Rapid Generation of Selectable Marker-Free Transgenic Rice with Three Target Genes by Co-Transformation and Anther Culture

The ＇double T-DNA＇ binary vector p13HSR which harbored two independent T-DNAs, containing hygromycin phosphotransferase gene （hpf） in one T-DNA region and three target genes （hLF, SB401, RZ10） in another T-DNA region, was used to generate selectable marker-free transgenic rice by Agrobacterium-mediated transformation. The regenerated plants with both the three target genes and the selectable marker gene hpt were selected for anther culture. RT-PCR analysis indicated that target genes were inserted in rice genomic DNA and successfully transcribed. It took only one year to obtain double haploid selectable marker-free transgenic plants containing the three target genes with co-transformation followed by anther culture technique, and the efficiency was 12.2%. It was also noted that one or two target genes derived from the binary vector were lost in some transgenic rice plants.

Effect of ^(137)Cs Gamma Rays to Panicles on Rice Anther Culture

Panicles of an indica rice line TM7-5 were subjected to radiation with 137^Cs gamma rays at 0 （control）, 5, 10, 15 and 20 Gy respectively, and then its anthers were cultured. There were slight differences among the treatments in peak emerging time of callus initiation, from 38 to 44 days after inoculation （DAI） as well as the frequency of callus initiation （2.3-3.5%）. About two thirds calli were induced before 44 DAI, and calli derived beyond 60 DAI lost the regeneration ability. Green plant regeneration frequency was significantly stimulated from two- to three-fold by irradiation of the 1370S gamma rays compared with the control, and the maximum was 22,81% （15 Gy）. The culture ability based on callus initiation and green plantlet regeneration was 0.19% for the control while it was over 0.45% for all the irradiated treatments, and the maximum was 0,59% for 15 Gy treatment. The advantages of panicle irradiation before anther culture and the potential application in rice anther culture, especially for recalcitrant indica rice, were discussed.

Preliminary Study on Anther in vitro Culture of Lily“Prato”

The effects of the microspore developmental stage,hormones and culture condition on anther in vitro culture of lily（Lilium spp.） were discussed.The results showed that when the flower buds were about 23-26 mm long,the microspores were at the uninucleate stage which was suitable for culture and the culture under the darkness would promote the callus induction of anther.The induction frequency could reach 42.5% in the optimized medium which was MS＋[6-BA（0.5）＋KT（2.0）＋2,4-D（1.0）] mg·L-1.The rate of callus differentiation could reach 31.57% in the optimized medium which was MS＋ NAA（1.5,2.0） mg·L-1.

Anther Culture and Plant Regeneration of Tetraploid Purple Coneflower (Echinacea purpurea L.)

Anthersisolated from tetraploid purple coneflowerplants were cultured in vitro. The highest callus induction rate was obtained when the medium was consisted of N6 basal elements, 4% sucrose, 0.5 mg?L?1 BA, and 0.10 mg?L?1 NAA. Various morphogenesis such as globular, heart-shape, torpedo-shapeand final state embryos as well asvarious texture calluses around were observed. Out of 110 plantlets regenerated, 104 were confirmed as diploid and the rest were as tetraploid. Plants of one diploid offspring strain presented aspecialcharacter in pot: unlike the original tetraploid plants, it grown tubular, bisexual ray florets. The results obtained in the present studies indicated that although the tetraploid purple coneflower plants produced only diploid microspores, the recovery of some useful mutants through in vitro anther cultures might be reasonably expected.

Anther culture of Transgenic rice plants

Anther culture is widely used in rice improve-ment and genome research.It is useful forgene transformation to stabilize foreign gene(s)and estimate the integrated copy number.We used two transgenic plants JB-3 andJB-4 as donors for anther culture.Their originwas a japonica cultivar Jingyin 119,whose im-mature embryos were transformed by particlebombardment with plasmid pCB1(a cecropin B

Effect of agar concentration in media on green plantlet regeneration frequency in rice anther culture

We studied the effect of agar concentration inmedia on callus induction rate and green plant-let regeneration frequency in rice.Materialswere Fgeneration of indica/indica or indica/japonica,which were 96E76(Hei’e/Zhaiye- qing 8),96E80[(IR 24/Guanglu＇ai 4//Zhongnan’ai)/Yifengzao],96E86(Zhong- munong 9/Zhaiyeqing 8).The induction mediaused was M8+2mg/L 2,4-D,and agar con-centrations were 0.6％,0.8％,and 1.0％,respectively.Regeneration media was MS+2mg/L KT+0.5mg/L IAA+0.5mg/LNAA,and agar concentrations were 0.6％ and1.0％.Results indicated that the calli induc-

Gametic selection in a doubled haploid population derived from anther culture of indica/japonica hybrid of Oryza Sativa L.

It is one of the key problems for application ofanther culture in hybrid breeding, geneticanalysis, and molecular mapping whether thedoubled haploid (DH) population derived fromanther culture of rice crosses represents a ran-dom array of the microspore population, i.e.whether gametic selection occurs in androgene-sis. A DH population including 132 lines de-

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 BAP （0.5 mg·L^-1）, KT （1.0 mg·L^-1）, IAA （0.25mg·L^-1） and NAA （0.5 mg·L^-1） for plant regeneration, the frequency of regeneration ranged from 0 to 54.17%, and the highest differentiation index reached 30.5 after sub-cultured for three times; the plants were rooted after transferred to 1/2MS medium with NAA （0.5 mg·L^-1） and IAA （0.5mg·L^-1）.

Exceptionally high genetic variance of the doubled haploid(DH)population of poplar

Doubled haploid(DH)plants have been widely used for breeding and biological research in crops.Pop ulus spp.have been used as model woody plant species for biological research.However,the induction of DH poplar plants is onerous,and limited biological or breeding work has been carried out on DH individuals or populations.In this study,we provide an effective protocol for poplar haploid induction based on an anther culture method.A total of 96 whole DH plant lines were obtained using an F1hybrid of Populus simonii×P.nigra as a donor tree.The phenotypes of the DH population showed exceptionally high variance when compared to those of half-sib progeny of the donor tree.Each DH line displayed distinct features compared to those of the other DH lines or the donor tree.Additionally,some excellent homozygous lines have the potential to be model plants in genetic and breeding studies.

Factors Affecting Embryogenic Callus Production and Plant Regeneration in Anther Culture of Bupleurum chinense

Objective To evaluate the influences of the genotypes,anther developmental stages,and cultural conditions on the efficiency of embryogenic callus induction and plant regeneration in the anthers culture of Bupleurum chinense.Methods The different effects such as four genotypes,plant growth regulators,and temperature condition were compared in the experiments.The histological study was performed with the process of the anther culture.Results The highest inducing rate of embryogenic calli were achieved for the genotypes Zhongcaiyihao(ZCYH),Z4,and Z5 at the early-to middle-uninucleate stages,except for genotype ZPM1 at the tetrad stage.Cold pretreatment increased the production of the embryogenic callus,in which 4-day cold pretreatment improved the production of embryogenic callus from 0% to 2.2% and 5.0% for genotypes ZPM1 and ZCYH,respectively.No embryogenic callus was induced in the medium containing less than 0.75 mg/L 2,4-dichlorophenoxyacetic acid(2,4-D) .The highest regeneration rate(34.6%) was obtained in 1/2 MS salts regeneration medium supplemented with 0.1 mg/L 6-benzylmaminopurine(BA) .The low concentration of BA was able to promote the embryogenic callus formation and subsequent plantlet regeneration via somatic embryogenesis.Chromosome counting of regenerated plantlets showed mostly diploid plant(2n = 12) with only one haploid plant(n = 6) .Because of the low rate of microspore embryo formation,we only tracked the process of embryogenesis from the connective tissue,instead of microspore by histological observations.Conclusion This study establishes an efficient system for embryogenic callus induction and plant regeneration system.This is the first report on the haploid plantlet through the anther culture of B.chinense.

Genetic Homozygosis of Resistance of Common Wild Rice (Oryza rufipogon) against Brown Planthopper

[ Objective] The stability of resistance heredity of common wild rice against brown planthopper was studied to screen wild rice germplasm with stable resistance, so as to provide resources for rice breeding with resistance against brown planthopper. [ Method] A total of 1 591 accessions of common wild rice, collected from 46 proterozoic growth locations of Guangxi, were screened to analyze resistance against brown planthopper, and genetic homozygosis in their offspring was detected using bag-selfing or anther culture methods. [ Result] The results showed that most of common wild rice germplasm were heterozygotic, and growth habit and awn of their first self-bred generation showed segregation. Among 1 591 accessions of common wild rice, only 30 accessions showed third to fifth grades of resist- ance against brown planthopper, and most of them showed fifth grade of resistance. Among selfing offspring of 11 resistant materials, the populations from Z1 to Z3 showed segregation of resistant genes, while some materials showed stable resistance in Z4. Out of 11 accessions of materials, resistance of five accessions increased from third grade to first, second and third grades; four accessions increased from fifth grade to first, second and third grades ; two accessions increased from fifth to third grade. Anther culture results showed that 125 accessions of callus from independent origins were obtained after induction by in vitro culture of 6 000 anthers; eight clusters of diploid and two clusters of haploid green seedlings were differentiated, with induction rate of callus as 2.0% and differentiation rate of green seed- lings as 6.4%. Out of eight offspring of anther culture, six showed fifth grade of resistance against brown planthopper and two showed third grade of resistance. [ Conclusion] The stable highly-resistant resources could be obtained and resistance level of common wild rice could be enhanced through bag-selfing and multigeneration identification of resistance against brown planthopper. Anther culture is beneficial to promote genetic homozygosis of resistance of common wild rice against brown planthopper and shorten the screening time.

Development and Characterization of Elite Doubled Haploid Lines from Two Indica Rice Hybrids

Despite significant yield advantage over inbred rice, the adoption rate of hybrid rice in India is very low due to the high seed cost and poor quality of the produce. To alleviate the problem, we initiated a doubled haploid （DH） breeding approach to develop new lines from two elite indica rice hybrids （CRHR5 and CRHR7） through rapid fixation of homozygosity in the recombinants. In vitro culture of the rice anthers resulted in 243 and 186 fertile DH lines of CRHR5 and CRHR7, respectively. Flow cytometric and pollen fertility analyses confirmed the DH ploidy status of the regenerations. Morpho-agronomic evaluation revealed 100% uniformity and stability for all the characters in the DH lines of both hybrids. Nineteen promising DH lines of each hybrid were advanced to A2 generation for yield evaluation. The yield levels of the DH lines ranged from 5 097-6 965 kg/hm^2 for CRHR5 and 5 141-7 235 kg/hm^2 for CRHR7, which were at par or higher than the parental hybrids. Physico-chemical characterization and cooking quality analyses revealed significant and acceptable values for grain length and width, alkali spreading value, amylose content and water uptake ratio of the selected DH lines. Three DH lines, CR5-10, CR5-49, CR5-61 from CRHR5, and four DH lines, CR7-5, CR7-7, CR7-12 and CR7-52 from CRHR7, showed significant grain yield and quality characteristics and have been recommended for multi-location trials for subsequent release as new indica doubled haploid rice varieties.

Genetic Diversity Analysis of Elite Doubled Haploid Rice Genotypes for Yield Attributing Traits in White Nile State, Sudan

The development of new rice varieties is highly dependent on genetic diversity in desirable agronomic traits. Therefore, this study aimed to identify potential genotypes having the characters of Korean varieties (Tongil-type) and japonica developed through doubled haploid (DH) technology to apply in our breeding materials. 35 elite DH lines derived from another culture of Korean and African rice along with two local checks were planted in a randomized complete block design with three replications during the two seasons of 2019 and 2020. All evaluated genotypes exhibited a wide and significant variation in the ten measured traits. The highest heritability related to high genetic advance was recorded for the number of tiller/plant, grain yield t/ha, number of filled grain per panicle, and thousand grain weights (g). Genotypic coefficient of variation and genetic advance were recorded for number of tiller/plant, number of filled grain per panicle, 1000 grain weight and grain yield t/ha in both seasons. Moreover, there was a highly significant and positive correlation of grain yield with number of filled grain per panicle (0.65), number of tiller/plant (0.64) and number of panicle per m2 (0.54). Cluster analysis based on grain yield components trait grouped the 37 rice genotypes into four clusters. Cluster B was the largest and consisted of 13 genotypes. Finally, it could be concluded that, based on number of productive tillers, number of filled grain/panicle, number of panicle per m2, and grain yield, the lines KF170506, KF170509, KF170542, KF170530, KF170543, KF170500 and KF170510 were high potential for further selection for new type of irrigated rice. In addition, hybridization of these 7 high-yielding could be used to achieve higher heterosis among the genotypes. Furthermore, this evaluation could be useful in developing reliable selection indices for improving rice breeding programs.

CO2 Exchange and Chlorophyll Fluorescence of Phosphoenolpyruvate Carboxylase Transgenic Rice Pollen Lines

To elucidate the photosynthetic physiological characteristics and the physiological inherited traits of rice （Oryza sativa L.） hybrids and their parents, physiological indices of photosynthetic CO2 exchange and chlorophyll fluorescence parameters were measured in leaves of the maize phosphoenolpyruvate carboxylase （PEPC） transgenic rice as the male parent, sp. japonica rice cv. 9516 as the female parent, and the stable JAAS45 pollen line. The results revealed that the PEPC gene could be stably inherited and trans- ferred from the male parent to the JAAS45 pollen line. Moreover, the JAAS45 pollen line exhibited high levels of PEPC activity, manifesting higher saturated photosynthetic rates, photosynthetic apparent quantum yield （AQY）, photochemical efficiency of photosystem II and photochemical and non-photochemical quenching, which indicated that the JAAS45 pollen line has a high tolerance to photo-inhibition/photooxidation under strong light and high temperature. Furthermore, JAAS45 was confirmed to still be a C3 plant by δ^13C carbon isotope determination and was demonstrated to have a limited photosynthetic C4 microcycle by feeding with exogenous C4 primary products, such as oxaloacetate or malate, or phosphoenolpyruvate. The present study explains the physiological inherited properties of PEPC transgenic rice and provides an expectation for the integration of traditional breeding and biological technology.

Breeding Rice lines for physio-functional food through indica‘Zhaxima’×japonica‘Nanjing 46’haploid technique

Resistant starch(RS)encompasses those forms of starch which are not accessible to human digestive enzymes and are fermented in the colons producing short chain fatty acids.The plant materials containing RS are few in the world.In this contribution,the culture ability of callus from anthers of F1 plants from,landraces,‘Zhaxima’(Oryza sativa var.indica,high-RS rice line with 7.705±0.142,g/100 g)בNanjing 46’(Oryza sativa var.japonica,rice variety with RS content(g/100 g)of 0.200±0.001 crosses were studied for obtaining high RS rice plants.The results showed that when M8 basic induction medium was added with 1.5 mg/L 2,4-D、2 mg/LNAA and 0.3 mg/L KT,the inductivity of callus was high as 32.14%for 21 d after pretreatment at 4°C for 3 d;When MS differentiation basic medium was added with 2 mg/LKT and 3 mg/L ABA,the frequency of regeneration for callus was 50.3%with only a regeneration frequency of 4.55%grown into green seedlings.The RS content in the seeds was between those of the two parents and was partially normally distributed,the highest RS contents of the regenerated plants was as high as 7.66±1.197%.This produced an efficient technology for regenerating stable rice lines with high RS and good eating quality using anthers culture.