N6 Medium Research Articles

Production of wheat doubled haploids by in vitro anther culture

The in vitro culture response of anthers, originating from high yielding and resistance to lodging F1 soft wheat hybrid plants selected under drought conditions, was investigated. The spikes were collected when the microspores were at the mid-uninucleate stage and after 3 days at 40 C for cold pre-treatment, the anthers were cultured in N6 and Gamborg- Eveleg B5 (B5) solid medium. Progeny from 17 different F1 families were used and 9 of them responded to in vitro anther culture and 7 of them produced fertile green plants. Doubled haploid green plants were fertile and produced seeds. Induction of aploid development depends on genotype, stage of male gametophyte development, composition of nutrient media and conditions of cultivation. The effect of the composition of media and different genotypes on wheat anther culture was investigated. From the genotypes produced regenerants, the high yielding genotypes from local breeding collection of wheat produced more albino plants (35.2%) compared to high yielding genotypes from CYMMIT collection (24.92%) ones. Independent of culture medium composition a ratio of doubled haploid/haploid was 4.57:1 and a ratio of haploid/aneuploid was 18.5:1, in particular 81.28 % of green plants were doubled haploid, 17.76% were haploid and the rest (0.96%) were aneuploid

Optimization of Media and Cold Pretreatment for Anther Culture Using Japonica/Indica and Indica/Indica Hybrids and their Callus Induction Comparisons in Different Rice Crosses

Rice ( Oryza sativa ) is pivotal to the lives of a large number of people around the world. Nowadays, anther culture is widely used in rice improvement as it can increase the selection efficiency. By optimizing the induction media and preservation time for cold pre-treatment, a significant improvement of embryogenic potential of the callus has been obtained in 3 different crosses of indica and japonica rice cultivars. It was observed that medium 2A was the best among the others as it was capable to induce a great number of calli while the addition of silver nitrate to the N6 medium doubled up the production of embryogenic calli. Analysis of variance indicated that there was a significant interaction between medium and cold pre-treatment. Seven days of preservation time showed much better callus production in N6 medium compared to MS medium. Significant differences of the callus induction were observed among indica cultivars. Therefore, seven days cold pre-treatment in the combination of silver nitrate in N6 medium can be used in practicing anther culture technique in indica rice. Anther culture technique was also used in twelve rice genotypes that consisted of three different rice crosses namely indica aromatic × japonica aromatic, indica aromatic × indica aromatic, and indica non-aromatic × indica aromatic to produce and comparisons of callus induction. It was found that MRQ50×Rambir Basmati genotype grew faster than other genotypes. Meanwhile, MR219×Rato Basmati took longest time to grow up and MR219×Sadri produced more callus than other genotypes. In addition, the indica non-aromatic× indica aromatic cross had the highest frequency in callus formation. Finally, the genotypes that produced more callus could be used as potential breeding material.

Genetic Analysis of F1 Hybrids Derived from Aromatic (Exotic) × Aromatic (Malaysian) Rice Crosses and Their Callus Induction Performance for Haploid Production

The aim of this study was basically to develop new aromatic rice lines through integrated breeding approaches. After screening and selection of superior parental materials, F1s were raised from four different crosses (MRQ50×Gharib, MRQ50×RMB, MRQ50×RTB and MRQ50×E13) and used for haploid production through Anther culture. Phenotypic coefficient of variation % (PCV), genotypic coefficient of variation % (GCV), heritability % (h2), genetic advance (GA) were estimated for yield and yield contributing traits such as for panicle length, grain per panicle, 1000 grain weight and yield per plant. In the cross MRQ50×RMB, the highest PCV was observed in grain per panicle (34.29) followed by yield per plant (31.73), 1000 grain weight (18.44), and panicle length (12.30). The same trends were occurred in cross MRQ50×RTB. The highest GCV was detected in grain per panicle (32.15) followed by yield per plant (30.90), 1000 grain weight (16.67) and panicle length (10.13). However, in the cross MRQ50×RTB, the highest GCV was obtained in yield per plant (37.89) followed by grain per panicle (30.54) and panicle length (9.33). Yield per plant was distinguished as the highest heritable trait in the cross MRQ50×RMB followed by grain per panicle (87.9), 1000 grain weight (81.7), and panicle length (67.8). On the contrary, in the cross MRQ50×RTB, this scenario was a bit different in which yield per plant was detected a highly heritable trait but the trends were different in grain per panicle, 1000 grain weight, and panicle length. Finally, the highest genetic advance was occurred in yield per plant followed by 1000 grain weight, grain per panicle, and panicle length. These trends were equal in both crosses. Study of callus induction ability of all hybrids studies showed that the best performance of callus induction was MRQ50×Gharib and N6 medium was found to be adequate for callus induction.

Conservation of (Lagunaria Patersonii ) with using in Vitro and in Vivo Propagation

Lagunaria (Lagunaria patersonii) is considered to be a nearly threatened tree in Egypt where there are only few trees facing a very high risk of extinction owing to the effects of pathogens, pollutants and parasites. Hence, this study was conducted to establish a fast in vitro propagation protocol. Seeds, lateral buds and shoot tips were cultured on MS, NN and N6 media with different concentrations of BAP and NAA and their combinations. Results indicated that the optimum medium for the measured characters was MS free growth regulators and The best rooting medium was 1/4 MS. RAPD analysis was also applied to detect the genetic polymorphism among Lagonaria plantlet and their original parents.

English

The objective of the present study was to develop an effective protocol for optimum callus induction, duration of culture and size of explants of primordial panicle of inter-genomic hybrid of Oryza sativa/ Oryza brachyantha. It was observed that, primordial panicle at very young stage (0.5 to 1.5 cm) takes hardly 5 to 7 days for callus initiation. The two basic media tested, the Murashige and Skoog (MS) medium and Chu’s N6 medium supplemented with 2-4-D, NAA and kinetin (2 mg L-1 2 mg L-1 and 1 mg L-1) was better for callus growth and proliferation. In this medium, callus was sub-cultured continuously for 12 passages (12 months) without loss of totipotency. Plantlet regeneration in MS medium was examined using two cytokinins (KIN and BA) along with one auxin NAA at various concentrations and combinations. Tests indicated that, good plant regeneration could be best effected without BA, but with 1.5 to 2.0 mg L-1 of KIN at fixed level of NAA (at 0.5 mg L-1).   Key words: Somaclonal, callus, subculture, wide cross, MS medium, N6 medium.

Comparative analysis of the efficiency of nutrient media for the induction of callus formation for rice hybrids

Data on the effectiveness of the cultivation of rice anthers on the six variants of nutrient media are given; four of them differ from the generally accepted N6 medium by the growth regulators content. It is shown, that RZ and C media in hybrid combinations with the Russian varieties increase callusogenesis six times as compared to N6 medium. The genotypes that are post responsive to the anther culture are allocated.

Doubled haploid production through culture of anthers in rice

Rice anthers were cultured in liquid N6 medium containing various concentration of growth harmones. Haploid plants were treated with different doses of colchicine to develop doubled haploid (DH) plants.The DH plants were transferred to soil and seeds were harvested from mature plants.The DH plants would be suitable for genetics and molecular breeding activities.

Callus Induction and Regeneration fromIn Vitroanther Culture of Rice (Oryza sativaL.)

The standardization of anther culture media for callus induction, and regeneration from anther derived callus of Azucena and Budda rice varieties. Anthers from panicles in which the distance between flag leaf and subtending leaf was 11cm in Azucena and 8 cm in Budda were used for anther culture. At this stage of development, anthers contained uni-nucleate pollen grains. Panicles were subjected to cold pretreatment of 5°C for 8 days. Callus induction frequency in different media combination ranged from 0.66% to 6.66% was observed in N6 medium supplemented with 1.0 mg−l,2, 4-Dichlorophenoxy acetic acid, 2.0 mg−l α-Naphthalene acetic acid and 0.5 mg−l Kinetin. In Azucena variety, highest callus induction (6.66%) while no callusing was found in Budda variety. Highest shoot regeneration (0.33%) from callus was observed in MS medium supplemented with 0.5 mg−l 6- Benzyl amino purine, 0.5 mg−l Kinetin and 80 mg−ladenine sulphate.

갈대(Phragmites communis Trinius) 성숙종자를 이용한 기내 식물체 재분화와 재분화체의 유전적 다양성

This study was carried out to address an efficient in vitro regeneration system from seed-derived callus of Phragmites communis, and to evaluate genetic variations of the regenerants using ISSR markers. Shoot regeneration via calli was greatly influenced by N6 medium compared with MS medium, and plant regeneration frequency was 90% in N6 supplemented with BA 0.25 mg/L and BA 0.5 mg/L. According to ISSR analysis of the thirty regenerants, out of 94 loci detected overall, 16 were identified to be polymorphic with a rate (PR) of 17.0%. The mean gene diversity (h) of different in vitro condition was 0.03 and ranged from 0.008 for N6 with BA 5 mg/L, to 0.040 for MS with IAA 0.1 mg/L+kinetin 2 mg/L. The results indicate that the regenerants have a low genetic variation, and ISSR analysis is effective to detect genetic variation of regenerants.

Molecular Characterization and Defoliation-Induced Expression of a Sucrose Transporter LcSUT1 Gene in Sheep Grass (Leymus chinensis)

Sucrose is a major photosynthetic product in plants’ leaves. Long-distance transport of sucrose requires sucrose transporter (SUT) to perform loading and unloading functions. In this study, a sucrose transporter gene LcSUT1 was cloned from sheep grass (Leymus chinensis (Trin.) Tzvel), a perennial grass of Gramineae: Poaceae. Bioinformatics analysis showed that the gene product LcSUT1 consisting of 12 predicted transmembrane domains and 11 loops belongs to the SUT1 clade. Heterologous expression of LcSUT1 in yeast proved that it was a functional sucrose transporter. Tissue-specific expression analysis showed that LcSUT1 was highly expressed in leaf and leaf sheath. The expression level of LcSUT1 was significantly up-regulated in leaf sheath after defoliation, but was not induced by wound signal. Furthermore, the level of LcSUT1 expression increased in callus, a model sink tissue, when grew on N6 medium without sucrose. Taken together, our results revealed a novel mechanism in which the increased expression of LcSUT1 in leaf sheaths after defoliation was caused by sucrose starvation rather than by wound signal.

Enhancing Agrobacterium tumefaciens-mediated transformation efficiency of perennial ryegrass and rice using heat and high maltose treatments during bacterial infection

Perennial ryegrass is one of the most widely cultivated grasses in temperate regions. However, it is recalcitrant for in vitro manipulation. In this study, various parameters affecting Agrobacterium tumefaciens-mediated infection were tested to optimize transformation efficiency in perennial ryegrass. The effects of heat shock and maltose concentration during Agrobacterium infection were evaluated along with variations in callus induction medium, bacterial infection media and callus age. Our results suggest that Agrobacterium infection at 42 °C for 3 min and co-cultivation of Agrobacterium-infected callus on a high maltose medium (6 %) significantly enhances the transformation efficiency in perennial ryegrass. The most optimal conditions proved to be use of four-month-old embryogenic callus induced on a modified N6 medium, infected with Agrobacterium grown on a modified Murashige and Skoog (MSM) medium, and a 42 °C heat shock treatment followed by the co-cultivation of the Agrobacterium and the callus on medium containing 6 % maltose (instead of 3 %). Using this optimized protocol, we were able to increase the transformation efficiencies for regenerated plants from approximately 1 % to over 20 %. Significant improvement in rice stable transformation efficiency was also observed when the optimized conditions were applied to this important cereal, indicating the method described here may apply to other monocots as well.

Optimization of Nutritive Factors in Culture Media for Growth and Tropane Alkaloid Production from Anisodus acutangulus Hairy Roots

Article history: Received on: 05/11/2012 Revised on: 29/11/2012 Accepted on: 18/12/2012 Available online: 28/02/2013 Anisodus acutangulus hairy roots were grown in N6 medium which was optimal for growth and alkaloid production. The cell biomass and alkaloid yield reached 2.7 g l and 3.9 mg l (dry weight) respectively in sucrose medium, higher than those obtained in other carbon source media. 90 mM nitrogen (NH4/NO3 = 4:1) gave the highest cell yield (4.5 g l) and the maximum alkaloid production (9.9 mg l). The cell yield (4.1 g l) of hairy roots grown at pH 6.5 was 2 times higher than that at pH 4.5. However, the maximum alkaloid production (7.2 mg l) was yielded at pH 4.5.

Effect of Maltose Concentration on Plant Regeneration of Anther Culture with Different Genotypes in Rice (<i>Oryza sativa </i>L.)

This study describes the impact of different concentrations of maltose on plant regeneration of anther culture for five genotypes of rice (Oryza sativa). N6 medium was used for calli induction, while N6 medium supplemented with different concentrations of maltose, 2.0 mg/L NAA and 0.5 mg/L kinase was used for plant regeneration. The result showed that during the initial stages of calli induction the anther cultures had varying rates of calli formation among genotypes, with the best frequency being observed for Dreami2/CaMsrB2-8-DH-1 with a calli frequency of 27.8%. Different genotypes of rice cultured in regeneration media showed varying plantlet regeneration on media supplemented with different concentrations of maltose, with low concentrations (0.04 g/L) leading to low frequency regeneration plantlet but high green plant production. Indeed, when Dreami2/CaMsrB2-8-DH-2 and Dreami2/CaMsrB2-8-DH-5 were cultivated under these conditions, 100% green plants were observed. Another genotype also showed a small rate of albino frequency in response to the lowest concentration of maltose, while increased maltose concentrations resulted in increased rates of albino plants. Overall, the results of this study should facilitate establishment of an efficient plant regeneration system from anther culture in rice.

English

Establishment of highly efficient and reproducible regeneration system would greatly influence the efforts of improvement of Elymus sp. through gene transfer technology. A suitable callus induction and efficient regeneration protocol for Dahurian wildrye grass ( Elymus dahuricus L.) was developed. It consisted of 3.0 mg/L 2,4 dichlorophenoxyacetic acid (2,4-D) or Dicamba + 1.0 g/L casein hydrolysate (CH) + 300 mg/L L-proline + 30 g/L sucrose in MS, and showed the highest percentage of callus induction. Highest (51%) regeneration was obtained from N6 medium containing 1.0 mg/L 2,4-D + 3.0 mg/L N 6 -benzylaminopurine (BA) + 1.0 g/L CH + 300 mg/ L L-proline + 30 g/L sucrose. Thus, optimization of regeneration frequency using mature seeds as explants material may offer a simple and efficient protocol for Dahurian wildrye grass that may improve molecular breeding of this species. Key words : Dahurian wildrye grass, Elymus dahuricus L., callus, plant regeneration.

Somatic embryogenesis from ovaries of sweet orange cv. Tobias

Callogenesis, somatic embryogenesis, and regeneration were obtained from tissues of unfertilized ovaries of sweet orange (Citrus sinensis Osbeck.) cv. Tobias. The influence of two modified basal media, woody plant medium (WPM) and N6 medium, to induce callus formation from pistils was determined. Overall, high frequencies of callogenesis were observed when either medium was used. However, initial culture of explants in WPM medium followed by transfer of callus to N6 medium resulted in higher frequency of callus induction (of 2.30 callus per explant that were larger than 0.5 cm in size), and of subsequent development of embryogenic callus (10%). A total of 125 somatic embryos were obtained. After 6 months of culture, 72% of somatic embryos germinated into plantlets. These plantlets were subsequently micrografted in vitro, and then acclimatized. Ploidy of these plants were determined using flow cytometry and TRAPS molecular markers were used to confirm their maternal origin.

국내개발 이탈리안 라이그라스 품종 성숙종자의 식물체 재분화

In order to improve forage characteristics of Italian ryegrass by genetic transformation, an efficient callus induction from mature seed and optimal plant regeneration system were established using a domestic cultivar 'Kospeed'. Addition of 5 mg/L of 2,4-D showed highest frequency of embryogenic callus induction from mature seeds. N6 medium showed higher frequency of both callus induction and plant regeneration as compared with MS and SH medium. The highest plant regeneration frequency 67% was obtained when embryogenic calli were transferred to N6 medium containing 1 mg/L 2,4-D and 5 mg/L BA. Supplementation of regeneration medium with sucrose at 30 g/L level maximized regeneration frequency as compared to the other concentrations. These data would be very helpful for molecular breeding of domestic Italian ryegrass cultivar through genetic transformation.

Siberian Wildrye Grass의 성숙종자 유래의 캘러스로부터 식물체 재분화

Ki-Won Lee, Ochirbat Chinzorig, Gi Jun Choi, Sei Hyung Yoon, Ki-Yong Kim,Hee Chung Ji and Sang-Hoon LeeABSTRACTSuccess in molecular breeding for better adapted varieties to environmental stresses depend upon the concerted efforts by various research including tissue culture, transformation, genetics and breeding. In order to optimize tissue culture conditions of Siberian wildrye grass, the effects of plant growth regulators on callus induction and plant regeneration were investigated with mature seeds. The highest callus induction frequency was observed when the mature seeds were cultured on MS medium supplemented with 5mg/L 2,4-D. The highest plant regeneration frequency was observed when callus was transferred to N6 medium supplemented with 1mg/L 2,4-D and 3mg/L BA. Regenerated plants were grown normally when shoots were transplanted to the soil. A short tissue culture period and regeneration system would be beneficial for molecular breeding of Siberian wildrye grass by the production of transgenic plant.(Key words:Siberian wildrye grass, Callus, Plant regeneration, Plant growth regulators)

English

To explore the possibility to improve N6 medium for callus induction and plant regeneration from maize immature embryos, comparative experiments were conducted to screen the optimal calcium concentration for the callus induction medium and the subculture medium modified from N6 medium and to examine the effects of uniconazole and ABT root promoting powder supplement on plant regeneration and root generation. According to the results, the calcium concentration of N6 medium was increased from 1.13 to 5 mmol/l for callus induction and subculture. Uniconazole from 0.25 and 0.5 mg/l and ABT root-promoting powder of 0.5 mg/l were suggested to be supplemented to the regeneration medium and the root generation, separately. On these improved media, the frequency of callus induction, the relative proliferation rate of callus subculture, the regeneration rate and the average root biomass of the regenerated plantlets increased significantly. All these improvements facilitate the establishment of acceptor system from elite parental inbred lines of commercial hybrids andprovide a solid basis for transgenic manipulation of maize.   Key words: ABT root-promoting powder, calcium, callus induction, immature embryo, maize, N6 medium, plant regeneration, uniconazole.

Regeneration and transformation of a maize elite inbred line via immature embryo culture and enhanced tolerance to a fungal pathogen Exserohilum turcicum with a balsam pear class I chitinase gene

Maize is one of the most important food crops worldwide and is subject to many diseases. Genetic engineering offers unique way to improve the current elite line via introduces single or multiple traits. In this report, we transformed an elite maize line, Jiao 51, with a balsam pear class I chitinase gene (Mcchit 1) driven by the maize ubiquitin-1 (ubi1) promoter. Conditions for callus induction from immature embryos were optimized using a Jiao 51 by culture on induction medium. The highest Type-I and Type-II callus induction efficiency of 91.33 and 89.10% were obtained on N6 media supplemented with 2.5 mg L-1 2, 4-D. Both types of calli grew fast. Type-I callus could easily change to Type-II callus. Agrobacterium-mediated transformation was used to generate eight transgenic lines that expressed the chitinase transgene. Assessing the functionality of the Mcchit1 protein in the transgenic plants was conducted using an excised leaf challenge assay. The transgenic maize expressing a balsam pear class I chitinase gene exhibited enhanced tolerance toExserohilum turcicum in greenhouse conditions. After 5 days inoculation, the number of lesions was significantly fewer, and the size of lesions was significantly smaller compared to the controls. Key words: Maize (Zea mays. L.), chitinase gene, transformants, fungal disease tolerance, Northern corn leaf blight, Exserohilum turcicum

Вплив складу живильного середовища на розвиток та проростання незрілих зародків кукурудзи в культурі in vitrо

The effect of mineral components of nutrient medium and abscisic acid (ABA) on isolated immature maize embryos is investigated with the aim to intensify their growth and to inhibit their early germination. Abscisic acid together with MS medium leads to the stasis of the development of both seedlings and embryos. The application of ABA along withN6 minerals inhibited the development of seedling’s organs in favour of embryos growth. For production of seedlings from immature embryos of 1 mm long their cultivation on modified N6 medium with addition of 12 mg/Lof abscisic acid is recommended.