F.  ×  bohemica (24 RAPD phenotypes for 32 sampled individuals) showed high genotypic diversity. Bayesian cluster analysis revealed three distinct genetic clusters. The majority of F . ×  bohemica individuals were assigned to a unique genetic cluster that differed from those of the parental species, while the other F . ×  bohemica individuals had different degrees of admixture to the three genetic clusters. At the regional scale, the occurrence of male-fertile F. sachalinensis coincided with the distribution of F . ×  bohemica plants showing a high percentage of assignment to both parental species, suggesting that they originated from hybridization between the parental species. In contrast, in regions where male-fertile F. sachalinensis were absent, F . ×  bohemica belonged to the non-admixed genetic group, indicating multiple introductions of hybrids or sexual reproduction among hybrids. We also found regional differentiation in the gene pool of F.  ×  bohemica , with individuals within the same region more similar to each other than to individuals from different regions.     

Effect of genotype on callus induction,shoot regeneration,and phenotypic stability of regenerated plants in the greenhouse of Primula ssp.

Genotypic differences between six genotypes of Primula vulgaris could be observed in callus induction rate, type of callus, root formation during the callus phase, and shoot regeneration rate. The shoot regeneration rate ranged from zero to 11.6 shoots per explant. There was no correlation between callus induction rate and shoot regeneration rate. Callus consistency and colour were an indicator of the organogenetic capacity of callus. An experiment with different periods of treatment with 4.0 mg l 2,4-dichlorophenoxyacetic acid and 2.0 mg l21 thidiazuron revealed that the shoot regeneration rate varied tremendously between genotypes. In two genotypes a period of 8 weeks on medium with plant growth regulators was sufficient to induce shoot regeneration. In three other genotypes a longer induction period was not able to overcome low regeneration capacity. However an increase in shoot regeneration rate was observed after 16iV32 weeks of induction. Phenotypic stability was also strongly dependent on genotype. In three genotypes the majority of regenerated plants looked normal and were diploid. Aberrations like abnormal growth habit, crinkly leaves, deviation of flower colour or lack of pollen formation occurred in only one genotype at a very low frequency (1.5 genotypes between 12.5 and 18.1 regenerants was tetraploid.     

Effect of genotype and medium composition on linseed (<Emphasis Type="Italic">Linum usitatissimum</Emphasis>) ovary culture

Breeding linseed (Linum usitatissimum L.) using haploid techniques allows breeders to develop new cultivars in a shorter time period. Many research groups successfully created new linseed genotypes through anther culture; however ovary culture has been the subject of only a few earlier studies. In the present study, the effect of genotype and growth regulators combination on callus induction and shoots regeneration in ovary culture of nine commercially important linseed cultivars was investigated. Ovaries were cultured on modified MS medium supplemented with three different combinations of plant growth regulators. Variable callogenic responses were expressed by all of the genotypes tested on different induction media. The results suggested that specific combination of growth regulators for callus induction must be designed for each genotype. Shoot regeneration from ovary derived callus is a critical phase of the whole gynogenetic process. Differences in adventitious shoot formation frequency among genotypes were demonstrated and four responsive genotypes have been selected. Ovary derived callus from cultivar ‘Mikael’ manifested the highest adventitious shoot formation frequency with a high number of shoots per explant. The optimum ratio of growth regulators for shoot regeneration was shown to depend on the genotype. Cultivars ‘Linola’, ‘Mikael’ and ‘Szaphir’ showed the highest shoot regeneration frequency when callus had originated on induction medium supplemented with 2 mg L⁻¹ BAP and 2 mg L⁻¹ NAA, while combination of 1 mg L⁻¹ BAP and 2 mg L⁻¹ IAA promoted shoot formation in ovary-derived callus of ‘Barbara’. The highest rate of shoots per explant has been obtained in second subculture.     

The use of phenotypic markers to identify Brassica oleracea genotypes for routine high-throughput Agrobacterium-mediated transformation

Doubled haploid (DH) genotypes from a genetic mapping population of Brassica oleracea were screened for ease of transformation. Candidate genotypes were selected based on prior knowledge of three phenotypic markers: susceptibility to Agrobacterium tumefaciens, shoot regeneration potential and mode of shoot regeneration. Mode of regeneration was found to be the most significant of the three factors. Transgenic plants were successfully obtained from genotypes that regenerated multiple shoots via a distinct swelling or callus phase. The absence of tissue culture blackening (associated with genotypes that formed callus) was found to be critical for transformation success. Transgenic shoots were obtained from genotypes that regenerated via an indirect callus mode, even when susceptibility to Agrobacterium was low. The most efficient genotype (DH AG1012) produced transgenic shoots at an average rate of 15% (percentage of inoculated explants giving rise to transgenic plants). The speed and efficiency of regeneration enabled the isolation of transgenic shoots 5–6 weeks after inoculation with A. tumefaciens. This line was also self-compatible, enabling the production of seed without the need for hand-pollination. A genetically uniform DH genotype, with an associated genetic map, make DH AG1012 highly desirable as a potential model B. oleracea genotype for studying gene function. The possibility of applying the same phenotypic tissue culture markers to other Brassica species is discussed.     

Effects of genotype,explant orientation,and wounding on shoot regeneration in tomato

Summary Effects of genotype and explant orientation on shoot regeneration from cotyledonary explants of tomato were studied using 10 commercially important cultivars. The explant orientation affected shoot regeneration in all the tested genotypes. Cotyledons placed in abaxial (lower surface facing down) orientation consistently produced better shoot regenerative response and produced greater numbers and taller shoots compared to those inoculated in adaxial (upper surface facing down) orientation. Genotypic variation in terms of shoot regeneration response, number of shoots, and shoot height was apparent. Wounding of cotyledonary explants increased shoot regeneration response. However, shoot height was much lower in shoots regenerated from wounded explants compared to those that originated from intact cotyledons. Shoots produced from wounded cotyledons were abnormal in their form and structure.     

Combination of thidiazuron and 2-isopentenyladenine promotes highly efficient adventitious shoot regeneration from cotyledons of mature sunflower (Helianthus annuus L.) seeds

Genetic improvement of sunflower (Helianthus annuus L.) through the use of biotechnological tools requires a reliable in vitro shoot regeneration system. Tissue culture protocols reported to date for sunflower suffer from low efficiency, poor reproducibility, genotype dependence and a tendency for flowering in vitro. The present study describes an efficient protocol system for shoot regeneration via direct adventitious shoot organogenesis from cotyledons of mature seeds of sunflower. About 169 media combinations comprising 12 different growth regulator combinations in various concentrations were assessed for induction of shoots from cotyledons derived from mature seeds and also from seedling tissues of 2?C20-day-old seedlings. Appearance of shoots from seedling tissues was sporadic and the frequency of shoot regeneration was low. Cotyledon explants from mature seeds were consistent with regard to frequency of adventitious shoot regeneration and number of shoots per explant. A high frequency (93.86?%) of adventitious shoot regeneration was obtained within 2?weeks of culture initiation on Murashige and Skoog (MS) medium supplemented with 9.84???M 2-isopentenyladenine (2-iP), 2.85???M indole-3-acetic acid (IAA) and 0.45???M thidiazuron (TDZ). Use of 2-iP in the shoot induction and elongation media prevented precocious flowering. Statistical analysis revealed significant effects of explant orientation, age of seedlings, and genotype on adventitious organogenesis. Maximum shoot regeneration was obtained when cotyledons from 0 and 1-day-old seedlings were placed with their adaxial surface in contact with the medium surface. The protocol developed was tested on 42 genotypes and found to be applicable to a wide range of genotypes. Histological studies indicated that the shoots originated predominantly through adventive organogenesis from the sub-epidermal and cortical regions.     

4种红景天植物的组织培养研究

以大花红景天、云南红景天、长鞭红景天和库页红景天的茎和叶为外植体进行组织培养,结果表明：大花红景天以茎为外植体诱导芽效果最好,其它3种红景天以叶为外植体诱导芽效果最好。云南红景天和长鞭红景天适合的芽诱导激素组合是0．1mg／L NAA和2．5mg／L 6-BA的组合,在该激素水平下两种红景天的出芽频率分别达到71％和84％;大花红景天和库叶红景天适合的芽诱导激素组合是0．5mg／L NAA和2．5mg／L 6-BA的组合,在该激素水平下两种红景天的出芽频率均达到80％。长鞭红景天和库叶红景天在添加IBA的培养基上容易生根形成完整植株,生根率分别达到87％和73％;经过炼苗后,长鞭红景天再生苗能够成功移栽,成活率达66％。     

Recovery from drought stress in Lolium perenne (Poaceae): are fungal endophytes detrimental?

Perennial ryegrass (Lolium perenne) is a cool-season, perennial species widely used for forage and turf. It is often infected by a clandestine, endophytic fungus (Neotyphodium lolii) that has the potential to affect host growth responses to abiotically stressful conditions. In some species, the grass-endophyte symbiosis is mutualistic, but the relationship is reported to be contingent on environmental conditions and host genotype in L. perenne. The objective of this research was to determine the potential effects of endophyte infection on recovery from severe drought stress in variable genotypes of a perennial ryegrass cultivar. Sixteen infected (+E) and 16 uninfected (-E) ramets were planted in the greenhouse for each of 10 ryegrass genotypes. Eight +E and eight -E plants per genotype were exposed to three sequential droughts where water was withheld for 11-14 d, resulting in <5% soil moisture; the others (control) were watered as needed. Response variables were tiller numbers 1 wk and 4 wk after drought, and leaf area and dry mass of shoots and roots 7 wk after drought. In both control and drought, -E plants had more tillers, and greater leaf area and total mass, than +E plants, suggesting a detrimental effect of endophytic fungi. Fungal hyphae survived the drought and were abundant in post-drought, +E plants. The effects of endophytes were specific for particular host genotypes, as exemplified by significant genotype × endophyte interactions. Root : shoot ratio and percent of mass allocated to tiller bases (a rough measure of resource storage) showed genotype × endophyte × drought interactions. There was plasticity for root : shoot ratio and genetic variation in the ability to restore root growth during recovery from drought. For 7 of 10 genotypes, -E plants showed an equal or greater allocation to tiller bases than +E plants following drought recovery, illustrating a cost to endophyte infection for some genotypes. The symbiotic relationship between L. perenne and its endophyte primarily benefits the fungus, not the host, under many environmental conditions.     

In vitro shoot regeneration of Populus deltoides: effect of cytokinin and genotype

Treatment differences were observed in the in vitro adventitious shoot regeneration response from internodal explants of three genotypes of Populus deltoides cultured on media supplemented with five concentrations each of the cytokinins 6-benzyladenine, 2-isopentyladenine, and zeatin. For each of the three genotypes, the greatest number of shoots were consistently regenerated on media containing the cytokinin zeatin. Tissue necrosis resulted when explants from any of the three genotypes were cultured on media supplemented with 6-benzyladenine. A zeatin concentration by genotype interaction was also observed. Genotypic differences in shoot regeneration were observed for 16 genotypes of Populus deltoides when cultured on medium supplemented with 0.5 mgL^–1 zeatin. Six genotypes were highly recalcitrant and failed to regenerate shoots. The percent of explants regenerating was greater than 50% for four genotypes.Abbreviations WNA modified Woody Plant Media - BA N⁶-benzyladenine - 2-iP 2-isopentenyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IBA indole-3-butyric acid - MS Murashige Skoog (1962) medium - NAA 1-naphthaleneacetic acid - PAR photosynthetically active radiation Journal Series No. 8938, Agricultural Research Division, University of Nebraska     

A rapid method to increase the number of F(1) plants in pea (Pisum sativum) breeding programs

In breeding programs, a large number of F(2) individuals are required to perform the selection process properly, but often few such plants are available. In order to obtain more F(2) seeds, it is necessary to multiply the F(1) plants. We developed a rapid, efficient and reproducible protocol for in vitro shoot regeneration and rooting of seeds using 6-benzylaminopurine. To optimize shoot regeneration, basic medium contained Murashige and Skoog (MS) salts with or without B5 Gamborg vitamins and different concentrations of 6-benzylaminopurine (25, 50 and 75 μM) using five genotypes. We found that modified MS (B5 vitamins + 25 μM 6-benzylaminopurine) is suitable for in vitro shoot regeneration of pea. Thirty-eight hybrid combinations were transferred onto selected medium to produce shoots that were used for root induction on MS medium supplemented with α-naphthalene-acetic acid. Elongated shoots were developed from all hybrid genotypes. This procedure can be used in pea breeding programs and will allow working with a large number of plants even when the F(1) plants produce few seeds.     

Genetic analysis of leaf explant regenerability inSolanum chacoense

An F₁ population consisting of 51 genotypes, derived from two unresponsive parental lines ofSolanum chacoense Bitt., was examined for shoot regeneration from leaf explants. Fourteen genotypes failed to respond whereas, among the responsive genotypes, four produced multiple shoots on over 90% of the explants. Estimates of broad-sense heritability were high for both frequency of responsive explants (83%) and the number of shoots per responsive explant (82%). The segregation of the F₁ hybrid progeny was in agreement with the theoretical ratios of a genetic model for tissue culture responsiveness involving three unlinked genes. This study confirms earlier findings concerning the genetic control ofin vitro shoot regeneration from leaf explants inS. chacoense.     

Clonal propagation byin vitro culture of Corchorus (jute)

Callus was produced on cotyledon, shoot tip, hypocotyl and root explants of twoCorchorus species on several media. Cytokinin was necessary for callus production on cotyledon explants. BothC.olitorius genotypes produced most callus on media with zeatin and either NAA or IAA, and theC.capsularis genotype produced most callus on media with IAA and either zeatin or BA. High frequencies of regenerated shoots were obtained from shoot tip explants of both species, from the apical meristem and from callus. Media with 2.0 mg 1⁻¹ BA were superior for both species, and media with zeatin were equally good forC.capsularis only. More regeneration was obtained for all genotypes after subculture of callus on media with 2.0 mg 1⁻¹ zeatin. Cotyledon callus produced less regeneration, also with differences between genotypes; explants of both genotypes ofC.olitorius produced regeneration on a medium with NAA and zeatin, and theC.capsularis genotype produced regeneration on a medium with IAA and BA. Limited regeneration from root explant callus was obtained forC.capsularis only on medium with BA and IAA. Regeneration was not obtained from hypocotyl callus. Further regeneration of shoots of both species was obtained from secondary callus after subculture, and from nodal segments of regenerated shoots and of seedling shoots cultured on basic MS medium without growth hormones. Roots were produced on about 80% of all shoots after transference to medium with 0.2 mg 1⁻¹ IBA, and rooted plantlets survived and flowered normally after transference to compost.     

Screening of Glycine max and Glycine soja genotypes for multiple shoot formation at the cotyledonary node

Summary To identify genotypes which may give better plant regeneration responses in vitro, multiple shoots were induced from 155 Glycine max and 13 Glycine soja genotypes from maturity groups 000 to VII on B5 medium supplemented with 1 or 5 mol benzylaminopurine (BAP). The average number of shoots formed show genotype specific and hormone concentration specific responses, with number of shoots ranging from 1 to 12 for different genotypes. The results were reproducible with different seed lots of the same genotype and genotypes with similar genetic backgrounds responded in a similar fashion. No hybrid vigor was observed, except in one instance of F₁ hybrids between low shoot producers where the number of shoots obtained were higher than either parent. The root forming ability of cuttings of soybean plants grown in vivo showed general agreement with shoot forming ability in vitro. The ability to form multiple shoots appears to be genetically controlled.This research was supported by funds from the Illinois Agricultural Experiment Station and Agrigenetics Research Associates     

大花萱草组培快繁体系的研究

以大花萱草的6个品种为试材,主要研究了其组织培养中的激素配比、外植体类型、基因型以及不定芽的生根条件。结果表明：适合于大花萱草紫蝶的最佳愈伤组织及不定芽诱导培养基为MS+1 mg·L^-1 6-BA+0.1 mg·L^-1 NAA,出愈率、分化率和平均出芽数分别为63.33%、91.11%和5.86;但是,上述指标在基因型间表现出差异;花茎是诱导不定芽的最佳外植体;1/2MS+0.2 mg·L^-1 NAA是紫蝶较适宜的生根培养基,生根率81.11%,平均生根数6.08;本实验建立了3个品种的再生体系,平均出芽数均在4个以上,其中“金娃娃”最高,是6.88。     

In vitro regeneration of cereals based on multiple shoot induction from mature embryos in response to thidiazuron

The in vitro competency of mature cereal embryos (winter, spring and durum wheats, oat, barley and triticale) was assessed for direct multiple shoot production on culture media containing the plant growth regulators, thidiazuron (TDZ) and/or 6–benzylaminopurine (BAP). Mature embryos of CDC Dancer oat showed the best response, with 69 shoots per explant on culture medium containing a combination of 4.5 μM TDZ and 4.4 μM BAP. TDZ alone induced about 16 shoots per explant from the oat. Among the wheat genotypes, durum wheat showed the most number of shoots (35) per explant on culture medium containing 4.5 μM of TDZ and 4.4 μM of BAP. With TDZ alone, shoot regeneration for durum wheat ranged from 27–32 shoots per explant. The regeneration frequency from the three winter wheat genotypes ranged from 11–25 shoots per explant and was highest on culture medium containing 9.1 μM TDZ and 4.4 μM BAP. The latter culture medium was also effective for a triticale genotype, inducing 34 shoots per explant. The regeneration from mature embryos of barley genotypes ranged from 5–9 shoots per explant. The mature embryos of all the cereals tested could be used for in vitro regeneration with TDZ and TDZ+BAP combinations.     

<Emphasis Type="Italic">In vitro</Emphasis> plant regeneration through somatic embryogenesis and direct shoot organogenesis in <Emphasis Type="Italic">Pennisetum glaucum</Emphasis> (L.) R. Br.

An efficient in vitro plant regeneration protocol through somatic embryogenesis and direct shoot organogenesis has been developed for pearl millet (Pennisetum glaucum). Efficient plant regeneration is a prerequisite for a complete genetic transformation protocol. Shoot tips, immature inflorescences, and seeds of two genotypes (843B and 7042-DMR) of pearl millet formed callus when cultured on Murashige and Skoog (MS) medium supplemented with varying levels of 2,4-dichlorophenoxyacetic acid (2,4-D; 4.5, 9, 13.5, and 18 μM). The level of 2,4-D, the type of explant, and the genotype significantly effected callus induction. Calli from each of the three explant types developed somatic embryos on MS medium containing 2.22 μM 6-benzyladenine (BA) and either 1.13, 2.25, or 4.5 μM of 2,4-D. Somatic embryos developed from all three explants and generated shoots on MS medium containing high levels of BA (4.4, 8.8, or 13.2 μM) combined with 0.56 μM 2,4-D. The calli from the immature inflorescences exhibited the highest percentage of somatic embryogenesis and shoot regeneration. Moreover, these calli yielded the maximum number of differentiated shoots per callus. An efficient and direct shoot organogenesis protocol, without a visible, intervening callus stage, was successfully developed from shoot tip explants of both genotypes of pearl millet. Multiple shoots were induced on MS medium containing either BA or kinetin (4.4, 8.8, 17.6, or 26.4 μM). The number of shoots formed per shoot tip was significantly influenced by the level of cytokinin (BA/kinetin) and genotype. Maximum rooting was induced in 1/2 strength MS with 0.8% activated charcoal. The regenerated plants were transferred to soil in pots, where they exhibited normal growth.     

In vitro culture methods in sorghum with shoot tip as the explant material

Twenty-four diverse genotypes of sorghum were evaluated for response to callus induction and plant regeneration with two media viz., MS and NBKNB using shoot tips as the start material to identify a model genotype. None of the genotypes tested showed promising results. Therefore, alternative methods of in vitro pathways using shoot meristem isolated from shoot tips were explored. Shoot apical meristems were isolated and were induced to multiple shoots or multiple shoot buds pathway by manipulation of thidiazuron (TDZ), 6-benzyl adenine (BAP) and 2, 4-dichlorophenoxy acetic acid (2, 4-D). Choice of the pathway whether large-scale multiplication of shoots or production of target tissues for transformation can be exercised based on the needs and applications. A simple procedure, for large scale handling of shoot tips is described in detail. Electron microscopic studies revealed that meristems isolated from 7-day-old seedlings are superior because of possessing greater number of transformation competent cells.     

Sugar beet (Beta vulgaris L.) morphogenesis in vitro: effects of phytohormone type and concentration in the culture medium, type of explants, and plant genotype on shoot regeneration frequency

In vitro regeneration techniques have been optimized for seven strains and cultivars of sugar beet (Beta vulgaris L.) bred in Russia. The frequency of shoot regeneration from somatic cells and tissues of sugar beet varies from 10 to 97% depending on the explant type, culture-medium composition, and genotype. The in vitro regeneration potential has been estimated in plants with different genotypes. The effect of medium composition (phytohormones and carbohydrates) on the frequency of the formation of a morphogenic callus competent for plant regeneration has been determined. The effect of the types and concentrations of various cytokines (zeatin, kinetin, and 6-benzylaminopurine) on direct shoot regeneration from cotyledon nodes has been estimated. The culture-medium composition has been optimized for direct shoot regeneration from petioles. The effects of different concentrations of abscisic acid on the frequency of shoot regeneration from a morphogenic callus has been studied. Micropropagation has been used to obtain petiole explants and reproduce the shoots obtained by direct regeneration from cotyledon nodes, petioles, and calluses. Improved shoot-regeneration methods can be used for both agrobacterial and bioballistic genetic transformation of the sugar beet genotypes studied.     

Architecture and size relations: an essay on the apple (Malus x domestica, Rosaceae) tree

The influence of tree size independent of age on some architectural features (annual shoot length, lateral branching, flowering) was investigated on 4-yr-old apple (Malus × domestica) trees either own-rooted or grafted on the dwarfing rootstock M.9, giving rise to large and small trees, respectively. Tree size significantly affected the length of the first annual shoot of bottom branches with a lesser effect on the subsequent annual shoots of the same branches and on branches situated higher in the tree canopy. The linear regression parameters, i.e., slopes and intercepts, between annual shoot length and number of growing laterals were affected by the genotype and, depending on genotype, by tree size. Flowering was generally lower, delayed, and more irregular on large trees compared to small trees, with on average similar ranking of genotypes regardless of tree size. This study provides evidence for a specific effect of tree size, as affected by the root system, on architectural development of the apple tree regardless of the genotype. From an architectural viewpoint, the dwarfing mechanism could be interpreted as a faster physiological aging essentially related to the reduction in length of the first annual shoot of bottom branches and the high flowering on this shoot.