Resistant Calli Research Articles

Влияние 10-(6'-пластохинонил) децилтриметилфосфония (SkQ1) на образование каллусов и регенерацию растений, и их устойчивость к гипоксии

This work investigated the effect of 10-(6'-plastoquinonyl) decyltrimethylphosphonium (SkQ1) on the regeneration, growth and development of cell and tissue cultures of such plants as sugar cane (Saccharum officinarum L.), spring wheat (Triticum aestivum L.), corn (Zea mays L.), tobacco (Nicotiana tabacum L.), alfalfa of two species (Medicago glutinosa L.) and (Medicago falcata L.), cornflower (Thalictrum minus L.) and stephania (Stephania glabra Miers.). The stimulating effect of SkQ1 in concentrations from 0.5 to 10 nM has been shown, promoting better shoot and root formation in calli, an increase in growth rate, expressed in the cell mass of calli and their growth index. In addition, the effect of SkQ1 on the resistance of sugarcane calli to hypoxia was studied by assessing the growth index of calli of sensitive and hypoxia-resistant sugarcane lines when incubated under anaerobic conditions. It was shown that in both sensitive and resistant calli grown on a medium supplemented with 10 nM SkQ1, the growth index was 4 and 3.3 times higher, respectively, than in calli cultivated in a medium not containing SkQ1. Keywords: ANTIOXIDANTS, PLASTOQUINONE, 10-(6'-PLASTOQUINONYL) DECYLTRIMETHYLPHOSPHONIUM (SkQ1), CALLUS, EMPLANTS, REGENERATION, SUSPENSION CULTURE, HYPOXIA

A Comparative Transcriptome Analysis of Avocado Embryogenic Lines Susceptible or Resistant to Rosellinia necatrix Exudate

Avocado embryogenic cultures were selected for resistance to the culture filtrate (CF) of Rosellinia necatrix, the causal agent of White Root Rot disease. A resistant callus line was obtained through recurrent selections in progressively increasing concentrations of fungal CF (from 60% to 80%). RNA sequencing (RNA-Seq) technology was used to compare the transcriptomic profiles of the avocado embryogenic-callus-resistant line L3 (capable to survive in the presence of 80% CF) and control line AN-9 (not exposed to CF), after 24 h of growth in a medium containing 40% CF. A total of 25,211 transcripts were obtained, of which 4,918 and 5,716 were differentially expressed in the resistant and control line, respectively. Interestingly, exposure of embryogenic callus lines to 40% of R. necatrix exudates induced genes previously reported to be related to avocado defense against fungal diseases (lignin biosynthesis, Pathogenesis Related (PR) proteins, WRKY (WRKYGQK) Transcription Factor (TF), NAC (NAM, ATAF1/2, and CUC2) TF, proteinase inhibitors and Ethylene Response Transcription Factor (ERF), among others), which were accumulated in greater amounts in the resistant line in comparison to the susceptible one. This research will contribute to the understanding of avocado defense against this pathogen, thereby aiding in the selection of resistant avocado rootstocks.

English

We used Agrobacterium-mediated infection of callus induced from the cotyledonary nodes of Amorpha fruticosa L. to study the β-glucuronidase gene (GUS)-integrated genetic transformation system. Transformed calluses were selected under 40 mg·L−1 kanamycin, differentiated into resistant adventitious buds, and developed into transformants. A single copy of gus was integrated in the genome of most T0 generation plants. Gus chemical staining analysis showed blue color in resistant calluses, adventitious buds, and the roots and leaves of transformed plants. This indicated gus overexpression driven by the 35S promotor and resultant β-glucuronidase activity. The genetic transformation system in this study could be used to study other functional genes of A. fruticosa and facilitate transgenic breeding for strain improvement. © 2022 Friends Science Publishers

Establishment of efficient callus genetic transformation system for Pyrus armeniacaefolia

The purpose of the present study was to establish an efficient and stable callus genetic transformation system for Pyrus armeniacaefolia. Fruits 15 days after flowering were used for callus inducing. Major factors that influence growth state of callus (concentrations of plant growth regulators, subculture cycle) and transformation (Agrobacterium tumefaciens concentration, infection time, co-culture time, AS and kanamycin concentrations) were examined. The results showed that the optimum mediums for callus induction was MS supplemented with 3.0 mgL−1 2,4-Dichlorophenoxyacetic acid (2, 4-D) and 0.5 mgL−1 6-Benzylaminopurine (6-BA). The appropriate proliferation medium was MS containing 2.5 mgL−1 2,4-D and 0.5 mgL−1 6-BA with 21 d subculture cycle. Transformation was successfully achieved using the following protocol: callus were soaked in A. tumefaciens strain EHA105 (OD600 = 0.8) harboring pBI121 plasmid for 15 min. After 3 d co-culture on proliferation medium supplemented with 20 mgL−1 AS, the callus were then screened on the selection medium (proliferation medium containing 25 mgL−1 kanamycin and 200 mgL−1 cefotaxime) to select the resistant callus. The GUS-positive rate was 96%. And the GUS-positive callus were further identification at DNA and transcription levels. We believe that the present study provides technical support for functional researches on fruit quality related genes.

Реакция семян и эксплантов льна на осмотический стресс

Abstract. Purpose. Obtaining experimental data for the development of methods for obtaining fiber flax forms resistant to stress factors (drought) by cell selection methods. Methods. The work was carried out in the Tver region in the laboratory of breeding technologies in 2017–2019. The flax varieties Varbara, Belinka, LM-98, Svetoch, Diplomat, Symfonia were used as objects of research. The seeds were obtained from the National Flax Collection Federal state budgetary scientific institution “Federal scientific center of lubya kultur”. The effect of sucrose solution on the length of the primary root was detected at concentrations of 0, 8.7, 14.9 %. To assess the energy of seed germination under osmotic stress, the concentration of sucrose was reduced and the range from 0 (control) to 9 % was considered. Results. Sucrose as a selective agent in the culture of immature flax embryos in vitro at a concentration of 5.0–7.0 % can be selective only for certain genotypes, for example, the Svetoch variety. When culturing callus tissues, using mannitol as an osmotic at concentrations of 0; 30.0; 36.4; 37.0; 37.4; 38.0 mg/l, selection of resistant callus cells with subsequent formation in meristematic foci of adventive buds and shoots can be carried out on media containing 30.0 or 36.4 mg/l osmotic. The scientific novelty of the research lies in the fact that the method for obtaining in vitro resistant explants to osmotic stress for flax is being developed for the first time.

Selection of vascular wilt resistance cumin callus to culture filtrate of Fusarium equiseti and regeneration of plants

A method is presented for cellular selection of Cumin plant resistant to Fusarium wilt. In vitro selection of resistant cells using culture filtrates of Fusarium equiseti is attempted which could lead to the development of new Cumin cultivars resistant to Fusarium wilt. In vitro selection of resistant cells using culture filtrates of Fusarium equiseti was attempted in order to obtain cellular selection of Cumin plant resistant to Fusarium wilt. MS basal medium with 1.0 μM 2, 4 D and 10 μM Kinetin was found most suitable for high callus induction. Cell suspension cultures were derived from these calli by transfer to liquid MS medium with 1.0 μM 2, 4 D and 10 μM Kinetin. These cells growing in suspension culture were plated on MS medium gelled with agar and containing 1–10% dilutions of Fusarium equiseti wilt toxin. After 3–4 weeks incubation, Survival calli (Clumps) were selected from 8% CF and put into 5–50% CF of Fusarium equiseti wilt toxin. In next phase resistant calli were obtained from 30% CF. Shoot and root proliferation was induced in these wilt toxin resistant calli in presence of culture filtrate (30%), to maintain the selection pressure to the regenerating shoots and plantlets obtained after rooting. The plants regenerated were hardened in coco-peat. Again the selection pressure was maintained on the plants growing further by irrigating them with nutrients mixed with culture filtrate till maturity. Thus the plants were continuously exposed to the fungal toxin to establish Fusarium equiseti wilt resistant selections.

ИСПОЛЬЗОВАНИЕ САХАРОЗЫ И МАННИТА ДЛЯ ДИФФЕРЕНЦИАЦИИ ГЕНОТИПОВ ЛЬНА ПО УСТОЙЧИВОСТИ К ОСМОТИЧЕСКОМУ СТРЕССУ

The studies were carried out with the aim of studying the effect of various concentrations of sucrose and mannitol on seeds, immature embryos, and callus cultures of flax to develop a method for obtaining genotypes resistant to osmotic stress. The work was carried out in the Tver region in the laboratory of breeding technologies in 2017–2019. Flax varieties Barbara, Belinka, LM-98, Aurore, Tverskoy, Svetoch, Diplomat, Symfonia were used as objects of research. The seeds were obtained from the National Flax Collection of the Federal Scientific Center for Bast Crops. The effect of sucrose solution on the length of the primary root was detected at concentrations - 0; 8.7; 14.9%. To assess the germination energy of seeds under osmotic stress, the concentration of sucrose was reduced and the range 0 (control) ... 9% was considered. Immature embryos removed from the capsules on the 10th day after pollination were cultivated on MS medium with sucrose, as a selective agent, at a concentration of 5.0 ... 7.0%. Callus tissues were cultured using mannitol as an osmotic at concentrations of 0; 30.0; 36.4; 37.0; 37.4; 38.0 mg/l. Concentrations of 5.0, 6.0 and 7.0% of sucrose can be used as an osmotic differentiator for seeds (10 ... 80% of seeds germinated in the Belinka variety, 80 ... 100% in the Varbara variety, 80 ... 90% in the variety LM-98). Sucrose, as a selective agent, in a culture of immature flax embryos in vitro at a concentration of 5.0 ... 7.0% can be selective only for certain genotypes, for example, the Aurore variety. The selection of resistant callus cells, followed by the formation of adventive buds and shoots in the meristematic foci, can be carried out on media containing 30.0 or 36.4 mg / L of osmosis, which allows obtaining morphogenic callus, buds, shoots in all studied genotypes, as well as in the Aurore variety 1.1 ... 1.2 byp./callus, in the Tverskoy variety - 0.6 ... 0.8, in the Barbara variety - 1.0 ...1.1

Development of Plant Prime-Editing Systems for Precise Genome Editing

Prime-editing systems have the capability to perform efficient and precise genome editing in human cells. In this study, we first developed a plant prime editor 2 (pPE2) system and test its activity by generating a targeted mutation on an HPT-ATG reporter in rice. Our results showed that the pPE2 system could induce programmable editing at different genome sites. In transgenic T0 plants, pPE2-generated mutants occurred with 0%–31.3% frequency, suggesting that the efficiency of pPE2 varied greatly at different genomic sites and with prime-editing guide RNAs of diverse structures. To optimize editing efficiency, guide RNAs were introduced into the pPE2 system following the PE3 and PE3b strategy in human cells. However, at the genomic sites tested in this study, pPE3 systems generated only comparable or even lower editing frequencies. Furthemore, we developed a surrogate pPE2 system by incorporating the HPT-ATG reporter to enrich the prime-edited cells. The nucleotide editing was easily detected in the resistant calli transformed with the surrogate pPE2 system, presumably due to the enhanced screening efficiency of edited cells. Taken together, our results indicate that plant prime-editing systems we developed could provide versatile and flexible editing in rice genome.

A CRISPR-Cas9-mediated domain-specific base-editing screen enables functional assessment of ACCase variants in rice.

A CRISPR-Cas9-mediated domain-specific base-editing screen enables functional assessment of ACCase variants in rice.

Establishment of Sorghum BTx623 Immature Embryos Genetic Transformation and Regeneration System

Sorghum is one of the world's important crops after wheat, rice, maize, and barley. Although the sorghum genome had been well-sequenced, genetic breeding and functional genome research in sorghum cultivar BTx623 is still limited due to the lack of efficient and stable genetic transformation and regeneration system in sequencing. In this study, the immature embryos of sorghum genome-sequencing cultivar BTx623 was used as the explants material, and the bar gene resistant to phosphoglyphosate was used as the screening marker for Agrobacterium -mediated sorghum genetic transformation. By screening the adaptability of callus to different concentrations of phosphoglyphosate, the appropriate concentration of phosphoglyphosate in the genetic transformation of sorghum cultivar BTx623 was determined to be 2.5 mg/L, and BTx623 immature embryo was used as explants to obtain resistant callus. After screening, regenerated plants were obtained by treating resistant callus with 0.0067 mg/L ZNC in regeneration medium. Therefore, this study successfully obtained resistant callus and regenerating plants, and established a genetic transformation and regeneration system in sorghum cultivar BTx623, which may have great significance for functional genome research and genetic breeding in sorghum.

Development of partial abiotic stress tolerant Citrus reticulata Blanco and Citrus sinensis (L.) Osbeck through Agrobacterium-mediated transformation method

BackgroundRecent studies indicate that farmers are facing several challenges due to biotic and abiotic stresses like diseases, drought, cold, and soil salinity which are causing declined Citrus production. Thus, it is essential to improve these varieties which would be resistant against biotic and abiotic stresses as well as high yielding. The transformation of abiotic stress tolerant genes in Citrus species is essential for using areas affected by abiotic stresses. This study was aimed to improve resistance of Citrus reticulata Blanco and Citrus sinensis (L.) Osbeck to abiotic stresses by transferring PsCBL and PsCIPK genes through Agrobacterium-mediated transformation. ResultsAbiotic stress tolerant PsCBL and PsCIPK genes isolated from Pisum sativum were transformed into two Citrus species, Citrus reticulata Blanco and Citrus sinensis (L.) Osbeck, through Agrobacterium-mediated transformation method. Mature seed-derived calli of two Species were infected with Agrobacterium tumefaciens LBA4404 harboring PsCBL and PsCIPK genes. The infected calli were co-cultured in dark condition and later on washed with antibiotic solution and transferred to selection medium. Preliminary resistant calli were recovered and regenerated to plantlets. Maximum regeneration rate was 61.11 ± 1.35% and 55.55 ± 1.03%, respectively. The genetic transformation was confirmed by performing β glucuronidase (GUS) assays and subsequent PCR amplification of the GUS gene. The transformation rates of the two cultivated species were higher than previous reports. Maximum transformation frequencies were found when bacterial OD600 was 0.5 and concentration of acetosyringone was 150 μM. In-vitro evaluation of drought and salt tolerance of transgenic plantlets were done, and transgenic plantlets showed better performance than the control plants. ConclusionsThe present study demonstrates that transformation of Citrus plants with PsCBL and PsCIPK genes result in improved abiotic stress tolerance.

Use of fluorescent reporter genes in olive (Olea europaea L.) transformation

Fluorescent proteins (FPs) can be used for different purposes in plant transformation studies such as the evaluation and improvement of transformation parameters or the isolation of transgenic cells in the absence of selective agents. In this research, the applicability of green (GFP) and red (DsRed) fluorescent proteins in olive transformation has been investigated. Olive embryogenic callus was transformed with Agrobacterium tumefaciens AGL1 strain carrying pXK7FNF2 (harbouring the gfp gene), pXK7RNR2 (DsRed), or pXK7S*NF2 (gfp and β-glucuronidase) binary plasmids. After 3 months of selection in the presence of paromomycin, several resistant calli were recovered for each construct, obtaining transformation rates in the range of 2–8%. The expression of FPs was studied during the different stages of olive plant regeneration using epi-fluorescence and confocal laser scanning microscopy. GFP from pXK7SN*F2 plasmid could be easily detected in olive somatic embryos (SE) during proliferation whereas SE transformed with pXK7FNF2 showed weak GFP signal. After embryo conversion, plants transformed with both vectors were analysed, but GFP could be detected neither in leaves nor in roots. By contrast, DsRed was highly expressed in SE and could also be visualized in leaf and root tissues of regenerated plants using confocal laser microscopy and epi-fluorescence zoom microscope, respectively. In addition, pXK7RNR2 was used to transform a different olive embryogenic line, detecting DsRed expression in SE transformed from this genotype. These results show that FPs can be a useful tool in genetic transformation of olive embryogenic cells, being DsRed gene more useful than gfp for this purpose.

Use of a Mutated Protoporphyrinogen Oxidase Gene as an Effective In Vitro Selectable Marker System that Also Conveys in planta Herbicide Resistance in Sugarcane

Genetic engineering can be used to introduce economically important traits in sugarcane cultivars. Part of any transformation process involves the selection of genetically transformed cells. In this study, an efficient sugarcane in vitro selection system was developed using mutated protophorhyrinogen oxidase (PPO) genes as selectable markers. Two PPO genes, that encode proteins targeted either to the mitochondria or plastid, were isolated from tobacco and maize. Site-directed mutagenesis was used to alter the nucleotide sequence of these genes so that the resulting proteins are less sensitive to diphenylether type herbicides. Sugarcane callus was genetically transformed through particle bombardment with constructs allowing expression of either transgene, and putative transgenic calli were selected on fomesafen. It took approximately 4 weeks to select herbicide resistant calli clones on 10 mg/l fomesafen in the presence of light, which increased the selection pressure, and a further 8 weeks to regenerate resistant plantlets. PCR analysis confirmed that all regenerated putative transgenic sugarcane plants contained the transgene. All transgenic plants showed levels of herbicide resistance when planted in soil.

Development of salt tolerant callus cultures by somatic hybridization between Oryza sativa and mangrove grass Myriostachya wightiana

Abstract Rice is one of the agronomically and nutritionally important cereal crops. It provides food for more than 50% of the world's population. However, the yield of rice crop is decreased by the salinity stress. This can be overcome by the development of salt-resistant rice varieties. In this study salt resistant callus cultures were developed by the somatic hybridization between Oryza sativa and Myriostachya wightiana. To optimize the isolation and fusion of protoplasts, the experiments were carried out with three different enzyme mixtures and different incubation periods. To know the salt tolerant ability, growth and total antioxidant activity were measured in the calli of Oryza sativa, Myriostachya wightiana, and hybrid at the different NaCl concentrations. In Oryza sativa high viable protoplasts yield was obtained by Enzyme mixture B (6.1 ± 0.2 × 106) whereas, Myriostachya wightiana gives high viable protoplasts yield by Enzyme Mixture C (3.4 ± 0.2 × 106). Optimum digestion time for the Oryza sativa was 12 h, which gave the highest number that is, 6.36 ± 0.15 × 106 protoplasts/g FW and viability were 91.2 ± 1.05%. Optimum digestion time for the Myriostachya wightiana was 24 h, which gave the highest yield that is, 3.49 ± 0.08 × 106 protoplasts/g FW and viability were 60.3 ± 1.72%. The fusion was performed with 50% (w/v) PEG-MW4000. The optimal incubation time was found as 30 and 45 min, which gave the highest number of fused viable protoplasts. A maximum number of binary fusions was achieved at 45 min incubation. The use of liquid culture medium resulted in a range of 5–20 calli per Petri dish which continued their growth. The hybrid calli show better growth in all NaCl concentrations. Hence, it was concluded that the hybrids have a better tolerance to salt stress. These results are useful in the development of salt tolerant rice varieties.

Stable plastid transformation of rice, a monocot cereal crop

Rice is one of the most important cereal crops and its biotechnology has been pursued to meet the food demand of ever-growing global population. Rice plastid transformation has been a great challenge to achieve homoplastomic plants due to its low efficiency of regeneration. In this experiment, Japonica rice line 19 was chosen to be the receptor for plastid transformation. A vector harboring smGFP gene was constructed and transferred into rice plastid genome by bombardment. The resistant callus was obtained after long-lasting multiple selections and proved to be in homoplastomic status by molecular testing. The plantlet was regenerated from homoplastomic callus and grown to seeding stage. This is the first case so far to achieve the homoplastomic rice and will be helpful to transform plastid genome of monocotyledonous crops with recalcitrant nature.

In Vitro Induced Resistance of Fusarium Wilt Disease (Fusarium oxysporum f.sp. cepae) by Salicylic Acid in Shallot CV ‘Bima Brebes’

Fusarium wilt caused by Fusarium oxysporum f.sp.cepae (Foc) is one of the most important diseases of onion in Indonesia. Induced resistance was one of the effective techniques to acquire resistance to fusarium wilt in shallot. Salicylic acid (SA) had been inferred to be in endogenous signal in the systemic acquired resistance response of plants. This research aimed to study the effect of exogenously added SA to resistance in shallot callus cv ‘Bima Brebes’ to fusarium wilt disease in vitro; and to determine the effective concentration of SA to induce resistance. A group of shallot callus was grown in MS medium containing varying SA concentration (0, 15, 20 and 25 ppm) in vitro for 2 weeks. Then, the callus were treated with toxin of Fusarium oxysporum, namely fusaric acid, to have observe the resistance response. In vitro selection was done twice in different fusaric acid concentration. Application of exogenous SA at all concentration did not suppres time of appearance of disease symptom yet. Toxic symptom in the callus was shown by the browning or blackening (off) of callus. Salicylic acid at concentration of 20 ppm effectively reduced the toxic symptom up to 16.66% and supported callus regeneration better than the concentration of 15 ppm and 25 ppm. The number of resistant callus regenerated was 66.67% at pretreatment of 20 ppm of SA.

In vitro selection of resistant/tolerant mutants lines of Citrus jambhiri Lush. using crude culture filtrate of Phytophthora parasitica and their randomly amplified polymorphic DNA analysis

AbstractThis study deals with in vitro‐induced mutagenesis and selection of Phytophthora tolerant lines of Citrus jambhiri and their regeneration. For in vitro‐induced mutagenesis, cotyledons were treated with ethyl methane sulfonate (EMS) 100, 200 and 300 mm for different time durations viz. 1, 3, 6 and 9 hr. Callus cultures were established from EMS treated cotyledon explants on MS medium supplemented with 2.0 mg/L of 2,4‐D and 500 mg/L of malt extract. Calli derived from cotyledon were challenged in vitro on selective MS medium containing 5%–100% of culture filtrate (CF) of the Phytophthora parasitica. Selected mutagen‐treated calli showed resistance in vitro on media containing CF. Calli treated with 100 mm EMS for 6‐hr duration showed tolerance (24%) up to 75% CF after 4th selection cycle. While, calli treated with 200 mm for 6‐hr duration showed maximum tolerance (76%) up to 75% CF. Resistant calli were then transferred to MS regeneration medium for shoot bud regeneration. A dose‐dependent decrease in the regeneration capacity of the selected calli was observed with the increasing concentration of the CF. In randomly amplified polymorphic DNA analysis, plantlets showed different banding pattern in comparison with the control plant, which confirms the presence of variations at genetic level.

Metabolite Profiles of Arsenic Tolerant Plants Regenerated from Stem Calli of Andrographis paniculata (Burm.f.) Nees using HPLC and 1D 1H NMR

In vitro culture provide a suitable condition for regeneration of arsenic tolerant plants from stem calli of Andrographis paniculata (Burm.f.) Nees. The regenerated plants could be valuable material applicable to soil remediation. In vitro culture of stem explants on MS basal salts, 3.0% sucrose, 0.8% agar medium fortified with various concentrations of As2O3 (0.0-9.0 µM) along with NAA (3.5 mg L-1) and KIN (1.0 mg L-1) influenced resistant callus formation. Growth of callus was slightly inhibited with increased resistance up to 22% (53±0.8 mg fwt and 27±1.5 mg dwt) on 7.0 µM As2O3 selective medium. The resistant callus line inoculated on MS medium supplemented with different concentrations of As2O3 along with BA (2.5 mg L-1) and NAA (3.0 mg L-1) induced the development of shoots. Shoot organogenesis was slightly inhibited by arsenic metal stress. However the growth tolerance has increased up to 14.5% on medium with 7.0 µM As2O3. The rate of adventitious rooting of plantlets was gradually decreased with more tolerance (11.1±1.4 rootlets per plantlets) in 7.0 µM As2O3 selected media. After acclimatization, about 40% of plants were survived as arsenic tolerance than control plants in pots containing soil treated with 7.0 µM As2O3 solutions. The level of arsenic detectability was 0.96 ppb and 4.67 ppb in control and 7.0 µM As2O3 treated plants, respectively by AAS analysis. Moreover, the production of Andrographolide was found quite high (4.41 mg/g) in tolerant plants grown at 7.0 µM As2O3 treatment than control by HPLC analysis. 1D 1H NMR profile revealed the metabolic changes significantly in control and 7.0 µM As2O3 treated plant samples. This is the first report confirming the suitability of in vitro selection for obtaining of vigorous and proliferative clones of A. paniculata plants tolerant to elevated arsenic concentration.

Herbicide resistant mutants selection in wild relatives wheat

In order to obtain herbicide resistant germplasms in wild wheat relatives, mutants selection were carried out on MS culture mediums with different dosage of Dalapon with Tove (Tolium.perenneL.), R123 (E.elongata(Host)Nevski), Napoleon (Tolium.perenneL.), Y100 (Ae.OvataL.) and Ae42 (Ae.Tauschii). The survive callus were improved having stable Dalapon-resistance by shuttle treatment of testing experiments. Resistant plants were obtained from the resistant callus of all the 5 cultivars and the resistance was improved by spraying Dalapon. Seeds and seelings were treated with Dalapon as a seeds level selection, 0.195% resistant plants of a wild species of R.ciliaris(Trin) Nevski. were obtained. All these results indicated that there is certain frequency of natural Dalapon-resistant mutants in wheat relatives.

Generation of Stable Transgenic Rice (Oryza sativa L.) by Agrobacterium-Mediated Transformation.

Rice (Oryza sativa L.) is the staple food for more than half of the world's population, and has also become an important model monocot. As a result, numerous genetic transformation protocols have been developed to improve and better understand this particular agronomic plant. Here we introduce a convenient transformation method using Agrobacterium. The explants used are embryogenic calli derived from mature seeds, which are easily obtained and can be used all year round. After selection and regeneration, transformants are obtained from resistant calli cultured on the regeneration medium. This protocol has been used to generate transgenic rice (Oryza sativa L.) in as little as 4 months. © 2016 by John Wiley & Sons, Inc.