EFFICIENT CALLUS INDUCTION AND PLANT REGENERATION FROM IMMATURE EMBRYO CULTURE OF TRITICUM AESTIVUM L. - THINOPYRUM INTERMEDIUM ALLEN DISOMIC ADDITION LINES

An efficient plant regeneration system was developed from the immature embryos of Triticum aestivum L. Thinopyrum intermedium alien disomic addition lines, which resistant to powdery mildew. The protocol was based on a series of experiments involving the callus induction and differentiation. The experiment studied the effects ofmature embryos. We found that the embryembryo size on callus induction and differentiation of the immature embryos. We found that the embryo size is critical for the establishment of embryogenic callus. Immature embryos (0.8 ~ 1.5 ram) showed high ability to produce embryogenie callus capable of regenerating green plants. The medium Murashige and Skoog‘ s (MS) added with 2mg/L 2, 4-diehlo-rophenoxyacetic acid (2, 4-D) gave the best embryogenic callus induction, maintenance and regeneration. The embryogenic callus maintained high regeneration during six subcultures in the callus induction medium. Suitable time ofpartialdesiccation could effectively improve the regeneration capacity of the callus cultured for 3-4 month. Bud green spot and root green spot were observed during the differentiation of callus and the difference between them was described. Regenerated shoots were rooted on half-strength MS medium containing 0.2 mg/L Naphthalene acetic acid (NAA). Plants were successfully transferred to soil and grew well. This efficient plant regeneration system provides a foundation for the study of somaclonal variation of Triticum aestivum L.- Thinopyrum intermedium alien disomic addition lines.     

Study on Plant Regeneration of Wheat Mature Embryos Under Endosperm-Supported Culture

To reveal the suitability of using mature embryos as an explant source in wheat tissue culture, mature embryos from eight common wheat cultivars （Triticum aestivum L. cv.） were cultured with or without endosperm to test their efficiency of callus induction and plant regeneration. When embryos were cultured together with endosperm （endosperm-supported culture, ES）, the percentage of callus induction was significantly lower than that when embryos were cultured in the absence of endosperm （non-endosperm-supported culture, NES）. This pattern was evident in most genotypes, regardless of whether 2 or 8 mg L^-1 2,4-D was added in the NES culture. However, in ES culture, more induced calli were differentiated into distinct green spots and they further developed into plantlets. Thus, more plants were regenerated in ES culture than in the NES treatment. Most of the eight tested genotypes showed a significant difference in callus induction rate and plantlet regeneration in both ES and NES cultures. In addition, the enzymatic activity of oxalate oxidase in the callus of ES culture condition was obviously higher than that in the callus of NES culture condition, suggesting that the activity of oxalate oxidase may be a parameter for selection of calli with potential for plantlet regeneration. These results indicate that wheat mature embryos are valuable explants for highly efficient callus induction and plant regeneration, if proper treatment and medium are used.     

The Comparison in Tissue Culture Ability of Mature Embryo in Different Cultivars of Rice

In order to study the regeneration technology of mature embryos in different rice varieties,nine japonica,nine indica and eleven hybrid rice varieties of two line or three line or superiority combinations were selected as explants to study the callus induction,differentiation and regeneration rates on different media.The higher callus induction （61.7-89.2%） was observed in japonica rice,when cytokinin was added at lower concentration （0.3 mg L-1 6-BA） in M8 basal medium,supplemented with 30 g L-1 sucrose,8 g L-1 agar and 2 mg L-1 2,4-D.Further,the addition of two cytokinins （2 mg L-1 6-BA,0.5 mg L-1 KT） and 1 mg L-1 NAA in the M8 basal supplemented medium resulted in 9.1-100% of the callus induction in indica rice.The percent callus induction in hybrid rice varieties was 40-86.3% when addition of 1 mg L-1 6-BA and 1 mg L-1 KT was added,and the cytokinins was required by the japonica and indica rice varieties in the M8 basal supplemented medium.It was observed that when the 0.5 mg L-1 2,4-D and 1 mg L-1 6-BA were added in japonica rice,and 0.2 mg L-1 2,4-D and 0.5 mg L-1 6-BA were added in indica and hybrid rice in the MS different media,the regeneration rates were 9.2-59.5%,3.6-87.5% and 17.2-43.2% for japonica,indica and hybrid rice,respectively.Thus,the regeneration technology with higher output is established in the mature embryos of similar rice varieties.     

Dicamba and Sugar Effects on Callus Induction and Plant Regeneration from Mature Embryo Culture of Wheat

To establish a highly efficient plant regeneration system for wheat genetic transformation, the effects of three different concentrations of dicamba and two different sugar types on callus induction and plant regeneration from mature embryo cultures were evaluated. Callus induction and plant regeneration were obtained from mature embryos of two commercial cultivars Zhoumai 18 and Yumai 34 （Triticum aestivum L.） cultured on L3 basal medium. The results showed that the efficiency of mature embryo culture was significantly influenced by the genotypes, sugar types and dicamba concentrations. 4 mg L^-1 dicamba proved the best effective for inducing embryogenic callus and also gave the highest proportion of plants regenerated across the two cultivars. Substitution of maltose by sucrose significantly improved the plant regeneration efficiency in both cultivars. There was a significant interaction between genotype-by-sugar types, and sugar types-bydicamba concentrations. Overall, Zhoumai 18 gave the highest frequency of plant regeneration （82.65%） when dicamba concentration was 4.0 mg L^-1 and with sucrose in initial callus induction. These results will facilitate genetic transformation work with elite wheat.     

Optimization of Agrobacterium tumefaciens-Mediated Immature Embryo Transformation System and Transformation of Glyphosate-Resistant Gene 2mG2-EPSPS in Maize（Zea mays L.）

Since maize is one of the most important cereal crops in the world,establishment of an efficient genetic transformation system is critical for its improvement.In the current study,several elite corn lines were tested for suitability of Agrobacterium tumefaciens-mediated transformation by using immature embryos as explants.Infection ability and efficiency of transformation of A.tumefaciens sp.strains EHA105 and LBA4404,different heat treatment times of immature embryos before infection,influence of L-cysteine addition in co-cultivation medium after transformation,and how different ways of selection and cultivation influence the efficiency of transformation were compared.Glyphosate-resistant gene 2mG2-EPSPS was transformed into several typical maize genotypes including 78599,Zong 31 and BA,under the optimum conditions.Results showed that the hypervirulent Agrobacterium tumefaciens sp.strain EHA105 was more infectious than LBA4404.Inclusion of L-cysteine（100 mg L-1） in co-cultivation medium,and heating of the immature embryos for 3 min prior to infection led to a significant increase in the transformation efficiency.Growth in resting medium for 4-10 d and delaying selection was beneficial to the survival of resistant calli.During induction of germination,adding a high concentration of 6-BA（5 mg L-1） and a low concentration of 2,4-D（0.2 mg L-1） to regeneration medium significantly enhanced germination percentage.Using the optimized transformation procedure,more than 800 transgenic plants were obtained from 78599,Zong 31 and BA.By spraying herbicide glyphosate on leaves of transgenic lines,we identified 66 primary glyphosate-resistant plants.The transformation efficiency was 8.2%.PCR and Southern-blot analyses confirmed the integration of the transgenes in the maize genome.     

Establishment and Optimization of the Regeneration System of Mature Embryos of Maize （Zea mays L.）

A reliable system was developed for regeneration from mature embryos derived from callus of four maize inbred lines （Liao 7980, Dan 9818, Dan 340, and Dan 5026）. The protocol was mainly based on a series of experiments involving the composition of culture medium. We found that 9 pM 2,4-dichlorophenoxyacetic acid in MS medium was optimum for the induction of callus. The induction frequency of primary calli was over 85% for four inbred lines tested. The addition of L- proline （12 mM） in subculture medium significantly promoted the formation of embryogenic callus but it did not significantly enhance growth rate of callus. Efficient shoot regeneration was obtained on regeneration medium containing 2.22 μM 6- benzylaminopurine in combinations with 4.64 μM Kinetin. Regenerated shoots were rooted on half-strength MS medium containing 2.85 μM indole-3-butyric acid. This plant regeneration system provides a foundation for genetic transformation of maize.     

Somatic Embryogenesis in Lily Bulb Scale Cultures

Abstract： Somatic embryogenesis from lily bulb scales has not been studied in details, although tissue culture methods have been applied to the propagation for decades. The effects of different kinds and concentration of auxins for oriental lily somatic embryogenesis were investigated （Lilium hybrida var. Sorbonne）. 2, 4-dichlorophenoxyacetic acid （2, 4-D）, thidiazuron （TDZ） and α-naphthaleneacetic acid （NAA） media with benzyladenine（6-BA） and lactalbumin hydrolysate （LH） were used for embryogenic callus in the darkness. The best response on embryogenic callus formation was obtained on MS media supplemented 2, 4-D 2.0 mg·L^-1, 6-BA 0.5 mg·L^-1 and LH 300 mg·L^-1. Transfer embryogenic callus to the media with TDZ, 6-BA, kinetin （KT） supplemented 2, 4-D. The highest number of somatic embryos has been produced on medium with 0.5 mg·L^-1 2, 4-D and 0.3 mg·L^-1 KT. Germinated embryos with shoot axes were changed to MS media with 6-BA 0.5 mg·L^-1. The results suggest that in vitro culture of somatic embryogenesis from lily bulb scales can be used for plant regeneration.     

Somatic Embryogenesis and Plant Regeneration from Leaflets of Fritillaria ussuriensis M.

A method for the production of somatic embryos and subsequent plant regeneration for fritillaria ussuriensis M.is described.Whole leaflet explants,derived from plantlets grown in vitro,formed light yellowith embryogenic calli within one month of culture in the dark.Somatic embryogenesis was obtained after a 28d incubation on MS induction medium supplemented with 2mg/L 2,4-D 0.5mg/L BA,0.5mg/L KT and 500mg/L CH followed by transfer to a second N medium containing 0.5mg/L KT and 100mg/L CH.Somatic embryos were transferred to MS medium with 0.1mg/L NAA placed in the light for regeneration ,After two weeks,mature somatic embryo developed into whole phantlet.     

Study on Effects of Two Kinds of Auxin in vitro Regeneration of Cyclamen

Cyclamen leaves and petioles explants were cultured on MS media supplemented with different concentrations of 2, 4-dichlorophenoxyacetic acid (2, 4-D) or 1-naphthaleneacetic acid (NAA) to induce callus. The effect of 2, 4-D on shoot regeneration was also studied. Either in media containing 2, 4-D or in media containing NAA, callus was observed, but the quality or quantity of callus induced by 2, 4-D or NAA were different. The callus induced by 2,4-D was white, compact and having powerful multiplication capacity. The callus was inclined to browning then was poorly organogenetic. While the callus induced by NAA was yellowish in appearance. It was pultaceous and proliferated bradytelicly. The callus usually can give rise to many shoots. But the frequency of inducing callus of 2, 4-D is higher than that of NAA. The regenerative plantlets derived from the callus respectively induced by 2, 4-D or NAA were transferred into rooting medium. The frequency of rooting were no difference.     

Studies on Factors Affecting the in vitro Germination and Growth of Taxus cuspidata Embryos

Factors affecting the in vitro germination and growth of Taxus cuspidata embryos were exam-ined.DCR medium was the best among 6 basal media tested;embryos from stage Ⅱ seeds were the optimal developmantal stage for in vitro germination; as the seeds approached maturity,germinability of embryos decease ,Embryos ,cultured in darkness tended to from callus and de-crease the frequency of germination;inoculation method obviously affected frequency of embryo germination,Embryos cultured in optimal conditions developed into seedilings in 2 months.     

Effects of CuSO4 and Uniconazole on Mature Embryo Culture in Japonica Rice

In order to establish the system of high frequency plant regeneration for japonica rice mature embryos, the effects of different concentrations of CuSO4 and uniconazole on in vitro culture of mature embryos were studied using three rice cultivars of Kongyu 131, Longjing 24, and Dongnong 425 as test materials. The results showed that callus induction and differentiation of japonica rice mature embryos were apparently improved on the medium with 10-15 μmol·L-1 CuSO4 and 0.50-1.00 mg·L-1 uniconazole. Induction and differentiation rates of different genotype rice mature embryos displayed different sensitivities to CuSO4 and uniconazole. For the callus induction frequency of three varieties, the optimal concentration of CuSO4 was 15.0 mol·L-1. When the concentration of CuSO4 was 15 μmol·L-1, the plantlet differentiation rates of Kongyu 131 and Dongnong 425 got to the highest, while the concentration of CuSO4 was 10 μmol·L-1 for Longjing 24. For the callus induction and plantlet differentiation rates of Kongyu 131 and Dongnong 425, the ideal concentration of uniconazole was 0.50 mg·L-1 and for Longjing 24 was 1.00 mg·L-1.     

Tissue Culture System for Different Hybrid of Indica Rice

The effect of varieties and media compositions on callus induction from rice anther and subsequent plant regeneration was studied. The results showed that the callus induction was not significantly different in different media, but mostly depended on genotypes; mean frequency of callus induction on F1 hybrid varieties showed that the medium supplemented with 100 mg · L-1 concentration of the proline was useful for callus induction; when the anther derived callus sub-cultured on MS medium supplemented with B...     

Effect of Different Treatment on Anther Callus Formation and Browning in Balsam Pear （Mornordica charantia L.）

Brown callus derived from anther limited the application of anther culture in balsam pear. In order to establish a more perfect regeneration system from anther cultuer, the effects of low temperature pretreatment, 2,4-dichlorophenoxyacetic acid （2,4-D）, vitamin C （Vc） and silver nitrate （AgNO3） on callus induction and browning in anther culture of balsam pear （Momordiea charantia L.） were investigated. The results showed that after pretreatment at 4 ℃ for 1 day, callus induction rate was the highest and browning rate was the lowest. Anthers on MS medium supplemented with 2,4-D 0.5 mg/L formed more and better callus. The medium supplemented with Vc or AgNO3 was advantageous to the induction of callus and reduction of browning. When cultured on medium supplemented with 50 mg/L Vc or 5 mg/L AgNO3, callus induction rate was the highest and browning rate was rather low.     

Efficient Plant Regeneration with Arabinogalactan-Proteins on Various Ploidy Levels of Cereals

To determine the most effective dose of arabinogalactan-protein (AGP) in regeneration medium, mature embryos of genotypes in three different ploidy levels (Triticum aestivum L. cv. Ikizce-96, Triticum durum Desf. cv. Mirzabey and Hordeum vulgare L. cv. Tokak) were used to establish an efficient plant regeneration system for cereals. Percentage of callus production, capacity of regeneration were calculated, and also culture effect, root, stem, and total plant length of regenerant plants were observed in six different regeneration media (MS control, MS+2, 5, 7, 10, 12 mg L-1 AGP) in these three different genotypes. According to the results, the highest amount of callus production was found in Ikizce-96 as 93.75% using 5 mg L-1 dicamba and 1 mg L-1 kinetin in induction medium. However, the most improved callus was observed in diploid barley Tokak as 179.95 mg in weight and 6.18 mm in diameter, respectively. The highest regeneration capacity was observed in the dose of 5 mg L-1 AGP in MS of all the genotypes and hexaploid wheat Ikizce-96 gave the best results with the highest regeneration capacity and culture effects (94.86 and 92.5%) in the same dose of AGP. These results indicated that effective dose of AGP in regeneration medium increase plant regeneration in calli derived from cereal mature embryos.     

Conservation of Iris bismarckiana Regel （Iridaceae） Using Plant Regeneration via Somatic Embryogenesis

In an attempt to propagate and conserve the rare, showy bulbous plants of Iris bismarckiana, newly recorded to the flora of Jordan and to contribute to the conservation the wild lris species in Jordan, a simple rapid, time consuming protocol has been developed using plant regeneration via somatic embryogenesis. Somatic embryogenesis was induced in zygotic embryo culture on Murashige and Skoog （MS） medium supplemented with 2,4-dichlorophenoxy acetic acid （8 mg/L） as the sole plant growth regulator, where both embryogenesis calli and somatic embryos were induced. Separation of embryos from embryo clusters was necessary to enhance the frequency of germination. Germination was stimulated by separation of embryos successfully from embryo clusters and transferred onto fresh MS medium. Data obtained were analyzed as a complete random design with three replications. Calli fragments that were transferred to embryogenesis induction medium （EIM） produced white embryo-like globular structures within two weeks. Within three more weeks, clusters of structures at various stages of development could be found on the same callus. The applied technique is rewarding and encouraging for further research on the endangered wild species of Iris in Jordan.     

Effects of inter-culture,arabinogalactan proteins,and hydrogen peroxide on the plant regeneration of wheat immature embryos

The regeneration rate of wheat immature embryo varies among genotypes, howbeit many elite agriculture wheat varieties have low regeneration rates. Optimization of tissue culture conditions and attempts of adding signal molecules are effective ways to increase plant regeneration rate. Inter-culture is one of ways that have not been investigated in plant tissue culture. Moreover, the use of arabinogalactan proteins(AGPs) and hydrogen peroxide(H2O2) have been reported to increase regeneration rate in a few plant species other than wheat. The current research pioneeringly uses inter-culture of immature embryos of different wheat genotypes, and also investigates impacts of AGP and H2O2 on the induction of embryogenic calli and plant regeneration. As a result, high-frequency regeneration wheat cultivars Kenong 199(KN199) and Xinchun 9(XC9), together with low-frequency regeneration wheat line Chinese Spring(CS), presented striking increase in the induction of embryogenic calli and plant regeneration rate of CS through inter-culture strategy, up to 52.19 and 67.98%, respectively. Adding 50 to 200 mg L–1 AGP or 0.005 to 0.01 ‰ H2O2 to the callus induction medium, enhanced growth of embryogenic calli and plant regeneration rate in quite a few wheat genotypes. At 50 mg L–1 AGP application level in callus induction medium plant regeneration rates of 8.49, 409.06 and 283.16% were achieved for Jimai 22(JM22), Jingdong 18(JD18) and Yangmai 18(YM18), respectively; whereas at 100 mg L–1 AGP level, CS(105.44%), Chuannong 16(CN16)(80.60%) and Ningchun 4(NC4)(62.87%) acted the best. Moreover CS(79.05%), JM22(7.55%), CN16(101.87%), YM18(365.56%), Yangmai 20(YM20)(10.48%), and CB301(187.40%) were more responsive to 0.005 ‰ of H2O2, and NC4(35.37%) obtained the highest shoot regeneration rates at 0.01 ‰ of H2O2. Overall, these two methods, inter-culture and AGP(or H2O2) application, can be further applied to wheat transgenic research.     

Effects of inter-culture,arabinogalactan proteins,and hydrogen peroxide on the plant regeneration of wheat immature embryos

The regeneration rate of wheat immature embryo varies among genotypes, howbeit many elite agriculture wheat varieties have low regeneration rates. Optimization of tissue culture conditions and attempts of adding signal molecules are effective ways to increase plant regeneration rate. Inter-culture is one of ways that have not been investigated in plant tissue culture. Moreover, the use of arabinogalactan proteins(AGPs) and hydrogen peroxide(H2O2) have been reported to increase regeneration rate in a few plant species other than wheat. The current research pioneeringly uses inter-culture of immature embryos of different wheat genotypes, and also investigates impacts of AGP and H2O2 on the induction of embryogenic calli and plant regeneration. As a result, high-frequency regeneration wheat cultivars Kenong 199(KN199) and Xinchun 9(XC9), together with low-frequency regeneration wheat line Chinese Spring(CS), presented striking increase in the induction of embryogenic calli and plant regeneration rate of CS through inter-culture strategy, up to 52.19 and 67.98%, respectively. Adding 50 to 200 mg L–1 AGP or 0.005 to 0.01 ‰ H2O2 to the callus induction medium, enhanced growth of embryogenic calli and plant regeneration rate in quite a few wheat genotypes. At 50 mg L–1 AGP application level in callus induction medium plant regeneration rates of 8.49, 409.06 and 283.16% were achieved for Jimai 22(JM22), Jingdong 18(JD18) and Yangmai 18(YM18), respectively; whereas at 100 mg L–1 AGP level, CS(105.44%), Chuannong 16(CN16)(80.60%) and Ningchun 4(NC4)(62.87%) acted the best. Moreover CS(79.05%), JM22(7.55%), CN16(101.87%), YM18(365.56%), Yangmai 20(YM20)(10.48%), and CB301(187.40%) were more responsive to 0.005 ‰ of H2O2, and NC4(35.37%) obtained the highest shoot regeneration rates at 0.01 ‰ of H2O2. Overall, these two methods, inter-culture and AGP(or H2O2) application, can be further applied to wheat transgenic research.     

Study on Cell Suspension Culture of Floribunda Rose

Friable callus was induced when immature seeds of floribunda rose were inoculated on MS medium supplemented with 2,4-D 3.0 mg.L^-1. When transfered onto subculture media, friable callus developed into embryogenic callus, which was used to establish cell suspension lines. Cell suspensions had to be subcultured at a interval of 4-5 days at the first several culture cycles. The best subculturing cycle for the stable cell suspensions was 8-10 days. The best inoculum quantity was 1 mL PCV（Packed Cell Volume） per 40 mL culture fluid.     

Induction and Genetic Identification of Embryogenic Calli from Hybrids of Shatian Pummelo

Shatian pummelo （Citrus grandis L. Osbeck cv. Shatian） is an elite variety in China, and the regeneration of the embryogenic callus is difficult. Diploid Shatian pummelo was used as the female and crossed with the allotetraploid somatic hybrid NS （Nova Tangelo ＋ Succari Sweet orange）, [ （ C reticulata Blanco x C. paradisi Macf.） cv. Nova ＋ C sinensis L. Osbeck cv. Succari]. About 90 days after pollination, the embryos obtained from crosses were cultured on the solid media of MT ＋ ME （malt extraction, 500 mg L^-1） and MT ＋ GA3 （1 mg L^-1）. The embryogenic callus was initiated from the embryoids and plantlets＇ hypocotyls and could be subcultured. Flow cytometry and SSR analysis verified that the callus was from the triploid hybrids. The callus had embryogenesis capacity and produced a large number of embryoids on MT ＋Lactose （50 g L^-1） medium after being subcultured for two years. It is comparatively easier to obtain the callus from the hybrid embryo than from Shatian pummelo itself. The callus is valuable for the conservation and utilization of Shatian pummelo.     

Analysis of Gene Effect on Four Characters of Immature Embryo Culture in Maize

This study was to find the regularity in the hereditary variation for the main culturing characters of the immature embryo culture in maize. Two kinds of inbred-line, R18-599 （red） with very excellent embryo culturing capacity and R15 with very poor embryo culturing capacity, were used as P1 and P2 for obtaining six generations. By culturing immature embryos of the six generations, four culturing characters, namely embryonic callus induction efficiency, nonembryonic callus induction efficiency, cloning ability of the embryonic callus, and number of regenerating plants, were analyzed using the general mean analysis and generation joint analysis. Results showed that the embryonic callus induction efficiency accorded with two major additive-dominance-epistatic genes and polygene-mixed additive-dominance-epistatic inheritance model. The induction efficiency of the nonembryonic callus accorded with two major additive-dominance-epistatic genes. The number of regenerating plants accorded with one major gene and polygene-mixed additive-dominance inheritance model. The cloning ability of the embryo callus accorded with two major genes and polygene-mixed inheritance model, whereas the effect of epistatic gene on this character was identified results of the two methods, generation joint analysis may genetic information. to be different using the two methods. By comparison of the not only raise experimental precision but also provide more