In vitro shoot-tip grafting improves recovery of cotton plants from culture

A rapid in vitro shoot-tip grafting (STG) technique was adapted to increase recovery of intact cotton plants from shoots developed in culture. Induction of root organogenesis in cotton shoots is genotype dependent and unreliable. The resulting loss of regeneration potential due to failure to form roots can vary from 30 to 80% according to genotype and represents a significant bottleneck in the overall recovery of plants from culture. If the non-rooting shoots are transgenic, the loss in regenerated plant material can be substantial. In vitro grafting of cotton shoots to seedling rootstock proved to be a simple and reliable method allowing 90–100% recovery of non-rooting shoots from culture. Success of any given graft was directly related to scion size (0.8–1.0 cm) and age (14–35 days) of the seedling rootstock. The method appeared to be genotype independent, and varietal differences between rootstock and scion did not effect the rate of plant recovery from culture. This revised version was published online in June 2006 with corrections to the Cover Date.     

High-frequency regeneration via multiple shoot induction of an elite recalcitrant cotton (Gossypium hirsutum L. cv Narashima) by using embryo apex

Cotton (Gossypium hirsutum L.) is one of the most commercially important fiber crops in the world. Compared with other crops, cotton represents a recalcitrant species for regeneration protocols. The development of efficient and rapid regeneration protocol for elite Indian cotton variety could help improve the quality characteristics and biotic or abiotic stress tolerance. Here we report a novel regeneration protocol in Indian cotton cultivar Narashima. The maximum number of multiple shoots obtained was 16 per explants, performance which has never been achieved in any prior reports. The embryo apex explants were isolated from 2 d old in vitro growing seedlings. Explants were cultured on MS medium containing different plant growth regulator combinations in order to induce multiple shoots. Among the tested combinations, the 2 mg/l of 6-benzylaminopurine (BAP) and 2 mg/l kinetin (KIN) proved to be most suited for achieving the maximum number of multiple shoots. The elongation of multiple shoots was obtained in media supplemented with gibberellic acid (GA3). The regenerated plants were successfully hardened in earthen pots after adequate acclimatization. This method avoids callus tissue, the stage of regeneration which may lead to somaclonal variation. The important feature of the presented method is shortening of regeneration time, as well as the induction of a high number of multiple shoots per explants. The present protocol may provide an efficient and rapid regeneration tool for obtaining more stable transformants from embryo apex explants of Indian cotton cultivar Narashima.     

Effect of light intensity on in vitro multiple shoot induction and regeneration of cotton (Gossypium hirsutum L. cv Khandawa-2)

Cotyledonary nodes taken alongwith shoot apex from seedlings of cotton (G. hirsutum) proliferated into shoots on nutrient agar medium supplemented with cytokinins. In the presence of optimal plant growth regulators, low light intensity enhanced the number of shoots initiated per explant in cotton. An average of 33.5 +/- 2.9 shoots were obtained from a single explant cultured for 8 weeks which is about four fold higher than the values reported in earlier protocols. The isolated shoots were rooted on nutrient agar medium supplemented with alpha-naphthalene acetic acid and transferred to soil after acclimatization. Regenerated plants were morphologically identical to the seed-germinated plants and were fertile.     

NaCl胁迫下棉花体内 Na~+ 、K~+分布与耐盐性

采用盐化土壤方法 ,选择苗期耐盐性较强的陆地棉品种枝棉 3号和中棉所 1 9及耐盐性较弱的品种泗棉 2号和苏棉 1 2号 ,研究了盐胁迫下棉苗体内 Na+、K+的运输和分配与耐盐性的关系。结果表明 ,耐盐品种根系具有一定的截留 Na+作用。棉花地上部盐分器官水平上的区域化分布特征明显 :2 0 0 mmol/L Na Cl胁迫下 ,枝棉 3号叶片中的 Na+含量显著低于泗棉 2号 ,茎及叶柄中的 Na+含量显著高于泗棉 2号 ;棉株地上部茎、叶柄、叶片中的 Na+含量分别由下而上逐渐减小 ,相同节位的茎、叶柄中的 Na+含量大于叶片 ,枝棉 3号更显著。1 0 0 mmol/L和 1 50 mmol/L Na Cl胁迫下 ,枝棉 3号和中棉所 1 9K+/Na+显著高于泗棉 2号和苏棉 1 2号。Na+在茎和叶柄中滞留和积累 ,根中的 K+向地上部选择性运输 ,以维持叶片中较高的 K+/Na+,是棉花耐盐性的一个重要特点     

Rapid in-vitro plant regeneration of cotton (Gossypium hirsutum L.)

A rapid, clonal propagation procedure has been developed to regenerate mature cotton (Gossypium hirsutum L.) plants from pre-existing meristems that were excised from in-vitro-grown tissues. This plant regeneration procedure was applicable to diverse cotton germplasms and required specific concentrations of 6-benzylaminopurine (BA) depending on the origin of the meristems. All shoots regenerated directly without a callus phase. Screening BA concentrations (0.0–10.0 μm) demonstrated that shoot meristems (apices), secondary leaf nodes, primary leaf nodes, and cotyledonary nodes derived from in-vitro-grown 28-day-old seedlings (Paymaster HS26) varied in their ability to form elongated shoots depending on the level of BA. Indicative of a germplasm-independent procedure, a BA concentration screen (0.0, 0.3, 1.0 μm) demonstrated that explants with pre-existing meristems, excised from diverse germlines, were also able to form elongated shoots at 0.3 μm BA. In most cases, elongated shoots derived from this procedure were rooted by a two-step process: an in-vitro maturation step (Murashige and Skoog medium-activated charcoal) followed by planting into soil after basal application of Rootone. This BA plant regeneration procedure was rapid, reproducible, and highly efficient for Stoneville 7A, Paymaster HS26, and other high-fiber-yielding germlines. Regenerated plants were phenotypically normal and all of the mature plants regenerated to date have initiated flowers and set viable R₁ seeds. Received: 15 March 1997 / Revision received: 28 August 1997 / Accepted: 5 September 1997     

Induction of multiple shoots and plant regeneration from ‘accessory buds’ of nodal segments from field-grown mature cotton plants (Gossypium hirsutum L.)

Summary Sprouting of a single shoot was obtained from nodal segments of field-grown cotton plants (Gossypium hirsutum L. cv. DCH-32 and NHH-44) when cultured on Murashige and Skoog's (MS) basal medium devoid of plant growth regulators. Pruning of the sprouted shoot followed by re-culturing of the explant on MS basal medium supplemented with 2.22 μM benzylaminopurine triggered the dormant ‘accessory buds’ present in the node to proliferate and differentiate into multiple shoots. The shoots elongated on the same medium and rooted on MS basal medium devoid of growth regulators. Plantlets were hardened under greenhouse conditions and transferred to the field. Flowering and boll formation were observed in these plants at maturity. This technique for successful clonal propagation from mature cotton tissues should permit the rapid multiplication of plants selected based on specific phenotypic or genotypic characteristics.     

Multiple Shoot Induction and Plant Regeneration from Embryo Axes of Six Cultivars of Gossypium hirsutum

The report describes in vitro plant regeneration from embryo axis explants of six cultivars of cotton. Induction of a maximum number of multiple shoots in all six cultivars could be achieved on Murashige and Skoog's (MS) salts and Gamborg's (B5) vitamins supplemented with 0.4 μM benzyladenine (BA) and 0.1 μM napthaleneacetic acid (NAA). Elongated shoots could be rooted on half strength medium supplemented with 0.5 μM NAA. Rooted shoots survived (92 %) after hardening in the greenhouse and grew to maturity (100 %) after transfer to field. This revised version was published online in July 2006 with corrections to the Cover Date.     

In vitro proliferation of shoots and regeneration of cotton

Shoot proliferation from different explants of several Indian cultivars of cotton was studied in culture. Cotyledonary nodes taken along with the shoot apex of seedlings produced multiple shoots on modified MS nutrient agar supplemented with cytokinins. 6-Benzyladenine was most effective in inducing growth of multiple shoots. Explants of several genotypes formed organogenic masses that differentiated to secondary shoots on repeated subculture. The isolated shoots were rooted on basal medium supplemented with naphthaleneacetic acid and were transferred to soil after acclimatization. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro induction of multiple shoots and plant regeneration in cotton (Gossypium hirsutum L.)

Induction of multiple shoots in cotton (Gossypium hirsutum L. cv. Anjali-LRK 516) has been achieved with cotyledonary nodes devoid of cotyledons and apical meristems. Explants from 35-day-old seedlings yielded the maximum number of shoots (4.7 shoots/explant) using Murashige and Skoog (MS) basal medium supplemented with 6-benzylaminopurine and kinetin (2.5 mg/1 each). Explants from 35-day-old seedlings raised in glass bottles produced a higher number of multiple shoots (8.3 shoots/explant) than those grown in glass tubes and cultured on the same shoot induction medium. Elongation of multiple shoots was obtained on liquid or agar MS basal medium without phytohormones. In vitro shoots were rooted on half-strength agar-solidified MS basal medium or with 0.05 or 0.1 mg/1 naphthaleneacetic acid. Hardening and survival of tissue culture plantlets was 95% under greenhouse conditions.Abbreviations BAP 6-Benzylaminopurine - GA₃ Gibberellic acid - MS Murashige and Skoog medium - NAA -Napthaleneacetic acid     

Habitat cues synergize to elicit chemically mediated landing behavior in a specialist phytophagous insect,the grape berry moth

Many phytophagous insects locate their host plant using mixtures of volatile compounds produced by the plant. A key behavior in the host location process that has been the focus of decades of behavioral research is optomotor anemotaxis. Another key step in host location is landing on (or near) the odor source. In previous work, rubber septa emitting a synthetic blend of volatiles extracted from young shoots of grape plants, Vitus spp. (Vitaceae), elicited equivalent levels of oriented upwind flight by female grape berry moths (GBM), Paralobesia viteana (Clemens) (Lepidoptera: Tortricidae), as did actual (control) grape shoots. However, in contrast to the shoots, females did not land on the odor source. In this study, we used flight tunnel assays to investigate the landing response of GBM females with respect to chemical and visual stimuli, as well as differences in relative humidity. When stimuli were presented individually, only the synthetic blend of host plant volatiles elicited equivalent levels of oriented upwind flight compared to the plants. Interestingly, wet cotton strips elicited low but consistent levels of upwind flight. In paired assays, only the synthetic blend paired with wet cotton strips elicited landing, although at significantly lower levels than that elicited by grape shoots. To achieve landing rates equivalent to live grape shoots, grape berry moth females required all three stimuli we tested: host odor cues, moisture, and visual cues simulating a grape shoot. These results suggest the cues have a synergistic effect, and that landing behavior requires complex sensory processing using multiple sensory inputs. Furthermore, these results suggest that moisture plays an important role in the host plant location process.     

A procedure for biolistic transformation and regeneration of transgenic cotton from meristematic tissue

We have optimized methods for transformation of cotton meristem tissue using the Bio-Rad PDS/1000/He gene gun, selection of transformed tissue, and regeneration of transformed cotton plants. We have used either single or multiple bombardments of cotton tissue with 1.6-Å particles at rupture pressures of 90 or 110 kg/cm². The distance between the tissue and the source of particles can be varied between 3 and 6 cm. After bombardment, transformed cotton tissue is identified by selection for growth on media supplemented with 50 μg/mL kanamycin. Tissue sections that form leaves, shoots and at least two roots are then transferred to media supplemented with 100 mg indoleacetic acid (IAA) to favor formation of extensive root systems. The plantlets are then transferred to soil, hardened off, and grown in the greenhouse. These plants have been confirmed to be transgenic by western-blot analysis of leaf protein extracts with polyclonal antiserum to the neomycin phosphotransferase II gene product.     

Efficient genotype-independent cotton genetic transformation and genome editing

Cotton(Gossypium spp.) is one of the most important fiber crops worldwide. In the last two decades, transgenesis and genome editing have played important roles in cotton improvement. However,genotype dependence is one of the key bottlenecks in generating transgenic and gene-edited cotton plants through either particle bombardment or Agrobacterium-mediated transformation. Here, we developed a shoot apical meristem(SAM) cell-mediated transformation system(SAMT) that allowed the transformation of r...     

Influence of explants, genotypes and culture vessels on sprouting and proliferation of pre-existing meristems of cotton (Gossypium hirsutum L. and Gossypium arboreum L.)

Summary A simple and efficient method for multiple shoot induction and proliferation was achieved in six Indian cotton cultivars from the pre-existing meristems of 21-d-old in vitro-grown seedlings. Combinations of naphthalene acetic acid (0.3–10.7 μM) and 6-benzylaminopurine (BA; 2.2 or 4.4 μM) were used for induction of shoots. The shoots proliferated and were maintained on MS (Murashige and Skoog) medium supplemented with 4.4 μM BA. Simultaneous elongation of shoots was obtained in the same medium. Optimum multiplication was observed in cv. LRK-516 (19.7±4.6), in cotyledonary nodes isolated from the adjoining node and cultured individually in 250 ml flasks. This indicates lateral inhibition of adjoining meristems. A positive influence of culture flasks as opposed to test tubes on the proliferation of multiple shoots was observed in all six cultivars tested. The morphogenic response varied with genotype and the nature of explants. Rooting of elongated shoots was achieved on MS medium devoid of growth regulators. The plantlets were transferred to the field after hardening in the greenhouse. All plants flowered and formed bolls on maturity.     

Multiple shoot induction and plant regeneration from embryonic axes of cotton

Cytokinins are involved in shoot development of plants. Events of multiple bud formation and shoot development in apical embryonic axes of cotton treated for 2 or 20 days with the cytokinin benzyladenine (BA), were compared with the development of untreated control axes. Meristematic regions (supernumerary vegetative buds) were observed in axes treated for 20 days with BA. An average of 3.4 shoots per embryonary axis was obtained when explants were cultured on medium supplemented with 3 mg l-1 BA. Higher and lower concentrations of the growth regulator yielded fewer shoots per explant. Results shown in this report suggest that BA is directly responsible for re-programming the embryonic apical meristem axes of cotton toward the production of multiple buds and subsequent shoot development. This revised version was published online in June 2006 with corrections to the Cover Date.     

Availability of sparingly soluble phosphorus sources to cotton (Gossypium hirsutum L.), wheat (Triticum aestivum L.) and white lupin (Lupinus albus L.) with different forms of nitrogen as evaluated by a 32P isotopic dilution technique

Background & Aims

Previous studies revealed that cotton plants grown on soils with low available-P were accessing significant non-fertilizer P sources. This suggests that cotton can access stable-P pools from soil. This study examined cotton??s ability to utilize sparingly soluble P sources in comparison with wheat and white lupin.

Methods

Plants were grown for 45 days in a Vertosol supplied with AlPO₄ and hydroxyapatite, and NH₄-N or NO₃-N. A ³²P dilution technique was used to determine the availability and plant uptake of P from these P sources.

Results

Three species differed substantially in P acquisition from the P sources. When averaged over N sources, the proportion of P in shoots sourced from AlPO₄ was 89%, 54% and 19% for wheat, cotton and white lupin, respectively. When supplied hydroxyapatite, white lupin sourced 75% from the added P, in contrast to 36% for wheat and 17% for cotton. NH₄-N nutrition increased the availability of hydroxyapatite to all the species and AlPO₄ to cotton and white lupin.

Conclusion

Cotton is inefficient in utilizing sparingly soluble P while wheat is efficient in mobilising AlPO₄ and white lupin is efficient in using hydroxyapatite. The superiority of wheat in AlPO₄ utilization may be related with its high root length density.     

Effects of fusicoccin on intact cotton plants (Gossypium hirsutum L.)

Fusicoccin (FC) was applied as a spray to shoots of intact field- and glasshouse-grown cotton plants. Distortions of shoot morphology resulted. Stems and petioles of FC-treated plants were irregular in diameter and twisted, whereas leaf laminae were curled and crinkled. Shoot elongation was inhibited by FC; the effect was dependent upon the concentration and timing of the applications.Abbreviation FC fusicoccin     

Slow desiccation leads to high-frequency shoot recovery from transformed somatic embryos of cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L. cv. Coker 310 FR)

In Agrobacterium-mediated genetic transformation of cotton (Gossypium hirsutum L. cv. Coker 310FR) the frequency at which somatic embryos were converted to plantlets was significantly improved by subjecting the embryos to slow physical desiccation. We used Agrobacterium strain GV3101 containing the binary vector pGSFR with the nos-nptII gene for in vitro selection and the 35S gus-int fragment as a reporter to optimize the transformation protocol. Although the concentration of kanamycin was reduced during embryogenesis and embryo maturation, even at the lower levels somatic embryos were predominantly abnormal, showing hypertrophy and reduced or fused cotyledons or poor radicle ends. A majority of these embryos (more than 75%) were beta-glucuronidase (GUS)-positive. Embryos with an abnormal appearance showed a very poor conversion to plantlets. However, these embryos, when subjected to slow physical desiccation followed by transfer to fresh medium, regenerated single or multiple shoots from the cotyledonary end. These shoots could be grafted on wild-type seedling stocks in vitro, which, following their transfer to soil, developed normally and set seeds. Regenerated plants tested positive for the transgene by Southern analysis. An overall scheme for the high-frequency production of cotton transgenics from both normal and abnormal appearing somatic embryos is presented.     

Verticillium wilt of cacao in Uganda: symptoms and establishment of pathogenicity

Pathogenicity tests have shown that Verticillium dahliae Kleb. causes a vascular wilt disease of cacao (Theobroma cacao L.) in Uganda. Isolates of V. dahliae from cacao, okra (Abelmoschus esculentus Medik.), and cotton (Gossypium hirsutum L.) induced similiar symptoms on artificially inoculated cacao seedlings. External symptoms of the disease include acute wilting, foliar chlorosis and abscission, stunting of roots and shoots, and production of suckers at lower nodes of severely affected plants. The internal symptoms are vascular discoloration, tylosis, and presence of gum deposits and mycelium of V. dahliae in xylem vessels.     

我国陆地棉基础种质表型性状的遗传多样性分析

选用43份陆地棉基础种质为研究材料,随机区组排列种植,并进行果枝数、铃数、株高等田间性状调查和衣分、铃重、纤维品质等测定.按照不同时期、不同来源、不同生态区对这些基础种质分别进行表型性状的遗传多样性分析.结果表明:基础种质间在产量、品质、农艺性状等表型性状上差异显著或极显著,遗传多样性指数为0.88;3期基础种质大部分性状差异不显著,但第2、3期基础种质比第1期的纤维长、整齐度高、细度好、衣分增加、早熟性提高、抗病和耐旱性增强,第2期基础种质遗传多样性和遗传丰富度最高;来自不同棉区的基础种质表型性状差异较大,黄河流域棉区基础种质综合性状较好,长江流域棉区产量性状较高,北部特早熟棉区早熟性好,美国引进种质抗黄萎病性较强;国内基础种质比国外品种在纤维长、强、细上的变异系数均有不同程度降低,但国内基础种质表型多样性比引进品种高.以上研究说明引进品种经过长期的环境适应、自然选择和人工选育后,产生了表型变异较为丰富的基础种质类型.     

Immunochemical detection with rabbit polyclonal and mouse monoclonal antibodies of different pools of phytochrome from etiolated and green Avena shoots

While two monoclonal antibodies directed to phytochrome from etiolated oat (Avena sativa L.) shoots can precipitate up to about 30% of the photoreversible phytochrome isolated from green oat shoots, most precipitate little or none at all. These results are consistent with a report by J.G. Tokuhisa and P.H. Quail (1983, Plant Physiol. 72, Suppl., 85), according to which polyclonal rabbit antibodies directed to phytochrome from etiolated oat shoots bind only a small fraction of the phytochrome obtained from green oat shoots. The immunoprecipitation data reported here indicate that essentially all phytochrome isolated from green oat shoots is distinct from that obtained from etiolated oat shoots. The data indicate further that phytochrome from green oat shoots might itself be composed of two or more immunochemically distinct populations, each of which is distinct from phytochrome from etiolated shoots. Phytochrome isolated from light-grown, but norflurazon-bleached oat shoots is like that isolated from green oat shoots. When light-grown, green oat seedlings are kept in darkness for 48 h, however, much, if not all, of the phytochrome that reaccumulates is like that from etiolated oat shoots. Neither modification during purification from green oat shoots of phytochrome like that from etiolated oat shoots, nor non-specific interference by substances in extracts of green oat shoots, can explain the inability of antibodies to recognize phytochrome isolated from green oat shoots. Immunopurified polyclonal rabbit antibodies to phytochrome from etiolated pea (Pisum sativum L.). shoots precipitate more than 95% of the photoreversible phytochrome obtained from etiolated pea shoots, while no more than 75% of the pigment is precipitated when phytochrome is isolated from green pea shoots. These data indicate in preliminary fashion that an immunochemically unique pool of phytochrome might also be present in extracts of green pea shoots.Abbreviation ELISA enzyme-linked immunosorbent assay - mU milliunit - Pfr far-red-absorbing form of phytochrome - Pr red-absorbing form of phytochrome