A rice exocyst subunit gene SEC3A is required for anther dehiscence

Anther dehiscence controls optimal interaction between pollen and stigma,thereby determining the successful sexual reproduction.The regulators or mechanisms of this process remain elusive.Here,two CRISPR/Cas9 mutants of a rice exocyst subunit gene SEC3A,sec3a-1 and sec3a-2,showed anther indehiscence at anthesis and male sterility at maturity.Pollen viability and germination in the mutants were partly defective,whereas their female gametes undergone a normal development.Hybrid or self-pollinated seeds could be produced by artificial pollination,suggesting potential use of a weak sec3a mutant as a female line during hybrid breeding.SEC3A is widely expressed in various tissues,including anther walls.Further results showed an excessive IAA accumulation and no endothecium lignification in sec3a-1/2 anthers.Our findings suggest that SEC3A appears to regulate anther dehiscence by modulating auxin signaling,providing insights into regulation of anther dehiscence and function of exocyst in plants.

R2R3-type Lo MYB21 affects jasmonate-regulated development and dehiscence of anthers in lily(Lilium oriental hybrids)

Lilies are widely cultivated for cut flowers,but their large anthers carry a considerable amount of colored pollen that is dispersed easily.Studying the molecular mechanism of anther development and dehiscence could help solve this problem.LoMYB21,encoding a putative R2R3v-myb avian myeloblastosis viral oncogene homolog(MYB)transcription factor,was identified from oriental lilies(Lilium‘Siberia’).Real-time quantitative PCR analysis showed that LoMYB21 was mainly expressed in the anther,filament and stigma and had high expression during the late stages of lily anther development.LoMYB21 had transactivation activity and was located in the nucleus through yeast one-hybrid assays and transient expression in Nicotiana benthamiana.Suppression of LoMYB21 by virus-induced gene silencing(VIGS)in Lilium‘Siberia’led to anther indehiscence and reduced the expression of genes related to Jasmonate acid(JA)biosynthesis and signal transduction.Induction of LoMYB21 in DEX::LoMYB21 transgenic Arabidopsis caused procumbent inflorescences that became infertile,accompanied by higher expression of JA biosynthetic and signaling genes.These results demonstrated that JA content and signaling were abnormal in silenced lily and transgenic LoMYB21 Arabidopsis,which affected anther development.Our study indicated that LoMYB21 could regulate lily anther dehiscence through JA biosynthesis and signaling during the late stages of anther development.

ZmMs33 promotes anther elongation via modulating cell elongation regulators,metabolic homeostasis,and cell wall remodeling in maize

Plant cell elongation depends on well-defined gene regulations,adequate nutrients,and timely cell wall modifications.Anther size is positively correlated with the number and viability of pollen grains,while little is known about molecular mechanisms underlying anther cell elongation.Here,we found that properly activated cell elongation regulators at transcriptional levels in loss-of-function ZmMs33 mutant(ms33-6038)anthers failed to promote maize anther elongation.ZmMs33 deficiency disrupted metabolic homeostasis mainly by inhibiting both photosynthesis in anther endothecium and lipid accumulation in anther tapetum.Importantly,ms33-6038 anthers displayed ectopic,premature and excessive secondary cell wall thickening in anther middle layer,which constrained cell elongation structurally and blocked nutrient flows across different anther wall layers.The metabolic disorder was only found in ms33-6038 mutant rather than several representative male-sterility lines at transcriptional and post-translational levels.Collectively,the disordered metabolisms and blocked nutrient flows defeated the activated cell elongation regulators,and finally inhibited anther elongation and growth with a unique‘‘idling effect”in ms33-6038 mutant.

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.

Study on Effect of Iron in Anther Media of Early Japonica Rice in Cold Region

In this study, through vitro culturing anthers of 7 F1 progenies of early Japonica rice in cold region on medium with different Fe2+ contents, it was found that Fe2+ content generated greater impacts on the induction rate and green plantlet differentiation. The result demonstrated that if Fe2+ increased from 32 to 40 mg/kg, the induction rate of early Japonica rice anther culture in N6 culture media was more then 1.4 times higher than that in N6 culture media containing 5.6 mg/kg Fe2+. In this concentration range, the induction rate increased with the increase of Fe2+ content, while if the concentration was over this concentration range, the induction rate decreased with the increase of Fe2+, showing single peak distribution. When the Fe2+ was 40 mg/kg in differentiation medium, the differentiation rate decreased dramatically. The green plantlet differentiations of callus which were induced on culture media containing 32-40 mg/kg Fe2+ were different, when they were cultured on MS culture media, and 85.7% materials could increase green plantlet productivity to about 7.8%. Therefore, increasing Fe2+in induction media properly could increase anther culture efficiency of early Japonica rice in cold region.

Immunohistochemical Analysis of IAA in Anthers of the Photoperiod-sensitive Genic Male-sterile Rice

The immunohistochemical localization of IAA and the comparison of their relative levels were carried out for the first time in the anthers of Nongken 58S and its wild type Nongken 58 (Oryza sativa subsp. japonica) after long_day and short_day treatments. The distribution of free_IAA in anthers and its dynamic variation could be reflected by this method. The results showed that the IAA level in the anthers of Nongken 58S after long_day treatment was much lower than that in short_day_treated Nongken 58S and those in wild type Nongken 58 in five stages from pistil and stamen primordia formation to late uninucleate stage. The possible reasons for IAA deficiency in Nongken 58S_LD anthers and its relationship with fertility alteration were also discussed.

Effects of Four Culture Media on Callus Induction Rate of Wheat Anther

Effects of four culture media including MS, N6, C17 and K on wheat anther callus induction in vitro culture were studied. The results showed that the callus in- duction rate of four kinds of culture medium was in the order of K〉C17〉N6〉MS.

Preliminary Study on the Anther Culture in Lycium barbarum L.

To investigate the culture technique in anther of Chinese wolfberry,we optimized the culture medium(including hormone combination)and culture conditions.The results showed that calluses were induced from all the six tested Chinese wolfberry materials,but the induction rate of callus varied toward the materials with different genotypes.When the experimental materials were cultured on medium appended with 2,4-D 1.0 mg/L and KT 1.0 mg/L under dark,the callus induction rate reached 20.0 % in this study,and this hormone combination should be the optimum for anther culture of Chinese wolfberry.With MS appended with 6-BA 0.5 mg/L and NAA 0.1 mg/L as differentiation medium and that appended with NAA 0.1 mg/L,the plants could be yielded in 20 days.

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.

Application of Plant Growth Regulator TDZ in Strawberry Anther Culture

[Objective] The aim was to investigate the application of plant growth regulator thidiazuron（TDZ）in strawberry anther culture.[Method]Different combinations of TDZ and naphthalene acetic acid（NAA）auxine with different concentrations were used for induction culture,and then its effect on callus induction and plant regeneration were observed.[Result] The combination of 1.0 mg/L TDZ＋0.5 mg/LNAA was effective to accelerating the differentiation and regeneration of strawberries,and the differentiation ratio was as high as 75%.However,TDZ showed insignificant effect on strawberries anther induction.[Conclusion]This research had provided reference for large-scale production of strawberry through anther culture.

Anther Culture about Hybrid F_1 by Solanum torvum SW.(♂) × Solanum melongena L.(♀)

Objective] This study was conducted to investigate the effects of different hormone combinations and concentrations on cal us induction and pIant regeneration of F1 hybrid of Solanum torvum SW. ＆#215; Solanum melongena L. [Method] Media suppIemented with different combinations and concentrations of hormones were pre-pared for anther cal us induction, proIiferation, differentiation and regeneration. [Re-sult] MS＋1.0 m/L 2, 4-D＋0.5 mg/L 6-BA was the optimum medium for anther cal us induction. MS＋1.0 mg/L KT＋0.1 mg/L NAA was the optimum medium for cal us proIiferation. MS＋2.0 mg/L 6-BA＋0.3 mg/L NAA was the optimum medium for in-duction of adventitious buds. MS＋0.3 mg/L IAA was the optimum medium for root-ing of adventitious buds. The highest rooting rate was onIy 37.5 because induction of rooting was a difficuIt work. [Conclusion] The resuIts wiI provide references for outcross fertiIity and genetic improvement of eggpIant.

Changes of Oxidative Stress and Soluble Sugar in Anthers Involve in Rice Pollen Abortion Under Drought Stress

Two rice maintaining lines with different drought tolerances, viz., Jin 23B （tolerant） and Zhenshan 97B （sensitive）, were used to study the oxidative stress and soluble sugar in rice anthers and pollen viability under drought stress during flowering stage. Higher antioxidant enzyme activities and lower malonaldehyde （MDA） content in rice anthers were observed in Jin 23B than in Zhenshan 97B under drought stress. Further, a great increase in the content of soluble sugar in rice anthers of Jin 23B was observed across the whole drought exposure, while Zhenshan 97B showed significant decrease in soluble sugar during 9-12 d after drought stress （DADS）. Accordingly, a marked decline of pollen fertility and activity, pollen numbers in an anther and pollen numbers on a stigma was observed in Zhenshan 97B, whereas little difference was found in Jin 23B. Thus, we suggest that pollen abortion caused by drought stress may be related with the reciprocity between oxidative stress and soluble sugar content in rice anthers.

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.

Maize MS2 encodes an ATP-binding cassette transporter that is essential for anther development

The anther cuticle and pollen exine play a critical role in male gametophyte development. The sporopollenin precursors and cuticular lipid monomers are transported to the surface of the microspores and the epidermis by lipid transport proteins(LTPs) and ATP-binding cassette G(ABCG) transporters for the formation of the pollen wall and anther cuticle, respectively. However, the function of ABCG transporters in maize anther development is unclear. Here, we cloned the MS2 gene from the maize male sterile2 mutant using map-based cloning and determined that it encodes an ABCG transporter. MS2 protein was experimentally confirmed to be located on the cell membrane. The quantitative real-time PCR(qRT-PCR)results showed that MS2 was ubiquitously expressed in all vegetative and reproductive tissues, whereas a high transcriptional level of MS2 was observed in anthers, especially at the young microspore stage. Gas chromatography-mass spectrometry(GC–MS) analysis showed decreased accumulation of cutin and wax components in ms2 anthers, indicating that MS2 plays a role in the transport of lipid molecules to anther cuticle and pollen exine. To our knowledge, MS2 is the first reported ABCG transporter gene that participates in anther development in maize.

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.

Identification and Characterization of a LEA-like Gene, CaMF5,Specifically Expressed in the Anthers of Male-fertile Capsicum annuum

To improve the understanding of molecular mechanisms of anther and/or pollen development in Chili pepper, in the present study, fulllength cDNA and DNA sequences of the pollen development-related gene CaMF5 were obtained from the anthers of a Capsicum annuum nuclear male-fertile line. Sequence analysis indicated that the full length of CaMF5 was 747 bp, containing a maximum opening reading frame of 447 bp.Amino acid sequence alignment and phylogenetic analysis revealed that CaMF5 shared approximately 37%–77% homology with a series of uncharacterized or hypothetical proteins and late embryogenesis abundant(LEA) proteins from other plants. However, no LEA structural domain was detected in CaMF5, which indicated that it might be a new type of LEA gene. CaMF5 was only expressed in flower buds at stages 7 and 8 and in open flowers of the male-fertile line, whereas it exhibited no expression in any examined organs of the male-sterile line. In addition, CaMF5 showed the highest transcript abundance in the anthers of the male-fertile line, with no expression being detected in any other examined organs, such as the sepals, petals, pistils, roots, stems, or leaves. Taken together, our results suggest that CaMF5 is an anther-specific gene that might encode a new type of LEA protein related to anther and/or pollen development in C. annuum.

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.

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

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

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.

Cytological Observation of Pollen Abortion of Lycium barbarum Haploids from Anther Culture

To reveal the sterility characteristics of Lycium barbarum haploids, cytological observations were made on the anthers of Ningqi No.1 and its haploids obtained from anther culture. The results showed that there were no significant differences in anther development between Ningqi No.1 and its haploids at the stage of pollen mother cell, and tetrads were formed successfully after the meiosis stage. The tetrads of Ningqi No.1 could release microspores. At the same time, the tapetal cells can provide nutrition for the development of the microspores, which eventually developed into mature pollen grains. Although the haploids could also release microspores at the tetrad stage, the tapetal cells degraded in advance, which made the released microspores unable to develop into mature pollen grains, resulting in pollen abortion of haploids.