Increased Callus Induction Research Articles

Optimized medium composition in Physalis alkekengi callus culture altered nitric oxide level for inducing antioxidant enzyme activities and secondary metabolites

Physalis alkekengi L. is a valuable medicinal plant from the Solanaceae family and has multiple therapeutic applications. This study aimed to develop an optimized protocol for callogenesis in P. alkekengi to obtain friable calluses with high biomass. The effect of different concentrations of picloram, casein hydrolysate (CH), basal media (Murashige and Skoog (MS) and Gamborg (B5)), and static magnetic field (SMF) were investigated on the callus induction and growth, signaling molecules, and enzymatic and non-enzymatic antioxidants. Results showed that CH (200 mgL−1) and SMF4 mT for 90 min increased callus induction and fresh weight in P. alkekengi, while different concentrations of picloram reduced callogenesis. Hypocotyl explants showed various callogenesis and metabolic responses depending on the basal medium type. The 2B5 medium supplied with CH 200 (mgL−1) induced friable and cream calluses with high biomass (0.62 g) compared to the MS medium (control). The maximum activity of superoxide dismutase and catalase activities was identified in the 2B5 medium and peroxidase in the 2MS medium. The highest total phenolic (129.44 µg g−1DW) content and phenylalanine-ammonia lyase activity were obtained in the 2MS medium, and total withanolides (49.86 µg g-1DW) and DPPH radical scavenging activity were observed in the 2B5 medium. The 2MS medium boosted the hydrogen peroxide and nitric oxide levels, while their contents alleviated in the 2B5 medium, although these parameters were higher than the control. The findings of this study suggest that an effective protocol for successful callogenesis in P. alkekengi and the nutrient composition of culture medium by affecting the level of signaling molecules can control the antioxidant defense system and callus growth.

The interaction effect of Salt stress and Lysine Concentrations in some properties of Fenugreek callus

The callus induction by use 1.5mg/l of α-naphthalene acetic acid (NAA) as auxin and 2mg/l of BA as cytokinins were added to MS medium that prepared as above before sterilize step. Sodium Chloride and Lysine effect on callus induction and growth of Fenugreek (Trigonella foenum-graecum L.).Five concentration of Sodium Chloride Salts (0, 2, 4, 6 and 8) were added to the culture medium and five concentrations of lysine (0, 10, 20, 30and 40mg/l) added to cultured medium. Results showed that NaCl at (2g/l) increased callus induction, fresh weight, dry weight and potassium ions of callus. The study also showed that (2 g \\l) of NaCl was signification increased Sodium and Chloride ions of callus. Lysine (Lys) effect on callus characteristics:-The treatment 40mg\\l outperformed of the rest treatments including control by gave the highest values of callus fresh and dry weights, as well as ions, soluble carbohydrates

Influence of Epichloë coenophiala, a seed-borne endophyte, on the micropropagation of tall fescue

Fil: Regalado Gonzalez, Jose Javier. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Ciudad Universitaria. Instituto de Micologia y Botanica. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Micologia y Botanica; Argentina

Use of gamma radiation to induce mutations in rice (Oryza sativa L.) and the selection of lines with tolerance to salinity and drought

One of the strategies for genetic improvement of Oryza sativa L. (rice) is the use of radiation-induced mutagenesis to search for materials suitable for production in marginal conditions, such as drought and salinity. In this present study, indica rice ‘CR-5272’ embryogenic calluses and seeds were exposed to gamma radiation. Survival and growth of plants derived from embryogenic calluses and seeds were evaluated in vitro in the presence of selection factors for salinity and drought (NaCl and sorbitol). Plants regenerated from irradiated calluses and seeds were screened for tolerance to these conditions and promising lines were identified. Culture of plants regenerated from irradiated calluses (M1) allowed the selection of 60, 58 and 15 promising lines with tolerance to salinity, drought, and both factors, respectively. Six promising salt-tolerant lines and four drought-tolerant lines were selected from plants grown from irradiated seeds (M2). A hormetic response to irradiation was observed in seeds; seeds irradiated at 50 Gy showed increased callus induction and callus weight. This study allowed advancements in the improvement of rice ‘CR-5272’ through the production of promising lines that are being evaluated in field studies.

Antimitotic and hormone effects on green double haploid plant production through anther culture of Mediterranean japonica rice

Rice double haploid (DH) plants are produced mainly through anther culture. In order to improve the anther culture protocol, microspores of two japonica rice genotypes (NRVC980385 and H28) were subjected to three growth regulator combinations and four colchicine treatments on induction medium. In addition, a post anther culture procedure using colchicine or oryzalin was tested to induce double haploid plantlets from haploid plantlets. A cold pre-treatment of microspores for 9 days at 10 °C increased callus induction 50-fold in the NRCV980385 genotype. For both genotypes, 2 mg L−1 2,4-D and 1 mg L−1 kinetin on colchicine-free induction medium gave the best culture responses. The culturability of both genotypes changed on colchicine-supplemented induction media. A high genotype dependency was recorded for callus induction, callus regenerating green plantlets and regeneration of green double haploid plantlets. Colchicine at 300 mg L−1 for 48 h enhanced callus induction 100-fold in H28. Colchicine-supplemented media clearly improved green double haploid plantlet regeneration. We showed that the post-anther culture treatment of haploid plantlets at 500 mg L−1 of colchicine permitted fertile double haploid plantlets to be generated. Finally, an enhanced medium-throughput flow cytometry protocol for rice was tested to analyse all the plantlets from anther and post anther culture.

Robust genetic transformation of sorghum (Sorghum bicolor L.) using differentiating embryogenic callus induced from immature embryos

BackgroundSorghum (Sorghum bicolor L.) is one of the world’s most important cereal crops grown for multiple applications and has been identified as a potential biofuel crop. Despite several decades of study, sorghum has been widely considered as a recalcitrant major crop for transformation due to accumulation of phenolic compounds, lack of model genotypes, low regeneration frequency and loss of regeneration potential through sub-cultures. Among different explants used for genetic transformation of sorghum, immature embryos are ideal over other explants. However, the continuous supply of quality immature embryos for transformation is labour intensive and expensive. In addition, transformation efficiencies are also influenced by environmental conditions (light and temperature). Despite these challenges, immature embryos remain the predominant choice because of their success rate and also due to non-availability of other dependable explants without compromising the transformation efficiency.ResultsWe report here a robust genetic transformation method for sorghum (Tx430) using differentiating embryogenic calli (DEC) with nodular structures induced from immature embryos and maintained for more than a year without losing regeneration potential on modified MS media. The addition of lipoic acid (LA) to callus induction media along with optimized growth regulators increased callus induction frequency from 61.3 ± 3.2 to 79 ± 6.5% from immature embryos (1.5–2.0 mm in length) isolated 12–15 days after pollination. Similarly, the regeneration efficiency and the number of shoots from DEC tissue was enhanced by LA. The optimized regeneration system in combination with particle bombardment resulted in an average transformation efficiency (TE) of 27.2 or 46.6% based on the selection strategy, 25% to twofold higher TE than published reports in Tx430. Up to 100% putative transgenic shoots were positive for npt-II by PCR and 48% of events had < 3 copies of transgenes as determined by digital droplet PCR. Reproducibility of this method was demonstrated by generating ~ 800 transgenic plants using 10 different gene constructs.ConclusionsThis protocol demonstrates significant improvements in both efficiency and ease of use over existing sorghum transformation methods using PDS, also enables quick hypothesis testing in the production of various high value products in sorghum.

Potensi Padi Bukit sebagai Alternatif kepada Padi Sawah

Our country imported rice to sustain demand from the public. In Malaysia 2 types of rice are grown; wetland rice which is widely cultivated while upland rice is cultivated on a smaller scale mostly by farmers in Sabah and Sarawak. Through plant biotechnology, micropropagation method via tissue culture and quantitative breeding traits are able to contribute to research on upland rice in Malaysia. Prior to the establishment of efficient in vitro propagation of upland rice, study on embryogenic callus induction is crucial. Our findings showed that NAA was not suitable for callus induction. Nevertheless, the highest callus induction was observed when cultivars Wai (75%) and Panderas (90%) were cultured on MSB5 media supplemented with 2 mg/L 2,4-D. Furthermore, addition of 20 g/L sorbitol had increased callus induction of 70% and 80% for Wai and Panderas, respectively. In other assessment, comparative study on ex vitro quantitative breeding trait showed that the height of plants without any fertilizer treatment were higher than commercial EM-treated plant. Rice plant treated with EM commercial soil had the shortest height and eventually died. However, rice planted with commercial EM produced the highest number of grains per panicle, shorter maturity period as compared to control.

An Improve Method for Somatic Embryogenesis of Schisandra chinensis (Turcz.) Baillon

A somatic embryogenesis protocol for plant regeneration of Schisandra chinensis (Turcz.) Baillon was established. Increased callus induction was obtained from mature zygotic embryos on Murashige and Skoog (MS) medium supplemented with 1.0 mg L(-1) N-phenyl-1,2,3-thidiazol-5-yl-urea (TDZ) or 2.0 mg L(-1) 2,4-dichlorophenoxyacetic acid (2,4-D). Addition of Zeatin (Zt) promoted the formation of embryogenic calli. To induce somatic embryogenesis, 2,4-D, TDZ and Zt were incorporated in the medium alone or in combination. Development of the maximum number of somatic embryos (81 globular, 37 heart, 52 torpedo and 37 cotyledon-stage) and germination of the highest number of embryos (50%) was observed on 1/2 MS medium supplemented with 1.0 mg L(-1) TDZ and 0.2 mg L(-1) Zt. Further development of somatic embryos into plantlets was completed in 1/2 MS medium free of plant growth regulators.

CALLUS INİTİATİON FOR INDİRECT POLLEN EMBRYOGENESİS İN ANEMONE CORONARİA

In the present study, anther culture method was used in Anemone coronaria, known as Manisa Tulip in Turkiye, in order to obtain haploid plants. The anthers with uninucleate microspores of the flower buds, collected from an A. coronaria var. coccinea population, were used as explant. In the treatments, two basic tissue culture media (NN and B5) were tested with 4 different auxin concentrations and also 4 different auxin:cytokinin ratios. Anthers were subjected to a chilling pre-treatment for 7 days at +7°C in the dark and then incubated under continuous dark conditions at 25 +1°C. The use of cytokinin together with auxin increased callus induction frequency. It was also found that the treatments with the combinations consisting of both kind of growth regulators in B5 medium showed more successful performance.

Influence of genotype, growth regulators, sucrose level and preconditioning of donor plants on flax (Linum usitatissimumL.) anther culture

The effect of genotype, growth regulators and preconditioning of donor plants on callus induction in anther culture of flax was investigated. Anthers were cultured on modified MS medium supplemented with five different combinations of plant growth regulators. The results suggested that specific combinations of growth regulators must be designed for each genotype. Major differences between the present results and previous reports are discussed. The influence of sucrose concentration was also investigated. For flax cultivar, 'Mikael', callus induction was higher in medium supplemented with 1 mg l(-1) BAP and 2 mg l(-1) 2,4D containing 6% sucrose, while this combination of growth regulators significantly increased callogenesis in cultivars 'Lirina', 'Barbara' and 'Szaphir' when supplemented with 9% or 12% sucrose. The preconditioning of donor plants influenced callogenesis in subsequently isolated anthers. Anthers from donor plants grown at a lower temperature (18/14 degrees C) significantly increased callus induction over those from plants grown at a higher temperature (22/18 degrees C), although each genotype still required optimization of growth regulator combinations in the induction medium. Only 'Mikael' regenerated shoots when the callus was from induction medium supplemented with 2 mg I(-1) BAP and 1 mg l(-1) NAA.

벼 약배양에 효과적인 배지조성 및 저온처리 방법

In spite of potential benefits of anther culture, low productivity of plant regeneration in some genotypes; e.g. tonsil and indica rice, is one of the major obstacles for practical use of anther culture. This study was conducted to improve cold shock method and carbohydrate source for increasing the efficiency of anther culture in rice. The most common carbon source, sucrose was replaced to maltose, which has two molecules of glucose. Maltose increased callus induction 1.4-to 1.8-fold higher in japonica rice, 3.2-to 11.6-fold in tongil types and 2.7-fold in indica rice IR50. Callus induction was increased from 0.2％ to 12.5％ in maltose medium compared to the medium supplemented with sucrose plus glucose in indica rice Tetep. A simple procedure of vacuum packaging of panicles during cold shock treatment prolonged not only anther viability more than 15 days but also increased callus induction more than 2-fold compared to open-air storage (conventional method). Combining of above two methods, callus induction was increased 28 to 56％ in japonica, 13 to 33％ in tonsil type and 12 to 31％ in indica rice. Plant regeneration was increased 14 to 35％ in japonica, 10 to 20％ in tonsil and 4 to 15％ in indica rice, respectively.

Variations in green plant regeneration response from anthers of indica rice and their hybrids with japonica cv. Taipei 309

The green plant regeneration ability from anthers of BR-7, a high yielding indica cultivar, Binnatoa (BA), a salt tolerant indica land race and IR-43 was tested in N6, M8, He2 and R2 media. The response was calculated on the basis of number of anthers producing green plants. The number of green plants per responding anther was also recorded. The response of BR-7 and BA was poor compared to the indica cultivar IR-43 in three of the media that were tested. In N6 medium, green plant regeneration of BA and BR-7 was respectively 10-fold and 100-fold less than the japonica cultivar Taipei 309 (T-309). No anther-derived green regenerant was obtained from another salt tolerant indica land race, Rajashail (RAJ). The N6 medium was selected to test green plant regeneration frequency from anthers obtained from the F1 crosses of T-309 × BR-7, T-309 × BA, T-309 × RAJ and T-309 × BR-7 AC regenerants backcrossed with BA. Our objective was to combine the salt tolerant trait of BA and the high yield of BR-7 in a single line. The intermediate crossing step with T-309 was performed to increase the green plant regenerability of the anthers. All F1 progeny from the crosses with T-309 showed significantly increased callus induction compared to the indica parent although the values were lower than the midparent means. Green plant regeneration compared to their respective indica parents either increased or decreased but never approached the level of T-309.

Ethylene precursors and antagonists increase embryogenesis of Hordeum vulgare L. anther culture

The role of ethylene in microspore embryogenesis and regeneration was analyzed by studying the effects of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) and the ethylene antagonists silver nitrate and silver thiosulphate on the androgenic response of in vitro cultured anthers of seven genotypes of barley. Incorporation of either ACC or silver salts in the culture medium lead to a significant increase in callus induction for five of the seven genotypes tested. The treatment that increased callus induction depended upon genotype. Only anthers cultured on 1 mg l(-1) silver thiosulphate gave rise to fertile plants in all seven genotypes tested.

Improvement of Anther Culture Methods for Haploid Production in Wheat

The yield of haploid production in wheat (Triticum aestivum L.) anther culture has been extremely low with the currently employed methods. The objective of this study was to determine whether variation in anther culture media would affect the yield of haploids. After a 48‐h cold pretreatment, anthers from six spring wheat cultivars and one winter wheat cultivar were cultured on liquid and agar‐solidified media, and in some cases the liquid medium was supplemented with Ficoll (Sigma Chemical Co., St Louis, MO). Liquid culture significantly increased callus induction, which averaged 98.3 calli per 100 anthers for the seven cultivars tested, compared with 5.1 calli per 100 anthers for agar culture. Although the calli from liquid media usually had lower plantlet regeneration than calli from agar‐solidified media, regeneration ability was improved by adding 200 g/L Ficoll 400 to the liquid induction medium, or by changing the plant growth regulator composition of the regeneration medium. By employing the liquid culture method and the modified regeneration medium, it is now more feasible to use doubled haploids in plant breeding practice and genetic research.

Lycopersicon esculentum: Trifoliate plants recovered from anther cultures of heterozygous tftf plants

The effects of the genotype and growth medium composition on callus induction and shoot regeneration from tomato (Lycopersicon esculentum Mill) anthers were studied. Five male sterile varieties, homozygous for the recessive gene ms 10(35), their isogenic fertile counterparts, and nineteen sterile mutants from an F2 population segregating for ms 10(35), were tested. Callus induction and shoot formation were found to be affected by the genotype. The presence of the mutant gene ms 10(35) greatly increased callus induction. A significant interaction concerning callus induction was found between the ms 10(35) gene and the general genetic background. In most of the plants shoot regeneration from the anthers was associated with various degrees of callus production. However, there was no correlation between callus production and the ability to regenerate plants from that callus. Anthers isolated from plants which were heterozygous for the recessive leaf marker trifoliate, regenerated diploid plants with trifoliate leaves. The plants retained the trifoliate phenotype for over six months in culture under non-aseptic condition. Since the trifoliate phenotype appears only in the homozygous recessive state, the evidence that these trifoliate plants are doubled haploids of sporogenic origin is discussed.