Influential Factors of Restorer of New Cytoplasmic Male Sterile(NER) on Anther Culture

Some influential factors of anther culture were studied preliminarily by conducting anther culture of the restorers of new cytoplasmic male sterile （NER）. Several results were obtain from this experiment and they were listed as follow：① MS cultrure medium with such hormones as 2,4-D 2 mg/L,6-BA 0.5 mg/L, NAA 0.5 mg/L was the best suitable for callus induction of NER. ②The difference of induction rate was significantly different between different plant age groups. From the 110th day to 141th day,the induction rate was increased with the increase of age and the difference of induction rate reached 0.01 significant difference level. The induction rate reached the highest value in the 141th day then it declined gradually. ③The combined use of 2, 4-D and 6-BA with proper increase of 2,4-D was good for inducing callus. ④The green plantlet induction rate of NER was increased when the concentration of 6-BA increased from 2 mg/L to 4 mg/L. Adding ZT from 0.5 mg/L to 2 mg/L. 6-BA would led 2.47% increase of green plantlet olantlet induction rate.

Influences of Carbon Sources and Plant Growth Regulators on Anther Culture Efficiency of Pepper

[Objective]In order to increase anther culture efficiency of pepper.[Method]MS culture media and Bolajiaohong were used in this experiment to study the influences of carbon sources and concentrations on anther callus induction of pepper.Jiayu was taken as a material to study influences of plant growth regulators and concentrations on anther callus induction of pepper according to L16（4^5） orthogonal design.[Result]The average callus and embryoid induction rates of maltose at all concentrations were higher than these of sucrose but the difference was not significant.Taking maltose or sucrose as a carbon source,3% to 6% concentration was good for increasing induction frequencies of calli and embryoids.However,If the concentration was over 6%,the induction rates were declined dramatically with the increase of sugar concentration.The influences of growth regulators on induction rate of calli were listed as 2,4-D﹥ZT﹥NAA﹥KT﹥6-BA;the influences on induction rates of embryoids were listed as 2,4-D﹥NAA﹥ZT﹥KT﹥6-BA.The 2,4-D,ZT,NAA and KT had signficant or extremely significant influences on induction rates of calli and embryoids.2,4-D,ZT at 1.0 mg/L and NNA,KT at 0.5 mg/L had the best effects.The influences of ZT on calli and embryoids were better than those of KT and 6-BA.1.0 mg/L 2,4-D ＋1.0 mg/L ZT ＋0.5 mg/L KT ＋0.5 mg/L 6-BA was the best regulator combination for induction culture of Jiayu anther.[Conclusion]The experiment provided research basis for anther culture of pepper.

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.

Pollen-embryogenesis and chromosomal variability in anther culture of Brassica hirta Moench (Sinapis alba L)

The anther cultures of Brassica hirta underwent pollenembryogenesis and callusing,which showed a wide range of chromosome numbers varying from 9 (n=12) to a highly polyploid.For embryogenesis,pretreatment of floral buds in 0.4 M sucrose solution for 72 hrs at 4℃ was superior to freshly cultured anthers.Culture temperature of 30℃ for 14 days before maintenance of cultures at 25℃ was significantly beneficial for embryo yield in comparison to cultures continuously incubated at 25℃.Dark treatment during culture was more effective for pollen-embryo yield.

Anther Culture of Chinese Radish(Raphanus sativus L. var. Longinnatus Bailey):Response of Different Genotypes to the Embryogenesis and the Traits of Regenerated Plantlets

[Objective] The aims were to ① conduct anther culture of Chinese radish varieties; ② observe the development of embryos from anther culture; ③ study the response of different genotypes to embryogenesis in anther culture; ④ observe the morphology of regenerated plantlets; ⑤ analyze the ploidy level of regenerated plantlets arising from the anther culture process. [Method]Anthers of 15 genotypes with diverse genetic backgrounds of Chinese radish have been cultured in vitro and induced to undergo embryogenesis and plant formation. [Result] Of 15 genotypes evaluated,four produced embryos. The genotype was the main factor to influence the embryogenesis. The morphology and the ploidy of the regenerated plantlets were observed,and the anther-derived plantlets included a mix of haploids,diploids and hexaploids. Of the plants that regenerated from anther embryos 60% were diploid. [Conclusion] The plantlets had the high ability to double spontaneously.

Effect of Growing Conditions of Rice Donor Plants on Anther Culture in Vitro

An experiment was conducted to find the effect of three types of donor plants growing conditions （growth chamber, open space ＋ growth chambers and open space） on callus induction and subsequent plant regeneration and productive shoot regeneration from the anthers culture in vitro of four rice hybrid （1-2, 2-1, 7-1, 13-3） developed by Primorsky Scientific Research Institute of Agriculture. Five variants of N6 medium （N6-I, N6-2, N6-3, Mix-l, N6-4） were used as basal medium. Mean value of callus induction frequency on three types of conditions ranged from 5.68% to 9.44% and the difference was non-significantly. In general, callus derived from donor plants grown on condition of open space ＋ growth chambers showed significantly better performances for plant regeneration （0.23 green regenerants on anther and 3.77 green regenerants on callus） and productive shoot regeneration （0.06 productive regenerants on anther and 0.56 productive regenerants on callus）. Favourable conditions for donor plant growth in open space positively affect on callus induction and regenaration. It is possible to get assured results on many hybrids, but not the highest. In growth chamber, frequency of callus induction can be the maximal only on some samples, few hybrids are resulted in deficiency of callus induction.

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-

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.

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.

Contributions of Chinese Botanists to Plant Tissue Culture in the 20th entury

This paper looks back to the development of plant tissue culture in China in the last century. Since 1934, tissue culture studies in China has kept up with the international development in the fields. Progress has been made by Chinese in nearly every branches of tissue culture, including in vitro organogenesis, shoot tip culture, anther culture, ovary culture, endosperm culture, protoplast culture as well as mass cell culture. On the basis of reviewing the articles written by Chinese on plant tissue culture, the internationally recognized contributions are specially mentioned. The applications of plant tissue culture to agriculture and industry in China are also introduced.

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.

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.

Genetic Analysis of Monosomic Alien Addition Line MAAL8 of O. officinalis(CC)-O. sativa(AA)

[Objective] By carrying out the anther culture on the monosomic alien addition line MAAL8 of O.officinalis-O.sativa and the back crossing with O.sativa H1493,the genetic characteristics of monosomic alien addition line were studied.[Method] The phenotype analysis was used to study the separation proportion of progeny.Moreover,SSR and the methylation analysis were used to study the transmission behavior of nonhomologous chromosome.[Result] 78 plants of 145 backcross progenies preserved the rolled leaf mark trait of MAAL8.In 32 anther culture plants,five plants had the normal rolled leaves,and two plants had the extremely rolled leaves.The rest had the flat leaves.14 couples of SSR markers were used to analyze,and it indicated that all the rolled-leaf plants could obtain the characteristic band type of O.officinalis,but the flat-leaf plants showed none of them.11 polymorphic RFLP markers were used to carry out Methylation-Sensitive Southern analysis.It showed that the methylation variation manners of eight markers between AA and CC genomes were different.[Conclusion] The nonhomologous chromosome of MAAL8 could pass to the progenies independently and integrally via the meiosis,and the phenotype characteristics didn＇t change.Moreover,the methylation manner of O.officinalis could inherit stably in the hybrid progeny as the addition of single chromosome.The stability of methylation might have the certain effect on the relatively independent inheritance of nonhomologous chromosome in the genome environment of O.sativa.

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 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.