Selection of culture conditions for callus induction and proliferation by somatic embryogenesis of Pinus koraiensis

The induction and proliferation of embryogenic callus are key steps for large-scale propagation of somatic embryogenesis pathway and long-term preservation of coniferous germplasm.Callus can be induced from immature embryos of Korean pine(Pinus koraiensis Sieb.et Zucc.;Pinaceae)as explants,but there are problems,such as low proliferation efficiency,loss of embryogenicity,poor vigor;thus,best conditions for proliferation and culture of immature embryos of Korean pine are not yet clear.To solve the problems with somatic embryogenesis of Korean pine and determine the best culture conditions for callus induction and proliferation,we varied hormone concentration,subculture cycle of proliferation and other plant growth regulators combinations in media to induce callus formation by megagametophytes of three Korean pine families at different developmental stages,then analyzed the effects on embryogenic callus retention and cell proliferation using a quadratic regression orthogonal rotation design.The results showed that the family origin and collection date of explants significantly affected callus induction(induction rate reached 1.67%).Embryogenic maintenance and callus proliferation were best on DCR medium supplemented with 0.25 mg L^(−1)6-benzyl adenine,1 mg L^(−1)naphthaleneacetic acid,30 g L^(−1)sucrose,500 mg L^(−1),L-glutamine,500 mg L^(−1)casein hydrolysis and 6.5 g L^(−1)agar.In addition,the combination of 2,4-dichlorophenoxyacetic acid+6-benzyl adenine also had a better proliferative effect on callus.The effects of different combinations of growth regulators on callus proliferation efficiency were significantly different.Transfer to new medium every 13–15 days not only maintained robust callus vigor,but also yielded a larger proliferation coefficient.The techniques and conditions for embryogenic callus induction and proliferation of Korean determined here will serve as a foundation for establishing a large-scale system for somatic embryogenesis and propagation of Korean pine.

Production of Embryogenic Callus and Plant Regeneration from Elite Guizhou Waxy Maize Inbred Lines

Immature embryos from three elite Guizhou waxy maize inbred lines （W21019, B7, and QCL5036） were evaluated for their ability of forming callus and regeneration into plants. Immature embryos harvested at different physiological stages were used as explants to initiate callus on N6 basal medium with 0-3.5 mg L-1 of 2,4-dichlorophenoxy acetic acid （2,4-D）. The concentration of 2,4-D, physiological age of immature embryos and genotype had a significant effect （P0.05） on the percentage of embryogenic callus formed. The optimum 2,4-D concentration for the initiation of embryogenic callus was varied among the waxy maize genotypes from 2.0 mg L-1 （B7 and QCL5036） to 3.0 mg L-1 （W21019）. The shoots were generated from embryogenic callus which were transferred into the regeneration medium supplemented with 0-2.5 mg L-1 of 6-benzylaminopurine （6-BA）. 6-BA in the medium significantly promoted the regeneration of embryogenic callus. Embryogenic size was also an important factor that affected regeneration capacity. 0.6-0.7 cm was proved to be the best size for regeneration from embryogenic callus and the mean number of shoots per primary callus in all genotypes achieved the highest number. The ability of the plant regeneration was also affected by genotype. W21019 had the highest number of shoots formed per primary embryogenic callus. With the optimum condition, the highest mean number of shoots per primary callus was up to 12.13, 5.73, and 3.33 in line W21019, B7, and QCL5036, respectively. The successful regeneration of the two inbred lines provides a basis for development of genetic transformation to improve priority traits such as enhanced insects and drought tolerance.

Improved Culture Media for Embryogenic Callus Generation in Sorghum [Sorghum bicolor (L.) Moench]

Many attempts on optimization of sorghum[Sorghum bicolor(L.)Moench]tissue culture induction media have been made,but the culture system remains with some bottlenecks compared to that of other crops.This study aimed at assessing the suitability of various induction media to produce embryogenic callus(yellow and friable)with high induction rates and reduced phenolic exudation.The six culture medium modifications:3 based on Murashige and Skoog(MS)medium and one each based on Chu N6,Gamborg B5 and 190-2 media respectively were applied in the culture of mature embryos from 10 sorghum genotypes.Although there was a genotype influence on the attainment of a yellow callus,friability of the callus was determined to be dependent on the culture medium and not the genotype.Half strength MS medium with 0.2 mg/l 2,4-D with 2.8 g/l Gelrite®as the gelling agent modified with 1.0 g/l KH_(2)PO_(4),1.0 g/l L-proline,1.0 g/l L-asparagine and 0.16 mg/l CuSO_(4)·5H_(2)O(type E)was found to be the most effective resulting in about 60%yellow coloured callus induction with 25%friability.Addition of CuSO_(4)·5H_(2)O,KH_(2)PO_(4),L-proline and L-asparagine significantly reduced the phenolic production.Half strength MS medium was observed to contribute to quality callus production when compared to full strength MS media modified with the compounds.The half strength MS medium was also observed to suppress phenolic production.Medium 190-2 produced the highest regeneration frequency(40%)among the 3-regeneration media tested.The results provide information on a suitable sorghum callus induction medium necessary for embryogenesis.

Effects of Auxins and Media on Callus Induction of Chinese Spring Wheat(Triticum aestivum L.)

The effects of auxins and media on callus induction from the mature and immature embryos of Chinese spring wheat （Triticum aestivum L.） varieties were investigated. It was found that genotype, medium, auxin source and concentration had the significant effects on the induction of embryogenic callus, explants germination and the increment of callus fresh weight. For immature embryos cultured on MS medium, 2 mg L^-1 of 2, 4-D was optimal, and the highest frequency of embryogenic callus （33.50%） was observed. For the mature embryos on N6 medium, 4 mg L^-1 of 2, 4-D was optimal. The frequency of embryogenic callus and increment of callus fresh weight on 2, 4, 5-T media were higher than those on 2, 4-D media, and in the presence of 2, 4, 5-T the precocious germination of explants for all genotypes were significantly suppressed. These results indicated that 2, 4, 5-T was superior to 2, 4-D and NAA in the culture of immature embryos. This is the first report about the effect of 2, 4, 5-T and NAA on wheat tissue culture, particularly in comparison with 2, 4-D in detail.

Efficient Somatic Embryogenesis and Plant Regeneration Through Callus Initiation From Seedling-derived Leaf Materials of Maize (Zea mays L.)

While being one of the world's most important crops,maize ( Zea mays L.) is still difficult to regenerate in tissue culture which severely limits its improvement by genetic engineering.Currently,immature zygotic embryos provide the predominantly used material for regeneration and transformation.However,the procedures involved are often laborious,time-consuming and season-dependent.Here,we further improved an efficient tissue culture and plant regeneration system that uses maize leaf segments of young seedlings as an alternative explant source.Embryogenic calli were evaluated by morphology,proliferation and regeneration capacity.All these indicated that seedling-derived leaf materials have the potential to replace immature embryos for tissue culture and regeneration.

Effect of Abscisic Acid on NaCI Stressed Callus Proliferation and Plant Regeneration in Rice

In rice, 2,4-Dichlorphenoxyacetic acid （MS2） has been considered best for callus induction and combination ofNAA, IAA, ABA （MS3） for somatic embryogenesis. Efficient plant regeneration was observed when MS basal salts supplied with 3% sorbitol and 3% maltose as carbon sources without hormones （MSac）. During this experiment, effect of sodium chloride （NaCl） and abscisic acid （ABA） was assessed on callus growth, plant regeneration and root induction efficiency of rice （Oryza sativa L.） varieties IR-6 and Basmati-370. Callus proliferation rate was highly decreased in both varieties on MS2b （100 mol.m3 NaCl ＋ 0.5 mg＇L1 ABA） than MS2a （100 mol＇m3 NaCl） cultures significantly. Proline, glycine-betaine and reducing sugars were increased significantly in MS2a and MS2b callusing cultures. However, total proteins were decreased in MS2a, while slightly increased in MS2b. Maximum plant regeneration （9.42 ±0.54 and 10.67 ±0.50 plantlets.callus1） from somatic embryos was observed on MS4c in IR-6 and Basmati-370, while 1.56 ± 0.06 （IR-6） and 0.95 ±0.05 （Basmati-370） plantlets callus1 were observed on MSab （100 mol·m3 NaCl）. No plant regeneration was observed on MS4b （100 mol·m3 NaCl ＋ 0.5 mg·L-1 ABA） medium in both varieties. Inhibition of root induction efficiency was high in MSsb （100 mol·m3 NaCl ＋ 0.5 mg·L-1 ABA） than MS5a （100 mol·m3 NaCl） in the stressed cultures （P 〈 0.05）. In this experiment, it was concluded that ABA involved in somatic embryogenesis and elevation of NaCl stress, while it causes inhibition of cell＇s growth as well as its regeneration into plantlets from somatic embryos.

Sugarcane Callus Culture and Cytological Characteristics

In order to obtain sugarcane embryogenic calli for transgenosis, the effects of different soaking time of explants, medium, culture conditions and sampling time on sugarcane callus culture were investigated. The results showed that soaking ex- plants with MS liquid medium for 20 min could significantly reduce the browning rate; MS＋3.0 mg/L 2.4-D was appropriate for induction and proliferation of embryo- genic calli; dark condition was suitable for the culture of sugarcane calli; calli differ- entiated from materials collected in August exhibited low browning rate, high callus induction rate and high embryogenic callus induction rate. Calli with different pheno- types were stained, prepared into slides and observed under a microscope to ana- lyze the cytological characteristics. The resultsshowed that milky-white granular em- bryogenic calli had closely arranged cells with large nucleus and exhibited strong differentiation capacity, which were appropriate receptor materials for genetic trans- formation of sugarcane.

Comparative transcriptome study provides insights into acquisition of embryogenic ability in upland cotton during somatic embryogenesis

Background： The conversion from non-embryogenic callus （NEC） to embryogenic callus （EC） is the key bottleneck step in regeneration of upland cotton （Gossypium hirsutum）, and hinders the transgenic breeding of upland cotton. To investigate molecular mechanisms underlying acquisition of embryogenic potential during this process, comparation analysis of transcriptome dynamics between two upland cotton cultivars with different somatic embryogenesis abilities was conducted. Results： Differentially expressed genes involved in the transformation from NEC to EC were detected in the two different cultivars. Principal component analysis based on DEGs showed that the NEC tissues of the two cultivars were highly heterogeneous, whereas the derived EC tissues were similar, which suggested the homogeneousness of EC between different lines. In the highly embryogenic cultivar CCRI 24, more of these genes were down-regulated, whereas, in the recalcitrant cultivar CCRI 12, more were up-regulated. Bioinformatics analysis on these DEGs showed that the vast majority of differentially expressed genes were enriched in metabolism and secondary metabolites biosynthesis pathways. Flavonoid biosynthesis and phenylpropanoid biosynthesis pathways were enriched in both cultivars, and the associated genes were down-regulated more in CCRI 24 than in CCRI 12. We deduced that vigorous secondary metabolism in CCRI 12 may hinder primary metabolism, resulting in tardiness of cell differentiation. Interestingly, genes involved in the plant hormone signal transduction pathway were enriched in the recalcitrant cultivar CCRI 12, but not in CCRI 24, suggesting more radical regulation of hormone signal transduction in the recalcitrant cultivar. Signal transduction rather than biosynthesis of plant hormones is more likely to be the determining factor triggering NEC to EC transition in recalcitrant cotton lines. Transcription factor encoding genes showed differential regulation between two cultivars. Conclusions： Our study provides valuable information about the molecular mechanism of conversion from NEC to EC in cotton and allows for identification of novel genes involved. By comparing transcriptome changes in transformation from NEC to EC between the two cultivars, we identified 46 transcripts that may contribute to initiating embryogenic shift.

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.

In vivo protective effect of late embryogenesis abundant protein(ApSK3 dehydrin)on Agapanthus praecox to promote post-cryopreservation survival

Dehydrins(DHNs),as members of the late embryogenesis abundant protein family,play critical roles in the protection of seeds from dehydration and plant adaptation to multiple abiotic stresses.Vitrification is a basic method in plant cryopreservation and is characterized by forming a glassy state to prevent lethal ice crystals produced during cryogenic storage.In this study,ApSK3 type DHN was genetically transformed into embryogenic calluses(EC)of Agapanthus praecox by overexpression(OE)and RNA interference(RNAi)techniques to evaluate the in vivo protective effect of DHNs during cryopreservation.The cell viability showed a completely opposite trend in OE and RNAi cell lines,the cell relative death ratio was decreased by 20.0%in ApSK3-OE EC and significantly increased by 66.15%in ApSK3-RNAi cells after cryopreservation.Overexpression of ApSK3 increased the content of non-enzymatic antioxidants(AsA and GSH)and up-regulated the expression of CAT,SOD,POD,and GPX genes,while ApSK3-RNAi cells decreased antioxidant enzyme activities and FeSOD,POD,and APX genes expression during cryopreservation.These findings suggest that ApSK3 affects ROS metabolism through chelating metal ions(Cu^(2+)and Fe^(3+)),alleviates H_(2)O_(2)and OH·excessive generation,activates the antioxidant system,and improves cellular REDOX balance and membrane lipid peroxidation damage of plant cells during cryopreservation.DHNs can effectively improve cell stress tolerance and have great potential for in vivo or in vitro applications in plant cryopreservation.

橡胶树热垦525胚性悬浮细胞系的建立及其胚性能力的维持

易碎胚性愈伤组织和胚性悬浮细胞系都是基因工程和细胞工程的理想受体材料。然而橡胶树(Hevea brasil-iensis)易碎胚性愈伤组织诱导频率低、耗时长,且其胚性能力通常随继代次数的增加而下降甚至完全丧失,限制了相关研究的持续开展。因此,快速获得易碎胚性愈伤组织,建立具有高效体胚发生能力的胚性悬浮细胞系,并尽可能较长时间维持其胚性能力是当前橡胶树基因工程和细胞工程研究的重要内容。本研究以橡胶树热垦525未成熟花药为外植体进行愈伤组织的诱导和胚性悬浮细胞系的建立,分析比较固液2种长期继代方式对维持其胚性能力的影响。结果表明:花药外植体在愈伤诱导培养基中获得的黄色致密初代愈伤组织体胚发生能力低,分散性差,不适合进行悬浮培养。将初代愈伤组织转移至胚性愈伤组织诱导培养基中培养70~80 d后,可观察到有胚性结构的形成和早期体细胞胚发生,同时周围长出鲜黄色小颗粒状易碎胚性愈伤组织。通过常规方法建立的Ⅰ型胚性悬浮细胞系具备胚性细胞的典型特征,体胚发生能力显著高于胚性愈伤组织,但在体胚发生过程中容易重新愈伤化;而通过筛选特定形态的胚性愈伤组织低密度启动悬浮培养建立的Ⅱ型胚性悬浮细胞系,在显微镜下可观察到细胞处在有序的胚性结构中,其体胚发生频率可达100%。在含2 mg/L2,4-D的液体培养中持续继代2 a后细胞明显老化且增殖缓慢,体胚发生能力几近丧失;而在去除2,4-D并添加低浓度脱落酸(0.1 mg/L)和水解酪蛋白(0.5 g/L)的固体培养基上持续继代2 a后,体胚发生能力仍能维持在较高水平。所建立的Ⅱ型胚性悬浮细胞系可为橡胶树遗传转化及原生质体培养等相关研究长期提供优质充足且状态相对稳定的材料来源。

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.

抗性湿地松体细胞胚的发育、成熟及萌发

为加快抗松针褐斑病湿地松体细胞胚胎发生技术的产业化应用进程,本文研究了肌醇浓度、脱落酸(ABA)及其添加方式、基本培养基、聚乙二醇(PEG-6000)浓度、琼脂糖种类及浓度、活性炭浓度、液体悬浮培养等因素对抗性湿地松体细胞胚的发育、成熟及萌发的影响。结果表明:添加8 g/L肌醇较适宜,过高浓度的肌醇则不利于湿地松体细胞胚发育;ABA可以采用高压灭菌;基本培养基、ABA、PEG-6000互作对湿地松体细胞胚的成熟影响很大,基本培养基以LP为最佳,ABA的最适浓度为10m g/L,PEG-6000以添加25、50 g/L为宜;在湿地松体细胞胚的成熟培养过程中,PEG-6000的浓度不能高于100 g/L;在湿地松体细胞胚的成熟培养基中,添加60 g/L蔗糖最合适;湿地松体细胞胚的成熟培养基中添加0.5 g/L或1 g/L的活性炭较适宜;培养基状态对体细胞胚的高频率诱导具有一定的影响,悬浮培养以100 mL三角瓶装培养液30 mL、摇床转速130 r/min为宜,液体转固体时吸取培养液1 mL为宜;未建立成熟的体细胞胚发生体系前,以采用固体培养为佳。最佳成熟培养基、激素及部分添加物组合为LP+10 mg/L ABA+50 g/L PEG-6000+60 g/L蔗糖+1.0 g/L活性炭。

秋石斛胚性愈伤组织诱导及再生体系的建立

秋石斛(Phalaenopsis-hybrid Dendrobium)花姿优美、花色丰富,是热区重要观赏植物,具有极高的产业价值。本研究以秋石斛杂交种迷你(Den.Yaya Victoria)、水蜜桃(Den.Swirl)、三亚阳光(Den.Sonia Hiasakul)原球茎和无菌苗幼嫩茎段为外植体,探讨不同植物生长调节剂组合对不同品种秋石斛胚性愈伤诱导、不定芽分化及植株再生的影响。结果表明:在胚性愈伤组织诱导阶段,原球茎和生长2个月的幼苗茎段作为外植体诱导效果最佳,暗培养30 d即可获得胚性愈伤。3个品种的愈伤组织诱导最佳培养基是以MSD(MS+30 g/L葡萄糖+1 g/L花宝1号+8 g/L琼脂)为基本培养基,添加0.5 mg/L KT和0.2~0.5 mg/L 2,4-D。迷你、水蜜桃、三亚阳光的胚性愈伤诱导率最高分别为31.11%、22.78%和50%,其中迷你品种仅需15 d即可获得胚性愈伤。最佳胚性愈伤增殖培养基为MSD+0.5 mg/L IBA+0.5 mg/L KT,胚性愈伤状态保持良好且不发生分化,增殖倍数0.9。最佳分化培养基为1/2 MS+0.5 mg/L IBA+0.15 mg/L KT,在光照条件下,接种后的胚性愈伤组织能够快速诱导形成芽点,30 d内分化形成2~3片叶的幼苗,相同培养基中保持60 d能够自行生根获得完整再生苗。最佳生根培养基为1/2 MS+30 g/L蔗糖+0.5 mg/L NAA+8 g/L琼脂,生根率为100%,平均生根数5条,根长约1.3 cm。本研究所使用的胚性愈伤组织诱导、增殖和分化培养基具有一定的广谱性,可为后续秋石斛胚再生途径及分子育种研究奠定基础。

葡萄VvMET1-1基因在葡萄和拟南芥中的功能研究

DNA甲基化修饰对于植物的生长发育十分重要,而MET1是植物中重要的甲基转移酶,能维持植物的CG位点甲基化修饰。为了研究葡萄MET1基因的功能及对植物发育的影响,将葡萄VvMET1-1基因转入葡萄胚性愈伤组织和拟南芥中进行研究。本研究选择35S::VvMET1-1-GFP表达载体转入葡萄胚性愈伤组织后检测到明显的GFP信号,且该胚性愈伤组织能被成功诱导为体细胞胚,表明VvMET1-1首次被成功转入葡萄胚性愈伤组织中并且不影响胚性愈伤组织的发育和分化。VvMET1-1被转入拟南芥中发现,该基因能影响拟南芥叶片的长度和宽度,但是不影响植株形态和种子发育;转录组数据分析表明,许多逆境胁迫、生长素、种子萌发、果实成熟、脱落酸和茉莉酸合成相关的基因差异表达;甲基化组结果显示,拟南芥过表达VvMET1-1后CG和CHH位点甲基化水平分别发生了明显的上调和下调,且DNA甲基化差异区(DMR)的类型主要是CG和CHH型,因此推测VvMET1-1可能通过调控DNA甲基化水平的变化影响下游基因的表达,进而调控拟南芥的生长发育。

生长素和细胞分裂素诱导植物胚性愈伤组织研究进展

植物胚性愈伤组织在植物研究与应用中是一种重要的试材,有很多关于植物胚性愈伤组织诱导方法研究的报道。生长素和细胞分裂素被广泛应用于胚性愈伤组织的诱导,是胚性愈伤组织诱导的关键因子。阐述了生长素和细胞分裂素在植物胚性愈伤组织诱导及体细胞胚胎发生过程中的作用,讨论了其在植物胚性愈伤组织诱导中的作用、使用方法与原则,为愈伤组织诱导、愈伤组织胚性化诱导等研究中植物生长调节剂的使用提供依据。

黄瓜胚性愈伤组织的诱导保存和再生

【目的】对黄瓜胚性愈伤组织的诱导保存和再生进行研究,为黄瓜高频率遗传转化奠定基础。【方法】以欧洲温室型黄瓜自交系14-1子叶节为外植体,在MS培养基上附加1.5 mg/L 2,4-D,进行25 d的胚性愈伤组织诱导培养后,取胚性愈伤组织在添加30,60,90,100,110,120,130,140和150 g/L蔗糖及1.5 mg/L 2,4-D的MS培养基进行继代培养,每30 d继代1次,观察胚性愈伤组织的褐变情况及胚性分化能力,并用电子天平在超净工作台中记录胚性愈伤组织质量的变化。继代培养60 d后,将保存的胚性愈伤组织和体细胞胚移至含1.5 mg/L 2,4-D的MS培养基上,待出现体细胞胚后移至MS培养基进行萌发,观察再生小植株的生长情况。【结果】将欧洲温室型黄瓜自交系14-1的子叶节,接种到附加1.5 mg/L 2,4-D的MS培养基上进行诱导培养后,子叶节一端的愈伤组织集中聚集于下胚轴处,之后有黄色胚性愈伤组织产生。在继代培养过程中,当培养基中添加的蔗糖为60~150 g/L时,胚性愈伤组织能保持胚性愈伤状态达60 d。之后将继代培养60 d后的胚性愈伤组织转接至附加1.5 mg/L 2,4-D的MS培养基上,在蔗糖质量浓度为60 g/L条件下保存的胚性愈伤组织可诱导出正常胚状体,且能形成健康小植株。【结论】由黄瓜子叶节诱导出的胚性愈伤组织可在MS+60 g/L蔗糖的培养基上保存达60 d,之后能正常萌发形成胚状体,进而形成正常小植株。

低温处理对红松胚性愈伤组织诱导生理机制的影响

为探究低温预处理对红松胚性愈伤组织诱导及生理机制的影响,采用红松(Pinus koraiensis)未成熟种子为外植体,探究低温预处理时间对胚性愈伤组织诱导率、未成熟种子中抗氧化酶活性、贮藏物质及植物激素质量分数的影响。结果表明:经4℃低温预处理21~28 d,对提高红松未成熟种子的胚性愈伤组织诱导率效果最佳,其中,2号家系经4℃低温预处理28 d,胚性愈伤组织诱导率可达25.00%,5号家系经4℃低温预处理21 d,胚性愈伤组织诱导率可达14.00%。经低温预处理后,未成熟种子中的淀粉、可溶性糖和可溶性蛋白质量分数和过氧化氢酶(CAT)活性随预处理时间的延长呈下降趋势,而超氧化物歧化酶(SOD)和过氧化物酶(POD)的活性则显著上升(P<0.05)。同时,4℃低温预处理21~28 d显著提高了红松未成熟种子中玉米素核苷(ZR)质量分数和生长素(IAA)质量摩尔浓度,降低脱落酸(ABA)质量分数。相关性分析结果表明,胚性愈伤组织诱导率与超氧化物歧化酶活性、过氧化物酶活性、玉米素核苷质量分数和生长素质量摩尔浓度呈极显著正相关,与脱落酸质量分数呈极显著负相关(P<0.01)。因此,低温处理主要通过调控红松未成熟种子中抗氧化酶活性及植物激素水平,促进胚性愈伤组织诱导能力。

日本落叶松瞬时转化体系的优化及初步应用

[目的]优化农杆菌介导的日本落叶松胚性愈伤组织瞬时转化体系。[方法]以液体增殖培养7 d的日本落叶松胚性愈伤组织为受体材料,利用携带β-葡糖醛酸酶基因(GUS)的pCAMBIA1305.1载体进行瞬时转化,根据GUS的表达量及酶活性,筛选最佳侵染液浓度、侵染时间和共培养时间。并利用筛选出的转化体系,分析落叶松scarecrow-like 6(LaSCL6)启动子的活性。[结果]瞬时转化后,GUS表达明显。当侵染液浓度OD600为0.2,侵染5 min,共培养72 h时,GUS的表达量最高,△CT值为-2.274 2;当侵染液浓度OD600为0.05,侵染5 min,共培养72 h时,GUS酶活性最高,为25.728 6 U·L^(-1)。LaSCL6启动子的活性是CaMV35S启动子的1.55倍。[结论]综合考虑GUS的表达量和酶活性,当侵染液浓度OD600为0.05,侵染5 min,共培养24 h时,GUS的表达量和酶活性较高,这一条件可以用来进行日本落叶松胚性愈伤组织的高效转化。

柑橘不同倍性种质胚性愈伤组织诱导及无病毒植株再生

【目的】通过离体培养败育胚珠诱导柑橘二倍体和四倍体种质的胚性愈伤组织,为柑橘多倍体研究提供成对离体材料;继续诱导胚性愈伤组织再生无毒种苗实现柑橘无核品种提纯复壮。【方法】从柑橘成熟果实中挑取败育胚珠,接种于3种愈伤诱导培养基中,在离体培养过程中诱导并驯化胚性愈伤组织;再诱导愈伤组织分化为胚状体和不定芽,将不定芽嫁接到枳橙砧木形成完整植株;通过InDel标记和病毒检测鉴定再生植株的遗传来源和脱毒效果。【结果】离体培养诱导出13个柑橘种质的胚性愈伤组织,其中8个愈伤组织完成驯化,分别为红橘二倍体/四倍体(2x/4x)、W.默科特橘橙2x/4x、鄂柑1号椪柑2x/4x、日辉橘2x和早红脐橙。基因型影响柑橘胚性愈伤组织诱导效率,早红脐橙诱导率高达74.73%,而红橘诱导率仅6.85%;柑橘四倍体愈伤组织诱导率(0.56%~22.61%)低于对应二倍体(6.85%~37.75%)。3种不同培养基的胚性愈伤组织诱导效率不同,其中MGS培养基对温州蜜柑在内的5个品种的诱导率较高(14.13%~63.01%),MK培养基对温州蜜柑在内的4个品种的诱导率较低(21.62%~69.51%),但MK培养基对椪柑2x和红橘2x的诱导率分别为70.21%和17.31%。分别获得早红脐橙、伦晚脐橙、国庆1号温州蜜柑、大分4号温州蜜柑和兴津温州蜜柑5个无核品种的愈伤组织再生苗23、22、20、10和15株;嫁接嵌合体早红脐橙的愈伤组织再生苗经InDel标记鉴定实为罗伯逊脐橙;国庆1号温州蜜柑、伦晚脐橙和早红脐橙再生苗经PCR检测证明未感染黄脉病和衰退病病毒。【结论】柑橘胚性愈伤组织诱导率受品种基因型和培养基影响;同一品种的不同倍性种质,二倍体愈伤组织诱导率和胚状体发生率均较高;胚性愈伤组织再生植株实现了病毒完全脱除,是柑橘无核品种提纯复壮的有效手段。